Journal of Research in Biology Volume 3 Issue 3
Upcoming SlideShare
Loading in...5
×
 

Like this? Share it with your network

Share

Journal of Research in Biology Volume 3 Issue 3

on

  • 537 views

 

Statistics

Views

Total Views
537
Views on SlideShare
537
Embed Views
0

Actions

Likes
0
Downloads
2
Comments
0

0 Embeds 0

No embeds

Accessibility

Categories

Upload Details

Uploaded via as Adobe PDF

Usage Rights

CC Attribution-NonCommercial LicenseCC Attribution-NonCommercial License

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment

Journal of Research in Biology Volume 3 Issue 3 Document Transcript

  • 1. Journal of Research in Biology www.jresearchbiology.com An International Scientific Research Journal for Biology Volume 3 Issue 3 ISSN: 2231 –6280 EISSN: 2231- 6299
  • 2. Aim and Scope Journal of Research in Biology is an international scientific journal committed to the development and spread of research in Biological sciences. It accepts research articles with affiliation to biological science from all around the globe and publishes them in the journal. The submitted articles are peer-reviewed by experts in the field and editorial board members. Make the most of your research by publishing articles in Journal of Research in Biology. Journal of Research in Biology works as a portal for biological scientific research publication. It works to promote the use of biological sciences knowledge in the world public policy, and to develop and advance science policy that serves the needs of scientific research and education communities, particularly the biological sciences. The journal has been uniquely positioned to help members of the scientific community; become effective advocates for their science and to be better known for the public that relate to or impact the biological sciences. Call for Papers Journal of Research in Biology seeks Research Articles, Short Communications and Mini reviews. The Journal will accept and review submissions in English from any author, in any global locality. A body of international peers will review all submissions with potential author revisions as recommended by reviewers, with the intent to achieve published papers that: Relate to the field of Biology Represent new, previously unpublished work Advance the state of knowledge of the field Conform to a high standard of presentation. Disclaimer: Journal of Research in Biology is not responsible for the content of individual manuscripts. Manuscripts available in this journal were peer reviewed. Manuscripts accepted in the issues conform to the editorial policies. But more details regarding the nature of their research, conflicts in their workplace, plagiarisms, stealing of others property, manipulation of data, illegal formulation of a paper from other allied papers etc., were all not known to us. Any details, queries regarding the manuscripts should be only dealt with the authors and not with the publisher. The concept of peer review can only limit the plagiarism to a small extent where as it is the work of the public and the individuals to identify and stop the illegal formulation of new articles from the other. The publisher invites all details regarding the plagiarism of an article published in the journal provided with the original data and supplementary files for confirmation. On identifying plagiarism issues in an article, the article published will be removed from the journal website and further on the citation of the same will be debarred. Provided the author of the manuscript will be prohibited to publish his/her other studies in our journal or throughout the journals under our portal.
  • 3. List of Editors of Editors in the Journal of Research in Biology Managing and Executive Editor: Abiya Chelliah [Molecular Biology] Publisher, Journal of Research in Biology. Editorial Board Members: Ciccarese [Molecular Biology] Universita di Bari, Italy. Sathishkumar [Plant Biotechnologist] Bharathiar University. SUGANTHY [Entomologist] TNAU, Coimbatore. Elanchezhyan [Agriculture, Entomology] TNAU, Tirunelveli. Syed Mohsen Hosseini [Forestry & Ecology] Tarbiat Modares University (TMU), Iran. Dr. Ramesh. C. K [Plant Tissue Culture] Sahyadri Science College, Karnataka. Kamal Prasad Acharya [Conservation Biology] Norwegian University of Science and Technology (NTNU), Norway. Dr. Ajay Singh [Zoology] Gorakhpur University, Gorakhpur Dr. T. P. Mall [Ethnobotany and Plant pathoilogy] Kisan PG College, BAHRAICH Ramesh Chandra [Hydrobiology, Zoology] S.S.(P.G.)College, Shahjahanpur, India. Adarsh Pandey [Mycology and Plant Pathology] SS P.G.College, Shahjahanpur, India Hanan El-Sayed Mohamed Abd El-All Osman [Plant Ecology] Al-Azhar university, Egypt Ganga suresh [Microbiology] Sri Ram Nallamani Yadava College of Arts & Sciences, Tenkasi, India. T.P. Mall [Ethnobotany, Plant pathology] Kisan PG College,BAHRAICH, India. Mirza Hasanuzzaman [Agronomy, Weeds, Plant] Sher-e-Bangla Agricultural University, Bangladesh Mukesh Kumar Chaubey [Immunology, Zoology] Mahatma Gandhi Post Graduate College, Gorakhpur, India. N.K. Patel [Plant physiology & Ethno Botany] Sheth M.N.Science College, Patan, India. Kumudben Babulal Patel [Bird, Ecology] Gujarat, India. CHANDRAMOHAN [Biochemist] College of Applied Medical Sciences, King Saud University. B.C. Behera [Natural product and their Bioprospecting] Agharkar Research Institute, Pune, INDIA. Kuvalekar Aniket Arun [Biotechnology] Lecturer, Pune. Mohd. Kamil Usmani [Entomology, Insect taxonomy] Aligarh Muslim university, Aligarh, india. Dr. Lachhman Das Singla [Veterinary Parasitology] Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India. Vaclav Vetvicka [Immunomodulators and Breast Cancer] University of Louisville, Kentucky. José F. González-Maya [Conservation Biology] Laboratorio de ecología y conservación de fauna Silvestre, Instituto de Ecología, UNAM, México. Dr. Afreenish Hassan [Microbiology] Department of Pathology, Army Medical College, Rawalpindi, Pakistan. Gurjit Singh [Soil Science] Krishi Vigyan Kendra, Amritsar, Punjab, India. Dr. Marcela Pagano [Mycology] Universidade Federal de São João del-Rei, Brazil. Dr.Amit Baran Sharangi [Horticulture] BCKV (Agri University), West Bengal, INDIA. Dr. Bhargava [Melittopalynology] School of Chemical & Biotechnology, Sastra University, Tamilnadu, INDIA. Dr. Sri Lakshmi Sunitha Merla [Plant Biotechnology] Jawaharlal Technological University, Hyderabad. Dr. Mrs. Kaiser Jamil [Biotechnology] Bhagwan Mahavir Medical Research Centre, Hyderabad, India. Ahmed Mohammed El Naim [Agronomy] University of Kordofan, Elobeid-SUDAN. Dr. Zohair Rahemo [Parasitology] University of Mosul, Mosul,Iraq. Dr. Birendra Kumar [Breeding and Genetic improvement] Central Institute of Medicinal and Aromatic Plants, Lucknow, India. Dr. Sanjay M. Dave [Ornithology and Ecology] Hem. North Gujarat University, Patan. Dr. Nand Lal [Micropropagation Technology Development] C.S.J.M. University, India. Fábio M. da Costa [Biotechnology: Integrated pest control, genetics] Federal University of Rondônia, Brazil. Marcel Avramiuc [Biologist] Stefan cel Mare University of Suceava, Romania. Dr. Meera Srivastava [Hematology , Entomology] Govt. Dungar College, Bikaner. P. Gurusaravanan [Plant Biology ,Plant Biotechnology and Plant Science] School of Life Sciences, Bharathidasan University, India. Dr. Mrs Kavita Sharma [Botany] Arts and commerce girl’s college Raipur (C.G.), India. Suwattana Pruksasri [Enzyme technology, Biochemical Engineering] Silpakorn University, Thailand. Dr.Vishwas Balasaheb Sakhare [Reservoir Fisheries] Yogeshwari Mahavidyalaya, Ambajogai, India. Dr. Pankaj Sah [Environmental Science, Plant Ecology] Higher College of Technology (HCT), Al-Khuwair. Dr. Erkan Kalipci [Environmental Engineering] Selcuk University, Turkey. Dr Gajendra Pandurang Jagtap [Plant Pathology] College of Agriculture, India. Dr. Arun M. Chilke [Biochemistry, Enzymology, Histochemistry] Shree Shivaji Arts, Commerce & Science College, India. Dr. AC. Tangavelou [Biodiversity, Plant Taxonomy] Bio-Science Research Foundation, India. Nasroallah Moradi Kor [Animal Science] Razi University of Agricultural Sciences and Natural Resources, Iran T. Badal Singh [plant tissue culture] Panjab University, India
  • 4. Dr. Kalyan Chakraborti [Agriculture, Pomology, horticulture] AICRP on Sub-Tropical Fruits, Bidhan Chandra Krishi Viswavidyalaya, Kalyani, Nadia, West Bengal, India. Dr. Monanjali Bandyopadhyay [Farmlore, Traditional and indigenous practices, Ethno botany] V. C., Vidyasagar University, Midnapore. M.Sugumaran [Phytochemistry] Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram District. Prashanth N S [Public health, Medicine] Institute of Public Health, Bangalore. Tariq Aftab Department of Botany, Aligarh Muslim University, Aligarh, India. Manzoor Ahmad Shah Department of Botany, University of Kashmir, Srinagar, India. Syampungani Stephen School of Natural Resources, Copperbelt University, Kitwe, Zambia. Iheanyi Omezuruike OKONKO Department of Biochemistry & Microbiology, Lead City University, Ibadan, Nigeria. Sharangouda Patil Toxicology Laboratory, Bioenergetics & Environmental Sciences Division, National Institue of Animal Nutrition and Physiology (NIANP, ICAR), Adugodi, Bangalore. Jayapal Nandyal, Kurnool, Andrapradesh, India. T.S. Pathan [Aquatic toxicology and Fish biology] Department of Zoology, Kalikadevi Senior College, Shirur, India. Aparna Sarkar [Physiology and biochemistry] Amity Institute of Physiotherapy, Amity campus, Noida, INDIA. Dr. Amit Bandyopadhyay [Sports & Exercise Physiology] Department of Physiology, University of Calcutta, Kolkata, INDIA . Maruthi [Plant Biotechnology] Dept of Biotechnology, SDM College (Autonomous), Ujire Dakshina Kannada, India. Veeranna [Biotechnology] Dept of Biotechnology, SDM College (Autonomous), Ujire Dakshina Kannada, India. RAVI [Biotechnology & Bioinformatics] Department of Botany, Government Arts College, Coimbatore, India. Sadanand Mallappa Yamakanamardi [Zoology] Department of Zoology, University of Mysore, Mysore, India. Anoop Das [Ornithologist] Research Department of Zoology, MES Mampad College, Kerala, India. Dr. Satish Ambadas Bhalerao [Environmental Botany] Wilson College, Mumbai Rafael Gomez Kosky [Plant Biotechnology] Instituto de Biotecnología de las Plantas, Universidad Central de Las Villas Eudriano Costa [Aquatic Bioecology] IOUSP - Instituto Oceanográfico da Universidade de São Paulo, Brasil M. Bubesh Guptha [Wildlife Biologist] Wildlife Management Circle (WLMC), India Rajib Roychowdhury [Plant science] Centre for biotechnology visva-bharati, India. Dr. S.M.Gopinath [Environmental Biotechnology] Acharya Institute of Technology, Bangalore. Dr. U.S. Mahadeva Rao [Bio Chemistry] Universiti Sultan Zainal Abidin, Malaysia. Hérida Regina Nunes Salgado [Pharmacist] Unesp - Universidade Estadual Paulista, Brazil Mandava Venkata Basaveswara Rao [Chemistry] Krishna University, India. Dr. Mostafa Mohamed Rady [Agricultural Sciences] Fayoum University, Egypt. Dr. Hazim Jabbar Shah Ali [Poultry Science] College of Agriculture, University of Baghdad , Iraq. Danial Kahrizi [Plant Biotechnology, Plant Breeding,Genetics] Agronomy and Plant Breeding Dept., Razi University, Iran Dr. Houhun LI [Systematics of Microlepidoptera, Zoogeography, Coevolution, Forest protection] College of Life Sciences, Nankai University, China. María de la Concepción García Aguilar [Biology] Center for Scientific Research and Higher Education of Ensenada, B. C., Mexico Fernando Reboredo [Archaeobotany, Forestry, Ecophysiology] New University of Lisbon, Caparica, Portugal Dr. Pritam Chattopadhyay [Agricultural Biotech, Food Biotech, Plant Biotech] Visva-Bharati (a Central University), India
  • 5. Table of Contents (Volume 3 - Issue 3) Serial No Accession No Title of the article Page No 1 RA0328 An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern India. Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, Joydeb Majumder and Basant Kumar Agarwala. 852-860 2 RA0327 Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms. Ujowundu CO, Nwaogu LA, Igwe KO, Ujowundu FN, Belonwu DC 861-869 3 RA0167 Effect of age, sex and hemoglobin type on adaptive and blood biochemical characteristics in Red Sokoto Goats. Akpa GN, Alphonsus C and Usman N. 870-875 4 RA0245 Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with special reference to its life table attributes in Tripura, India. Samit Roy Choudhury and Basant Kumar Agarwala. 876-885 5 RA0329 Anti-inflammatory activity of lycopene isolated from Chlorella marina on carrageenan-induced rat paw edema. Renju GL and Muraleedhara Kurup G. 886-894 6 RA0332 Identification of animal Pasteurellosis by PCR assay. Venkatesan PS, Deecaraman M and Vijayalakshmi. 895-899 7 RA0345 Source of light emission in a luminous mycelium of the fungus Panellus stipticus. Puzyr Alexey, Burov Andrey and Bondar Vladimir. 900-905 8 RA0274 Local people’s attitude towards conservation and development around Pichavaram mangrove ecosystem, Tamil Nadu, India. Lakshmi Kodoth and Ramamoorthy D. 906-910 9 RA0318 Biodegradation of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria. Nwanyanwu CE and Abu GO. 911-921 10 RA0317 Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria. Nwanyanwu CE, Nweke CO, Orji JC and Opurum CC. 922-931 11 RA0337 Effect of Chromolaena odorata leaf extract on haematological profiles in Salmonellae typhi infested Wistar rats. Nwankpa P, Ezekwe AS, Ibegbulem CO and Egwurugwu JN. 932-939
  • 6. JournalofResearchinBiology An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern India Keywords: Avifauna, biodiversity hotspot, Agartala, Tripura, north-east India . ABSTRACT: North-east India is a part of Indo-Burma hotspot and among the richest bird zones in India. Tripura lies in the border of Indo-Burma global biodiversity hotspot area but is poorly covered by ornithological works. Avifauna of Tripura state is known by 277 species but there is lack of information about their distribution, particularly in and around Agartala city, which is the capital of Tripura state and is a tourist destination along the border of Bangladesh for its natural landscapes, inland water species, and strong presence of green flora. With a view to enhance its value for tourist attraction and naturalists, a study was conducted to record the species of birds that occur in and around the City. In the present study 73 bird species were recorded from Agartala city and its adjacent areas belonging to 41 families and 14 orders. 852-860 | JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Journal of Research in Biology An International Scientific Research Journal Authors: Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, Joydeb Majumder and Basant Kumar Agarwala. Institution: Ecology & Biodiversity Laboratories, Department of Zoology, Tripura University, Suryamaninagar-799 022, Tripura, India. Corresponding author: Basant Kumar Agarwala. Email: bagarwala00@gmail.com Web Address: http://jresearchbiology.com/ documents/RA0328.pdf. Dates: Received: 28 Jan 2013 Accepted: 15 Feb 2013 Published: 10 Apr 2013 Article Citation: Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, Joydeb Majumder and Basant Kumar Agarwala. An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern India. Journal of Research in Biology (2013) 3(3): 852-860 Journal of Research in Biology An International Scientific Research Journal Original Research
  • 7. INTRODUCTION Avifauna contributes most significantly to the diversity of terrestrial vertebrates, which have a special role in conservation of biodiversity of a particular area (Daniels, 1994). Birds are very good indicator of environmental changes as they respond in the minute change in habitat structure and composition (Robert et al., 2001). Indian subcontinent harbour nearly 1300 species of birds, which is more than 13% of total bird species of the world (Grimmet et al., 2004), and more than 60% of Indian birds are found in north-east India (Choudhury, 2010). North-east India is one of the most significant biodiversity hotspots of the world and among the richest bird zones in India because of convergence of the Indo-Malayan, Indo-Chinese and Indian biogeographical realms. As a result, it is unique in providing an abundance of habitats that harbour diverse biota with a high degree of endemism (Chatterjee et al., 2006; Narwade et al., 2011). Tripura (22°56´- 24°32´ N and 91°10´- 92°21´ E, with an area of 10,490 km2 ) is a small state of north-east India bounded by Bangladesh on three sides and with Assam and Mizoram on the other side. It lies in the border of Indo-Burma global biodiversity hotspot area (Myers et al., 2000) but very poorly covered by ornithological works (Choudhury, 2010). Although avifaunal checklist for Tripura state listed 277 species (Choudhury, 2010) but little is known regarding the bird species found in the vicinity of Agartala city, situated by international boundary of Bangladesh. STUDY SITES Agartala city is situated in the western region of Tripura state with the latitude of 23°45' North and longitude of 91°45' East and an average elevation of 20.36 m above sea level. It is the capital town of Tripura with a mix of urban and semi urban complex and a rich green cover. Forests and farms adjoin the town on three sides, and therefore, it is also called ‘Green City’. The total city area is 62.02 km2 and is delimited on the west side by international boundary with Bangladesh. Climatic condition is of tropical monsoon type with an average annual rainfall of 220 cm. Average minimum and maximum temperature recorded in the region are 6.8°C in January and 37.70°C in June, respectively. Present study was carried out in eleven different sites (viz., College Tilla lake area, Golbazar, Pratapgarh, Dashamighat, Arundhutinagar, Shanmura, Bhubanban, Barjala, Jagannath Bari lake area, G B Bazar and Nandannagar) (Table 1, Figure 1) covering different sides of Agartala city and its adjacent areas. METHODOLOGY The study sites were visited fortnightly throughout the study period from 2009-2011. Data on Bhattacharjee et al., 2013 853 Journal of Research in Biology (2013) 3(3): 852-860 Sl. No Sites Coordinates Altitude (m) 1. College Tilla lake area 23°49'35.45" N; 91°17'42.28" E 17 2. Golbazar 23°49'38.30" N ; 91°16'57.15" E 16 3. Pratapgarh 23°49'08.98" N ; 91°17'17.10" E 16 4. Dashamighat 23°49'46.34" N ; 91°15'51.45" E 16 5. Arundhutinagar 23°49'01.44" N ; 91°16'21.68" E 31 6. Shanmura 23°50'51.98" N ; 91°16'07.20" E 15 7. Bhubanban 23°51'56.50" N; 91°15'73.70" E 21 8. Barjala 23°52'05.05" N ; 91°16'32.13" E 23 9. Jagannath Bari lake area 23°50'05.43" N; 91°16'53.70" E 14 10. G B Bazar 23°51'33.74" N ; 91°17'33.97" E 27 11. Nandannagar 23°51'43.68" N ; 91°17'57.00" E 28 Table 1: Geo-coordinate details of the study sites
  • 8. present bird species were collected by direct observations with the help of binoculars (VISTA LE 8 X 40). Almost all the species mentioned in the checklist were photographed. For this purpose, digital cameras of Canon Power shot SX 200 IS (12 X Digital zoom), Cannon SRL EOS 50D and Panasonic Lumix DMC FZ 40 were used. In lake areas birds were observed from the bank, peripheral areas, and urban areas were surveyed on foot regularly. Farm and forested areas in the vicinity of the city were surveyed to record the assemblages of different bird species. Most of visits were made in morning and afternoon time when birds are most active. Identification of birds was based on the field guides produced by Ali and Ripley (1995), Ali (1996 and 2002) and Grimmett et al., (2003). Status of the birds was classified as C-Common, MC-Most common, NC-Not common, S-Singleton, W- Winter visitor. RESULTS AND DISCUSSION In the present study 73 bird species were recorded from Agartala city and its adjacent areas belonging to 41 families and 14 orders (Table 2, Plate 1 and 2). There is no authentic information about the avifauna of Tripura except that by Blyth (1845, 1846), Ali and Ripley (1968-74) and International Waterfowl and Wetlands Research Bureau on Asian Waterfowl Census, 1989 (Scott and Rose 1989). Majumdar et al., (2002) recorded 259 species of birds, belonging to 56 families and 16 orders. Recently Choudhury (2010) recorded 277 species of birds, in the annotated checklist from Tripura, but avifaunal diversity of Agartala city is not yet available. Out of 14 orders. Passeriformes was found dominant with 22 families followed by Coraciiformes with 3 families and Pelecaniformes and Piciformes with 2 families each. Dominance of Passeriformes was also recorded by Choudhury (2010) and Majumdar et al., (2002) from the state and from Nagpur district of central India (Chinchkhede and Kedar, 2012). The resident birds such as Pond heron, Cattle egret, Lapwing, Blue rock pigeon, Spotted dove, Parakeets, Asian koel, Kingfisher, Bee eater, Lineated barbet, Woodpecker, Bush lark, Bulbul, Shrike, Robin, Tailorbird, Cinereous tit, Sunbird, Sparrow, Starling, Myna, Oriole, Black drongo and Crow etc were found regularly throughout the study period. Little Cormorant, Asian Openbill-Stork, Black headed Ibis, Lesser Whistling Duck, Crested Serpent Eagle, Red Junglefowl, Red Collared Dove, Yellow-footed Green Pigeon, Brown Fish Owl, Asian Palm Swift, Indian Roller, Coppersmith Barbet, Orange headed Thrush, Blue Rock Thrush, White-rumped Shama, Scarlet-backed Flower pecker, Tricoloured Munia etc were found less common in this study. Common Sandpiper and Black headed Ibis were observed during the winter season only in the paddy fields of peripheral areas of the city. Common Hoopoe Bhattacharjee et al., 2013 Journal of Research in Biology (2013) 3(3): 852-860 854 Figure 1. Showing the study sites in and around Agartala City.
  • 9. Bhattacharjee et al., 2013 855 Journal of Research in Biology (2013) 3(3): 852-860 Sl. No. Common name Scientific name Status IUCN Abundance Cormorants [Phalacrocoracidae] 1. Little Cormorant Phalacrocorax niger Vieillot, 1817 LC NC Herons & Egrets [Ardeidae] 2. Indian Pond Heron Ardeola grayii (Sykes, 1832) LC MC 3. Cattle Egret Bubulcus ibis (Linnaeus, 1758) LC MC 4. Median Egret Mesophoyx intermedia (Wagler, 1827) LC C Storks [Ciconiidae] 5. Asian Openbill-Stork Anastomus oscitans Boddaert, 1783 LC NC 6. Black headed Ibis Threskiornis melanocephalus (Latham, 1790) NT W, NC Ducks [Anatidae] 7. Lesser Whistling Duck Dendrocygna javanica (Horsfield, 1821) LC NC Hawks & Eagles [Accipitridae] 8. Crested Serpent Eagle Spilornis cheela Latham, 1790 LC NC 9. Black Kite Milvus migrans (Boddaert, 1783) LC C Pheasants [Phasianidae] 10. Red Junglefowl Gallus gallus (Linnaeus, 1758) LC NC Rails & Coots [Rallidae] 11. White-breasted Waterhen Amaurornis phoenicurus Pennant, 1769 LC C Lapwings [Charadriidae] 12. Red-wattled Lapwing Vanellus indicus (Boddaert, 1783) LC MC Sandpipers [Scolopacidae] 13. Common Sandpiper Actitis hypoleucos (Linnaeus, 1758) LC W, C Pigeons & Doves [Columbidae] 14. Blue Rock Pigeon Columba livia Gmelin, 1789 LC MC 15. Spotted Dove Streptopelia chinensis (Scopoli, 1768) LC MC 16. Red Collared Dove Streptopelia tranquebarica (Hermann, 1804) LC NC 17. Orange-breasted Green Pigeon Treron bicinctus (Jerdon, 1840) LC C 18. Yellow-footed Green Pigeon Treron phoenicoptera (Latham, 1790) LC NC Parakeets [Psittacidae] 19. Rose-ringed Parakeet Psittacula krameri (Scopoli, 1769) LC C 20. Red-breasted Parakeet Psittacula alexandri (Linnaeus, 1758) LC C Cuckoos & Coucals [Cuculidae] 21. Asian Koel Eudynamys scolopaceus (Linnaeus, 1758) LC MC 22. Greater Coucal Centropus sinensis (Stephens, 1815) LC C Owls [Strigidae] 23. Collared Scops Owl Otus lettia Hodgson, 1836 LC S 24. Spotted Owlet Athene brama (Temminck, 1821) LC C 25. Brown Fish Owl Bubo zeylonensis (Gmelin, 1788) LC NC Table 2: List of birds in and around Agartala city during 2009-2011
  • 10. Bhattacharjee et al., 2013 Journal of Research in Biology (2013) 3(3): 852-860 856 Swifts [Apodidae] 26. Asian Palm Swift Cypsiurus balasiensis Gray, 1829 LC NC 27. House Swift Apus affinis (J E Gray, 1830) LC C Kingfishers [Alcedinidae] 28. Common Kingfisher Alcedo atthis (Linnaeus, 1758) LC MC 29. Stork-billed Kingfisher Halcyon capensis (Linnaeus, 1766) LC C 30. White-throated Kingfisher Halcyon smyrnensis (Linnaeus, 1758) LC MC Bee-eaters [Meropidae] 31. Little Green Bee-eater Merops orientalis Latham, 1802 LC MC Rollers [Coraciidae] 32. Indian Roller Coracias benghalensis (Linnaeus, 1758) LC NC Hoopoe [Upupidae] 33. Common Hoopoe Upupa epops Linnaeus, 1758 LC S Barbets [Capitonidae] 34. Lineated Barbet Megalaima lineata (Vieillot, 1816) LC MC 35. Coppersmith Barbet Megalaima haemacephala Muller, 1776 LC NC Woodpeckers [Picidae] 36. Rufous Woodpecker Celeus brachyurus (Vieillot, 1818) LC C 37. Greater Flameback Chrysocolaptes lucidus (Scopoli, 1786) LC C 38. Fulvous-breasted Woodpecker Dendrocopos macei (Vieillot, 1818) LC C Larks [Alaudidae] 39. Singing bush lark Mirafra cantillans Blyth, 1844 LC C Pipits & Wagtails [Motacillidae] 40. Paddy field Pipit Anthus rufulus Vieillot, 1818 LC C 41. White Wagtail Motacilla alba Linnaeus, 1758 LC W,C Bulbuls [Pycnonotidae] 42. Red-whiskered Bulbul Pycnonotus jocosus (Linnaeus, 1758) LC C 43. Red-vented Bulbul Pycnonotus cafer (Linnaeus, 1766) LC MC Loras [Irenidae] 44. Common Lora Aegithina tiphia (Linnaeus, 1758) LC C Shrikes [Laniidae] 45. Brown Shrike Lanius cristatus Linnaeus, 1758 LC W, MC 46. Grey-backed Shrike Lanius tephronotus (Vigors, 1831) LC W, C Thrushes [Turdidae] 47. Orange headed Thrush Zoothera citrina (Latham, 1790) LC NC Flycatchers [Muscicapidae] 48. Blue Rock Thrush Monticola solitarius (Linnaeus, 1758) LC NC 49. White-rumped Shama Copsychus malabaricus (Scopoli, 1786) LC NC 50. Oriental Magpie Robin Copsychus saularis (Linnaeus, 1758) LC MC
  • 11. Bhattacharjee et al., 2013 857 Journal of Research in Biology (2013) 3(3): 852-860 Babblers [Timaliidae] 51. Rufous-necked Laughing- thrush Garrulax ruficollis (Jardine & Selby, 1838) LC C Warblers [Sylviidae] 52. Common Tailorbird Orthotomus sutorius (Pennant, 1769) LC MC Flycatchers [Stenostiridae] 53. Grey-headed Canary- flycatcher Culicicapa ceylonensis (Swainson, 1820) LC W, C Tits [Paridae] 54. Cinereous Tit Parus cinereus Vieillot, 1818 LC MC Flowerpeckers [Dicaeidae] 55. Scarlet-backed Flowerpecker Dicaeum cruentatum (Linnaeus, 1758) LC NC Sunbirds [Nectariniidae] 56. Ruby-cheeked Sunbird Anthreptes singalensis (Gmelin, 1788) LC C 57. Purple-rumped Sunbird Nectarinia zeylonica (Linnaeus, 1766) LC C 58. Purple Sunbird Cinnyris asiaticus Latham, 1790 LC MC White-eyes [Zosteropidae] 59. Oriental White-eye Zosterops palpebrosus (Temminck, 1824) LC C Munias [Estrildidae] 60. Scaly-breasted Munia Lonchura punctulata (Linnaeus, 1758) LC C 61. Tricoloured Munia Lonchura malacca (Linnaeus, 1766) LC NC Sparrows [Passerinae] 62. House Sparrow Passer domesticus (Linnaeus, 1758) LC MC Weavers [Ploceidae] 63. Baya Weaver Ploceus philippinus (Linnaeus, 1766) LC C Starlings & Mynas [Sturnidae] 64. Chestnut-tailed Starling Sturnus malabaricus (Gmelin, 1789) LC C 65. Asian Pied Starling Gracupica contra (Linnaeus, 1758) LC MC 66. Common Myna Acridotheres tristis (Linnaeus, 1766) LC MC 67. Jungle Myna Acridotheres fuscus (Wagler, 1827) LC MC Orioles [Oriolidae] 68. Black-hooded Oriole Oriolus xanthornus (Linnaeus, 1758) LC MC Drongos [Dicruridae] 69. Black Drongo Dicrurus macrocercus (Vieillot, 1817) LC MC 70. Greater racket-tailed Drongo Dicrurus paradiseus Linnaeus, 1766 LC NC Crows & Treepie [Corvidae] 71. Rufous Treepie Dendrocitta vagabunda (Latham, 1790) LC C 72. House Crow Corvus splendens Vieillot, 1817 LC MC 73. Jungle Crow Corvus macrorhynchos Wagler, 1827 LC MC Abbreviations: Status: LC = least concern; NT = near threatened; C = common; MC = most common; NC = not common; S = singleton; W = winter visitor.
  • 12. Bhattacharjee et al., 2013 Journal of Research in Biology (2013) 3(3): 852-860 858 Plate 1. A-Lineated barbet, B-Cattle egret, C-Red-wattled Lapwing, D-Common Hoope, E-Little cormorant, F-Stripe-breasted woodpecker, G-Rufous woodpecker, H-White throated kingfisher, I-Yellow footed green pegion, J-Asian open bill stork, K-Chestnut-tailed starling, L-Collared scops owl. Plate 2: M-Asian Koel, N-Crested serpent eagle, O-Common Tailorbird, P-Cinereous Tit, Q-Emerald dove, R-Little green bee-eater, S-Grey-baked shrike, T-Indian pond heron, U-Red collared dove, V-White-rumped shama, W-Singing bush lark, X-Black headed ibis.
  • 13. and Collared Scops Owl were sighted only once in the two years study. Brown Shrike, Grey-backed Shrike, Grey-headed Canary-flycatcher were observed in the winter season only (Table 2), which corroborates with the findings of Choudhury (2010) and Majumdar et al., (2002). CONCLUSION The present avifaunal survey of Agartala city and its adjacent areas revealed 73 bird species which is very important as it is the first ornithological record of the city and will give a baseline data for future study. Rich bird diversity is influenced by the topographical location of the city and adjacent areas of Bangladesh. Expansion of the city by construction activities, reducing forest and farm areas with population pressure, filling of pond and lake areas, dumping of wastes and garbage in the low lands, use of chemical pesticides in agricultural fields and hunting of birds are the major threats to the avifaunal diversity here which needs proper conservation management practices. ACKNOWLEDGEMENT Authors are thankful to Mr. Dipankar Kishore Sinha for his constant services, tireless field assistance and in capturing photographs during the study. REFERENCES Ali S. 1996. The book of Indian birds, Twelfth Revised Edition, Bombay Natural History Society Oxford University Press, Mumbai. Ali S. 2002. The book of Indian birds, Thirteenth Revised Edition, Bombay Natural History Society Oxford University Press, Mumbai. Ali S and Ripley SD. 1968-74. Pakistan 10 vols., Oxford University Press, Bombay. Ali S and Ripley SD. 1995. A pictorial guide to the birds of Indian Subcontinent. Bombay Natural History Society, Mumbai. BirdLife International 2009: IUCN 2011. IUCN Red List of Threatened Species. Version 2011.2. <www.iucnredlist.org>. Downloaded on 18 June 2012. Blyth E. 1845. Notices and descriptions of various new or little known species of birds, Journal of the Asiatic Society of Bengal, 14: 546-602. Blyth E. 1846. Notices and descriptions of various new or little known species of birds, Journal of the Asiatic Society of Bengal, 15: 1-54. Chatterjee S, Saikia A, Dutta P, Ghosh D, Pangging G and Goswami AK. 2006. Biodiversity significance of north east India: WWF-India. New Delhi. pp-71. Chinchkhede KH and Kedar GT. 2012. Avifaunal diversity of Koradi lake in Nagpur district of central India. Journal of Research in Biology, 2: 70-76. Choudhury A. 2010. Recent ornithological records from Tripura, north-eastern India, with an annotated checklist. Indian BIRDS 6(3): 66-74. Daniels RJR. 1994. A landscape approach to conservation of birds. Journal of Bioscience 19(4): 503- 509. Grimmet R, Inskip T and Islam MZ. 2004. Birds of Northern India. Christopher Helm A and C Bleak Publishers Ltd. London. Grimmett R, Inskipp C and Inskipp T. 2003. Pocket Guide to the Birds of the Indian Subcontinent, Oxford University Press, New Delhi. Majumdar N, Ray CS and Datta BK. 2002. Aves. In: Fauna of Tripura (Part 1) (Vertebrates). State Fauna Series 7, pp. 47-158 (Ed.: Director 2002). Kolkata: Zoological Survey of India. Bhattacharjee et al., 2013 859 Journal of Research in Biology (2013) 3(3): 852-860
  • 14. Submit your articles online at www.jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php. Myers N, Russell A, Mittermelert C, Mittermelert G, Gustavo AB and Fonseca KJ. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853- 858. Narwade S, Kalra M, Jagdish R, Varier D, Satpute S, Khan N, Talukdar G, Mathur VB, Vasudevan K, Pundir DS, Chavan V and Sood R. 2011. Literature based species occurrence data of birds of North-East India. In: Smith V, Penev L (Eds) e-Infrastructures for data publishing in biodiversity science. ZooKeys 150: 407-417. Robert A Fimbel, John AG and Robinson G. 2001. The Cutting Edge: Conserving Wildlife in Logged Tropical Forest. Scott DA and Rose PM. 1989. Asian Waterfowl Census. The International Waterfowl and Wetlands Research Bureau, 43-46. Bhattacharjee et al., 2013 Journal of Research in Biology (2013) 3(3): 852-860 860
  • 15. JournalofResearchinBiology Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms. Keywords: Oil and gas, Pollution, Phytochemicals, Vitamins, Oha, Okra. ABSTRACT: The phytochemical, proximate, mineral and vitamin contents of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms were investigated. Plant samples were harvested from Polluted Environment (PE) at Izombe in Oguta Local Government Area- an oil drilling and gas flaring environment. The results obtained were compared to identical vegetables harvested from Eziobodo in Owerri West Local Government Area, designated as Unpolluted Environment (UPE). Our result showed that A. esculentus and P. mildbraedii have excellent nutritional value, which can confer biochemical and physiological advantage to humans. The quantitative proximate composition showed that the carbohydrate and ash contents of samples harvested from PE differed significantly (P<0.05) from samples obtained from unpolluted environment. The protein, crude fibre, moisture and total fat contents of samples from PE differed non significantly (P<0.05) when compared with samples obtained from UPE. The phytochemical contents of A. esculentus and P. mildbraedii were significantly higher in samples from UPE than in samples from PE. The mineral and vitamin contents were also determined. The concentration of nutritionally important macro and micro elements indicates that the two vegetable samples studied are rich sources of minerals and, therefore, can be used to improve the diet of both humans and livestock. This study also showed that environmental pollutants emanating from the activities of oil and gas industries can impact negatively on some important chemical and nutritive compositions of edible vegetables. 861-869 | JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Journal of Research in Biology An International Scientific Research Journal Authors: Ujowundu CO1 , Nwaogu LA1 , Igwe KO1 , Ujowundu FN1 , Belonwu DC2 . Institution: 1. Department of Biochemistry, Federal University Technology Owerri, Nigeria. 2. Departmentof Biochemistry, University of Portharcourt, Nigeria. Corresponding author: Ujowundu CO. Email: ujowundu@yahoo.com Web Address: http://jresearchbiology.com/ documents/RA0327.pdf. Dates: Received: 16 Jan 2013 Accepted: 09 Feb 2013 Published: 11 Apr 2013 Article Citation: Ujowundu CO, Nwaogu LA, Igwe KO, Ujowundu FN, Belonwu DC. Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms. Journal of Research in Biology (2013) 3(3): 861-869 Journal of Research in Biology An International Scientific Research Journal Original Research
  • 16. INTRODUCTION Nigeria, a major producer of crude oil, benefits as well as suffers from the positive and negative effects of crude oil drilling and gas flaring (Adeniye et al., 1983). Gas flaring is the unscientific burning of excess hydrocarbons gathered in an oil/gas production flow station. Gas flaring is a major source of pollution in Nigeria's Niger Delta because it is the preferred means of disposing waste gas associated with oil exploitation in that region by many multinational oil companies that operate its fields. Gas flaring releases carbon monoxide, oxides of sulphur and nitrogen, hydrocarbons, soot and heavy metals (Coker, 2007; Ikoro, 2003). These pollutants actually interfere with growth and survival of living organisms in such environment especially in plants. It pollutes seedlings and fruits of plants which in turn have a devastating effect on humans who consume them. Such effects include respiratory or cardiovascular diseases. This study used Abelmoschus esculentus Moench and leaves of Pterocarpus mildbraedii Harms, two commonly consumed indigenous vegetables to evaluate the biochemical effects of these environmental pollutants. Abelmoschus esculentus Moench (local name, Okra) and leaves of Pterocarpus mildbraedii Harms (local name, Oha) are vegetables commonly consumed as a source of food and medication for their high content of nutrients and phytochemicals and mainly used for soup preparation. Consumption of vegetables provide taste, palatability, increases appetite and provides fiber for digestion and to prevent constipation. They play key roles in neutralizing acids produced during digestion of proteins and fatty foods and also provide valuable roughages which helps in movement of food in the intestine. Some of these vegetables possess the ability to reduce or reverse so many disease conditions and disorders such as those which require a reduced intake of glucose (diabetes) (Mcdowell, 2001: Ogbonnia et al., 2008). These vegetables can be deeply affected by pollution. The most glaring sight in gas production flow station is the ten-meter-high flame that burns continuously from vertical pipes at many facilities owned by oil companies. These vertical pipes are fed with gas given off during production. Gas flaring, for about four decades has contributed to the high pollution level, and the ecosystem of Izombe may have been impacted negatively. A good example of such negative effect is high soil acidity that creates chemical and biological conditions which may be harmful to the soil and plants (Nwaugo et al., 2006). One of these conditions is the reduction in the capacity of plants to absorb cations (Wild et al., 2005). The higher acidic nature of soil is attributable to high concentrations of sulphur dioxide and particulates from gases flared into the atmosphere which is washed back to the soil as acid rain. This study has two objectives, first to contribute to the knowledge of nutritional and antinutritional composition of A. esculentus Moench and P. mildbraedii Harms. Secondly, to evaluate the effect of environmental pollution resulting from crude oil exploration and exploitation and other industrial processes within the area of study. The arable nature and vast land mass of Izombe, confers it the status of food basket of Imo State, Nigeria. MATERIALS AND METHODS Collection and preparation of plant samples Samples of A. esculentus Moench and P. mildbraedii Harms were obtained at Izombe, in Oguta Local Government Area and at Eziobodo, in Owerri West Local Government Area both in Imo state, Nigeria and identified by a plant taxonomist in the Federal University of Technology, Owerri (FUTO). Izombe is a rainforest ecosystem, which hosts multinational industries specialized in crude oil exploration and exploitation. Flaring of gases constitutes the major method of waste gas disposal at these oil fields. Situate Ujowundu et al., 2013 862 Journal of Research in Biology (2013) 3(3): 861-869
  • 17. to these oil fields are communities of indigenous inhabitants whose occupation are subsistence and semi- commercial farming. Eziobodo is also within the rainforest region of Nigeria, occupied by indigenous and FUTO students population. It has no known industrial activities, except few automobiles that convey inhabitants in and out of the village. Samples were sorted by removing extraneous materials, spoilt and unhealthy ones. After washing, okro samples were carefully sliced. The samples were oven dried, macerated, sieved and properly stored. Evaluation of proximate composition The method described by James (1995) and Onwuka (2005) were used to determine crude fiber. Fat content was determined by the method of Min and Boft (2003). Moisture content was determined by the method of AOAC (1990). The sample’s total protein content was determined by microkjeldhal method described by James (1995). Protein concentration was obtained by determining total nitrogen and multiplied by the factor- 6.25. Carbohydrate contents was calculated using the arithmetical difference method described by Pearson (1976) and James (1995). Evaluation of phytochemical content Tannin content of samples were determined by Folin-Denis colorimetric method (Kirk and Sawyer, 1998). Saponin, alkaloid and flavonoid were done by method described by Harborne, (1973). The spectrophotometric method as described by Griffiths and Thomas (1981) was used for determining phytate content. Determinations were done in triplicates and results were expressed as averages of percent values on dry weight basis. Evaluation of vitamins content Retinol, ascorbic acid and α-tocopherol contents in the samples were determined using the method of Association of Vitamin Chemist as described by Kirk and Sawyer (1998). Evaluation of mineral content Some mineral contents were determined by atomic absorption spectrophotometer (James, 1995). The dry samples were burnt to ashes to remove all organic materials leaving inorganic ash. The resulting ash was dissolved in 10 ml of 2 M HCl solution and diluted to 100 ml with distilled water in a volumetric flask. The mixture was filtered and the resulting extract was used for the specific evaluation of copper, zinc and iron. Sodium, potassium, calcium and magnesium were determined with the aid of Jaway digital flame photometer. Phosphorus was determined as phosphate by the vanadomolybdate colorimetric method (Pearson, 1976) Ujowundu et al., 2013 Journal of Research in Biology (2013) 3(3): 861-869 863 Environment of sampling Carbo- hydrates Crude Protein Ash Crude Fibre Moisture Total Fat Polluted 32.47±2.22a 14.28±0.30a 13.78±0.40a 22.13±1.40a 10.52±0.89a 6.82±0.90a Unpolluted 37.94±1.78b 12.67±0.07a 7.99±0.16b 21.83±0.23a 12.71±1.71a 6.85±0.03a Table 1: Proximate composition (%) of Abelmoschus esculentus Moench Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different. Environment of sampling Carbo- hydrates Crude Protein Ash Crude Fibre Moisture Total Fat Polluted 29.38±1.24c 8.06±1.43c 19.40±0.57c 17.65±0.79c 22.37±1.18c 3.14±0.33c Unpolluted 34.82±0.30d 9.67±0.07c 12.05±0.18d 16.58±0.21c 23.67±0.29c 3.21±0.06c Table 2: Proximate Composition (%) of Pterocarpus mildbraedii Harms Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.
  • 18. Statistical analysis Data obtained were expressed as means± standard deviation. Statistical Package for the Social Sciences (SPSS) was used for the Analysis of Variance (ANOVA) for the test of significant difference between means (P<0.05). RESULTS The proximate contents of A. esculentus and P. mildbraedii are presented in Tables 1 and 2 respectively. Results obtained from unpolluted environment (UPE) showed that A. esculentus have higher content of carbohydrate, protein, fibre and total fat compared to P. mildbraedii. However, higher ash and moisture content were observed in P. mildbraedii when compared to A. esculentus from UPE. Carbohydrate content in samples obtained from polluted environment (PE) were significantly (P<0.05) lower than the UPE. But the ash contents were significantly higher in samples from PE. The protein, crude fiber, moisture and total fat contents of the samples showed no significant difference. Results of phytochemical analysis are presented in tables 3 and 4. The concentrations of phytochemicals were significantly higher (P<0.05) in samples from PE. Also, the phytochemical contents of P. mildbraedii from PE were higher than that of A. esculentus from PE. The highest concentrations of phytochemicals were observed in flavonoids (0.54±0.02%) and tannin (1.83±0.01%) from A. esculentus and P. mildbraedii respectively from PE. However, alkaloids and tannins contents were highest in A. esculentus and P. mildbraedii respectively from UPE. Vitamin contents were presented in tables 5 and 6. Vitamin A concentration in A. esculentus and P. mildbraedii were 627.59±0.47 mg/100g and 375.48±0.18 mg/100g respectively, indicating the highest vitamin content in the samples. Also, vitamin C and B5 contents in samples from UPE were significantly higher (P<0.05) when compared to samples from PE. Similarly P. mildbraedii have significantly higher value of vitamin B5 (189.33±2.31 mg/100g) compared to A. esculentus from UPE. In A. esculentus (table 5), all the vitamins determined were significantly (P<0.05) lower except vitamin B5, vitamin B9 and vitamin E in samples from PE. Also, samples of P. mildbraedii from PE when compared with samples from UPE showed significantly lower content in all the vitamins (table 6) except in vitamin B2, vitamin B3 and vitamin E. The mineral contents are shown in tables 7 and 8. The concentrations of copper, iron, zinc and lead in A. esculentus from PE were significantly higher (P<0.05) Ujowundu et al., 2013 864 Journal of Research in Biology (2013) 3(3): 861-869 Environment of sampling Saponins Tannins Phytates Alkaloids Phenols Flavonoids Polluted 0.33±0.03a 0.37±0.01a 0.23±0.01a 0.43±0.01a 0.26±0.04a 0.54±0.02a Unpolluted 0.12±0.05b 0.20±0.01b 0.09±0.01b 0.27±0.09b 0.11±0.00b 0.24±0.02b Table 3: Phytochemical Composition (%) of Abelmoschus esculentus Moench Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different. Environment of sampling Saponins Tannins Phytates Alkaloids Phenols Flavonoids Polluted 0.41±0.02c 1.83±0.00c 0.37±0.01c 0.57±0.01c 0.44±0.02c 0.63±0.01c Unpolluted 0.23±0.08d 1.56±0.14d 0.15±0.01d 0.28±0.12d 0.35±0.00d 0.45±0.08d Table 4: Phytochemical Composition (%) of Pterocarpus mildbraedii Harms Vegetables Values (mean ± SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.
  • 19. Ujowundu et al., 2013 Journal of Research in Biology (2013) 3(3): 861-869 865 Environment ofsampling VitaminAVitaminB1VitaminB2VitaminB3VitaminB5VitaminB6VitaminB9VitaminCVitaminE Polluted592.78±19.69a 0.02±0.02a 0.05±0.01a 0.83±0.10a 21.00±2.52a 0.58±0.02a 0.67±0.11a 68.41±2.31a 1.52±0.02a Unpolluted627.59±0.47b 0.08±0.01b 0.08±0.01b 1.12±0.01b 23.33±2.31a 0.81±0.10b 0.73±0.08a 78.03±1.02b 1.88±0.17a Table5:VitaminContent(mg/100g)ofAbelmoschusesculentusMoench Values(mean+SDoftriplicatedeterminations)withdifferentsuperscriptspercolumnaresignificantly(P<0.05)different. Environment ofsampling VitaminAVitaminB1VitaminB2VitaminB3VitaminB5VitaminB6VitaminB9VitaminCVitaminE Polluted302.57±7.01c 0.07±0.02c 0.04±0.01c 0.65±0.09c 175.22±6.97c 0.25±0.08c 0.53±0.17c 85.29±1.79c 1.97±0.18c Unpolluted375.48±0.18d 0.12±0.00d 0.06±0.01c 0.57±0.02c 189.33±2.31d 0.59±0.05d 0.89±0.02d 99.15±2.69d 2.27±0.17c Table6:VitaminContent(mg/100g)ofPterocarpusmildbraediiHarmsVegetables Values(mean+SDoftriplicatedeterminations)withdifferentsuperscriptspercolumnaresignificantly(P<0.05)different. Environment ofsampling MagnesiumCalciumPhosphorusSodiumPotassiumCopperIronZincLead Polluted22.17±1.39a 70.23±2.02a 57.35±0.59a 6.34±0.46a 107.63±4.45a 0.42±0.15a 0.95±0.26a 0.48±0.14a 0.65±0.07a Unpolluted39.60±1.39b 82.83±2.32b 68.28±0.46a 7.47±0.23a 130.40±6.55b 0.06±0.01b 0.74±0.25b 0.32±0.07b 0.35±0.10b Table7:MineralContent(mg/100g)ofAbelmoschusesculentusMoench. Values(mean+SDoftriplicatedeterminations)withdifferentsuperscriptspercolumnaresignificantly(P<0.05)different. Environment ofsampling MagnesiumCalciumPhosphorusSodiumPotassiumCopperIronZincLead Polluted48.67±4.38c 64.38±3.95c 409.89±0.43c 17.16±1.71c 250.73±4.29c 0.34±0.13c 0.77±0.18c 0.34±0.06c 0.50±0.22c Unpolluted52.80±2.40c 77.48±2.32d 413.89±12.48c 21.13±0.12d 284.27±2.44d 0.04±0.00d 0.58±0.09d 0.16±0.02d 0.19±0.13d Table8:MineralContent(mg/100g)ofPterocarpusmildbraediiHarmsVegetables. Values(mean+SDoftriplicatedeterminations)withdifferentsuperscriptspercolumnaresignificantly(P<0.05)different.
  • 20. when compared to samples from UPE. Results presented in table 8 showed that P. mildbraedii from UPE are excellent source of phosphorus (413.89±12.48), potassium (284.27±2.44), calcium (77.48±2.32) and magnesium (52.80±2.40). Also, the concentration of minerals in P. mildbraedii from PE and UPE were significantly different in all except in magnesium and phosphorus. DISCUSSION Gas flaring and other oil and gas activities for about four decades have contributed to pollution in Oguta, which have impacted on the ecosystem. Soots were seen on vegetation within the communities around the flaring site. Plants growing in such environment have over the years taken in varying doses of pollutants which invariably may affect the nutritional and chemical contents. Our result showed that A. esculentus had better nutritional value than P. mildbraedii with respect to protein and carbohydrate contents. Also, A. esculentus and P. mildbraedii showed higher values in proximate contents (except in protein) than A. hybridus as reported by Nwaogu et al., (2006). Carbohydrates provide energy to cells in the body, particularly to the brain, a carbohydrate dependent organ in the body. (Nelson and Cox, 2005). These vegetables can supplement the daily energy intake of humans (Bingham, 1998; Effiong et al., 2009). The crude fibre content, indicates that the vegetables are good sources of fibre, thus making them veritable source of roughage. The concentrations of carbohydrate were significantly reduced while ash contents were increased in plants from polluted environment when compared to plants from unpolluted environment (UPE). The reduced carbohydrate can be attributed to the effect of air pollutants as reported by Farzana (2005), in which he affirmed that it reduces photosynthesis in chloroplasts. The contents of protein, crude fiber and fat in samples from PE were lower than those from UPE but were not significantly different, which indicates that they were not adversely affected by the pollution. The phytochemical results indicate that A. esculentus and P. mildbraedii are good sources of these beneficial chemicals. They have antioxidative, hypocholesterolemic, chemoprotective and antibacterial properties (Price et al., 1987; Enechi and Odonwodo, 2003; Okwu, 2004). Both vegetables are rich in alkaloids, flavonoids and tannins which indicates that they have diuretic, antispasmodic, anti-inflammatory and analgesic effects (Owoyele et al., 2002; Nobre-Junior, 2007 ; Alisi et al., 2011). Comparatively, P. mildbraedii had higher content of the phytochemicals studied. Also, significantly higher amount of phytochemicals were observed in vegetables obtained from PE. The increase can be linked to their role in oxidative stress in plants. Phytochemicals are secondary metabolite of plants, known to exhibit diverse pharmacological and biochemical effects on living organisms. It has been reported that certain phytochemicals play important role in antioxidant defense systems of vegetative plants (Ugochukwu and Babady, 2003). Pollution by gas flaring is taught to generate free radicals in surrounding environment. Thus, it is expected that plants may increase synthesis of antioxidant defense compounds. These vegetables showed significantly high amount of vitamins especially vitamins A, B1, B2, B5, B6 and C in samples from UPE when compared to samples from PE. These vitamins are involved in intermediary metabolism of both plants and animals acting as part or whole coenzyme to some specific enzyme system and playing important role in both enzyme and non enzyme oxidative stress defense systems. The high concentrations of vitamins A and C will contribute significantly to the daily requirements in view of the reports of Murray (1998). Vitamin C maintains blood vessel flexibility and improves circulation in the arteries of smokers. The most important Ujowundu et al., 2013 866 Journal of Research in Biology (2013) 3(3): 861-869
  • 21. benefit of vitamins A and C is their involvement in free radical scavenging processes (Trumbo et al., 2004; Nwaogu et al., 2011). These chemically active radicals are byproducts of many normal biochemical processes. Their numbers are increased by environmental assaults such as chemicals and toxins. The lower concentrations of these vitamins in samples from PE suggest an inability of the plants to synthesize these vitamins in sufficiently large amount for their metabolic functions. Oxidative stress caused by gas flaring in Oguta community can interfere with the synthetic mechanisms of the plants in the environment (Farzana, 2005). Some of the mineral contents of A. esculentus Moench and P. mildbraedii Harms are comparable or higher than that reported for Amaranthus hybridus (Nwaogu et al., 2006) Mucuna utilis (Ujowundu et al., 2010), Commelina nudiflora and Boerhavia diffusa (Ujowundu et al., 2008). The values obtained for the minerals indicates that the samples are good sources of mineral and are of great nutritional importance. In animals, potassium and sodium are important electrolytes. Potassium is a major intracellular cation. Sodium is involved in the regulation of acid-base equilibrium, protection against dehydration and maintenance of osmotic pressure in living system. It plays a role in the normal irritability of muscles and cell permeability (Schwart, 1975). Copper (Cu) is essential for haemoglobin synthesis, normal bone formation and the maintenance of myelin within the nervous system (Passmore and Eastwood, 1986). In animals, the manifestations of copper deficiency include; anaemia, hypo-pigmentation, defective wool keritinization, abnormal bone formation with spontaneous reproductive and heart failure (Williams, 1982). In humans, it has been established that occurrence of Cu absorption disorder in after partial gastetomycin leads to severe malnutrition just as when protein is severely deficient in the diet; as in kwashiorkor (Davies, 1972). Calcium and phosphorus are important and indispensable for the synthesis of strong bones and teeth, kidney function and cell growth (Uddoh, 1988; Brody, 1994). Phosphorus and magnesium are also important in the regulation of acid-alkaline balance in the body (Fallon, 2001). The mineral contents, like Mg, Ca, P, S and K in vegetables from PE have significantly (P<0.05) reduced value compared to vegetables obtained from UPE. The release of pollutants such as oxides of sulphur and nitrogen, hydrocarbons and other volatile organic carbons can create chemical and biological conditions which may be harmful to plants and soil microorganisms. One of such conditions is the reduction in the capacity of plants to absorb cations (Wild et al., 2000). Crops grown in soil with low mineral contents exhibit various forms of mineral deficiency. In plants, potassium is an essential nutrient and has an important role in the synthesis of amino acids and proteins (Malik, 1982). Ca and Mg play significant role in photosynthesis, carbohydrate and nucleic acids metabolism (Russel, 1973). The reduced content of these minerals will definitely affect these important plant processes. Lead is yet to record any physiological role in the biological system and are known to be extremely toxic even at the slightest concentration. Their presence in the samples calls for serious concern This study has shown that A. esculentus Moench and P. mildbraedii Harms are good sources of nutrients and their consumption should be encouraged. Improved information on these plants will contribute to the awareness of their nutritive value, especially in this time of increased food insecurity. Also, gas flaring showed negative effects on these plants, which could affect animals that consume them. Similarly, the adverse health consequences on the inhabitants around the gas flare site are of great concern. Communities around such environment should be enlightened on the inherent dangers. Oil and gas industries should be compelled to upgrade their waste disposal technologies, with emphasis Ujowundu et al., 2013 Journal of Research in Biology (2013) 3(3): 861-869 867
  • 22. in gas disposal. This will reduce the detrimental effects on the health and well-being of inhabitants of Izombe in Oguta Local Government Area of Imo State. REFERENCES Adeniye EO, Olu-Sule R and Anyanye A. 1983. Environmental and Socio-economic Impacts of Oil Spillage in the Petroleum Producing Areas in Nigeria. The Petroleum industry and the Nigerian Environmental proceedings of the 1983 International oil seminar, NNPC 3:130-135. Alisi CS, Nwaogu LA, Ibegbulem CO and Ujowundu CO. 2011. Antimicrobial Action of Methanol Extract of Chromolaena Odorata-Linn is Logistic and Exerted by Inhibition of Dehydrogenase Enzymes. Journal of Research in Biology 1(3): 209-216. AOAC. 1990. Method of analyses of association of official analytical chemists. Journal of the Association of Analytical Chemists 25:516-524. Bingham S. 1998. Nutrition: A consumer’s guide to food eating. London: Transworld Publishers. 123-127. Brody T. 1994. Nutritional Biochemistry, San Diego, CA: Academic Press. Coker KA. 2007. Ludwig’s Applied Process Design for Chemical and Petrochemical Plants. Gulf Professional Publishing 1(4):732-737. Davies ITJ. 1972. Copper Absorption. The Clinical Significance of the Essential Biological Metals William Heine, Mann Medical Books Ltd, London 47. Effiong BN, Sanni A and Fakunle JO. 2009. Phytochemical and chemical composition of Combretum zenkeri. Human Science 86:301-307. Enechi OC and Odonwodo I. 2003. Assessment of the Phytochemical and Nutrient composition of Pulverized Root of Cissus quadrangularis. J. Biol. Res. Biotechnol., 1(1):63-68. Fallon S and Enig MG. 2001. Nourishing Traditions: The Cookbook that Challenges Policitally Correct Nutrition and the Diet Dictocrats. 40-45. Farzana P. 2005. Response of plant metabolism on air pollution and climate in sindh, Pakistan. Digital-verlg GmbH, 41:3-15. Harborne JB. 1973. Phytochemical methods: a guide to modern techniques of plant analysis. Chapman and Hall Publishers 3(1):5-29. Ikoro NJ. 2003. The Socio-economic Implications of Gas flaring in Nigeria. Du-France Communications, Yenagoa, Bayelsa 4(13):35-47. James CS. 1995. Analytical chemistry of foods. Backie Academic Journal 198:125-181. Kirk RS and Sawyer R. 1998. Pearson’s Composition and Analysis of Food. Addison Linsley Longman Limited 9:15-28. Malik CP and Srivastava AK. 1982. Text book of plant physiology. New Delhi: Ludhiana. Mcdowell IF. 2001. Folate, homocysteine, endothelial function and cardiovascular disease, what is the link. Biomedical Pharmacoether 55(8):425-433. Murray MJ. 1998. High quality vitamins, minerals and special supplements. Jama 279:1200-1205. Nelson DC and Cox MM. 2005. Integration and Control of Metabolic Processes. Lehninger Principles of Biochemitsry (4th ed) Worths Publishers. 780-783. Nobre-Junior HV. 2007. Chemoprotective actions of tannins. Journal of Herbs, Spices and Medicinal Plants 13(2):48-55. Nwaogu LA, Igwe CU, Ujowundu CO, Arukwe U, Ihejirika CE and Iweke AV. 2011. Biochemical changes in tissues of albino rats following subchronic exposure to crude oil. J. Res. Biol., 8: 617-623 Nwaogu LA, Ujowundu CO and Mgbemena AI. 2006. Studies on the nutritional and phytochemical composition of Amaranthus hybridus Leaves. Bio- Res., 4: 28-31. Nwaugo VO, Onyeagba RA and Nwachukwu NC. 2006. Effect of Gas Flaring on Soil Microbial Spectrum in parts of Niger Delta Area of Nigeria. Annual Journal of Biotechnology 5(19):1824-1826. Ogbonnia S, Odimegwu J and Enwuru VN. 2008. Clinical manifestation of diabetes. Department of Ujowundu et al., 2013 868 Journal of Research in Biology (2013) 3(3): 861-869
  • 23. Submit your articles online at www.jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php. Pharmacognosy, Nordestgaard 22(6):35-42. Okwu DE. 2004. Phytochemicals and Vitamin content of Indigenous Spices of South Eastern Nigeria. J. Sust. Agric. Environ., 6(1):30-37. Onwuka SK. 2005. Cortical metal and crude protein levels of certain vegetables. Brain Talk Communities 3:15-23. Owoyele BV, Oguntoye SO, Dare K, Ogunbiyi BA and Aruboula EA. 2002. Analgelsic, anti-inflammatory and antipyretic activities of flavonoid, alkaloid and tannin fractions of Chromolagna odorata. Journal of Medicinal Plants 2(9):219-225. Passmore R and Eastwood MA. 1986. In Davison Passmore R., Human Nutrition and Dietetics, Churchill Livingstone, London. 124-126. Pearson D. 1976. Chemical analyses of food. Church hill, Livingstone. 7:72-73. Price KR, Johnson IT, Fenwick GR. 1987. The chemistry and biological significance of saponins in food and feeding stuffs. CRC. Crit. Rev. Food Sci. Nutr., 26:27-135. Russel EW. 1973. Soil conditions and plant growth. Supergene Zone, M. Nedra, (in Russian). 19. Schwart MK. 1975. Role of trace elements in cancer. Cancer Res., 35:3481-3484. Trumbo P, Schlicker S and Yates AA. 2004. Roles vitamin A and C play as antioxidants. Science Metabolism 30:253-258. Uddoh CK. 1988. Nutrition: Macmillan Publishers Ltd. London and Basingstoke, 71-97,109. Ugochukwu NH and Babady NE. 2003. Antioxidant effects on Gongonemia catifolium in hepatocytes of rat model of non insulin dependent diabetes mellitus. Filoterapia 173(7-8):612-618. Ujowundu CO, Okafor OE, Agha NC, Nwaogu LA, Igwe KO and Igwe CU. 2010. Phytochemical and Chemical Composition of Combretum zenkeri Leaves. Journal of Medicinal Plants Research 4(10):965-968. Ujowundu CO, Igwe CU, Enemor VHA, Nwaogu LA and Okafor OE. 2008. Nutritive and Anti-Nutritive Properties of Boerhavia diffusa and Commelina nudiflora Leaves. Pak. J. Nutr. 7(1): 90-92. Wild E, Dent J, Barber JC, Thomas GO and Jones KC. 2005. Real Time Visualization and Quantification of Polycyclic Aromatic Hydrocarbon (PAH) Photodegradation on and within Plant Leaves” Environmental Science Technology 39(1):268-273. Williams DM. 1982. Clinical significance of copper deficiency and toxicity in the World population. In the clinical Bio-chemical and nutritional aspects of trace elements. Prasad, A.S., New York, 177. Ujowundu et al., 2013 Journal of Research in Biology (2013) 3(3): 861-869 869
  • 24. JournalofResearchinBiology Effect of age, sex and hemoglobin type on adaptive and blood biochemical characteristics in Red Sokoto Goats Keywords: Adaptive coefficient, heart rate, rectal temperature, blood biochemical characteristics. ABSTRACT: This study was conducted to evaluate the effect of haemoglobin (Hb) types, sex and age on adaptive and blood biochemical characteristics of Red Sokoto goats. Ninety four (94) goats were sampled from two locations: Dei-dei and Gwagwalada grazing reserved, Abuja. Data were collected on adaptive characteristics {heart rate (HR) and rectal temperature(RT) and adaptive coefficient (AC) was calculated from the HR and RT} and blood biochemical characteristics{ haemoglobin (Hb) types, Hb-concentration (Hb-conc), Potassium concentration (K-conc) and albumin concentration (alb-conc)}. The effects of haemoglobin type, sex and age on the adaptive and blood biochemical characteristics of the goats was analyzed by general linear model (GLM) procedure of SAS. The results showed that the mean RT of the sampled goats was 38.9°C with very minimal variations (CV=0.5). The mean HR of the goats was 76.1bpm, with min and max HR of 70 and 80bpm. The mean albumin, Hb and K concentration were 38.4g/l, 8.9g/dl and 4.0Mmol/l, respectively. The variation of Hb type with adaptive and blood biochemical characteristics was significant (P<0.05) except Hb concentration. Higher HR was observed in goats with Hb AA and AB. Age and sex had significant effect (P<0.05; P<0.01) on HR, AC and albumin concentration of the goats. Although there was no trend in the variation of HR and AC with age, but HR and AC were higher in the older goats than the younger, however the albumin concentration significantly decreased with progressive increase in age of the goats. 870-875 | JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Authors: Akpa GN, Alphonsus C and Usman N. Institution: Animal Science Department, Ahmadu Bello University, Zaria, Nigeria. Corresponding author: Alphonsus C. Email: mcdyems@gmail.com Web Address: http://jresearchbiology.com/ documents/RA0167.pdf. Dates: Received: 15 Dec 2011 Accepted: 01 Jan 2012 Published: 16 Apr 2013 Article Citation: Akpa GN, Alphonsus C and Usman N. Effect of age, sex and hemoglobin type on adaptive and blood biochemical characteristics in Red Sokoto Goats Journal of Research in Biology (2013) 3(3): 870-875 Journal of Research in Biology An International Scientific Research Journal Original Research Journal of Research in Biology An International Scientific Research Journal
  • 25. INTRODUCTION In recent years, advances in the field of biotechnology have opened up a completely new area at molecular levels with the introduction of techniques such as routine electrophoresis employed for detection of polymorphism at protein and enzyme loci as well as other serological and immunogenetic procedures for the measurement of variations (Salako et al., 2007). Data obtain from this type of study could be useful as genetic markers for important economic characteristics and could aid significantly in selection of superior animals for breeding purposes. Haemoglobin typing is very important as different Hb types may have selective advantage in different geographical regions (Ndamukong, 1995). Economic pressures of various kinds are forcing the production of livestock into climatic environments that are increasingly more remote from the considered ideal for optimal production and feed utilization. Thermal stress, which is one of the major factors that affect the productivity of many farm animals can be reflected in an easily observable changes in pulse rate, respiration rate, and rectal temperature, although the whole body reacts to thermal stress by an elaborate series of chain reactions (Ahmed, 2004). The most obvious index of thermal stress is body temperature response. Deviation from normal rectal temperature indicates that the animal is under stress, that its homeothermic mechanisms are overtaxed Ahmed, 2004). Adaptive characteristics of animals serve as a key to managing any livestock operation. Adaptive traits such as rectal temperature, heart rate, and flank movement have been documented to have some significant effect on genetic variations. Every normal animal has a range of individual adaptive traits in relation to a specific physiological pattern. Because study of environmental physiology involves so many variables and scientific discipline, much is being published on this subject especially as related to farm animals. Comprehensive reviews have appeared under the authorship of Alderson, 1992, Derman and Noakes, 1994, Tambuwal et al., (2002), Otoikhian et al., 2009, chukwuka et al., 2010, Opara et al., 2010, Gurcan et al., 2010 and a lot of others. Research results reported in this paper is intended to supplement data reviewed by authors listed. Eventually, accumulated data will permit specific recommendation on breeding, feeding and management of farm animals. This study therefore aimed at studying the effects of Haemoglobin type, sex and age on adaptive and blood biochemical characteristics of Red Sokoto goats. MATERIALS AND METHODS Location The study was conducted at the Federal Capital Territory Abuja, located within the Northern guinea Savanna zone of Nigeria. It is laying between latitudes 8.25° and 9.0° N of the equator and Longitude 6.45° and 7.39° E of the Greenwich Meridian. (Presentation Copyright@ falling Rain Genomics, 1996-2010). Data collection Ninety four (94) goats were sampled from two locations: Dei-dei and Gwagwalada grazing reserve, Abuja. Data were collected on the adaptive and blood biochemical traits. The adaptive traits were Heart rate (HR), Rectal temperature(RT) and Adaptive coefficient (AC) while the blood biochemical characteristics were haemoglobin (Hb) types, Hb-concentration (Hb-conc), Potassium concentration (K-conc) and albumin concentration (alb-conc). METHODS OF MEASUREMENTS Adaptive Traits Heart Rate (HR) Heart rate was taken by placing stethoscope on femoral artery of the hind limb of the goat to count the number of beat per minute. 871 Journal of Research in Biology (2013) 3(3): 870-875 Akpa et al.,2013
  • 26. Rectal Temperature (RT) This was taken using clinical thermometer which was inserted into the rectum of the goat and left for 45-50 seconds. It was then removed and the temperature level was read. The values read were recorded, and the process was repeated for the other goats. Blood Biochemical Characteristics Blood samples were taken from each of the experimental goats through the jugular vein. 5 mls of the blood was taken from each goat, from which 2 mls was put into heparinized vacutainer tubes containing anticoagulant ethylene diamine tetra acetic acid (EDTA). The remaining 3 mls of the blood was put into sterile vacutainer tubes (without anticoagulant). The samples were labeled accordingly. The blood samples in the sterile vacutainer tube were centrifuged in order to have a clear layer of serum. This serum was pipetted into another sterile bottle and store in a refrigerator. The blood samples were taken to the Haematological laboratory of Ahmadu Bello Teaching Hospital from where the analysis of blood biochemical characteristics was carried out. A spectrometer with wavelength capability of 600-650 nm (Zenway 5041 colorimeter) was used to analyzed for the albumin concentration, while the K concentration was analysed using Corning flame photometer 410. Electrophoresis and Cyanmethaemoglobin method was used to analyzed for Hb-types and Hb-conc, respectively. Data Analysis The adaptability of the goats were measured by determining the adaptive coefficient from the values of the Rectal Temperature (RT) and Heart Rate (HR) as thus Adaptive coefficient (AC) = (RT/38.33) + (HR/23.00) The effects of haemoglobin types, sex and age on the adaptive and blood biochemical characteristics of the goats were determined by general linear model (GLM) procedure of SAS, (2005). RESULTS AND DISCUSSION Rectal Temperature (RT) is directly affected by the surrounding and ambient temperature, and high ambient temperature has a negative effect on productivity of the animal. Chukwuka et al., (2010) reported that negative effect of high ambient temperature is direct in the form of stress suffered by the animal and the diversion of energy from the purpose of production to regulation of body temperature and indirectly by affecting the availability of feed resources upon which production is dependent. In this study, the mean RT of the goats was 38.9°C with minimum and maximum body temperature of 38.1 and 39.4 °C (Table 1). These values were within the reference range of previous study of goats in thermal neutral condition (Otoikhian et al., 2009) and this indicate that the goats used for this research showed no clinical signs of stress during the research period. The body temperature of the goats exhibited minimal variations (CV=0.5%), thus implying that goats are homoeothermic animals, they can maintain near constant body temperature under wide range of environmental conditions. The Heart Rate (HR) is the pulse that helps to know the beating rate of the heart which is measured Journal of Research in Biology (2013) 3(3): 870-875 872 Akpa et al.,2013 Characteristics N Mean±SE CV(%) Min Max Rectal Temperature (°C) 94 38.9±0.02 0.5 38.1 39.4 Heart Rate (bpm) 94 76.1±0.39 3.8 70.0 81.0 Adaptive coefficient 94 4.3±0.02 5.0 4.1 4.6 Albumin concentration (g/l) 94 38.4±0.34 8.5 31.0 48.0 Hemoglobin concentration (g/dl) 94 8.9±0.16 17.1 4.0 12.7 Potassium concentration (Mmol/l) 94 4.0±0.06 14.7 3.0 5.8 Table 1: Summary Statistics of the measured characteristics in Red Sokoto Goats
  • 27. in beats per minute (bpm) using stethoscope (Otoikhian et al., 2009). The mean HR of the goats used in this study was 76.1bpm, with the min and max HR of 70 and 80bpm. This is slightly higher than the range of 70-75bpm reported by Derman and Noaks (1994) in goats. The minor difference observed in the values of the HR may be explained by differences in geographical conditions, season or climate and physiological conditions of the sample goats. Rectal temperature and heart rate have been shown to be good indicators of the thermal stress and may be used to assess thermal adversity of the environment (Al- Haidary, 2004). The Adaptive Coefficient (AC) (which is the function of RT and HR) signifies the level of adaptability of the goats to the environments varied significantly (P<0.05) with Hb types, sex and age of the goats. The goats with Hb AA and AB had higher AC than those with BB and AC; likewise the bucks had higher AC than the does. Potassium is one of the intracellular elements that regulate the intracellular density of the cell. The amount of K-concentration is fairly high at intracellular membranes (Gurcan et al., 2010). The values of K-concentration reported by Opara et al., (2010) for WAD bucks and does were 17.8 and 6.9mmol/l, respectively. These values were higher than the mean value of 4.0mmol/l observed in this study; this is probably due to differences in breed and physiological conditions of the sampled animals. Researchers had identified the existence of different type of K in different species of animals, and that in sheep for instance, there are two types of K which is high and low K with the low K type dominant over the high K type (Soysal et al., 2003). Also Gurcan et al., (2010) reported a range of 4.23 to 11.69mmol/l for low K type in animals. The concentration of albumin in this study (38.4g/dl) was slightly higher than the 34.5g/dl reported by Opara et al., (2010). The variation of Hb type with adaptive and blood biochemical characteristics was significant (P<0.05) except Hb-concentration (Table 2). The relationship between Hb types and HR can be linked to the different Akpa et al.,2013 Characteristics Hemoglobin Type AA BB AB AC SEM LOS Rectal Temperature (°C) 39.0a 39.0a 39.0a 38.9b 0.02 * Heart Rate (bpm) 76.6a 75.3b 76.2a 75.1b 0.40 * Adaptive coefficient 4.4a 4.3b 4.4a 4.3b 0.02 * Albumin concentration (g/l) 37.6b 39.2a 38.6a 38.8a 0.34 * Hemoglobin concentration (g/dl) 9.1 8.6 8.8 8.6 0.16 ns Potassium concentration (Mmol/l) 3.8b 3.9a 4.0ab 4.4a 0.06 * Number of observations 30 11 41 12 94 ab : means within the same row with different superscripts differ significantly(P<0.05); ns:not significant; Table 2: Effect of hemoglobin type on adaptive and blood biochemical characteristics Characteristics Sex Buck Doe SEM LOS Rectal Temperature (°C) 39.0 39.0 0.03 ns Heart Rate (bpm) 78.5a 75.0b 0.49 ** Adaptive coefficient 4.4a 4.3b 0.02 ** Albumin concentration (g/l) 39.8a 37.7b 0.49 ** Hemoglobin concentration (g/dl) 9.1 8.7 0.26 ns Potassium concentration (Mmol/l) 3.9 4.0 0.08 ns Number of observations 30 64 94 ab : means within the same row with different superscripts differ significantly(P<0.01); ns:not significant; Table 3: Effect of Sex on adaptive and blood biochemical characteristics 873 Journal of Research in Biology (2013) 3(3): 870-875
  • 28. levels of oxygen carrying capacity of the different Hb types. In this study, higher HR was observed in goats with Hb AA and AB, and Hb A is known to be the haemoglobin allele with highest affinity for oxygen. This is in line with the earlier report of Huisman et al., (1959) who relates the preponderance of Hb A to it greater affinity to oxygen. This could also explain the high adaptive coefficient observed on goats with Hb types AA and AB since adaptive coefficient is a function of HR and RT. The variation of HR, AC and Albumin concentration with sex was highly significant (P<0.01; Table 3). The RT of the buck and does were similar however, the HR was higher in bucks (78.5bpm) than the does (75.0bpm) this is probably due to the high sexual activity of the bucks. There was no significant (P>0.05) difference between the bucks and does in Hb and K concentration. This is contrary to the study of opera et al., (2010) who reported significant differences between WAD bucks and does in there Hb and K concentration. This is probably due to differences in breeds and location of the animal, Hb type had been reported to vary with breed and location (Ndamukong, 1995, Abdussamad et al., 2004 Essien et al., 2011) Age significantly (P<0.05) influence HR, AC and albumin concentration but had no significant influence on the RT, Hb and K concentration (Table 4). Although there was no trend in the variation of HR and AC with age, but it was observed that the HR AC was higher in the older goats than the younger, however the albumin concentration significantly decreased with progressive increase in age of the goats. The observed significant influence of age on albumin concentration is at variance with the earlier studies of Piccione et al., (2009) and Opara et al., (2010) who reported non-significant effect of age on albumin concentration of WAD goats. CONCLUSION The mean body temperature (38.9°C) of the goats used was within the reference normal range for goats in thermal neutral condition and this indicates that the goats showed no clinical signs of stress during the research period. The albumin concentration, heart rate and adaptive coefficient of the goats had clear variation based on differences in haemoglobin type, sex and age of the animals. REFERENCES Abdussamad AM, Esievo KAN, Akpa GN. 2004. Haemoglobin types in the Nigerian Zebu and their crosses in Zaria. Proceedings of the Nigerian Society for Animal Production (NSAP). 29-31. Alderson GLH. 1992. genetic conservation of domestic livestock. CAD International, Wallingford, UK. 242. AL-Haidary. 2004. Physiological Responses of Naimey Sheep to Heat Stress Challenge under Semi-Arid Environments. International Journal of Agriculture &Biology 06(2):307-309. Journal of Research in Biology (2013) 3(3): 870-875 874 Akpa et al.,2013 Characteristics Age of goat 12mon 18mon 24mon 30mon SEM LOS Rectal Temperature (o C) 39.0 39.0 39.0 39.0 0.02 ns Heart Rate (bpm) 77.1b 74.4c 74.4c 78.5a 0.37 * Adaptive coefficient 4.4b 4.3c 4.3c 4.5a 0.02 * Albumin concentration (g/l) 39.2a 37.4b 36.7c 36.5c 0.32 * Hemoglobin concentration (g/dl) 9.1 8.7 8.3 8.5 0.16 ns Potassium concentration (Mmol/l) 4.0 4.0 4.0 4.0 0.06 ns Number of observations 55 25 12 2 94 ab : means within the same row with different superscripts differ significantly(P<0.05); ns:not significant; Table 4: Effect of Age on adaptive and blood biochemical characteristics
  • 29. Chukwuka OK, Okoli IC, Okeudo NJ, Opara MN, Herbert U, Obguewu IP and Ekenyem BU. 2010. Reproductive potentials of West African Dwarf sheep and goats. A Review, Research Journal of Vetrenary Sciences 3(2):86-10. Derman KD and Noakes TD. 1994. Comparative aspect of exercise physiology. In: Hodgson, D.R., Rose, R.J (Eds). The Athletic horse: Principle and practice of Equine Sports Medicine. Philadelphia. W.B. Saunders. 13-25. Essien IC, Akpa GN, Barje PP, Alphonsus C. 2011. Haemoglobin types in Bunaji cattle and their Friesian crosses in Shika, Zaria-Nigeria. Afri. J. Anim. Biomed. Sci., 6(1):112-116. Gurcan EK, Erbas C and Ozden C. 2010. Biochemical polymorphism of erythrocyte, Potassium and glutathione protein: the relationship with some blood parameters in Kivircik sheep breed. African Journal of Agricultural Research 2(10):1022-1027. Huisman THJ, Van Der Helm HJ, Visser HKA and Van Vilet G. 1959. Symposium on abnormal Haemoglobin. Blackwell Scientific Publication. 181. Ndamukong. 1995. Haemoglobin polymorphism in Grassland dwarf sheep and goats of the North West province of Cameroon. Bulletin of Animal Health and Production in Africa. 43:53-56. Opara MN, Udeyi N and Okoli IC. 2010. Haematological parameters and blood chemistry of apparently healthy West African Dwarf (WAD) goats in Owerri, South-eastern Nigeria. Tropical Animal Health and Welfare Research group. New York Science Journal 3(8). Otoikhian CSO, Orheruata MA, Imaseuen JA and Akporhuarho OP. 2009. Physiological response of local (West African Dwarf) and adapted Switzerland (White Bornu) goat breed to varied climatic conditions in South-south Nigeria. African Journal of General Agriculture. 5(1):1-6. Piccione G, Casella S, Lutri L, Vazzana I, Ferrantelli V, Caola G. 2009. Reference values of some Haematological, Haematochemical and electrophoretic parameters in the Girgentana goats. Faculty of Vetrinary Medicine, University of Messina, Italy. Presentation Copyright @Falling Rain Genomics, Inc 1996-2010. SAS. 2005. user guide for personal computers, statistical programme 9.01 windows version.(SAS Institute Inc. Cary, NC). Salako AE, Ijadunola TO and Agbesola YO. 2007. Haemoglobin polymorphism in Nigerian indigenous Small Ruminant population-preliminary investigation. African Journal of Biotechnology 6(22):2636-2638. Soysal ML, Gurcan EK, Ozkan E. 2003. Turkiye de yetistirilen cesitli koyun Irklrinda trim kan potasyum konsan trasyonu polimorfizmi vizerine arastirmalar. GAP III Tarim Kongresi, sanliurfa. Tambuwal FM, Agala BM and Bangana A. 2002. Haematological and Biochemical values of apparently healthy Red Sokoto goats. Proceeding of 27th Annual Conference, Nigerian Society of Animal Production (NSAP).FUTA, Akure, Nigeria. 50-53. 875 Journal of Research in Biology (2013) 3(3): 870-875 Akpa et al.,2013 Submit your articles online at www. jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php.
  • 30. JournalofResearchinBiology Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with special reference to its life table attributes in Tripura, India Keywords: Catopsilia pomona butterfly, Pieridae, eco-biology, life table, Tripura, north east India. ABSTRACT: Butterflies of the family Pieridae are common in tropical parts of the world. They are considered as major pollinators as well as pests of economically important plants. Catopsilia pomona is a dominant pierid butterfly found in association with wild plants of Tripura, northeast India. It is abundant throughout the year. Present study was conducted to document the eco-biology of Catopsilia pomona with special reference to its life table attributes in the state of Tripura. Survival rates of life cycle stages in the semi-natural as well as in the field were the maximum during the wet and hot season. Mortality (k value) of different life cycle stages as a proportion of individuals dying during development varied from 0.16 to 0.46 in different seasons. Results suggested that abiotic factors and mortality factors of egg significantly influenced the survival rate of C. pomona population. This butterfly depends on three species of Cassia plants, all shrubs, for their oviposition and larval development in the environment of Tripura. 876-885 | JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Journal of Research in biology An International Scientific Research Journal Authors: Samit Roy Choudhury and Basant Kumar Agarwala* Institution: Ecology & Biodiversity Laboratories, Department of Zoology, Tripura University, Suryamaninagar- 799022, Tripura, India. Corresponding author: Basant Kumar Agarwala Email: bagarwala00@gmail.com Phone No: 0091 381 237 9083/9123 Web Address: http://jresearchbiology.com/ documents/RA0245.pdf. Dates: Received: 22 May 2012 Accepted: 28 May 2012 Published: 17 Apr 2013 Article Citation: Samit Roy Choudhury and Basant Kumar Agarwala. Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with special reference to its life table attributes in Tripura, India. Journal of Research in Biology (2013) 3(3): 876-885 Journal of Research in Biology An International Scientific Research Journal Original Research
  • 31. INTRODUCTION Host selection for survival, development and reproduction in majority of insects often vary in space and time (van Nouhuys et al., 2003; Nylin et al., 2009) which, in turn, depends on the availability (minimum density per unit area) of closely related host plant species (Thorsteinson, 1960), and trade off between host preference by females for oviposition and larval performance of insects (van Nouhuys et al., 2003). However, adult butterflies and their caterpillars show preference for certain host plants for tender shoots, pollen and nectar as food source. Thus, butterfly diversity of a particular habitat generally reflects the overall plant diversity of that habitat. Butterflies are essential component of any natural ecosystem. Their value as indicators of biotope quality is being recognized because of their sensitivity to minor changes in micro- habitat, climatic conditions as well as seasonal changes (Kremen, 1992; Murugesan and Muthusamy, 2011). They are considered as ideal subject for ecological studies of terrestrial landscapes (Thomas and Malorie, 1985). North eastern region of India is blessed with vegetation rich landscapes that support diverse butterfly fauna and other insects (Alfred et al., 2002). The state of Tripura, being a part of this region, also contains large number of butterfly species which is evident from infrequent records of these taxa (Mandal et al., 2002; Agarwala et al., 2010; Majumder et al., 2011; Roy Choudhury et al., 2011). Butterflies of the family Pieridae are common in tropical parts of the world and are considered as major pollinators of crop plants (Borges et al., 2003), and a few of them are also considered as pests of economically important plants (Anonymous, 2007; Capinera, 2008). Despite their common occurrence, there is a lack of substantial study on the ecology, seasonal abundance, host preference and life history of the most common pierid species Catopsilia pomona F. found in association with wild plants of north east India, including Tripura. However, information on life table and host selections are available on other pierid species that feed and oviposit on crop plants (Chew, 1995). C. pomona, a dominant pierid butterfly, is found throughout the year in the state of Tripura (Agarwala et al., 2010; Majumder et al., 2011; Roy Choudhury et al., 2011). It prefers green and moist lands, pasture lands, farms, and edge of drains, moist deciduous forests, hillocks, and semi-arid areas with high abundance of grasses, small herbs and shrubs i.e. secondary type of vegetation (Atluri et al., 2004). Reported larval host plants of common emigrant comprise of Cassia fistula L., C. sophera L., C. occidentalis L., C. tora L., C. siamea (Lam.) Irwin et Barneby, Butea frondosa, and Bauhinia racemosa L. (Kunte, 2000; Atluri et al., 2004). Among these plants C. fistula, C. tora, C. occidentalis, C. sophera, and B. racemosa are important as medicinal plants (Anonymous, 2004; Danish et al., 2011; Harshal et al., 2011; Singh and Dubey, 2012), and C. siamea is used in social forestry (Atluri et al., 2004, Borikar et al., 2009). Hence, it is very important to document the seasonal occurrence and its host plant preference for oviposition and larval development of C. Pomona. With this view, the present study was conducted to know the eco-biology of Catopsilia pomona with special reference to its life history attributes in the state of Tripura. Study site Present study was conducted in Trishna Wildlife Sanctuary of south Tripura district (23°26.137’ N, 91°28.184’ E: 51-82 m asl), having an area of about 194.7 sq. km. Study location is characterized by patches of secondary moist deciduous forests and surrounded by swamp areas. Forest patches are rich in sal trees, garjan trees, bamboo bushes, herbs, shrubs and climbers. Trishna sanctuary is known by 230 tree species, 110 species of shrubs, 400 species of herbs, and 150 species of climbers (Economic review of Tripura, 2008-2009). Among the known host plants of C. pomona, the study Roy Choudhury and Agarwala, 2013 877 Journal of Research in Biology (2013) 3(3): 876-885
  • 32. area contains three species of Cassia only viz. Cassia sophera, C. tora and C. occidentalis which are considered to be the preferred hosts of larvae. Some part of the study area is used for rubber cultivation and paddy cultivation (Figure 1). The area has a tropical climate, with cold weather from November to February. Average daily temperature varies from the minimum of 6.8°C in January to the maximum of 37.7°C in June. The area receives, on an average, 3353.4 mm rainfall annually. MATERIALS AND METHODS Field census of eggs, larvae and oviposition preference of C. pomona Prior to the study a reconnaissance survey was made in the Trishna study area to locate the available host plants distribution of C. pomona. Walk census for leaves of host plants containing eggs and larvae were held at an interval of 7-days from March 2007 to February 2008. For this, two line transects (approx. 1 km long and 5 m wide) were set up in the study area. Thirty host plants, 10 plants each of C. sophera, C. tora and C. occidentalis, were randomly selected for the study along the length of transects and were marked with plastic tags. Thus, sixty plants from three species were selected from transects. Ovipositing females were followed in the selected host plants for recording number of eggs laid per female per leaf. Binoculars were used to observe the females from a distance (about 2 m) without disturbing them. The same host plant was also observed for presence of larvae. All the females seen ovipositing on the selected host plants was recorded during the transect walk. Two transects were walked in two consecutive days in a week. Ten apical leaves were observed within a selected plant for egg and larval counts which were made between 8.00 AM to 12.00 noon local time. When a female was found to either laying eggs or seen perching near a host plant, halt was made for approx. 8 to10 minutes, and then move to the subsequent host plants along the transect. Different host plants selected by females for oviposition were recorded, photographed, collected and later identified by comparing with the herbarium deposited in the gallery of Plant Taxonomy and Biodiversity Laboratories, Department of Botany, Tripura University. Roy Choudhury and Agarwala, 2013 Journal of Research in Biology (2013) 3(3): 876-885 878 Figure 1. Geographical map of Trishna and landscape of the Study area.
  • 33. Larval host range and seasonal variation in development Leaves of the host plant species found to contain freshly laid eggs of C. pomona in field were brought to the field station (3 km from the study area), and transferred individually to 10 cm diameter paired Petri dishes lined with corrugated papers. These were fed with surplus quantity of tender leaves of respective host plants from which they were actually collected. Twenty replicates were used for each host plant species. Food was changed every 24 hrs intervals. Petri dishes were cleaned at the time of food change. These were observed twice in a day at 11 am and again at 5 pm to record the incubation period of eggs, developmental time of larvae, and pupae. Mortality in development, if any, was also recorded. This was simultaneously done on each host plant, once in five different seasons to record the seasonal variation, if any. Experiments were set up at the field station (Temp: 18°C ~ 27°C, RH: 45~75%, and L: D: 16:8h) i.e. in the controlled environment. Larval development in field Selected plants with freshly laid eggs and subsequent developmental stages were provided with coloured tags and these were numbered for easy identification. Individual eggs, larvae and pupae were followed daily, and the disappearance of individuals or those that failed to develop in to the next stage at different life stages were recorded. Larvae were found to be slightly sluggish and females laid solitary eggs, usually one on each leaf. The study was repeated once in different seasons. Survival rate and K-factor analysis An age-specific life table was constructed following the method of Stiling (2002). To prepare the life table, records were made on the larval durations and survival rate at each developmental stage i.e. eggs to emergence of adults from pupae. For this purpose, 409 eggs and 317 eggs of C. pomona were studied in natural (in field) and in controlled conditions (ambient condition of field station), respectively. Meteorological data of Trishna study area were collected from the records maintained by the Department of Agriculture, Govt. of Tripura at Arundhuti Nagar, Agartala. Data analysis Field data on proportion of host plants used by C. pomona for laying of eggs and distribution of eggs per leaf of the different host species during a year were used to draw population curves. For this purpose, weekly data were pooled on monthly basis. Developmental time from egg to the eclosion of pupae on different host plants and between different seasons was subjected to one-way analysis of variation (ANOVA). Mean values of development time on different host plant species and between different seasons were compared by Tukey’s multiple comparison test. Differences in development time recorded in field and in field station were compared by Students t-test. A significance level of 0.05 was used to reject the null hypothesis. Field data on distribution of eggs on different host plant species were subjected to regression analysis to reveal the relationship between oviposition preference and host utilization. Based on the life table data, survival rate and K factor value that closely mirrors the overall population mortality was Roy Choudhury and Agarwala, 2013 879 Journal of Research in Biology (2013) 3(3): 876-885 Host plant No. of leaves observed No. of larvae counted Mean (+SEM) no of larvae/ leaf ANOVA No. of eggs counted Mean (+SEM) no of eggs/ leaf ANOVA C. sophera 4800 984 0.21 + 0.01 F = 6.909 , df = 2,14397, P = 0.0001 1237 0.26+0.02 F = 5.26, df = 2,14397, P = 0.006 C.occidentalis 4800 563 0.12 + 0. 02 899 0.19+0.03 C. tora 4800 647 0.13 + 0.01 816 0.17+0.02 Table 1. Oviposition preference of C. pomona females on different host plants in the study area
  • 34. determined. At each life stage, number of deaths (k value) was calculated as under: k = log Nt - log Nt+1, where Nt is the density of the population before it is subjected to the mortality and Nt+1 is the density afterward. Total generational mortality factor K is determined as the sum of the individual mortality factors k at egg, larval and pupal stage of the C. pomona species (Stilling, 2002). For interpretation of colleted data, the year was divided in to five seasons: spring (March, April), summer (May, June), rain (July, September), autumn (October, November), and winter (December- February). To determine the relationship between successful development (%) of C. pomona eggs and climatic factors in the study area regression analysis was carried out. Origin 7 software (www.originlab.com) was used for the analysis of data. RESULTS Egg abundance and oviposition preference Females of C. pomona laid solitary eggs at edges and on undersides of tender or young leaves (one egg/ leaf/female) of C. sophera, C. occidentalis and C. tora plants throughout the year (Table 1, Figure. 2). In the year-round census of 10000 m2 (1000 m long x 5 m wide x 2 transects @ 1 ha) which represents less than 0.5% of the study area (19.47 ha), 52.54% to 85.07% of C. sophera plants, 21.31% to 69.47% of C. occidentalis plants and 23.88% to 56.52% of C. tora plants were found with one or more eggs. Between the three host plant species, common emigrant females selected the highest proportion of C. sophera for oviposition during hot and wet months, and the maximum was recorded in the month of August (Figure 2). In comparison, distribution pattern of eggs on C. occidentalis plants showed marked difference from the distribution of eggs on C. sophera. Higher proportion of this host plant species was recorded during dry and cooler months, and the maximum was recorded in the month of January (69.47%) (Figure. 2). In case of C. tora, the trend of egg distribution was found to be nearly similar to that of C. sophera but the proportion of host use was found to be much lower than C. sophera (Figure. 2). Occurrence of eggs showed that 4800 leaves each of C. sophera, C. occidentalis and C. tora that were surveyed during the year, contained 1237, 899 and 816 eggs, respectively (mean + SEM: C. sophera: 0.26+0.02 eggs per leaf, C. occidentalis: 0.19+0. 03 eggs per leaf and C. tora: 0.17+0.02 eggs per leaf, ANOVA: F = 5.26, df = 2, 14397, P = 0.006) (Table 1). Larval host range Larvae of C. pomona were found to feed on tender leaves of the three host plant species, viz. C. sophera, C. occidentalis and C. tora. Higher proportion of C. sophera plants were used as food and maximum was recorded in the hot and wet month of August (26.70%). Incidence of larvae on C. occidentalis Roy Choudhury and Agarwala, 2013 Journal of Research in Biology (2013) 3(3): 876-885 880 Month N Mean + SEM value (days) C. sophera C. occidentalis C. tora March 36 24.50 + 0.26 1 a 24.75 + 0.25 1 a 24.74 + 0.33 1 a May 36 20.67 + 0.31 2 a 20.92 + 0.42 2 a 20.92 + 0.42 2 a August 36 18.92 + 0.23 3 a 19.42 + 0.63 3 a 19.00 + 0.28 3 a October 36 21.17 + 0.24 2 a 21.33 + 0.28 2 a 21.25 + 0.25 2 a December 36 30.67 + 0.47 4 a 30.83 + 0.41 4 a 31.00 + 0.41 4 a Dissimilar numbers following means in a column denote significant difference and similar letters accompanying means show no difference between them by Tukey’s multiple comparison range test at 5% significant level. Table 2. Development time (in days) of C. pomona on different host plant species
  • 35. plants was recorded to be the highest in January (20.61%) and lowest in August (1.64%), respectively. In case of C. tora host, the highest proportion was recorded in the month of June (17.24%) and the lowest in the month of January (5.33%) (Figure. 3). Occurrence of larvae showed that 4800 leaves each of C. sophera, C. occidentalis and C. tora that were surveyed during the year, contained 984, 563 and 647 larvae, respectively (mean + SEM: C. sophera: 0.21 + 0.01 larva per leaf, C. occidentalis: 0.12 + 0. 02 larva per leaf and C. tora: 0.13 + 0.01 larva per leaf, ANOVA: F = 6.909, df = 2,14397, P = 0.0001) (Table 1). Developmental time and seasonal variation Developmental time of different immature stages (egg to pupae) of C. pomona was found to vary in different seasons but did not show difference in any one season between different host species (Figure 4). Development time was recorded to be the longest at lower temperature and lower relative humidity corresponding to the month of December (controlled condition: average temperature=18°C, average relative humidity=51.33%) and shortest at higher temperature and higher relative humidity in August (average temperature=27.91°C, average relative humidity =77.07%) (Table 2). Survival rate and K factor analysis Results showed that in field about 30% of the eggs deposited by C. pomona developed in to pupae during the months of July and August (average temperature 31.09°C, average humidity 70%, mean rainfall 7.45 cm). Developmental success was limited to 13.04% in the month of December (average temperature 19.330 C, average humidity 51%, rainfall 0 cm). Regression analysis of survival rate showed positive correlations with average temperature (y =1.08 + 0.87x, Roy Choudhury and Agarwala, 2013 881 Journal of Research in Biology (2013) 3(3): 876-885 Figure 4. Development time (in days) of C. pomona on different larval host plants in different months of a year. Similar alphabets accompanying bars denote no significant difference between the mean values in that month. Figure 3: Mean number of larvae of C. pomona recorded on different host plants. Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Proportionofhostplantsselectedforoviposition Months C. occidentalis C. sophera C. tora Figure 2. Proportion of host plants of three Cassia species recorded with eggs of C. pomona in different months of the year in the study area.
  • 36. r2 =0.74) (Figure 5a), average relative humidity (y = 3.87 + 0.33x, r2 = 0.52) (Figure 5b), and with mean rainfall (y =20.07 + 1.64x, r2 = 0.64) (Figure 4c). Number of eggs that developed successfully in fields (24.03+1.46; n=240) and in semi natural condition (76.36+0.90; n=240) showed significant difference (t =30.54, df =478, P =0.000). K-value analysis showed maximum mortality in development (0.46) in the month of December and minimum (0.16) in the month of September. K - values of the eggs recorded in different seasons were found to be very high (0.21) and very low (0.09), respectively, during these two months. Analyses showed that mortality in the egg stage influenced the total K value the most (Figures. 6a, b). DISCUSSION Natural populations of phytophagous insects including butterflies frequently encounter wide choice of host plants of differing suitability (Badeness et al., 2004; Dennis et al., 2006). The dominant strategy among herbivorous insects involves specialization on a set of closely related plants that will maximize offspring survival and fitness (Ward and Spalding, 1993; Gibbs et al., 2006), and also to the phenological characteristics of host plants. It is evident from the present study that C. pomona butterflies utilize three species of Cassia for oviposition and larval development in Trishna study area. Among these host plants, maximum number of C. pomona eggs were found in C. sophera with higher proportion recorded during hot and wet months, and lowest in dry and cooler months of the year. During dry and cool months, females choose C. occidentalis in higher proportion for oviposition followed by C. tora. This might be due to the availability of more young leaves in C. occidentalis and C. tora compared to C. sophera in dry and cooler months of the year. Results indicated that common emigrants preferred C. sophera than the two other host plants but utilized three hosts throughout the year depending on the host plant phenology, and made the larval host range wider. Patterns of host use have several effects on butterfly Roy Choudhury and Agarwala, 2013 Journal of Research in Biology (2013) 3(3): 876-885 882 Figure 5. Regression analysis between successful development (%) of C. pomona eggs and climatic factors: (a) average temperature (o C), (b) average relative humidity (%), and (c) mean rainfall (cm).
  • 37. population dynamics (Hanski and Singer, 2001). Food plant-insect herbivore association is based on resource size and optimal synchronization of their respective life-cycles. If resource size in time and given space is large, insects will show monophagism. In comparison, if resource size is short and patchy, then insect herbivores are generally polyphagous or oligophagous (Price, 1997; Dixon, 1998; Nylin et al., 2009). In this study, availability of taxonomically closely related Cassia plants in time and given area under study on which C. pomona successfully completed their life history attributes might widen their host range. This finding is in conformity with the optimisation theory of species in relation to host plants in time and space (Begon et al., 1996; Scheirs and Bryn, 2002). Population of C. pomona showed strong relationships with climatic factors. They took longer time for development in the dry and cooler months when the suitable habitat for oviposition and larval development were minimum than in wet and hot months. But, developmental time on the different host plants did not differ during a particular season that suggested possible qualitative similarity between host plants. However, several studies showed that ovipositing females of phytophagous butterflies typically show a preference for host plants that are capable of supporting fast larval growth (Thompson, 1988a, b, c; Janz et al., 1994). Climatic factors are well known for their significant influence on population dynamics of animal communities (Leonard et al. 1998). Analysis of K-value in this study has revealed that the average temperature, the average relative humidity and the mean rainfall showed strong positive relationships with survival rate of C. pomona. In the present study no biotic factors such as parasites, predators were noticed which can also influence the population dynamics of C. pomona butterfly. CONCLUSION Results revealed that C. pomona females occurred and laid eggs throughout the year on three host plant species of Cassia. It preferred C. sophera host over C. occidentalis and C. tora for oviposition and larval development. Pattern of egg distribution i.e. oviposition was found to be linked with host plant phenology. Egg mortality was the major influencing factor in determination of survival rate. The k-value of egg mortality (k1) and total mortality factor (K) showed strong positive relationship. ACKNOWLEDGEMENT Authors are thankful to the Head, Department of Roy Choudhury and Agarwala, 2013 883 Journal of Research in Biology (2013) 3(3): 876-885 Figure 6. Key- factor analysis of development of C. Pomona: (a) mortality in developmental stages expressed as k values, (b) regression fit of mortality in egg stage (k1) to the total K value.
  • 38. Zoology, Tripura University for the laboratory facilities. REFERENCES Anonymous. 2004. Ethnomedical Information on Fedegoso (Cassia occidentalis). Raintree Nutrition, Inc. Carson City, Nevada 89701. Anonymous. 2007 Overview of forest pests – Thailand, 43. Agarwala BK, Roy Choudhury S and Raychaudhuri P. 2010. Species richness and diversity of butterflies in urban and rural locations of north-east India. Entomon. 35:87-91. Alfred JRB, Sinha N K and Chakraborty S. 2002. Checklist of Mammals of India, Records of Zoological Survey of India, Kolkata, Occ. Paper No. 199: 1-289. Atluri JB, Venkata Ramana SP and Reddi CS. 2004. Ecobiology of the tropical pierid butterfly Catopsilia pyranthe. Current Science, 86(3): 457-461. Badeness F, Shelton A and Nault B. 2004. Evaluating trap crops for diamond back moth, Plutella xylostella (Lepidoptera: Plutellidae). Journal of Ecological Entomologia. 97(4): 1365-1372. Begon M, Harper JL and Townsend CR. 1996. Ecology: Individuals, populations and communities. Blackwell Science Ltd., London. Borges RM, Gowda V and Zacharias M. 2003. Butterfly pollination and high contrast visual signals in a low-density distylous plant. Oecologia, 136(4): 571-573. Borikar VI, Jangde CR, Philip P and Rekhe DS . 2009. Study of Antiulcer Activity of Bauhinia racemosa Lam in rats. Veterinary World, 2(6): 217-218. Capinera JL. 2008. Encyclopedia of Entomology, 2nd Edition. Spinger. Chew FS. 1995. From weeds to crops: Changing habitats of Pierid butterflies (Lepidoptera: Pieridae). Journal of the Lepidopterists’ Society. 49: 285-303. Danish M, Singh P, Mishra G, Srivastava S, Jha KK and Khosa RL 2011 . Cassia fistula Linn. (Amulthus)- An Important Medicinal Plant: A Review of Its Traditional Uses, Phytochemistry and Pharmacological Properties. Journal of Natural Product Plant Resources. 1(1): 101-118. Dennis RLH, Shreeve TG and Van Dyck H. 2006. Habitats and resources: the need for a resource-based definition to conserve butterflies. Biodiversity and Conservation. 15(6): 1943-1966. Dixon AFG. 1998. Aphid Ecology, 2 nd edn. Chapman and Hall, London. Economic review of Tripura. 2008-2009. Directorate of Economics & Statistics Planning (Statistics) Department Government of Tripura, Agartala. Gibbs MLA Lace, Jones MJ and Moore AJ. 2006. Multiple host-plant use may arise from gender-specific fitness effects. Journal of Insect Science. 6: 04, available online: insect science. Org/6.04 Hanski I, Singer MC. 2001. Extinction-colonisation dynamics and host-plant choice in butterfly metapopulations. American Naturalist. 158: 341-353. Harshal A. Pawar I and Priscilla M. D’mello. 2011. Cassia tora Linn.: An overview. International Journal of Pharmaceutical Science, 2: 2286-2291. Janz NS, Nylin N, Wedell. 1994. Host plant utilization in the comma butterfly: sources of variation and evolutionary implications. Oecologia 99(1-2): 132-40. Kremen C. 1992. Assessing the indicator properties of species assemblages for natural areas monitoring. Ecological Applications. 2(2): 203-217. Kunte K. 2000. Butterflies of peninsular India, Universities Press (India) Ltd, Hyederabad. Leonard G, Levine JM, Schmidt P, Bertness MD. 1998. Flow-generated bottom-up forcing of intertidal community structure in a Maine estuary. Ecology 79: 1395-1411. Majumder J, Lodh R and Agarwala BK. 2011. Butterfly fauna of Rowa wildlife sanctuary, Tripura, North-East India. Proceedings of National Conference on water, energy and biodiversity with special reference to North-East region. Excel India Publishers, New Delhi, 1: 266-271. Mandal DK, Ghosh SK and Majumdar M. 2002. Roy Choudhury and Agarwala, 2013 Journal of Research in Biology (2013) 3(3): 876-885 884
  • 39. Zoological Survey of India, State Fauna Series 7: Fauna of Tripura. 3: 283-334. Murugesan S and Muthusamy M. 2011. Patterns of butterfly biodiversity in three tropical habitats of the eastern part of Western Ghats. Journal of Research in Biology 1(3): 217-222. Nylin S, Nygren GH, Soderlind L and Stefanescu C. 2009. Geographical variation in host plant utilisation in the comma butterfly: the roles of time constraints and plant phenology. Evolutionary Ecology. 23(5): 807-825. Price PP. 1997. Insect Ecology, Third ed. Wiley. Roy Choudhury S, Ray Choudhury P and Agarwala BK. 2011. Butterflies of Trishna wildlife sanctuary of north-east India with a note on their diversity and seasonality. Proceedings of National Conference on water, energy and biodiversity with special reference to North-East region. Excel India Publishers, New Delhi, pp. 261-265. Scheirs J and Bryn LC. 2002. Integrating optimal foraging and optimal oviposition theory in plant-insect research. Oikos 96(1): 187-191. Singh A and Dubey NK. 2012. An ethnobotanical study of medicinal plants in Sonebhadra District of Uttar Pradesh, India with reference to their infection by foliar fungi. Journal of Medicinal Plants Research. 6(14): 2727-2746. Stiling P. 2002. Ecology Theories and Applications. Prentice Hall of India Pvt Ltd (4th Edition). Thomas CD and Malorie HC. 1985. Rarity, species richness, and conservation: Butterflies of the atlas mountains in Morocco. Biological Conservation 33: 95- 117. Thompson JN. 1988a. Variation in preference and specificity in monophagous and oligophagous swallowtail butterflies. Evolution 42:118-28. Thompson JN. 1988b. Evolutionary genetics of oviposition preference in swallowtail butterflies. Evolution 42(1): 1223-34. Thompson JN. 1988c. Evolutionary ecology of the relationship between oviposition preference and performance of offspring in phytophagous insects. Entomologia Experimentalis et Applicata. 47(1): 3-14. Thorsteinson AJ. 1960. Host selection in phytophagous insects. Annual Review of Entomology 5: 193-218. van Nouhuys S, Singer MC, Nieminen M. 2003. Spatial and temporal patterns of caterpillar performance and the suitability of two host plant species. Ecological Entomology, 28(2): 193-202. Ward LK and Spalding DF. 1993. Phytophagous British insects and mites and their food-plant families: total numbers and polyphagy. Biological Journal of the Linnean Society 49(3): 257-276. Roy Choudhury and Agarwala, 2013 885 Journal of Research in Biology (2013) 3(3): 876-885 Submit your articles online at www.jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php.
  • 40. JournalofResearchinBiology Anti-inflammatory activity of lycopene isolated from Chlorella marina on carrageenan-induced rat paw edema Keywords: Microalgae, Chlorella marina, lycopene, anti-inflammation. ABSTRACT: Even though role of lycopene (all-trans) in controlling inflammation was reported, lycopene (cis and all-trans 40:60) isolated from green algae Chlorella marina was not reported so far. In this present study inflammation was induced in male Sprague dawley rats and edema was produced acutely by injecting 0.1 ml of carrageenan into the plantar region of the right hind paw of the rats subcutaneously. Intra peritoneal administration of algal lycopene (AL) at the dose of 10 mg/kg b.wt showed maximum (83%) inhibition on paw edema. The anti- inflammatory effect was significantly (P< 0.05) higher in rats fed with algal lycopene when compared to the standard drug voveran (71%) and all- trans tomato lycopene (TL) (63%). Carrageenan induced rats showed elevated levels of cyclooxygenase (COX) and lipoxygenase (LOX) activities in monocytes. Myeloperoxidase (MPO) in serum, C- reactive protein (CRP) and ceruloplasmin activity in plasma was also high in carrageenan induced rats when compared to normal. Lycopene from Chlorella marina showed significant effect in reducing the above parameters to that of the standard drug while tomato lycopene showed less effect when compared to algal lycopene. Therefore algal lycopene from Chlorella marina would be recommended for the treatment of anti-inflammatory disorders. 886-894 | JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Journal of Research in Biology An International Scientific Research Journal Authors: Renju GL and Muraleedhara Kurup G. Institution: Department of Biochemistry, University of Kerala, Trivandrum, India. Corresponding author: Muraleedhara Kurup G. Email: gmkbio@gmail.com. Phone: +919447251408. Fax: 91-471 2308078. Web Address: http://jresearchbiology.com/ documents/RA0329.pdf. Dates: Received: 02 Feb 2013 Accepted: 22 Feb 2013 Published: 23 Apr 2013 Article Citation: Renju GL and Muraleedhara Kurup G. Anti-inflammatory activity of lycopene isolated from Chlorella marina on carrageenan-induced rat paw edema. Journal of Research in Biology (2013) 3(3): 886-894 Journal of Research in Biology An International Scientific Research Journal Original Research
  • 41. INTRODUCTION Inflammation is a response which protects and heals the host tissue after infection or injury. (Nathan, 2002). However, it is frequent that the inflammatory response to several insults erroneously leads to the damaging of normal tissues. Prostaglandin-E2 is generated from arachidonic acid by the enzyme cyclooxygenase (COX) at sites of inflammation in substantial amounts and can mediate many of the pathologic features of inflammation (Serhan and Levy, 2003). One of the early cellular events in inflammation is the margination of leukocytes, primarily neutrophils and this can be measured by myeloperoxidase activity (Goulet et al., 1994). Currently, non steroidal anti-inflammatory drugs (NSAIDs) were used for inflammatory diseases. Even though this drugs transiently suppresses inflammation, but their long term use cause ulceration in the gastrointestinal tract and renal morbidity (James and Hawkey, 2003). However research focused on finding newer drugs with pharmacological actions without side effects. Several antioxidants have been reported to have anti-inflammatory and anti-arthritic activities (Maxwell et al., 2006). In the present study a culturable marine edible algae Chlorella marina was selected to evaluate the anti-inflammatory activity of lycopene. Generally tomatoes are the source of lycopene, but it has many disadvantages (Shi and Le mague, 2000). The content of lycopene in tomato is very less and the configuration of lycopene is all-trans. Even though lycopene from algae has been reported (Ishikawa and Abe, 2004), no attempt has been made so far for the commercialization of algal lycopene. It can be seen that marine sources especially algae are the least exploited for their bioactive molecules (Pinky and Goswai, 2012). Work in our laboratory has shown that the lycopene content in algae is comparatively high, when compared to tomato lycopene. The most interesting observation was that algal lycopene contain cis-configuration (5-cis, 9-cis, 13- cis and 15-cis). Recently it has been reported that the cis form of lycopene is more biologically active than the trans form (Stahl and Sies, 1996). MATERIALS AND METHODS Chemicals Lycopene, carrageenan, linoleic acid, Histopaque, arachidonic acid other fine chemicals were purchased from Sigma, St. Louis, MO, USA. Diclofenac sodium (Voveran) was obtained from Novartis, India. Salt and vitamin mixtures were purchased from Merck, Germany. All other chemicals and reagents were purchased from Sisco Research Laboratory Pvt.Ltd (SRL), India, and were of analytical grade. Algal source Marine algae Chlorella marina Butcher was collected from the Vizhinjam coast of Kerala, located at Latitude 08° 22’ North Longitude. 76° 59’ East on the south west coast of India and was cultured under laboratory conditions. The microalgae were identified by the botanist (Dr. G. Valsaladevi, Department of botany) and a voucher specimen (No. KUBH 5812) has been deposited in the Department of Botany, University of Kerala, India. Culture medium Walne’s medium (1970) was used as a basal medium for the cultivation of Chlorella marina. 5 g /L glucose was added to the basal medium. Flasks were incubated at 25°C with continuous illumination. The pH was adjusted to 7.5. Nicotine (10 µM/ L) was sterilized by autoclaving and was added to 5 days old cultures for the production of lycopene. Biomass harvest Chlorella marina cells were grown in suspension cultures up to 30 to 40 days. The cells were harvested at stationary phase by withdrawing the cultures in 50 ml polypropylene tubes and centrifuged at 5000 rpm for Renju and Kurup., 2013 887 Journal of Research in Biology (2013) 3(3): 886-894
  • 42. 10 minutes. Removed the medium and the pellets were freeze dried, weighed and stored under nitrogen at -20°C. Isolation of lycopene from Chlorella marina (AL) and analysis Harvested biomass (5g dry weight) was suspended with 5 ml of 80% cold acetone and kept overnight under 4°C for better and easy recovery of carotenoids. The mixtures were vortexed for 2 minutes and centrifuged at 5000 rpm for 20 minutes. After repeated extractions (4 times), the supernatants were pooled and the colorless cell pellets were discarded. The extracts were dried over anhydrous sodium sulphate and reduced to a minimum volume by evaporating the solvents using N2 stream. The crude extracts were kept for further separation of carotenoids in amber colored containers under nitrogen at -20°C. All operations were done at subdued light under nitrogen atmosphere. The absorbance in the solvent phase was quantified by spectrophotometric method at 470 nm as described by Lichtenthaler (1987). Isolation of all-trans lycopene from tomato (TL) Tomatoes obtained from the local market, Trivandrum, India were used. The all-trans lycopene from tomato was extracted and evaluated according to the procedure of Fish et al., (2002). Determination of lycopene by HPLC Lycopene extracted from algal cells and tomatoes were determined by HPLC method at 450 nm as described by Shaish et al., (1992). HPLC analysis of lycopenes were performed using a silia chrom® column (250 x 4mm + 5 x 4, NCLIOSIL 100-5-C18 5.0µm), K 1001 type pump and the UV detector type of K 2600, Germany. Elution was performed isocratically with methanol: acetonitrile (9:1) v/v at a flow rate of 1 ml min-1 . A UV detector with a wavelength of 450 nm was employed. Lycopene (95%) obtained from Sigma chemicals were used as standard. The retention time was recorded and peak areas of standards and tests were noted on each run and used for calculation of concentrations of different fractions. All samples were injected in duplicate. Experimental animals Male Sprague Dawley rats with the average body weight of 150- 200 g of the same breed were selected for the study. These animals were housed in the department animal house and provided standard pellet diet and water ad libitum and maintained with temperature at 25 ± 1°C, humidity (55-60%) and photoperiod (12:12 h) light and dark cycle. Experimental procedures conducted on rats were approved by the Animal Experiment Committee (218/CPCSEA) for animal care of Kerala University according to Government of Indian law on animal use and care. Induction of acute inflammation-Carrageenan induced rat paw edema Carrageenan-induced rat paw edema assay was conducted according to the procedure as described by Winter et al., (1962). Five groups of six rats were treated as AL and TL with doses 10 mg/kg and reference drug Voveran, a Diclofenac sodium preparation (20 mg/kg) were given orally and intraperitoneally (i.p), 1 h before the injection of carrageenan. Control rats were given 0.1 ml 1% carrageenan. Inflammation was induced by 0.1 ml, 1% carrageenan suspension in 0.9% NaCl solution was injected into the right hind paw after 1 hour. The volume of the right paw was measured by paw edema meter before and after injection in the third and fifth hour. The paw edema and inhibition was calculated by the equation: Activity= 100 - (100 × average drug treated/average for control). Treatment Protocol and Experimental Design in Acute Inflammation Edema was induced on rat right hind paw by aponeurosis injection of 0.1ml of 1% carrageenan in 0.9% saline. The experimental groups consisted of 30 rats were divided in to five groups. Group I: control (received saline only), Group II: carrageenan alone Renju and Kurup., 2013 Journal of Research in Biology (2013) 3(3): 886-894 888
  • 43. Group III: carrageenan + algal lycopene (AL groups, 10 mg/kg i.p) Group IV: carrageenan + tomato lycopene (TL groups, 10 mg/kg i.p) Group V: carrageenan + Voveran (VOV groups, 20 mg/kg i.p.) At the end of third hour, the animals were sacrificed by euthanasia. Blood was removed to ice cold containers for various biochemical analyses. Activity of Cyclooxygenase (COX) and Lipooxygenase (LOX) in Peripheral Blood Mononuclear Cells (PBMC) Mononuclear cells were isolated the procedure described by Radhika et al., (2007). Cox activity was measured by the method of Shimizu et al., (1984). 15-LOX activity was determined by the method of Axelrod et al., (1981). Biochemical analysis Serum myeloperoxidase (MPO) activity was measured by Mullane et al., (1985). CRP in plasma was determined by using Immunoturbidometric kit (Diasys Diagnostics, Germany). Ceruloplasmin was estimated by the method of Ravin (1961). Protein was determined by the methods of Lowry et al., (1951). Statistical analysis The Statistical package for social sciences (SPSS/PC+), version 11.5 (SPSS Inc; Chicago. IL, USA) was used to analyze the results for statistical significance using one-way ANOVA followed by Duncan’s test. P value < 0.05 was considered as significant. RESULTS AND DISCUSSION Sub plantar injection of carrageenan into the foot of rats caused a time-dependent increase in paw volume. The localized inflammatory response as evidenced visually by the edema reached a maximum intensity at third hour after carrageenan induction and this maximal effect was seen until the fifth hour. Administration of AL and TL has showed significant effects in decreasing carrageenan-induced paw edema. Algal lycopene showed maximum edema inhibition compared to all-trans tomato lycopene and drug. AL exhibited 70% and 83% edema inhibition at third/fifth hours, respectively. This effect was comparable to the reference drug Voveran which exerted 54% and 71% edema inhibition at third and fifth hour, respectively. TL showed 51% and 63% edema inhibition at third and fifth hour after carrageenan induction (Figure. 1). COX activity in PBMC was significantly (p<0.05) increased in carrageenan treated rats when compared to control rats (Figure. 2). Treatment with AL showed significant (p<0.05) decrease in COX activity when compared to carrageenan induced rats. Prostaglandin is formed by the interaction of two distinct but related enzymes, COX-1 and COX-2 and plays an important role in promoting the signs and symptoms of inflammation (Otterness and Bliven, 1985; Ibegbulem et al., 2012). The activity of COX in PBMC was decreased (p<0.05) in AL treated group when compared to TL and voveran treated group. Reduction of paw swelling and decreased activity of COX showed the immunological protection rendered by the algal lycopene. These results showed the anti-inflammatory potential of the AL. The activity of 5-LOX and 15-LOX in PBMC was significantly (p<0.05) increased in carrageenan induced rats when compared to normal rats (Figure.3 and 4). Algal lycopene treatment significantly reduced (p<0.05) in 5-LOX and 15-LOX activity, when compared to CII rats. The effect was significantly higher (p<0.05) than TL and drug treated groups. Lipoxygenases are a family of key enzymes in the biosynthesis of leukotrienes that are postulated to play an important role in the pathophysiology of several inflammatory diseases (Henderson, 1994; Yamamoto, 1992). In the normal situation, cellular leukotriene production is suppressed by selenium dependent peroxidases (Werz et al., 1997). On receiving Renju and Kurup., 2013 889 Journal of Research in Biology (2013) 3(3): 886-894
  • 44. inflammatory stimuli, leukotriene production is elicited through the arachidonic acid cascade, causing micro vascular injury, vasoconstriction and production of pro-inflammatory cytokines (Peskar, 1991). Studies have shown that LOX and leukotrienes have a profound role in carrageenan-induced inflammation (Henderson, 1994; Gamache et al., 1986). In the carrageenan-induced inflammation model, AL significantly reduced carrageenan-induced 5-LOX and 15-LOX activities in mononuclear cells, indicating decreased leukotriene production and hence a protective effect. MPO activity in serum was significantly increased (p<0.05) in carrageenan induced rats when compared to normal group (Table 1). Treatment with AL showed significant decrease (p<0.05) in MPO activity when compared to carrageenan induced rats. The MPO activity was significantly decreased when compared to TL and drug treated groups. The activity of MPO is a marker of neutrophil infiltration (Bradley, 1982), and was found to be significantly increased in the paw tissue of carrageenan-induced rats. AL significantly decreased (p<0.05) the elevated MPO activity, an indicator of neutrophil in inflamed paws, suggesting that inhibition of neutrophil infiltration might be another mechanism by which AL achieves its anti-inflammatory effect. Table 1 also shows the variations in serum CRP and ceruloplasmin level in the test animals compared to control. Serum CRP and ceruloplasmin levels were significantly increased (p<0.05) in carrageenan induced rats when compared to normal rats. Supplementation with AL significantly decreased (p<0.05) the serum CRP and ceruloplasmin levels when compared to carrageenan induced rats. The levels of CRP and ceruloplasmin were decreased significantly (p<0.05), when compared to TL and Voveran treated groups. C-reactive protein is an acute phase protein that has been identified as an important biomarker for various inflammatory, degenerative, and neoplastic diseases. Elevated levels of CRP have been found in the blood during virtually all diseases associated with active inflammation or tissue destruction, particularly in patients with rheumatoid arthritis (Pepys and Hirschfield, 2003; Kushner, 1991). In our study the increased levels Renju and Kurup., 2013 Journal of Research in Biology (2013) 3(3): 886-894 890 Figure 1: Effect of algal lycopene on carrageenan- induced paw edema in normal and experimental rats. Figure 2: Effect of algal lycopene on activity of COX in PBMC of normal and experimental rats COX activity is expressed as an optical density increase (OD increase) per mg protein per minute. Val- ues are expressed as mean ± SEM of six rats in each group. a – Statistical difference of Control group with CII group when p < 0.05. b – Statistical difference of CII group with group AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with group AL and group TL when p < 0.05. d –Statistical difference of TL group with AL when p <0.05.
  • 45. of CRP level was found to be significantly decreased in algal lycopene treatment when compared to TL and Voveran treatments. The serum protein, ceruloplasmin is a powerful free radical scavenger that oxidizes iron from the ferrous to ferric state. Ceruloplasmin levels increase under conditions leading to the generation of oxygen products such as the superoxide radical and hydrogen peroxides (Revnic, 1995). Serum ceruloplasmin level was significantly increased in carrageenan induced rats when compared to normal rats. Treatment with AL showed significant decrease in the concentration of ceruloplasmin. The increased levels of ceruloplasmin in carrageenan induced rats could be decreased significantly on treatment with algal lycopene when compared to TL and standard drug Voveran might be having a protective response against free radical mediated lipidperoxidation. Lycopene from edible marine microalgae C. marina showed higher anti-inflammatory activity than all-trans tomato lycopene and standard drug Voveran. These effects might be due to the presence of two isomeric form of lycopene (cis and all-trans) in the microalgae. Reports available indicate that the cis-lycopene has a high antioxidant potential when compared to all-trans lycopene (Stahl and Sies 1992; Clinton et al., 1996). Algal lycopene isolated from C. marina could reduce cell influx, oedema formation Renju and Kurup., 2013 891 Journal of Research in Biology (2013) 3(3): 886-894 Figure 3: Effect of algal lycopene on activity of 5- LOX in PBMC of normal and experimental rats 5-LOX activity is expressed as an optical density increase (OD increase) per mg protein per min- ute. Values are expressed as mean ± SEM of six rats in each group. a – Statistical difference of Control group with CII group when p < 0.05. b – Statistical difference of CII group with group AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with group AL and group TL when p < 0.05. d –Statistical difference of TL group with AL when p <0.05. Figure4: Effect of algal lycopene on activity of 15- LOX in PBMC of normal and experimental rats 15-LOX activity is expressed as an optical den- sity increase (OD increase) per mg protein per minute. Values are expressed as mean ± SEM of six rats in each group. a – Statistical difference of Control group with CII group when p < 0.05. b – Statistical difference of CII group with group AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with group AL and group TL when p < 0.05. d –Statistical difference of TL group with AL when p <0.05.
  • 46. and release of mediators associated with inflammatory condition, and therefore has the potential to be used as an anti-inflammatory agent. Further studies are in progress to evaluate the molecular mechanism of its anti-inflammatory activity. ACKNOWLEDGEMENT We express gratitude to Dr. Anantha Lekshmi, Veterinary Doctor, Department of Biochemistry, University of Kerala, Kariavattom, India for helping us with the animal experiments. REFERENCES Axelrod B, Cheesebrough TM and Laakso S. 1981. Lipoxygenase from soybean. Methods.Enzymol; 71:441- 453. Bradley PP, Priebat DA, Christensen RD and Rothstein G. 1982. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol; 78:206-209. Clinton SK, Emenhiser C, Schwartz SJ, Bostwick DG, Williams AW, Moore BJ and Erdman JW. 1996. Cis-trans lycopene isomers, carotenoids, and retinol in the human prostate. Cancer. Epidemiol. Biomar. Preven; 5 (10): 823–833. Fish WW, Perkins Veazie P and Collins JK. 2002. Extraction of lycopene from tomato paste. J. Food. Compo.Analy., 15: 309–317. Renju and Kurup., 2013 Journal of Research in Biology (2013) 3(3): 886-894 892 Groups MPO (µm/min/mg) CRP (mg/ml) Ceruloplasmin (mg/dl) Control 5.85 ± 0.37 22.0 ± 1.24 0.10 ± 0.006 CII 20.52 ± 1.11a 97.71 ± 3.80a 0.34 ± 0.014a AL 7.45 ± 0.37bc 45.94 ± 2.01bc 0.19 ± 0.007bc TL 13.75 ±0.48bcd 80.64 ±3.18bcd 0.28 ± 0.016bcd VOV 9.74± 0.39b 56.89 ± 2.42b 0.22 ± 0.010b Table 1: Levels of CRP, Ceruloplasmin in plasma and MPO in serum of experimental animals. Values are expressed as mean ± SEM of six rats in each group. a – Statistical difference of Control group with CII group when p < 0.05. b – Statistical difference of CII group with group AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with group AL and group TL when p < 0.05. d –Statistical difference of TL group with AL when p <0.05. Table 2: Statistical table of Myeloperoxidase in one way ANOVA followed by Duncan’s test MPO Sum of Squares df Mean Square F Between Groups 827.642 4 206.911 91.002 Within Groups 56.842 25 2.274 Total 884.485 29 Where df is degrees of freedom, F is F- ratio. Table 3: Statistical table of CRP in one way ANOVA followed by Duncan’s test Where df is degrees of freedom, F is F- ratio. CRP Sum of Squares df Mean Square F Between Groups 20984.623 4 5246.15 6 121.093 Within Groups 1083.082 25 43.323 Total 22067.705 29 Table 4: Statistical table of CRP in one way ANOVA followed by Duncan’s test Ceruloplas- min Sum of Squares df Mean Square F Between Groups .196 4 .049 59.148 Within Groups .021 25 .001 Total .217 29 Where df is degrees of freedom, F is F- ratio.
  • 47. Gamache DA, Povlishock JT and Ellis EF. 1986. Carrageenan induced brain inflammation. Characterization of the model. J Neurosurg., 65 (5): 679- 685. Goulet JL, Snouweart JN, Latour AM, and Coffman TM. 1994. Altered inflammatory responses in leukotriene-deficient mice. Proc. Natl. Acad. Sci; 91 (26):12852–12856. Henderson WR Jr. 1994. The role of leukotrienes in inflammation. Ann. Intern .Med; 121(9): 684-697. Ibegbulem CO, Egbung GE, Okoro, KK, Kalu NN, Nwaogyu LA, and Igwe Ko. 2012. Hypothesized biochemical modes of action of palm oils used in ethno- medicine. J. Res. Biol; 2(6): 596–601. Ishikawa E and Abe H. 2004. Lycopene accumulation and cyclic carotenoid deficiency in heterotrophic Chlorella treated with nicotine. J. Ind. Microbiol. Biotechnol; 31: 1367–5435. James MW and Hawkey CJ. 2003. Assessment of non- steroidal anti-inflammatory drug (NSAID) damage in the human gastrointestinal tract. Br. J. Clin. Pharmacol; 56 (2): 146 -155. Kushner I. 1991. C-reactive protein in rheumatology. Arth. Rheum; 34, 1065–1068. Lichtenthaler Hk. 1987. Chlorophylls and carotenoids: pigments of photosynthetic Biomembrane. Methods. Enzymol; 147: 350-382. Lowry OH, Rosebrough NJ, Farr AC and Randall RJ. 1951. Protein measurement with folin phenol reagent. J. Biol. Chem; 193:265–267. Maxwell SR, Payne RA, Murray GD and Webb DJ. 2006. Selectivity of NSAIDs for COX-2 and cardiovascular outcome. Br. J. Clin. Pharmacol; 62: 243- 245. Mullane KM, Kraemer R and Smith B. 1985. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischaemic myocardium. J. Pharmacol.Methods; 14 (3): 157-167. Nathan C. 2002. Points of control in inflammation. Nature, 420 (6917): 846-852 Otterness IG and Bliven ML. 1985. Laboratory models for testing non steroidal anti-inflammatory drugs: Non- steroidal Anti-inflammatory Drugs, Ed.by J.G.Lombardino Wiley.Newyork. 220: 111-114. Pepys MB and Hirschfield GM. 2003. C-reactive protein: a critical update. J .Clin .Invest; 111: 1805-1812. Peskar BM. 1991. Role of leukotriene C4 in mucosal damage caused by necrotizing agents and Indomethacin in rat stomach. Gastroenterol; 100 (3): 619–626. Pinky Baruah and Goswai UC. 2012. Characterization of carotenoid pigments in amphibian. J. Res. Biol; 2: 114 -118. Radhika A, Jacob SS and Sudhakaran PR. 2007. Influence of oxidatively modified LDL on monocyte- macrophage differentiation. Mol. Cell .Biochem; 305:133 -143. Ravin H A. 1961. An improved colorimetric enzymatic assay of ceruloplasmin. J. Lab. Clin. Med; 58: 161-168. Revnic F. 1995. The significance of Serum Ceruloplasmin in Diagnosis of Rheumatoid Arthritis. Toxicol. Lett; 78: 70-70. Renju and Kurup., 2013 893 Journal of Research in Biology (2013) 3(3): 886-894
  • 48. Serhan CN and Levy B. 2003. Success of prostaglandin E2 in structure–function is a challenge for structure- based therapeutics. Proc. Natl. Acad. Sci. 100:8609- 8611. Shaish A, Ben Amotz and Avron M. 1992. Biosynthesis of β carotene in Dunalialla. Methods. Enzymol; 213: 439-444. Shi J and Le Mague M. 2000. Lycopene in tomatoes: chemical and physical properties affected by food Processing. Critical Reviews in Food. Sci. Nutr; 40: 1–42. Shimizu T, Kondo K and Hayaishi O. 1984. Role of prostaglandin endoperoxides in the serum thiobarbituric acid reaction. Arch. Biochem. Biophys; 206:271–276. Stahl W and Sies H. 1992. Uptake of lycopene and its geometrical isomers is greater from heat Processed than from unprocessed tomato juice in humans. J. Nutr; 122 (11): 2161-2166. Stahl W and Sies H. 1996. Lycopene: a biologically important carotenoid for humans? Arch. Biochem. Biophy; 336, 1-9. Walne PR. 1970. Studies on the food value of nineteen genera of algae to juvenile bivalves of the genera Ostrea, Crassostrea, Mercenaria, and Mytilis. Fish. Invest; 26: 1–62. Werz O, Schneider N, Brungs M, Sailer E, Safayhi H and Ammon H. 1997. A test system for leukotriene synthesis inhibitors based on in vitro differentiation of human leukemic cell lines HL-60 and Mono Mac 6 Naunyn Schmiedebergs Arch .Pharmacol; 356 (4): 441- 445. Winter CA, Risley EA and Nuss GW. 1962. Carrageenan-induced edema in hind paws of the rats as an assay for anti-inflammatory drugs. Proc. Soc. Exp .Biol. Med; 111:544-547. Yamamoto S. 1992. Mammalian lipoxygenases: molecular structures and functions. Biochim. Biophys. Acta; 1128 (1-2): 117–131 Renju and Kurup., 2013 Journal of Research in Biology (2013) 3(3): 886-894 894 Submit your articles online at www.jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php.
  • 49. JournalofResearchinBiology Identification of Animal Pasteurellosis by PCR Assay Keywords: Culture lysate, genomic DNA, Pasteurella multocida, PCR . ABSTRACT: Diagnosis of pasteurellosis has become difficult, as there are five different capsular types and 16 somatic types. Molecular techniques like PCR are adapted nowadays for rapid and accurate diagnosis in early stage of the disease and also it provides useful information for epidemiological studies. The present study was conducted to study the efficiency of polymerase chain reaction (PCR) in the identification of P. multocida isolates and evaluation of different PCR methods viz., (i) PCR using genomic DNA (ii) PCR using culture lysate and (iii) PCR by colony touch method. In the present study P. multocida specific PCR was performed by using KMT1SP6 and KMT1T7 oligos. These oligos amplified the genomic DNA from P. multocida isolates only. All the three methods produced PCR amplified product at 460 bp and colony touch method was found to be the best method. 895-899 | JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Journal of Research in Biology An International Scientific Research Journal Authors: Venkatesan PS, Deecaraman M and Vijayalakshmi M. Institution: Department of IBT, Dr. M.G.R. Educational & Research Institute, Department of IBT, Maduravoyal, Chennai - 600095. Corresponding author: Venkatesan PS. Email: venkyvet74@gmail.com Web Address: http://jresearchbiology.com/ documents/RA0332.pdf. Dates: Received: 04 Feb 2013 Accepted: 05 Mar 2013 Published: 30 Apr 2013 Article Citation: Venkatesan PS, Deecaraman M and Vijayalakshmi. Identification of animal Pasteurellosis by PCR assay. Journal of Research in Biology (2013) 3(3): 895-899 Journal of Research in Biology An International Scientific Research Journal Original Research
  • 50. INTRODUCTION The various forms of pasteurellosis caused by Pasteurella multocida are the major health problem for livestock population worldwide. Diagnosis of pasteurellosis has become difficult, as there are five different capsular types and 16 somatic types. Molecular techniques like PCR are adapted nowadays for rapid and accurate diagnosis in early stage of the disease and also it provides useful information for epidemiological studies. Pasteurellosis has high impact on economic status of Indian farmers. The overall incidence rate of haemorrhagic septicaemia (HS) was reported as 6.4 per lakh population during 1974-86, resulting in losses exceeding ten million rupees annually (Dutta et al., 1990; singh et al., 1996). Isolation and identification of P. multocida from specimens like fresh tissues or heart blood followed by the performance of various biochemical and serological methods have been used to study P. multocida. These include catalase, indole, oxidase and sugar fermentation tests. Due to time consuming procedure and limitations of these methods, molecular techniques like polymerase chain reaction (PCR) were adapted nowadays. PCR has advantages over the conventional techniques in rapidity, sensitivity and specificity to identify the P. multocida. The present study was conducted to assess the efficiency of PCR in the identification of P. multocida from poultry and ruminants and to evaluate the different methods in PCR assay viz. PCR using genomic DNA, PCR using culture lysate and PCR by colony touch method. MATERIALS AND METHODS Isolation and Identification of P. multocida Fifty two samples were collected from various geographical areas of Tamil Nadu, India. Specimens such as heart blood swab, liver, spleen and long bones collected from various animals, were streaked directly onto 5% sheep blood agar and Pasteurella multocida selective agar as reported earlier (Moore et al., 1974) and incubated at 37°C with 5-10 % CO2 for 24-48 h. Plates were examined for colonies, the suspected colonies were subjected to grams staining, and biochemical test as per standard techniques. Standard vaccine strain of P. multocida P52 (B:2) was taken as reference strain. Pathogenicity test in mice were carried out for all the fifteen isolates and PCR was performed for all the isolates. Isolation and Purification of Genomic DNA A 900 µl cell suspension of each sample were resuspended in 100 µl of 10x Tris-EDTA (TE) buffer (pH 8.3) with 10 mg of lysozyme and were incubated at 37°C for 1.5 h. Bacterial cultures were treated with 10 µl of proteinase K (10 mg/ml) and incubated at 50°C for 1 h. The nucleic acid was extracted with phenol-chloroform-isoamyl alcohol followed by ethanol precipitation as per the method of Sambrook et al., (1989) and Sachithanandam et al., (2011). PCR Using Culture Lysate One Milliliter of 18 h broth culture or take few freshly grown pure colonies from blood agar plate and suspend in 500 µl sterile distilled water and centrifuge at 4000 g for 1 minute and collect the pellet. The pellet was washed with sterile distilled water, resuspended in 100 µl sterile distilled water and boiled for 10 min. The samples were centrifuged to sediment cell debris and 10 µl of the supernatant was used in the PCR reaction. PCR Using Colony Touch Method A single pure colony grown on agar plates was used to perform PCR. A pipette tip was lightly touched onto a colony and then suspend in PCR amplification mixture. PCR Technique The species-specific primers KMT1SP6 and KMT1T7 designed by Townsend et al., (1998) were used in this study to amplify the gene sequences in P. multocida. Primers 1 KMT1SP6 5’-GCT GTA AAC GAA CTC GCC AC- 3’ Venkatesan et al., 2013 896 Journal of Research in Biology (2013) 3(3): 895-899
  • 51. Primers 2 KMT1T7 5’- ATC CGC TAT TTA CCC AGT GG-3’ PCR mixture was prepared using PCR kit obtained from FINNZYME, Finland. The 50 µl of reaction mixture was prepared with 10 µl template DNA, 10ng of each primers, 200 µM concentration of each dNTPs, 10x PCR buffer and 1 unit Taq DNA polymerase. PCR amplification was carried out in an automated thermal cycler (Perkin Elmer Gene AMP PCR system 2400) with the following thermal programme. Initial denaturising at 95°C for 4 min followed by 30 cycles of denaturising at 95°C for 1 min., annealing at 55°C for 1 min., extension at 72°C for 1 min. and final extension at 72°C for 9 min, were carried out. After amplification, PCR products were checked in 1.5% agarose gel electrophoresis along with the standard molecular weight marker (Lambda DNA Hind III digest and ϕ X 174 DNA Hae III digest; FINNZYME, Finland). The biochemical tests were carried out as per the standard procedure followed in Arun kumar et al., (2012) RESULTS Out of total collection of 52 suspected samples, procured from cattle sheep, goat and poultry, 15 samples were confirmed as P. multocida based on biochemical tests (Table 1) and PCR. All the P. multocida isolates were pathogenic to mice and dies within 24 h. PCR was performed for all the 15 isolates by 3 methods viz., colony touch method, culture lysate and with genomic DNA. P52 strain of P. multocida, obtained from the Institute of veterinary preventive medicine (IVPM) Ranipet, Tamil Nadu, taken as a positive control and the following bacteria Escherichia coli, Clostridium chauvoei, Salmonella enteritidis, Salmonella typhimurium, Bacillus anthracis, Venkatesan et al., 2013 Journal of Research in Biology (2013) 3(3): 895-899 897 Tests Name of the Isolates D1P D2P FP GP HP KP LP NP OP AS CS TS YS BG MC Hemolysis on Blood agar - - - - - - - - - - - - - - - Growth on MacConkey agar - - - - - - - - - - - - - - - Motility - - - - - - - - - - - - - - - Gelatin Liquefaction - - - - - - - - - - - - - - - Methyl Red Test - - - - - - - - - - - - - - - H2S (Hydrogen sulphide) + + - - + + + - + + + + + + - Catalase + + + + + + + + + + + + + + + Oxidase + + + + + + + + + + + + + + + Nitrate Reduction + + + + + + + + + + + + + + + Indole + + + + + + + + + + + + + + + Lysine Decarboxylase - - - - - - - - - - - - - - - Ornithine Decarboxylase - + + + + + - + + + + + + + + Urease - - - - - - - - - - - - - - - Pyrase - - - - - - - - - - - - - - - Esculin Hydrolysis - - - - - - - - - - - - - - - VT (Voges Proskaeur - - - - - - - - - - - - - - - Phenylalanine - - - - - - - - - - - - - - - β-Galactosidase (ONPG) - - - - - - - - - - - - - - - β-Glucuronidase + + + + + + + + + + + + + + + α-Galactosidase + + + + + + + + + + + + + + + β-Xylosidase - - - - - - - - - - - - - - - N-acetyl β-D-glucosaminedase - - + + + + - + - - + + + + + Table 1. Biochemical Profiles for the Identification of Pasteurella multocida Isolates + : Positive, - : Negative,
  • 52. Venkatesan et al., 2013 898 Journal of Research in Biology (2013) 3(3): 895-899 Staphylococcus aureus and Klebsiella spp. used as negative controls. The expected amplification size of 460 bp was obtained in all the 15 isolates. PCR amplification was noticed at approximately 460 bp by all the three methods and in all the 15 isolates as like that of positive control (figure 1). No amplification product was observed in negative controls (figure 1). Molecular weight of PCR product was estimated based on the standard molecular weight marker. DISCUSSION The 15 isolates of P.multocida collected from different places and sources of origin produced approximately 460 bp amplified product as that of reference strain P52, but no amplified product was noticed among the negative controls. It is concluded that the primers were highly specific to P. multocida isolated from various sources. The above result agrees with the previous reports of earlier workers (Townsend et al., 1998; Hunt et al., 2000; Miflin and Blackall, 2000; OIE manual, 2000; Dutta et al., 2001). In this study the amplified product of approximately 460 bp was observed using three different methods viz. colony touch method, culture lysate method and purified genomic DNA method (figure 1). The intensity of the amplified PCR product varies (figure 1), due to the variation in DNA concentrations. Townsend et al., (1998) reported that PCR using colony touch method produced amplification Figure 1: Pasteurella multocida – specific PCR (PM-PCR) assay CS TS YS BG MC PC NC M 460bp→ D1P D2P FP GP HP PC NC M 460bp→ KP LP NP OP AS PC NC M These figures illustrate fragments specifically amplified by PCR in all the P. multocida isolates by means of the primers KMT1SP6 and KMT1T7. Variation in the intensity of the amplified product was observed, due to variation in DNA concentration of each sample. D1P, D2P, FP, GP, HP, KP, LP, NP, OP, AS, CS, TS, YS, BG, and MC are the names of P. multocida isolates.
  • 53. product approximately at 460 bp and the intensity of the amplified product varied due to inconsistency of the DNA concentration. Dabo et al., (2000) reported that the boiled cell extract method has the advantages of simplicity and rapidity in the identification of P. multocida isolates. Since the PCR amplified product of 460 bp was noticed in all samples of poultry and ruminants, using oligos KMT1SP6 and KMT1T7, the oligos are considered as specific to P. multocida affecting all species of poultry and ruminants. Considering the cost and time involved in the preparation and purification of genomic DNA, the colony touch method has advantages of simplicity and rapidity for epidemiological surveys involving large number of P. multocida isolates. PCR using colony touch method would be an adaptable easy to perform method in regional laboratories for rapid diagnosis of HS and FC from field cases without the need to obtain pure culture and extensive biochemical and serological tests. ACKNOWLEDGEMENT The authors thank the Head, department of microbiology, Madras Veterinary College, Chennai, for providing the facilities to carry out this work. REFERENCE Arun Kumar JM, Lakshmi A, Sangeetha Rani V and Sailaja B. 2012. Isolation and characterization of feather degrading bacteria from poultry waste. Journal of Research in Biology. 2(7): 676-682. Blackall PJ and Miflin JK. 2000. Identification and typing of Pasteuruella multocida; A Review. Avian Pathology 29(4):271-287. Dabo SM, Confer A and Lu YS. 2000. Single primer polymerase chain reaction fingerprinting for Pasteurella multocida isolates from laboratory rabbits. American Journal of Veterinary Research 61(3):305-309. Dutta J, Rathore BS, Mullick SG, Singh R and Sharma GC. 1990. Epidemiological studies on occurrence of haemorrhagic septicaemia in India. Indian Veterinary Journal 67(10): 893-899. Dutta TK, singh VP and Kumar AA. 2001. Rapid and Specific diagnosis of animal pasteurellosis by using PCR assay. Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases 22(1):43-46 Hunt ML, Alder B and Townsend KM. 2000. The molecular biology of Pasteurella multocida. Veterinary Microbiology 72(1-2):3-5. Manual of Standards for diagnostics tests and vaccines. 2000. Office International Des Epizootics Manual, France. 446-456. Sachithanandam V, Mohan PM, Dhivya P, Muruganandam N, Baskaran R, Chaaithanya IK and Vijayachari P. 2011. DNA barcoding, phylogenetic relationships and speciation of Genus: Plectropomus in Andaman coast. Journal of research in Biology. 1( 3): 179-183. Sambrook J, Fritisch EF and Mamiatis T. 1989. Molecular cloning: a laboratory manual, Cold spring harbor press, Plainview, N.Y. 2nd ed. 3. Singh VP, Kumar AA, srivastava SK and Rathore BS. 1996. Significance of Haemorrhagic Septicaemia in Asia: India. International workshop on diagnosis and control of Haemorrhagic Septicaemia. Bali, Indonasia. May 28-30. Townsend KM, Frost AJ, Lee CW, Papadimitrion JM and Dawkins HJS. 1998. Development of PCR assays for species and type specific identification of Pasteurella mutlocida isolates. Journal of Clinical Microbiolgy 36(4):1096- 1100. Venkatesan et al., 2013 Journal of Research in Biology (2013) 3(3): 895-899 899 Submit your articles online at www.jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php.
  • 54. JournalofResearchinBiology Source of light emission in a luminous mycelium of the fungus Panellus stipticus Keywords: Bioluminescence, Panellus stipticus, luminous mycelium, confocal microscopy. ABSTRACT: Mechanism of bioluminescence and light-emitting sources in higher fungi remain as an open question for a long time. We investigated the mycelium of cultivated luminous Panellus stipticus using confocal microscopy. No excitation light was imposed on the sample. Two types of sources of bioluminescence and their location were determined in the substrate mycelium. One were small 0.1-3 µm local formations disposed on the surface of hyphae, the other - relatively vast areas in bulk of the nutrient medium. No luminescence signal was recorded inside the hyphae. This may mean that the components of luminescent reaction are spatially separated within the cells, or the intracellular conditions block the reaction. The origin and formation of the light-emitting structures are discussed. 900-905 | JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Journal of Research in Biology An International Scientific Research Journal Authors: Puzyr Alexey, Burov Andrey and Bondar Vladimir. Institution: 1. Institute of Biophysics SB RAS, Krasnoyarsk. 2. Special Design- Technology Bureau "Nauka" KSC SB RAS, Krasnoyarsk. 3. Institute of Biophysics SB RAS, Siberian Federal University Krasnoyarsk. Corresponding author: Burov Andrey. Email: andrey@icm.krasn.ru Web Address: http://jresearchbiology.com/ documents/RA0345.pdf. Dates: Received: 02 Apr 2013 Accepted: 27 Apr 2013 Published: 06 May 2013 Article Citation: Puzyr Alexey, Burov Andrey and Bondar Vladimir. Source of light emission in a luminous mycelium of the fungus Panellus stipticus. Journal of Research in Biology (2013) 3(3): 900-905 Journal of Research in Biology An International Scientific Research Journal Original Research
  • 55. INTRODUCTION Bioluminescence in fungal cells, which involves the emission of light generated by a chemical reaction, has long attracted attention of scientists (Harvey, 1952; Shimomura, 2006; Desjardin et al., 2008). Researchers studying bioluminescence of fungi focus their efforts on three key areas: (i) methods of cultivation under laboratory conditions and characteristics of the light emission (Weitz et al., 2001; Prasher et al., 2012; Dao, 2009; Mori et al., 2011), (ii) the molecular organization of luminescence system and light emission mechanism (Shimomura, 2006; Airth and McElroy, 1959; Kamzolkina et al., 1983; Oliveira and Stevani, 2009; Bondar et al., 2011), (iii) - application of fungal luminescence in analytical techniques (Weitz et al., 2002; Mendes and Stevani, 2010). There has been little research conducted to determine sources of luminescent light in the fungal structures. To the best of our knowledge, only the mycelium of Panus stipticus and Armillaria fusipes, growing on agar were investigated for light source detection (Berliner and Hovnanian, 1963). The used photographic process allowed to record light from a single hypha. However, a low resolution of the technique limited by the emulsion grain size denied localizing the source of light. The authors of this, obviously, pioneer work, suggested that the light was emitted over the entire cell. Given the size of the objects under study, such research should employ methods of microscopic investigations. Calleja and Reynolds, who studied Panus stipticus and Armillaria mellea by optical microscope with EMI 4-stage image intensifier tube, came to the conclusion that light emission in an individual hypha was limited to a segment removed from the apical point (Calleja and Reynolds, 1970). Absence of later works related to structural and morphological studies of mycelium of luminous fungi with microscopy is astonishing as all known microscopic methods are widely used to investigate non-luminous fungi (Riquelme and Bartnicki-Garcia, 2008; Roberson et al., 2011; Steinberg and Schuster, 2011). In this report the mycelium of luminous Panellus stipticus was studied using confocal microscopy to determine and localize the source of light emission. In our opinion it is important to find in luminous fungi structures (or formations), which are the light-emitting sources, and their location. On the one hand, this can provide additional knowledge about morphology of luminous fungi, on the other - might give insight into molecular-cellular organization of fungal luminescent system and mechanism of light emission. MATERIALS AND METHODS In this work we studied the culture of Panellus stipticus luminous fungus (Bull:Fr.) Karst., IBSO 2301 (Figure 1). The mycelium was grown in plastic Petri dishes at temperature 22°С on a commercial nutrient medium Potato Dextrose Agar (HiMedia Laboratories Pvt., India), or on richer medium containing in 1 liter: 10 g of glucose, 5 g of peptone, 3 g of yeast extract, 3 g of malt extract, 20 g of agar-agar. The specimens exhibiting the highest light intensity were selected for the experiments. For confocal microscopy, a confocal laser scanning microscope (LSM-780 NLO, Carl Zeiss, Gottingen, Germany) equipped with a high sensitivity GaAsP was used. Bioluminescence was recorded in the accumulation mode with the 491–631 nm filter. The laser was turned off (laser power = 0.0%) so that no excitation light was imposed on the sample. This was done to avoid fungal autofluorescence - emission of light by biological substances such as flavins, lipofuscins and porphyrins when excited by ultraviolet, violet, or blue light (Zizka and Gabriel, 2008). Images were processed using ZEN 2010 software (version 6.0; Carl Zeiss). To prepare a specimen for microscopy a fragment of agar with mycelium was cut Puzyr et al., 2013 901 Journal of Research in Biology (2013) 3(3): 900-905
  • 56. out and transferred to the cover glass. RESULTS AND DISCUSSION Figure 2 shows a 3D projection of the mycelium by producing a Z-stack with 82 sections, 0.208 μm thick each. No bioluminescence was detected from the aerial mycelium. The light emission was recorded from the surface of specimen to a depth of ~ 16 μm with maximum intensity localized at the depth of Z= 6-8 μm where the main body of mycelium was located. Only isolated signals were detected at Z=8-16 μm that confirmed that the agar did not contribute to the observed bioluminescence. Two types of sources emitting luminescent signals could be distinguished. One light source were small 0.1-3 µm local formations, associated with the substrate hyphae, the other – vast areas in bulk of agar (Figure 3). Light intensity recorded in the agar was much higher than that of the local sites in the area of hyphae. The use of the larger magnification (Figure 4) and bright field microscopy (Figure 5a) suggests that the local luminous sites are cellular excretions located on the hyphae surface while vast luminescent areas are formed by their aggregation in agar. While presence of luminous sites on the surface of hyphae could be assumed, finding of luminescent areas in the agar came as a surprise. It is uncontroversial that the recorded bioluminescent signals result from the interaction of mixing light components synthesized by the fungal cells. Luminescent signals were recorded by the confocal microscope only when these components were outside the cells. No bioluminescence inside hyphae may mean that inside the cells the components of luminescent reaction are spatially separated and do not interact with each other, or the intracellular conditions (pH, oxygen concentration, presence of inhibitors, etc.) block the reaction. One could argue that the surface of glowing structures should be either hydrophobic or they have a membrane enclosing the internal volume. Only under these conditions components necessary for the luminescent reaction do not mix with the water phase contained within the nutrient medium. This suggestion is based on the sharp boundaries exhibiting by both small local formations on the walls of hyphae and vast areas in the nutrient agar (Figure 5b). So far it is not clear whether the luminous structures containing components necessary for the emission are formed within the fungal hypha or on/in their surface. In the first case it requires a transport system providing for the mechanism excreting the Puzyr et al., 2013 Journal of Research in Biology (2013) 3(3): 900-905 902 Figure 1 View of culture of Panellus stipticus (IBSO 2301) growing on agar in natural light (A) and in the dark (B).
  • 57. forming structures outside the cell. This is plausible because the Golgi apparatus, that synthesizes secretory vesicles containing products of vital functions and excretes them from the cell, is well known. In the second case on/in the wall cell there should exist structural elements performing specialized secretory function. On the basis of the results above we hypothesize the following. Cells of P. stipticus synthesize and localize the components required for bioluminescence in structures which can originate within the cell and then are moved on the outside surface of the hyphae by a mechanism analogous to the mechanism of transport via the Golgi complex. They can be also assumed to form directly on/in outside surface of the hyphae by structural elements of the cell possessing secretory function. Such enclosed structures make possible to concentrate the necessary components within a small volume. Separation of luminous structures from the surface of hyphae and their subsequent diffusion into the bulk of the nutrient medium produce the vast Puzyr et al., 2013 903 Journal of Research in Biology (2013) 3(3): 900-905 Figure 2 Fragment of 3D pattern of bioluminescence produced by P. stipticus. Figure 3 Confocal luminescence image of the P. stipticus mycelium. Figure 4 Confocal luminescence image of an individual hyphae. 20µm 5µm
  • 58. areas of luminescence in the agar. CONCLUSION Confocal microscopy due to its high resolution and ability to record low light signals offers new opportunities in investigation of fungal bioluminescence system. Using this technique the sources of light emission were identified for the first time in the mycelium of P. stipticus (IBSO 2301) cultivated on agar medium. One source were local formations disposed on the surface of the substrate hyphae, the other – vast areas in bulk of agar formed by aggregation of these luminous structures. Further study is required for a detail understanding whether the discovered structures are specific for this fungus or they are common among other luminous fungi. ACKNOWLEDGEMENTS The authors thank Mr. Barinov A.A. (OPTEC, Novosibirsk) and Dr. Baiborodin S.I. (TsKP for microscopic analysis of biological objects, SB RAS, Novosibirsk) for technical assistance with confocal microscopy. We are grateful to Dr. Medvedeva S.E. (IBP SB RAS, Krasnoyarsk) for the cultivation of luminescent fungi. This work was supported: by the Federal Agency for Science and Innovation within the Federal Special Purpose Program (contract No 02.740.11.0766); by the Program of the Government of Russian Federation «Measures to Attract Leading Scientists to Russian Educational Institutions» (grant No 11. G34.31.058); by the Program of SB RAS (project No 71). REFERENCES Airth RL and McElroy WD. 1959. Light emission from extracts of luminous fungi. J Bacteriol.;77(2):249-250. Berliner MD and Hovnanian HP. 1963. Autophotography of luminescent fungi. J Bacteriol. 86 (2):339-341. Bondar VS, Puzyr AP, Purtov KV, Medvedeva SYe, Rodicheva EK, Gitelson JI. 2011. The luminescent system of the luminous fungus Neonothopanus nambi. Doklady Biochem Biophys.;438(1):138-140. Calleja GB, Reynolds GT. 1970. The oscillatory nature of fungal bioluminescence. Trans Br Mycol Soc. 55:149- 154. Dao TV. 2009. Pilot culturing of a luminous mushroom Omphalotus af. illudent (Neonothropanus namibi). Biotechnology in Russia. 6:29-37. Desjardin DE, Oliveira AG, Stevani CV. 2008. Fungi bioluminescence revisited. Photochem Photobiol Sci.;7 (2):170-182. Harvey EN. Bioluminescence. New York: Academic Press. 1952. Puzyr et al., 2013 Journal of Research in Biology (2013) 3(3): 900-905 904 Figure 5 Confocal luminescence (A), bright field (B) and overlay (C) images of the substrate. Scale bar = 20 μm.
  • 59. Kamzolkina OV, Danilov VS, Egorov NS. 1983. Nature of luciferase from the bioluminescent fungus Armillariella mellea. Dokl Akad Nauk SSSR.;271:750- 752. Mendes LF and Stevani CV. 2010. Evaluation of metal toxicity by a modified method based on the fungus Gerronema viridilucens bioluminescence in agar medium. Environ Toxicol Chem. ;29:320-326. Mori K, Kojima S, Maki S, Hirano T, Niwa H. 2011. Bioluminescence characteristics of the fruiting body of Mycena chlorophos. Luminescence. 26(6): 604-10. Oliveira AG and Stevani CV. 2009. The enzymatic nature of fungal bioluminescence. Photochem Photobiol Sci. 8(10):1416-21. Prasher IB, Chandel VC, Ahluwalia AS. 2012. Influence of culture conditions on mycelial growth and luminescence of Panellus stipticus (bull.) P. Karst. J Res Biol. 2(3):152-9. Riquelme M and Bartnicki-Garcia S. 2008. Advances in understanding hyphal morphogenesis: ontogeny, phylogeny and cellular localization of chitin synthases. Fungal Biol. Rev.;22(2):56-70. Roberson RW, Saucedo E, Maclean D, Propster J, Unger B, Oneil TA, Parvanehgohar K, Cavanaugh C, Steinberg G, Schuster M. 2011. The dynamic fungal cell. Fungal Biol. Rev.;25(1):14–37. Shimomura O. Bioluminescence: chemical principles and methods. Singapore: World Scientific, 2006. Weitz HJ, Ballard AL, Campbell CD, Killham K. 2001. The effect of culture conditions on the mycelial growth and luminescence of naturally bioluminescent fungi. FEMS Microbiol Lett. 202(2):165-170. Weitz HJ, Colin D, Campbell CD, Killham K. 2002. Development of a novel, bioluminescence-based, fungal bioassay for toxicity testing. Environ Microbiol. 4(7): 422-429. Puzyr et al., 2013 905 Journal of Research in Biology (2013) 3(3): 900-905 Submit your articles online at www.jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php.
  • 60. JournalofResearchinBiology Local people’s attitude towards conservation and development around Pichavaram mangrove ecosystem, Tamil Nadu, India. Keywords: Mangrove ecosystem, Livelihood, Attitudes, Conservation, Development. ABSTRACT Studies in mangrove ecosystem are often focused on biological or ecological criteria and interdependency between mangroves and people is normally neglected. The situation is similar in Tamil Nadu; India which has a coastline of about 950 km. One of the major mangrove forests in Tamil Nadu is situated in Pichavaram, Cuddalore district. The present study was carried out in the seventeen hamlets, which are directly or indirectly dependent on the Pichavaram mangrove wetlands for their livelihood and survival. These seventeen hamlets consist of over 2600 households many of whom derive their principal income from fishing and related activities. Individual surveys were carried out for 10% of the households in each of the selected hamlets. Semi-structured questionnaires were used for surveys to study the attitude and perception of the community on the conservation and importance of mangrove wetlands and resources. The study was conducted to assess the awareness, attitudes and views of people dependent on the mangrove ecosystem towards conservation issues and development options. It was observed that a large percentage of the sampled population showed a positive inclination towards conservation of the ecosystem and were well aware of their responsibility towards it. 906-910 | JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Journal of Research in Biology An International Scientific Research Journal Authors: Lakshmi Kodoth and Ramamoorthy D. Institution: Department of Ecology & Environmental Sciences, Pondicherry University, Puducherry. Corresponding author: Lakshmi Kodoth. Email: lakshmi.kodoth@gmail.com Web Address: http://jresearchbiology.com/ documents/RA0274.pdf. Dates: Received: 08 Aug 2012 Accepted: 26 Aug 2012 Published: 06 May 2013 Article Citation: Lakshmi Kodoth and Ramamoorthy D. Local people’s attitude towards conservation and development around Pichavaram mangrove ecosystem, Tamil Nadu, India. Journal of Research in Biology (2013) 3(3): 906-910 Journal of Research in Biology Original Research An International Scientific Research Journal
  • 61. INTRODUCTION The Mangrove ecosystem has been studied extensively by scientists more in the ecological and biological sense. During the 1980s and early 1990s, more attention was given to research involving the human interactions with the forested wetlands (FAO, 1985; Hamilton et al., 1989; FAO, 1994; Cormier-Salem, 1999). Mangrove wetlands are a dominant feature of the intertidal areas of the tropical and subtropical regions in between 25°N and 25°S latitudes. The mangrove ecosystem provides a number of ecological services: provision of plant and animal products (Macnae, 1974; Rasolofo, 1997; Spaninks and Beukering, 1997), sediment trapping and nutrient uptake and transformation (Furukawa et al., 1997; Hussain and Badola, 2008), they provide detritus food for the aquatic fauna, harbour migratory and aquatic birds, serve as spawning ground for fishes, mussels and prawns. They also act as a natural shield against storms and tidal waves (Kathiresan and Rajendran, 2005). The coastal communities are largely dependent on the mangrove forests for firewood, timber, honey, fodder and for its fishery resources. Most coastal communities in the tropics are significantly dependent on the harvest of marine and coastal resources for sustaining their livelihoods (Kunstadter et al., 1986). The majority of people living near the mangrove areas derive their income predominantly from fishing and related activities. Hence, the present study was carried out as it is essential to understand people’s attitude and perception towards the mangrove ecosystem as they derive their livelihood from it; it helps us in formulating better policies and enhances the developmental plan for the ecosystem. Study Area India has a coastline of 7,516 km of which Tamil Nadu has about 950 km. Extensive mangrove wetlands are located in two places namely, i) in Pichavaram, Cuddalore district and ii) Muthupet in Thivarur and Tanjore districts. The Pichavaram mangrove wetland is located in the northern extreme of Cauvery delta between the Vellar and Coleroon estuaries (figure 1). Geographically, it is located between 79°47’E longitude and 11°27’N latitude. The Pichavaram mangrove forests have an area of about 1,350 ha, which are colonised by 13 true mangrove species. Rhizophora Sp and Avicennia Sp are the predominant mangrove species present in the Pichavaram mangrove forests. Pichavaram mangrove wetland is also rich in its fishery resources (figure 2). Annually about 245 tons of fishery resources are harvested from this mangrove wetland, of which prawns alone contribute 85% of the catch (Selvam et al., 2003). Methods People belonging to 17 hamlets surrounding the Kodoth and Ramamoorthy, 2013 907 Journal of Research in Biology (2013) 3(3): 906-910 Figure 1 Glimpse of the Pichavaram mangrove forest Figure 2 Fishing in the mangrove backwaters
  • 62. Pichavaram mangroves wetland were selected for survey. For each selected hamlet 10% of the households were picked up randomly for the household survey. Using semi-structured questionnaires, information on the demography, land use, income and occupational pattern as well as local dependence on the mangrove resources were gathered (Badola and Hussain, 2003; Glaser,2003) . Few open ended questions were also included to determine the attitude and perception of villagers towards development and conservation issues. A total of 324 households were surveyed. The responses we got were mostly in terms of yes, no and we don’t know. RESULTS AND DISCUSSION Assess the awareness and views towards conservation The results (Table 1) showed that majority of the respondents i.e. 91% (n=324) were aware that Pichavaram mangrove was as declared protected area and this awareness was gained largely because of, NGO’s working in that area and the forest department. An overwhelming percentage (84%) of the local population felt responsible towards the protection of the mangrove ecosystem and another 76.7% are in favour of eco-development projects in the area. Out of the 324 respondents, 67% of the people are willing to cooperate with the forest department for the same. Only a small percentage of people feel their rights being violated because of the protected area status if the ecosystem. When questioned regarding their views on eco-development initiatives and its implementation, a majority of the respondents (44.7%) were in favour of the community led initiatives. 32% felt that NGO’s should take lead in eco-development and the rest 23% felt that the government should take up eco-developmental projects by itself ( Table 2). The importance of the mangrove forests to the local population was emphasized when a majority of people were against cutting down of the forests. A majority of the respondents (71%) felt that more mangrove plantations need to be carried out, while 28.4% felt that the present conditions of the mangrove forests were good (Table 3). Kodoth and Ramamoorthy, 2013 Journal of Research in Biology (2013) 3(3): 906-910 908 Table 1: Attitude of people towards Pichavaram Mangrove Ecosystem and conservation (n= 324) Questions Yes (%) No (%) Don’t Know (%) Are you aware that Pichavaram Mangrove Ecosystem is declared as Protected area? 91 9 - Do you feel any sense of responsibility for the protection of the ecosystem? 84 13.8 2.2 Do you feel your rights have been violated after the declaration of PA? 11.9 80.5 7.6 Do you face any problem because of PA? 15.8 78.6 5.6 Are you in favour of the implementation of an ecodevelopment project? 76.7 15.3 8 Would you like to co-operate with the forest department with regard to the ecodevelopment project? 67 23 10 Table 2: View of respondents towards Eco-Development itiatives (n = 324) Views Frequency Percentage Want through govt. initiative 75 23.1 Want through Community initiative 145 44.7 Want through NGO initiative 104 32 Table 3: View of local people towards various management alternatives (n = 324) Management Alternatives Responses (%) Forests should be cut and land used for other purposes 0.6 Current situation of protecting the forests is good 28.4 Increase in mangrove plantations needed 71
  • 63. The findings in this study are similar to that of the study in Bitarkanika mangrove ecosystem in Orissa (Badola and Hussain, 2003) which shows that the villagers are well aware of their responsibility to the ecosystem and willing to participate in the conservation efforts of both the government and NGO’s. Developmental Options Recently, eco tourism has been promoted to a large extent in Pichavaram mangrove forests. Majority of the respondents (76%) were in favour of developing eco tourism as it will improve job opportunities for the local population. Shrimp farms are not favoured in the area as 83% of the responses were against setting up of such farms. This is primarily due to the fact that shrimp farms in the area are the reason for increase in salinity of the canal water (Table 4). Ecological functions and values identified by local community The respondents were given a list of ecological functions to find out how much they were aware of the functions and its direct or indirect importance in their livelihoods. Table 5 shows ranking of use values, 76% gave highest ranking to contribution of mangroves towards fishing. 63% gave agriculture as their second preference. Incase of ranking ecological functions performed by the Pichavaram mangrove ecosystem, 77.8% of the responses favoured Tsunami/cyclone mitigation. 67.2% gave second preference to nutrient cycling (Table 6). The results show that the respondents were aware of both the direct and indirect benefits of the mangrove ecosystem. It is evident from the results that people value the uses or function which more beneficial to them in their day today lives. CONCLUSION The results showed that in general people have a positive attitude towards conservation and are aware of their responsibility in sustaining these mangrove forests. The socio economic and market conditions influence the people’s attitude towards the resources. Eco developmental plans were in favour with the local population since it will be helpful in formulating sustainable policies for ecosystem. The promotion of eco tourism in the area had a largely positive response hence it should be capitalised on to improve local economy. Inclusion of the local people in decision making process can lead to successful management of the Pichavaram mangrove ecosystem. REFERENCE Badola R and Hussain SA. 2003. Valuation of the Bhitarkanika mangrove ecosystem for ecological security and sustainable resource use. Study report. Wildlife Institute of India, Dehra Dun. Kodoth and Ramamoorthy, 2013 909 Journal of Research in Biology (2013) 3(3): 906-910 Table 5: Ranking of the use values in Percentage (n=324) Use values Rank 1 (%) Rank 2 (%) Rank 3 (%) Fishing 76 18 6 Agriculture 26 63 11 Tourism 35 56 9 Table 6: Percentage ranking of various functions (n=324) Ecological functions Rank 1 (%) Rank 2 (%) Rank 3 (%) Fish 59.4 34.3 6.3 Aesthetic 38 59 3 Tsunami/cyclone mitigation 77.8 22.2 0 Nutrient 32.2 67.2 0.6 Table 4: View of local people towards various developmental options (n = 324) Queries Yes (%) No (%) Don’t know(%) Are you in favour of developing eco tourism in the area 76 16 8 Are you in favour of shrimp farms 8 83 9 Has Shrimp farms been useful to you? (n=14) 47 46 7
  • 64. Cormier-Salem MC. 1999. The Mangrove: an area to be cleared…for social scientists. Hydrobiologia. 413: 135-142. FAO. 1985. Mangrove management in Thailand, Malaysia and Indonesia. FAO Environment Paper 4, Food and Agriculture Organization of the United Nations, Rome. FAO. 1994. Mangrove forest management guidelines. FAO Forestry Paper 117, Food and Agriculture Organization of the United Nations, Rome. Glaser M. 2003. Interrelations between mangrove ecosystem, local economy and social sustainability in Caete Estuary, North Brazil. Wetland Ecology and Management. 11:265-272. Furukawa K, Wolanski E and Mueller H. 1997. Currents and sediment transport in mangrove forests. Estuarine, Coastal and Shelf Science. 44:301-310. Hamilton LS, Dixon JA and Miller GO. 1989. Mangrove forests: an undervalued resource of the land and of the sea. In: Borgese EM, Ginsburg N, Morgan JR. (Eds.), Ocean Yearbook 8. University of Chicago Press, Chicago. 254-288. Hussain SA and Badola R. 2008. Valuing mangrove ecosystem services: linking nutrient retention function of mangrove forests to enhanced agroecosystem production. Wetlands Ecology and Management. 16:441-450. Kathiresan K and Rajendran N. 2005. Coastal mangrove forests mitigated tsunami. Estuarine, Coastal and Shelf Science. 65:601-606. Kunstadter P, Bird ECF and Sabhasri S. (Eds.). 1986. Man in the Mangroves. United Nations University, Tokyo. Macnae W. 1974. Mangrove forest and fisheries. FAO/ UNDP Indian Ocean Fishery Programme. Indian Ocean Fishery Commission. Publication IOFCDev. 74:34-35. Rasolofo MV. 1997. Use of mangroves by traditional fishermen in Madagascar. Mangroves Salt Marshes. 1:243-253. Selvam V, Ravichandran KK, Gnanappazham L and Navamuniyammal M. 2003. Assessment of community-based restoration of Pichavaram mangrove wetland using remote sensing data. Current Science. 85:6,794-798. Spaninks F and Beukering PV. 1997. Economic Valuation of Mangrove Ecosystems: Potential and Limitations. CREED Working 14. Kodoth and Ramamoorthy, 2013 Journal of Research in Biology (2013) 3(3): 906-910 910 Submit your articles online at www.jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php.
  • 65. JournalofResearchinBiology Biodegradation of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria Keywords: Biodegradation, phenol, bacteria, Okrika River. ABSTRACT: Assessments on biodegradation at low and high doses of phenol by bacterial strains indigenous to Okrika River in Niger Delta of Nigeria were carried out. Growth at low dose of 0.01 µg/l phenol showed that highest and lowest cell density values of OD540nm of 0.15 and 0.09 in Pseudomonas sp. SD1 and Citrobacter sp. RW1 while at 1.0 µg/l phenol concentration the highest cell density values of OD540nm of 0.28 was observed in Staphylococcus sp. RW2. The highest specific growth rate of 0.019 h-1 at 500 mg/l of phenol was obtained for Pseudomonas sp. SD1 while Citrobacter sp. RW1 had the lowest specific growth rate of 0.014 h-1 at 500 mg/l of phenol. The specific phenol degradation rate ranges from 55.35 to 130.98 mg/(L.h.OD). The order of specific phenol consumption rate at 1000 mg/l by the organisms is: Bacillus sp. SD3>Pseudomonas sp. SD1>Citrobacter sp. RW1>Staphylococcus sp. RW2. Citrobacter sp. RW1 exhibited highest growth yield in 250 mg/l of phenol with the growth yield of 6.24 (x 10-4 A540 unit.l/mg). The results showed that the test organisms might be the most suitable bacterial strains for removal of phenols at low and high doses in phenolic polluted media. 911-921| JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Journal of Research in biology An International Scientific Research Journal Authors: Nwanyanwu CE 1* Abu GO2 . Institution: 1.Department of Microbiology, Federal university of Technology, P.M.B.1526, Owerri, Nigeria. 2.Department of Microbiology, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Nigeria. Corresponding author: Nwanyanwu CE. Email: cnwanyanwu2000@yahoo.com Web Address: http://jresearchbiology.com/ documents/RA0318.pdf. Dates: Received: 26 Dec 2012 Accepted: 17 Jan 2013 Published: 06 May 2013 Article Citation: Nwanyanwu CE and Abu GO. Biodegradation of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria. Journal of Research in Biology (2013) 3(3): 911-921 Journal of Research in Biology An International Scientific Research Journal Original Research
  • 66. INTRODUCTION Contamination of aquatic environment brought about by the discharge of wastewater resulting from anthropogenic activities clearly continues to be a major environmental issue. Effluents are very important sources of chemicals entering aquatic ecosystems. They may contain hundreds, or even thousands of chemicals, but only a few of them are responsible for effluent toxicity (Tisler et al., 1999). High strength wastewaters have been reported to be associated with chemical processing industries. Wastewaters generated from these processing industries such as petrochemical, oil refineries, coke-processing plants, etc contain a large number of organic and inorganic pollutants at high concentrations that exhibit adverse effect on the environments when released (Papadimitriou et al., 2009). The presence of high level of these contaminants formed the major pollutant in the water body as a result of continuous discharge of effluents by industries into the ecosystem. In water these pollutants of the discharged effluent sorbs onto particulate materials and if not degraded eventually end up in sediments. As an ultimate respiratory of most xenobiotic contaminants that enter water bodies, sediments act as both carrier and sources of contaminants in aquatic environment (Akan et al., 2010). Thus, the contaminated sediments may represent a continual threat of recontamination of the aquatic environment as the adsorbed pollutants if not degraded, in turn lead to the exposure of aquatic life to organic pollutants such as phenol (Mort and Dean-Ross, 1994). On the other hand, the release of contaminants from sediments could increase the amount of toxic compounds in the waters making them more available to organisms and affecting their life cycles, reproduction, metabolism and physiology. Microorganisms being ubiquitous in nature exploit many carbon and energy sources in its niche for growth. Several species of micro-organisms inhabiting hostile ecological niche have been reported by Colwell and Walker (1977), Atlas (1981), Heinaru et al. (2000) and Polymenakou and Stephanou (2005). Microorganisms indigenous to aquatic environment are crucial for the biodegradation of organic matter and the cycling of nutrients, while these microorganisms are susceptible to toxic pollutants from industrial effluent discharges, especially petroleum refinery. Therefore, perturbations of aquatic microbial communities could have consequences for the higher trophic levels and for the overall aquatic environment. The composition of effluents from petroleum refineries varies according to their origin, storage and treatments as these wastewaters are enriched with different pollutants. Phenol and its derivatives along with other organic and inorganic compounds is one of the most common contaminants present in refinery effluents (Jena et al., 2005) which renders refinery effluents its toxic nature. Phenols as constituents of industrial effluents may remain in water body for much longer period if it is continually or consistently released into the aquatic environments from sources thereby increasing its elevation in the environment. The toxic nature of phenol and its derivatives to microbial cells is well documented (Kahru et al., 2002; Keweloh et al., 1990). Owing to toxic nature of phenol, its contact with microorganisms always results in the decrease of microbial enzyme activity (Nwanyanwu and Abu, 2011) as well as leading to death of organisms at higher concentration. A large number of microbial genera possess the capability to degrade organic pollutants. Among the bacterial genera implicated in the degradation of phenol include Pseudomonas, Bacillus, Corynebacterium species etc. The ability of organisms to degradation phenol and other toxicants is related to adaptation of the microorganisms to the compound of concern and adaptation is associated with synthesis of new enzymes capable of transformation of the toxicant to harmless substances (Jaromir and Wirgiliusz, 2007). The resultant effect of biodegradation of phenol and other organic compounds is growth as the organic pollutants are used Nwanyanwu and Abu, 2013 912 Journal of Research in Biology (2013) 3(3): 911-921
  • 67. as the source of carbon and energy. This research assessed the growth and utilization of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria. MATERIALS AND METHODS Chemical reagents All chemical reagents used in the study were of analytical grade and were obtained from sigma chemical company, St Louis Missouri, USA, BDH chemicals, Poole, England and HACH chemical company. Sample collection and analysis The Okrika River is a small tidal river that empties into Bonny estuary in Niger Delta of Nigeria. The River is highly polluted as a result of effluent discharges from Port Harcourt petroleum refinery industry sited along its bank (IAIA09 Conference Proceeding, 2009). Sediment and water samples were collected from the river as described by Nweke et al., (2007) and the samples analyzed within few hours of collection. The results of the physicochemical analysis of the samples are as shown in Table 1. Isolation and identification of bacterial strains The bacterial strains used in this work were isolated from the samples by spreading one tenth of decimally diluted sediment suspension and water samples on mineral salt agar-phenol (2.5 mM) medium and the isolated organisms identified as described elsewhere (Nwanyanwu et al., 2012). The isolates were designated according to their sources (RW for River water, SD for sediment) and were then maintained on nutrient agar slants. Preparation of inoculum The bacterial strains used for the assay were grown in 100 ml of sterile nutrient broth media for 48 h. The turbid culture medium were harvested, washed and suspended in deionized distilled water then followed by standardization of the suspensions spectrophotometrically to an optical density of 0.4 at 540 nm and used as inocula. Assay for isolates growth in very low concentrations of phenol The ability of the isolates to grow and utilize phenol at low concentrations (0-1.0 µg/l) was assessed in sterile Bushnell Haas (BH) mineral salt broth medium. The assay was carried out as described by Nwanyanwu et al., (2012) with little modification. The medium without agar was used instead for the assay. After inoculation of the flasks, growth profile of the organisms was monitored by the optical density (OD540nm) on daily basis. Growth and biodegradation of phenol at high concentration Degradation of phenol at high concentration by the organisms was carried out in sterile BH medium contained Erlenmeyer flasks. The flasks were supplemented with aliquot of sterile phenol (2000 mg/l) to bring the final phenol concentrations in the flasks to 250, 500, 750 and 1000 mg/l. The flasks after inoculation with the test organisms were incubated at 30o C in an incubator. At predetermined time, samples were withdrawn to determine cell growth and phenol concentration. Controls, one without phenol and another without cells in BH medium were set up. At predetermined time, samples were removed and used to measure for cell growth (optical density, OD540nm) and Nwanyanwu and Abu, 2013 Journal of Research in Biology (2013) 3(3): 911-921 913 Table 1: Physicochemical characteristics of Okrika River Parameter/unit Sample source Water Sediment pH 8.90 6.90 Elect. conduc. (µscm-1 ) 364 615 Oil and grease (mg/l) 16.0 103.0 BOD (mg/l) 8.16 - COD (mg/l) 84.0 - PO4 (mg/l) 0.15 0.90 SO4 (mg/l) 118 117 Phenol (mg/l) 6.1 15.5 Zn (mg/l) 0.03 3.48 Cu (mg/l) <0.01 0.06 Pb (mg/l) <0.01 <0.01
  • 68. phenol residue (4-amino antipyrine) in cell free samples. Analytical methods C e l l g r o w t h w a s d e t e r m i n e d spectrophotometrically while phenol was analyzed by photometric method using 4-aminoantipyrine as the colouring agent and measuring the absorbance at 500 nm (Folsom et al., 1990). Data Analysis Specific growth rate The specific growth rate (µ) for each concentration of phenol was calculated from the slope of linear logarithmic plots of optical density against time as expressed in equation 1 (Gokulakrishnan and Gummadi, 2006): Specific degradation rate The specific degradation rate (Qs) was determined through the relationship of equation 2 (Loh and Wang, 1998): Where: [Ph] denotes phenol concentration (mg/l), t denotes incubation time (h) and X denotes cell concentration (optical density, OD540 nm). Yield factor Yield factor (Y) of the biomass was calculated using equation 3 (Bajaj et al., 2009):   Where dX is the change in cell biomass related to the change in substrate concentration dS. X was replaced with the OD at 540 nm. RESULTS AND DISCUSSION The phenol content of Okrika River water and sediment were 6.1 and 15.5 mg/l while oil and grease of the River water and sediment were 16.0 and 103.0 mg/l respectively (Table 1). This level of oil and grease as well as phenol in the River water and sediment were much higher than the previously reported levels of 10.56 and 15.23 mg/l (oil and grease) and 5.13 and 16.0 mg/l (phenol) (Otokunefor and Obiukwu, 2005). This indicated that these compounds have accumulated in Okrika River over time and pose the major pollutants of the river. Figure 1 shows the growth of the test organisms in low concentration of phenol amended mineral salt medium. All the organisms showed progressive growth in low phenol concentration medium. Highest growth of the organisms was observed in phenol concentration of 1.0 µg/l followed by 0.1 µg/l. The least growth was observed in 0.01 µg/l. Among the test organisms, Staphylococcus sp. RW2 showed the highest growth in 0.1 and 1.0 µg/l of phenol with optical density (OD) values of 0.23 and 0.28 respectively while Citrobacter sp. RW1 showed the least growth in all the low concentrations (0.01, 0.1 and 1.0 µg/l) of phenol amended medium with OD values of 0.09, 0.11 and 0.13 respectively. Growth of microorganisms especially bacterial species at phenol concentration as low as microgram per litre have been reported by many authors. Chesney et al., (1985) have reported growth of water microorganism in water sample supplemented with 0.001 to 1.0 µg/ml of phenol. Also Goldstein et al., (1985) have reported the growth of Pseudomonas sp. in a 1 12 12 tt XXIn 2 / X dtPhd Qs 3 dS dX Y Nwanyanwu and Abu, 2013 914 Journal of Research in Biology (2013) 3(3): 911-921 Table 2: Yield factor (Y) of biomass after growth at different initial phenol concentrations Bacteria Yield factor, Y(x 10-4 A540 unitsa . l/mg) Phenol concentration (mg/l) 250 500 750 1000 Citrobacter sp. RW1 6.24 4.46 2.69 3.11 Staphylococcus sp.RW2 4.96 3.80 3.28 3.00 Pseudomonas sp. SD1 4.96 3.80 3.28 3.00 Bacillus sp. SD3 3.28 4.46 2.69 3.11 a A540 units = optical density at 540 nm
  • 69. medium amended with 1.0 and 10.0 µg/l concentration of 2, 4-dichlorophenol. Pahm and Alexander (1993) found that Pseudomonas sp. K, Flavobacterium sp. M4, Flavobacterium sp. M1 and Pseudomonas sp. SP3 grown in p-nitrophenol (PNP) of concentration of 0.1 µg/l reached a total viable count of 105 and 106 cells/ml. Figures 2 and 3 showed typical profiles of cell growth and biodegradation of phenol at high concentrations by bacterial strains of Okrika River ranging from 250 to 1000 mg/l. The lag phase of the organisms in phenol fortified medium was short. The short in lag phase period depends on the pre-exposure of the organism. Phenol was completely utilized by the isolates within 180 h of incubation. Phenol concentrations of 500, 750 and 1000 mg/l was degraded completely within 96, 132 and 156 h by Pseudomonas sp. SD1 while same concentrations of phenol was degraded completely within 108, 144 and 180 h by other test organisms. Time-dependent degradation of organic compounds has been reported to be linked with concentration of the organic compound as observed by many authors (Colwell and Walker, 1977; Kotresha and Vidyasagar, 2008). This may be due to changes in the transport mechanism of the substrate across the cell membrane in response to high phenol concentration hence diminished capacity to catabolize phenol. This is in line with the reports of Gilbert and Brown (1978), Keweloh et al., (1990), Collins and Daugulis (1997) and Nwanyanwu and Abu, 2013 Journal of Research in Biology (2013) 3(3): 911-921 915 Figure 1: Growth profile of the bacteria in mineral salt medium fortified with phenol concentrations Time (h) Absorbance(A540nm)
  • 70. Nwanyanwu and Abu (2011) who observed the toxic effect of phenol at the membrane level, thereby disrupting the activity of enzymes in phenol-utilizing bacteria. Also, Joseph and Joseph (1999) and Ye and Shen (2004) reported that phenol toxicity depends on the sensitivity as well as source of organism. The growth profiles of the pure cultures expressed as optical density and phenol residues at different initial concentrations are shown in figures 2 and 3. The cells gradually increase in number as the phenol residues of the medium progressively decreased. This may be due to high phenol concentration made available more carbon to the organism for growth. Pseudomonas sp. SD1 degraded 1000 mg/l of phenol in 160 h with a cell biomass (OD540nm) of 0.363. The dependence of specific growth rate on phenol concentration is shown in Figure 4. From this plot, the specific growth rate increased with increase in the initial phenol concentration upto 250 mg/l and then a progress decrease started with increase in phenol Nwanyanwu and Abu, 2013 916 Journal of Research in Biology (2013) 3(3): 911-921 Absorbance(A540nm) Figure 2: Biodegradation and cell growth profile of planktonic bacteria of Okrika River in high phenol concentrations Time (h) Phenol(mg/l)
  • 71. concentration. In the present study, at 500 mg/l of phenol concentration, the specific growth rate of Pseudomonas sp. SD1 is increased (highest µ =0.017 h-1 ). For concentration higher than 500 mg/l, the specific growth rate of Pseudomonas sp. SD1 decreases and became almost constant at 750 mg/l (µ = 0.011 h-1 ) and 1000 mg/l (µ = 0.011 h-1 ) of phenol. This is quite similar to the result obtained by Dey and Mukherjee (2010) who observed increase in specific growth rate (0.093 h-1 ) of mixed microbial culture up to 300 mg/l of initial phenol concentration and then started decreasing to a constant (0.057 h-1 ) at 600 and 700 mg/l of phenol. This trend suggested that the phenol is an inhibitory substrate. Thus the parameter has been found to be a strong function of initial phenol concentration. At 250 and 500 mg/l, the highest specific growth rate values of 0.026 and 0.017 h-1 were observed in Citrobacter sp. RW1 and Pseudomonas sp. SD1 respectively while the lowest specific growth rate of 0.016 and 0.014 h-1 at the same concentration of phenol was observed in Pseudomonas sp. SD1 and Nwanyanwu and Abu, 2013 Journal of Research in Biology (2013) 3(3): 911-921 917 Figure 3: Biodegradation and cell growth profile of sediment bacteria of Okrika River in high phenol concentrations Time (h) Phenol(mg/l)Absorbance(A540nm)
  • 72. Citrobacter sp. RW1 respectively. However, the growth rates of the test organisms are similar to that of Pseudomonas aeruginosa and Pseudomonas pseudomallei degrading phenol in saline solutions (Afzal et al., 2007). The specific rate of phenol degradation of the organisms is depicted in figure 5. The specific degradation rate (Qs), was estimated by correlating phenol concentration versus culture time using regression technique in Microsoft Excel to obtain the equation of best fit of the degradation curve. The correlation were differentiated with respect to time and then divided by the cell mass (Loh and Wang, 1998). The specific degradation (consumption) rate of a compound was suggested to be a measure of microbe performance. The highest specific consumption rate of phenol was observed in Bacillus sp. SD3 with specific degradation rate value of 130.98 mg/(L.h.OD) at 1000 mg/l while Staphylococcus sp. RW2 showed the least specific consumption rate of phenol with a specific degradation rate value of 99.83 mg/(L.h.OD) at the same concentration. The organisms in this work showed a robust decrease in specific degradation rate as the phenol concentration decreases. This is in line with the work of Cho et al., (2000) who observed an increase in specific degradation rate as phenol concentration increases in their assessment of influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardioides sp. NSP41. Agarry and Solomon (2008) also made similar reports in their work on kinetics of batch microbial degradation of phenols by indigenous Pseudomonas fluorescence. Table 2 shows the growth yield of the test organisms expressed as absorbance, A at 540nm unit litre of cells produced per mg of phenol substrate utilized. The growth yield varied among the test organisms ranging from 2. 69 to 6.24 (x 10-4 A540 units. l/mg). High growth yield were obtained at low concentration of toxicant (phenol) while low values of growth yield were obtained at high phenol concentration. At 250 mg/l highest and lowest growth yield were observed in Citrobacter sp. RW1 and Bacilllus sp. SD3 with cell yield coefficients of 6.24 and 3.28 (x 10-4 A540 units.l/mg) respectively. The higher value of Y observed in Citrobacter sp. RW1 indicate that phenol was degraded very efficiently by the organism. All the growth yields Nwanyanwu and Abu, 2013 918 Journal of Research in Biology (2013) 3(3): 911-921 Phenol, So (mg/l) Figure 4: Specific growth rate of the organisms at different initial phenol concentrations Specificgrowthrate(h-1 ) Figure 5: Specific degradation rate at different initial phenol concentrations by the bacterial strains Phenol, So (mg/l) Specificdegradationrate(mg/(L.h.OD))
  • 73. reported here were lower than those reported by other authors. Yield coefficients of 0.14 and 0.16 have been reported (Bajaj et al., 2009). The yield coefficients reported by Yoong et al., (1997) are 0.16 and 0.27. As Citrobacter sp. RW1, Staphylococcus sp. RW2, Pseudomonas sp. SD1and Bacillus sp. SD3 shown high specific phenol consumption rate, they have demonstrated strong potential to utilize and grow in phenol of low and high phenol concentrations of upto 1000 mg/l. This indicated that these strains have great potential for application in the treatment of phenolic wastewater and in the bioremediation of phenol impacted media. REFERENCE Afzal M, Iqbal S, Rauf S and Khalid ZM. 2007. Characteristics of phenol biodegradation in saline solutions by monocultures of Pseudomonas aeruginosa and Pseudomonas pseudomallei. J. Hazard. Mat. 149(1): 60 - 66. Agarry SE and Solomon BO. 2008. Kinetics of batch microbial degradation of phenols by indigenous Pseudomonas fluorescence. Int. J. Environ. Sci. Tech. 5 (2): 223 – 232. Akan JC, Abdulrahman FI, Sodipo OA, Ochanya AE and Askira YK. 2010. Heavy metals in sediments from River Ngada, Maiduguri Metropolis, Borno State, Nigeria. J. Environ. Chem. Ecotoxicol. 2(9): 131 - 140. Atlas RM. 1981. Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiology Rev. 45(1): 180 – 209. Bajaj M., Gallert C and Winter J. 2009. Phenol degradation kinetics of an aerobic mixed culture. Biochem. Eng. J. 46(2): 205 – 209. Chesney RH, Sollitti P, Rubin HE. 1985. Incorporation of Phenol Carbon at Trace Concentrations by Phenol- Mineralizing Microorganisms in Fresh Water. Appl. Environ. Microbiol. 49: 15 – 18. Cho YG, Rhee SK, Lee ST. 2000. Influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardioides sp. NSP41. Biodegradation 11(1): 21 – 28. Collins LD and Daugulis AJ. 1997. Characteristics and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol. Appl. Micobiol. Biotechnol. 48(1):18 - 22. Colwell RR and Walker J.D. 1977. Ecological Aspects of Microbial Degradation of Petroleum in the Marine Environment. Crit. Rev. Microbiol. 5(4): 423 - 445. Folsom BR, Chapman PJ, Pritchard PH. 1990. Phenol and trichloroethylene degradation by Pseudomonas cepcia GA: Kinetics and interaction between substrates. Appl. Environ. Microbiol. 56(5): 1279 – 1285. Gilbert P and Brown MRW. 1978. Influence of growth rate and nutrient limitation on the gross cellular composition of Pseudomonas aeruginosa and its resistance to 3- and 4- chlorophenol. J. Bactriol. 133(3): 1066 - 1072 Gokulakrishnan S and Gummadi SN. 2006. Kinetics of cell growth and caffeine utilization by Pseudomonas sp. GSC 1182. Proc. Biochem. 41: 1417 – 1421. Goldstein RM, Mallory LM, Alexander M. 1985. Reasons for Possible Failure of Inoculation to enhance biodegradation. Appl. Environ. Microbiol. 50(4): 977 – 983. Heinaru E, Truu J, Stottmeister U, Heinaru A. 2000. Three types of phenol and p-cresol catabolism in phenol and p-cresol-degrading bacteria isolated from River water continuously polluted with phenolic compounds. FEMS Microbiol. Ecol. 31(3): 195 – 205. Nwanyanwu and Abu, 2013 Journal of Research in Biology (2013) 3(3): 911-921 919
  • 74. IAIA09 Conference Proceedings. 2009. Environmental Pollution: A case study of the impact of the Port Harcourt Oil Refinery Company (PHRC), Nigeria. Impact Assessment and Human Well-Being 29th Annual Conference of the International Association for Impact Assessment, 16-22 May 2009, Accra International Conference Center, Accra, Ghana (www.iaia.org) Jaromir M and Wirgiliusz D. 2007. Phenols transformations in the environment and living organisms. Curr. Topics in Bioph. 30 (suppl. A): 24 - 36 Jena HM, Roy GK and Meikap BC. 2005. Development and comparative study of a semi-fluidized bed bioreactor for treatment of wastewater from process industries. Proc. Plant Eng. 23(1): 70 ‐ 75 Joseph V and Joseph A. 1999. Acclimation of algal species following exposure to phenol. Bull. Environ. Contam. Toxicol. 62(1): 87 - 92. Kahru A, Maloverjan A, Sillak H. and Pollumaa L. 2002. The toxicity and fate of phenolic pollutants in the contaminated soils associated with the oil-shale industry. Environ Sci. Pollut Res. 1: 27 – 33. Keweloh H, Weyrauch G, Rehm HJ. 1990. Phenol- induced membrane changes in free and immobilized Escherichia coli. Appl. Microbiol. Biotechnol. 33(1): 66 - 71. Kotresha D, Vidyasagar GM. 2008. Isolation and characterisation of phenol-degrading Pseudomonas aeruginosa MTCC 4996. World J. Microbiol. Biotechnol., 24(1): 541-547. Loh KC and Wang SJ. 1998. Enhancement of biodegradation of phenol and a nongrowth substrate 4-chlorophenol by medium augmentation with conventional carbon sources. Biodegradation 8(5): 329 – 338. Mort SL and Dean-Ross D. 1994. Biodegradation of phenolic compounds by sulphate reducing bacteria from contaminated sediments. Microb. Ecol. 28: 67 – 77. Nwanyanwu CE, Nweke CO, Orji JC. 2012. Growth responses of petroleum refinery effluent bacteria to phenol. J. Res. Biol. 3: 167 - 177 Nwanyanwu CE and Abu GO. 2011. Assessment of viability responses of refinery effluent bacteria after exposure to phenol stress. J. Res. Biol. 1(8): 594 - 602 Nweke co, Alisi CS, Okolo JC and Nwanyanwu CE. 2007. Toxicity of zinc to heterotrophic bacteria from a tropical river sediment. Appl. Ecol. Environ. Res. 5(1): 123-132 Otokunefor TV and Obiukwu C. 2005. Impact of refinery effluent on the physicochemical properties of a water body in the Niger Delta. Appl. Ecol. Environ. Res. 3 (1): 61 - 72. Pahm MA and Alexander M. 1993. Selecting inocula for the biodegradation of organic compounds at low concentrations. Microb. Ecol. 25(3): 275 – 286. Papadimitriou CA, Samaras P, Sakellaropoulos GP. 2009. Comparative study of phenol and cyanide containing wastewater in CSTR and SBR activated sludge reactors. Biores. Technol. 100: 31 – 37. Polymenakou PN and Stephanou EG. 2005. Effect of temperature and additional carbon sources on phenol degradation by an indigenous soil Pseudomonad. Biodegradation. 16(5): 403 – 413 Tisler T, Zagorc-Koncan J, Ros M, Cotman M. 1999. Biodegradation and toxicity of wastewater from industry producing mineral fibres for thermal insulation. Chemosphere 38(6): 1347 – 1352. Ye FX and Shen DS. 2004. Acclimation of anaerobic sludge degrading chlorophenols and the biodegradation kinetics during acclimation period. Chemosphere. 54 Nwanyanwu and Abu, 2013 920 Journal of Research in Biology (2013) 3(3): 911-921
  • 75. (10): 1573 – 1580. Yoong ET, Lant PA, Greenfield PF. 1997. The influence of high phenol concentration on microbial growth. Wat. Sci. Tech. 36(2-3): 75 – 79. Submit your articles online at www.jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php. Journal of Research in Biology (2013) 3(3): 911-921 921 Nwanyanwu and Abu, 2013
  • 76. JournalofResearchinBiology Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria Keywords: Phenol, heavy metals, refinery effluent bacteria. ABSTRACT: Bacterial isolates from petroleum refinery effluent were evaluated for growth in increasing doses of phenol and heavy metal ions. All the test organisms were able to grow in mineral salt medium with phenol concentration of 15.0 mM (≈ 1412.0 mg/l) except Pseudomonas sp. RBD3. Aeromonas sp. RBD4, Staphylococcus sp. RBD5 and Pseudomonas sp. RBD10 showed the highest tolerance to 15.0 mM of phenol followed by Corynebacterium sp. RBD7 while Escherichia coli RBD2 and Citrobacter sp. RBD8 showed the least tolerance to 15.0 mM of phenol. The minimum inhibitory concentrations (MICs) ranged from 1.0 mM for mercury and 4.5 mM for chromium, nickel, lead and copper. The bacterial strains were most susceptible to mercury toxicity. Viable counts of the organism on mineral salt-phenol agar showed a typical growth pattern for inhibitory substrate. The threshold concentration is 0.5 mM for Bacillus sp. RBD1, Escherichia coli RBD2, Bacillus sp. RBD6, Citrobacter sp. RBD8, Streptococcus sp. RBD9, Pseudomonas sp. RBD11 and Escherichia coli RBD12 and 1.0 mM for Pseudomonas sp. RBD3, Aeromonas sp. RBD4, Staphylococcus sp. RBD5, Corynebacterium sp. RBD7 and Corynebacterium sp. RBD10. The results suggest that microorganisms isolated from petroleum refinery effluent are potentially useful for detoxification of phenol impacted systems in the presence of heavy metals. 922-931 | JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Journal of Research in Biology An International Scientific Research Journal Authors: Nwanyanwu CE, Nweke CO, Orji JC, Opurum CC. Institution: Department of Microbiology, Federal University of Technology, P.M.B. 1526, Owerri, Nigeria. Corresponding author: Nwanyanwu CE. Email: cnwanyanwu2000@yahoo.com Web Address: http://jresearchbiology.com/ documents/RA0317.pdf. Dates: Received: 24 Dec 2012 Accepted: 09 Jan 2013 Published: 10 May 2013 Article Citation: Nwanyanwu CE, Nweke CO, Orji JC, Opurum CC. Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria. Journal of Research in Biology (2013) 3(3): 922-931 Journal of Research in Biology An International Scientific Research Journal Original Research
  • 77. INTRODUCTION Petroleum refinery effluents are wastes liquids that resulted from the refining of crude oil in petroleum refinery. The effluents are composed of oil and grease along with many other toxic organic and inorganic compounds (Diya’uddeen et al., 2011). Among the toxic components of these effluents are heavy metals. Heavy metals include cobalt, chromium, nickel, iron, manganese, zinc, etc. They usually form complexes with different non metal donor atoms which account for their participation in various microbial metabolisms in the environment (Kamnev, 2003). Some of these heavy metals such as cobalt, chromium, nickel, iron manganese, zinc, etc. are required in trace amount by microorganisms at low concentration as nutrients, since they provide vital co-factors for metalloproteins and enzymes and are known as essential metals while others such as cadmium, mercury, lead, etc have no physiological functions and are known as nonessential metals (Sevgi et al., 2010). At high concentration both essential and nonessential heavy metals exert an inhibitory action on microorganisms by impairing the essential functional groups as well as modifying the active conformation of biological molecules. This results in reduction of microbial activity leading to increased lag phase as well as slow growth rate (Aleem et al., 2003). It is expected that petroleum refinery effluents will contain some of these metals in reasonable quantity as well as aromatic compounds such as phenols. Organic and inorganic mixed pollutants are known to be commonly present in industrial effluents and also other contaminated sites. In this case, apart from affecting the viability of the microbiota, the metal activity may have synergistic effect on biodegradation processes of the aromatic compounds. Thus studies related to the association of the bacterial tolerance properties to metals and degradation of phenolic compounds may be relevant to applications in bioremediation processes (Silva et al., 2007). Discharge of these metals into natural waters at increased concentration in refining operations can have severe toxicological effects on aquatic environment and humans. Heavy metals as well as phenol are known to be harmful pollutants emanating from industrial wastewaters that have negative effects on microorganisms. These metals are in the form of inorganic and metallo-organic compounds while phenol appears to be a soluble component of the industrial effluents (Nwanyanwu and Abu, 2010; Hernandez et al., 1998). These environmental pollutants which are environmentally mobile tend to accumulate in organisms, and become persistent because of their chemical stability or poor biodegradability (Emoyan et al., 2005). Contamination of wastewater with high concentration of heavy metals caused a significant decrease in the numbers of bacteria in biological system (Otokunefor and Obiukwu, 2005). It is obvious that heavy metals are one of the toxic contaminants in wastewaters and causes disorder in biological wastewater treatment (Sa’idi, 2010). Microorganisms being ubiquitous in nature have been reported to be found in inhospitable habitats such as petroleum refinery effluents, coke effluents, etc (El-Sayed et al., 2003; Hidalgo et al., 2002) as the effluents are characterized by the presence of phenols, metal derivatives, surface active substances and other chemicals (Suleimanov,1995). Bruins et al., (2000) in their work reported that organisms in such inhospitable environment must have developed metal resistance systems in an attempt to protect sensitive cellular components. On the other hand, utilization of phenol and other pollutant is enhanced by adaptation and production of appropriate enzymes by organisms for the removal of the toxicants (Nwanyanwu et al., 2012). This study investigated the tolerance to heavy metals and phenol by bacterial population in petroleum refinery effluent. Nwanyanwu et al., 2013 923 Journal of Research in Biology (2013) 3(3): 922-931
  • 78. MATERIALS AND METHODS Sample collection Petroleum oil refinery effluent was collected from Biological treatment plant unit (Rotary biodisk, RBD) of Port Harcourt oil refinery complex and transported to the laboratory for physicochemical analysis which includes pH, total dissolved solids, biological oxygen demand (BOD), chemical oxygen demand (COD), phosphate (PO4), nitrate (NO3), oil and grease, phenol, electrical conductivity and heavy metals content. The methods used for the analysis were as shown elsewhere (Nwanyanwu et al., 2012). Microbiological analysis Microbiological counts were estimated by plating 0.1 ml of the 102 - 106 decimally diluted effluent samples in physiological saline on appropriate agar plates. Total heterotrophic bacterial count was done on nutrient agar plates while phenol-utilizing bacterial count was done on phenol-agar medium of Hill and Robinson (1975). The inoculated plates were incubated for 24 h at 30o C for the heterotrophic bacterial count and 72 h for phenol- utilizing bacteria count. Isolation and identification of bacterial strains The discrete bacterial colonies that developed on phenol-agar medium were purified, characterized biochemically and identified as described by Nwanyanwu et al., (2012). Preparation of inoculum The organisms were grown in nutrient broth medium contained in Erlenmeyer flasks (100 ml) at 28±2o C for 48 h. Thereafter, the cells were harvested and washed in sterile deionized distilled water. The cell suspensions were standardized by adjusting the turbidity to an optical density of 0.1 at A540. Screening of isolates for phenol tolerance Into 5.0 ml mineral salt broth medium contained in 15.0 ml screw capped glass culture tubes were added aliquots of phenol stock solution (200 mM). The tubes were sterilized by autoclaving at 121o C for 15min and allowed to cool at room temperature (28±2o C). Thereafter, 0.1 ml aliquot of cell suspensions were seeded into the tubes and incubated at 30o C for 96 h. The final concentrations of phenol in the tubes ranged from 0.1-100 mM. Controls included cells in mineral salt medium without phenol and mineral salt medium supplemented with phenol but without cells. Development of turbid culture depicted tolerance to phenol stress. Isolates that exhibited phenol tolerance from 5.0 mM and above were used for further phenol and heavy metal toxicity assay. Nwanyanwu et al., 2013 Journal of Research in Biology (2013) 3(3): 922-931 924 Table1: Physicochemical and microbiological analyses of biological treatment unit of petroleum refinery wastewater Parameter/ unit Value pH 8.18 Elect. conduct (µs/cm) 485 Oil and grease (mg/l) 15.0 TDS (mg/l) 250 BOD (mg/l) 8.0 COD (mg/l) 76.0 Phenol (mg/l) 13.6 PO4 2- (mg/l) 0.14 NO3 - (mg/l) 1.20 Metal concentration Zn2+ (mg/l) 0.02 Cu2+ (mg/l) <0.02 Cr2+ (mg/l) 0.05 Pb3+ (mg/l) <0.01 Ni2+ (mg/l) 0.02 Cd2+ (mg/l) <0.01 Microbial load THBC (CFU/ml) 2.52 x 108 TPUBC (CFU/ml) 1.14 x 108 % TPUBC (%) 45.24 THBC = Total Heterotrophic bacterial count; TPUBC = Total phenol-utilizing bacterial count
  • 79. Growth on phenol-mineral salt agar The isolates were tested for their ability to grow on mineral salt agar medium (MSM) amended with increasing phenol concentrations. An aliquot (100 µl) of decimally diluted standardized inoculum of each isolate in physiological saline was spread plated onto surface of MSM plates with 2.0-20 mM of phenol concentrations. Control included cells in MSM plates without phenol. The culture was incubated at 30o C for 72 h (Kahru et al, 2002). The number of the colony that developed was enumerated as colony forming unit per ml (CFU/ml). Minimum inhibitory concentration (MIC) determination Stock solutions of Cd, Zn, Hg, Cu, Pb, Ni, Co and Cr as salts of CdCl2, ZnSO4, HgCl2, CuSO4, PbCl2, Ni(NO3)2, CoCl2.6H2O and K2Cr2O7 were prepared in deionized distilled water. All the chemicals used were analytical reagent grade. The minimum inhibitory concentrations (MIC) of eight heavy metal ions at which no growth was observed were determined at pH 7.2 against each bacterial isolate using tube dilution method (Hassen et al., 1998) with little modifications. Graded concentrations of each heavy metal ranging from 0.05 mM to 10.0 mM were prepared in tryptone soy broth (TSB) contained in screw capped culture tubes. The supplemented TSB-heavy metal medium was sterilized by autoclaving at 121o C for 15 min. On cooling to room temperature (28±2o C), the tubes were seeded with 100 µl of the bacterial suspension and incubated at 30o C for 72 h. Inoculated medium free of heavy metal ions and uninoculated medium with metal ions served as positive and negative controls respectively. The MIC of the metal to the test isolates is the lowest concentration that totally inhibited growth of the organisms. RESULTS AND DISCUSSION The physicochemical and microbiological properties of the petroleum refinery effluent are shown in Table 1. Phenol-utilizing bacteria represented 45.24% of the microbial load of biodisk effluent. The high population of phenol-utilizing bacteria obtained could be related to natural selection and adaptation to phenol at the unit. The concentration of heavy metals in the effluent present in the effluent may be as a result of physicochemical treatment (oxidation and reduction, chemical precipitation, etc) given to the raw wastewater before been channeled into the biological treatment unit. The result of screen test for phenol tolerance is shown in Table 2. With the exception of Pseudomonas sp. RBD3 that tolerated phenol up to 10 mM, all the organisms are able to tolerate phenol stress up to 15.0 mM. The growth of the isolates in the medium with phenol concentrations above 10.0 mM may be attributed to previous exposure to phenolic raw wastewater influent into the biological treatment unit (RBD). This is in line with the report of Santos et al., (2001) in which they related the growth of Trichosporom sp. in phenolic amended medium of 10.0 mM concentration to previous phenolic wastewater shock load from stainless steel industry. Moreso, the tolerance of the organisms to high concentration of phenol (15.0 mM) may be the ease with which the isolates open the phenol ring for its subsequent uptake as carbon and energy source (Ajaz et al., 2004). Gurujeyalakshmi and Oriel (1989) in their work have reported that Bacillus stearothermophilus strain BR219 could grow on phenol at levels up to 15 mM. In contrast, growth inhibition of Bacillus, Pseudomonas and Citrobacter species at phenol concentration above 1.0 mM has been reported by many authors (Obiukwu and Abu, 2011). Janke et al., (1981) reported inhibition of phenol hydroxylase activity in Pseudomonas species at 0.25 mM phenol concentration. Yang and Humphrey (1975) found that the growth of Pseudomonas putida was strongly inhibited above phenol concentration of 0.5 mM. Buswell and Twomey (1975) reported that growth of Nwanyanwu et al., 2013 925 Journal of Research in Biology (2013) 3(3): 922-931
  • 80. Bacillus stearothemophilus was inhibited at phenol concentration above 5.0 mM. The effect of increasing doses of phenol (0.05 - 15.0 mM) on the population of the test organisms are shown in Figure 1. Generally, the viable counts increased with the concentration of phenol until a certain concentration when the growth of the organisms was inhibited. The growth of the organisms on phenol followed a substrate inhibition pattern. Increasing phenol concentration resulted in decrease in microbial growth and eventually very minimal growth was detected at the highest phenol concentration (15.0 mM) in all the test organisms. The growth of Bacillus sp. RBD1, Escherichia coli RBD2, Bacillus sp. RBD6, Citrobacter sp. RBD8, Streptococcus sp. RBD9, Pseudomonas sp. RBD11 and Escherichia coli RBD12 with a total viable count of 7.1 x 106 , 8.0 x 106 , 7.2 x 106 , 7.8 x106 , 7.5 x 106 , 8.8 x 106 , 7.4 x 106 and 7.4 x 106 CFU/ml respectively were stimulated at phenol concentrations up to 0.5 mM (≈ 47.06 mg/l). Similarly, at phenol concentration up to 1.0 mM (≈ 94.11 mg/l), the growth of Pseudomonas sp. RBD3, Aeromonas sp. RBD4, Staphylococcus sp. RBD5, Corynebacterium sp. RBD7 and Corynebacterium sp. RBD10 with a total viable count of 8.4 x 106 , 7.5 x 106 , 7.2 x 106 and 8.4 x 106 CFU/ml respectively, were stimulated. Thereafter, the total viable counts progressively decreased as the phenol concentration increases. This growth pattern is typical of in an inhibitory substrate like phenol. The inhibition of bacterial growth by phenol is well-documented. However, some bacteria are more tolerant to phenol than others. For instance, the growth inhibition constant (Ki) for bacteria degrading phenol have been reported as 54.1mg/l (0.57 mM) (Monteiro et al., 2000), 129.79 mg/l (1.379 mM) (Kumar et al., 2005), 2434.7 mg/l (25.87 mM) (Arutchelvan et al., 2006) and 7.818 mM (Wei et al., 2008). In this study, all the test organisms tolerated phenol up to 10.0 mM (≈ 941 mg/l) and with the exception of Pseudomonas sp. RBD 3, all the bacterial strains tolerated 15 mM (≈ 1412 mg/l). This is in line with the report of Worden et al., (1991) that Bacillus stearothermophilus BR219 Nwanyanwu et al., 2013 Journal of Research in Biology (2013) 3(3): 922-931 926 Table 2: Phenol tolerance of the test isolates in different concentrations of phenol Bacteria Growth in mineral salt broth with added phenol Phenol concentration (mM) 0.1 0.2 0.5 1 2 5 10 15 20 50 100 Bacillus sp. RBD1 + + + + + + + + - - - Escherichia coli RBD 2 + + + + + + + + - - - Pseudomonas sp. RBD 3 + + + + + + + - - - - Aeromonas sp. RBD 4 + + + + + + + + - - - Staphylococcus sp. RBD 5 + + + + + + + + - - - Bacillus sp. RBD 6 + + + + + + + + - - - Corynebacterium sp. RBD7 + + + + + + + + - - - Citrobacter sp. RBD8 + + + + + + + + - - - Streptococcus sp. RBD9 + + + + + + + + - - - Pseudomonas sp. RBD10 + + + + + + + + - - - Corynebacterium sp. RBD11 + + + + + + + + - - - Escherichia coli RBD12 + + + + + + + + - - - + = growth ; - = no growth
  • 81. tolerated phenol concentration of 15.0 mM. Similarly, Corynebacterium species was reported to resist 15 mM phenol while Staphylococcus, Corynebacterium, Bacillus and Proteus were found to resist 10 mM of phenol (Ajaz et al., 2004). However, many authors have reported inhibition of microorganisms at such high phenol concentration (Hossein and Hill, 2006; Kotturi et al, 1991). Li and Humphrey (1989) as well as Gurujeyalakshmi and Oriel (1989) have reported microbial growth inhibition at relatively low concentrations of 2.0 mM and 0.25 mM respectively. 927 Journal of Research in Biology (2013) 3(3): 922-931 Nwanyanwu et al., 2013 Phenol (mM) TotalViableCount(x106 CFU/ml) Figure 1: Growth of bacteria on mineral salt agar medium supplemented with increasing doses of phenol. 0 2 4 6 8 10 Pseudomonas sp.RBD3 0 2 4 6 8 10 0 4 8 12 16 Citrobacter sp. RBD8
  • 82. The tolerance levels of refinery wastewater phenol-utilizing bacteria to heavy metals expressed as minimal inhibitory concentrations (MIC) are shown in Table 3. The test isolates in this study showed similar trend of susceptibilities to heavy metal ions based on minimal inhibitory assay. The high MIC values obtained in the study may be as a result of long term exposure of the organisms to metal ions in the refinery effluent. Highest MIC values were exhibited in Chromium, Copper and Nickel while the least MIC was shown in mercury among the isolates with a maximum value of >3.0 mM and minimum value of <2.0 mM. Pseudomonas sp. RBD3 showed maximum MICs value range of 1.5 - 4.5 mM whilst Escherichia coli RBD12 showed minimum MICs value range of 1.0 - 3.5 mM in all the metals tested. The MICs are higher than that reported by El-Deeb (2009) for some phenol-degrading bacteria. However, the MIC values are similar to the values reported elsewhere (Nieto et al., 1989, Nweke et al., 2006a, Akinbowale et al., 2007). The MIC of metal ranging from 0.5 - 2.5 mM, 1.25 - 2.5 mM, 5.0 - 12.0 mM, 1.0 - 1.25 mM, 0.25 - 1.0 mM and 1.25 - 5.0 mM against hydrocarbon-utilizing bacteria was reported for cadmium, chromium, lead, cobalt, mercury and copper respectively (Nweke et al., 2006a). These reported MICs in most cases corroborates the values observed in this study. The MIC in growth inhibition assay is analogous to the concentration of metal ion that exhibited 100 % inhibition in dehydrogenase activity assay. Thus, the MIC of zinc against river water planktonic bacteria have been reported as 1.558 ± 0.037 mM, 1.283 ± 0.068 mM, 2.469 ± 0.045 mM and 1.328 ± 0.094 mM for Escherichia, Proteus, Micrococcus and Pseudomonas species respectively (Nweke et al., 2006b). Likewise, the concentration of zinc that gave 100% inhibition of dehydrogenase activity in sediment Bacillus and Arthrobacter species are 1.442 ± 0.062 mM and 1.199 ± 0.042 mM respectively (Nweke et al., 2007). Also, Hassen et al., (1998) have reported MIC values of 0.1, 0.8, 1.5, 1.6 and 1.8 mM for Mercury, Cobalt, Zinc and Cadmium, Copper and Chromium respectively on Pseudomonas aeruginosa, Citrobacter freundii, Staphylococcus aureus, Streptococcus sp. and Bacillus thurieniensis. Hassen et al., (1998) in their work reported 3.0 mM chromium as the MIC for Nwanyanwuet al., 2013 Journal of Research in Biology (2013) 3(3): 922-931 928 Organism MIC of metal (mM) Cd Zn Hg Cu Pb Ni Co Cr Bacillus sp. RBD1 3.5 2.0 1.5 4.0 4.5 3.5 2.0 4.0 Escherichia coli RBD2 3.5 2.5 1.0 4.0 3.0 4.0 2.5 3.5 Pseudomonas sp. RBD3 4.0 3.0 1.5 4.5 3.0 4.5 3.0 4.5 Aeromonas sp. RBD4 3.5 3.0 1.0 3.0 4.0 4.0 2.0 4.0 Staphylococcus sp. RBD5 4.0 2.0 1.0 3.5 3.0 4.0 3.0 4.0 Bacillus sp. RBD6 3.0 2.5 1.5 3.5 4.0 4.0 3.0 4.0 Corynebacterium sp. RBD7 3.0 2.0 1.5 3.0 3.5 3.5 2.5 3.5 Citrobacter sp. RBD8 3.5 1.5 1.0 2.5 3.0 3.0 2.0 2.5 Streptococcus sp. RBD9 4.0 2.0 1.0 3.5 2.5 4.0 3.0 2.5 Pseudomonas sp. RBD10 3.5 3.0 1.5 4.5 4.0 4.5 3.5 4.0 Corynebacterium sp. RBD11 2.5 2.0 1.0 4.0 4.0 3.0 4.0 4.5 Escherichia coli RBD12 3.0 1.5 1.0 3.0 3.5 3.5 2.5 3.0 Table 3: Minimal inhibitory concentrations of heavy metals
  • 83. Pseudomonas aeruginosa S8 and Citrobacter freundii S24. The variation in the tolerance of heavy metal could be attributed to the bacterial strain involved, assay technique or culture conditions. However, the study has proved that heavy metals such as mercury, zinc and lead do indeed have toxic effect on bacteria. Although it may vary from one species to another, there is no doubt that heavy metals do inhibit bacterial growth. Metals as toxic contaminants of various environmental sites have been reported to have adversely affected potential biodegradation processes occurring in the environment (Said and Lewis, 1991). Amor et al., (2001) reported that the level of metal inhibition of microbial growth depends on concentration as well as nature of the metal and the type of microbial species. Sandrin and Maier (2003) reported that metals such as copper, zinc, cadmium, chromium, nickel, mercury and lead are known to inhibit biodegradation of organic pollutants by microorganisms. Phenol biodegradation have also been reported to be inhibited by metals (Nakamura and Sawada, 2000; Alves de Lima et al., 2007, El-Deeb, 2009). Due to accumulative behaviour of heavy metals, the effluents from petroleum refinery industries could constitute enriched media to propagate and spread microbial populations which are resistant to metallic ions. Thus, microorganisms isolated from petroleum refinery effluent having combined abilities to grow in high concentration of phenol medium and resistance to metals is potentially useful for detoxification of phenolic wastewater co-contaminated with heavy metals. REFERENCES Ajaz M, Noor N, Rasool SA, Khan SA. 2004. Phenol resistant bacteria from soil: identification- characterization and genetical studies Pak. J. Bot. 36(2): 415 - 424. Akinbowale OL, Haihong P, Peter G, Barton MD. 2007. Antibiotic and heavy metal resistance in motile aeromonads and pseudomonads from rainbow trout (Oncorhynchus mykiss) farms in Australia. Inter. J. Antimicrob. Agents 30: 177–182 Aleem A, Isar J, Malik A. 2003. Impact of long-term application of industrial wastewater on the emergence of resistance traits in Azotobacter chroococcum isolated from rhizospheric soil .Biores. Technol., 86: 7 - 13. Alves de Lima A., Pereira MP, Filho RGS, Hofer E. 2007. Utilization of phenol in the presence of heavy metals by metal-tolerant nonfermentative gram-negative bacteria isolated from wastewater. Rev. Latinoam. Microbiol., 49 (3 - 4): 68 -73. Amor L, Kennes C, Veiga MC. 2001. Kinetics of inhibitionin the biodegradation of monoaromatic hydrocarbons in the presence of heavy metals. Biores. Technol., 78: 181 - 185. Arutchelvan V, Kanakasabai V, Elangovan R, Nagarajan S and Muralikrishnan V. 2006. Kinetics of high strength phenol degradation using Bacillus brevis, J. Haz. Mat., B129(1-3): 216 - 222. Bruins MR, Kapil S, Oehme FW. 2000. Microbial resistance to metals in the environment. Ecotoxicol. Environ. Saf., 45:198 - 207. Buswell JA, Twomey DG. 1975. Utilization of Phenol and Cresols by Bacillus stearothermophilus strain Ph24. J.Gen. Microbiol., 87: 377 - 379. Diya’uddeen BH, Wan Daud WMA, Abdul Aziz AR. 2011. Treatment technologies for petroleum refinery effluents: A review. Proc. Saf. Environ. Protection 89: 95 - 105. El-Deeb B. 2009. Natural combination of genetic systems for degradation of phenol and resistance to heavy metals in phenol and cyanide assimilating Nwanyanwu et al., 2013 929 Journal of Research in Biology (2013) 3(3): 922-931
  • 84. bacteria. Malaysian J. Microbiol. 5(2):94 -103. El-Sayed WS, Ibrahim MK, Abu-Shady M, El-Beih F, Ohmura N, Saiki H, Ando A. 2003. Isolation and characterization of phenol-catabolizing bacteria from a coking plant. Biosc. Biotechnol. Biochem., 67(9): 2026 - 2029. Emoyan OO, Ogban FE, Akarah E. 2005. Evaluation of Heavy metals loading of River Ijana, Nigeria. J. Appl. Sci. Environ. Manag., 10(2): 121 - 7. Gurujeyalakshmi G, Oriel P. 1989. Isdoation of phenol -metabolizing enzymes in Trichosporon cutaneum. Arch. Microbiol., 130:54 -58. Hassen A, Saidi N, Cherif M, Boudabous A. 1998. Resistance of environmental bacteria to heavy metals. Biores. Biotechnol., 64: 7 - 15. Hernandez A, Mellado RP, Martinez J L. 1998. Metal accumulation and vanadium-induced multidrug resistance by environmental isolates of Escherichia hermannii and Enterobacter cloacae. Appl. Environ. Microbiol., 64: 4317 - 4320. Hidalgo A, Jaureguibeitia A, Prieto MB, Rodriguez- Fernandez C, Serra JL, Llama MJ. 2002. Biological treatment of phenolic industrial wastewaters by Rhodococcus erythropolis UPV-1. Enz. Microb. Technol.,31: 221 - 226. Hill GA, Robinson CW. 1975. Substrate inhibition kinetics: Phenol degradation by Pseudomonas putida. Biotech. Bioeng., 17: 1599 -1615. Hossein N and Hill GA. 2006 .Closure Effects on Oxygen Transfer and Aerobic Growth in Shake Flasks. Biotechnology and Bioengineering. 95: 34 -41. Janke D, Pohl R and Fritsche W. 1981. Regulation of Phenol Degradation in Pseudomonas putida. Z. Allg. Mikrobiol., 21: 295 - 303. Kahru A, Maloverjan A, Sillak H, Pollumaa L. 2002. The toxicity and fate of phenolic pollutants in the contaminated soils associated with the oil-shale industry. ESPR-Environ. Scie. Pollut. Res. 1: 27 - 33. Kamnev AA. 2003. Phytoremediation of heavy metals: an overview. In: M. Fingerman, R. Nagabhushanam (Eds.), Recent Advances in Marine Biotechnology. Volume 8: Bioremediation. Science Publishers, Inc., Enfield (NH, USA), pp. 269-317. Kotturi G, Robinson CW, Inniss W.E. 1991. Phenol degradation by a psychrotrophic strain of Pseudomonas putida. Appl. Microbiol. Biotechnol. 34: 539-543. Kumar A, Kumar S, Kumar S. 2005. Biodegradation kinetics of phenol and catechol using Pseudomonas putida MTCC 1194 Biochemical Engineering Journal 22 (2005) 151 - 159. Li JK and Humphrey AE. 1989. Kinetic and fluorimetric behaviour of phenol fermentation. Biotechnol Lett 11:177 - 182. Monteiro AAMG, Boaventura RAR, Rodigues AE. 2000. Phenol biodegradation by Pseudomonas putida DSM 548 in a batch reactor Biochemical Engineering Journal 6: 45-49. Nakamura Y and Sawada T. 2000. Biodegradation of phenol in the presence of heavy metals. J. Chem. Technol. Biotechnol., 75: 137 - 142. Nieto JJ, Fernández-Castillo R, Márquez MC, Ventosa A. Quesada E. Ruiz-Berraquerro F. 1989. Survey of metal tolerance in moderately halophilic eubacteria. Appl. Environ. Microbiol., 55(9): 2385 - 2390. Nwanyanwu CE, Nweke CO, Orji JC. 2012. Growth responses of petroleum refinery effluent bacteria to phenol. J. Res. Biol., 3: 167 - 177. Nwanyanwu et al., 2013 Journal of Research in Biology (2013) 3(3): 922-931 930
  • 85. Submit your articles online at www.jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php. Nwanyanwu CE and Abu GO. 2010. In vitro effects of petroleum refinery wastewater on dehydrogenase activity in marine bacterial strains. Rev. Amb. Agua. 5: 21 - 29. Nweke CO, Alisi CS, Okolo JC, Nwanyanwu CE. 2007. Toxicity of zinc to heterotrophic bacteria from a tropical river sediment. Appl. Ecol. Envriron. Res., 5(1): 123 - 132. Nweke CO, Mgbachi LC, Nwanganga C, Nwanyanw CE. 2006a. Heavy metal tolerance among hydrocarbon utilizing bacteria isolated from oil-contaminated soil. Nigeria J. Microbiol., 20(2): 1057 - 1065. Nweke CO, Okolo JC, Nwanyanwu CE, Alisi CS. 2006b. Response of planktonic bacteria of New Calaber River to zinc stress. Afr. J. Biotechnol., 5(8): 653 - 658. Obiukwu CE and Abu GO. 2011. Toxicity of phenol to bacteria isolated from a petroleum refinery waste treatment plant. Int. Sci. Res. J., 3: 10 - 14. Otokunefor TV and Obiukwu C. 2005. Impact of refinery effluent on the physicochemical properties of a water body in the Niger Delta. Appl. Ecol. Environ. Res., 3 (1): 61 - 72. Said WA and Lewis DL. 1991. Quantitative assessment of the effects of metals on microbial degradation of organic chemicals. Appl. Environ. Micrbiol., 57:1498 - 1503. Sa’idi M. 2010. Experimental studies on effect of Heavy Metals presence in Industrial Wastewater on Biological Treatment. Int. J. Environ. Sci., 1: 666 - 676. Sandrin TR and Maier RM. 2003. Impact of metals on the biodegradation of organic pollutants. Environ. Health Perspectives. 111(8): 1093-1101. Santos VL, Heilbuth NM, Linardi VR. 2001. Degradation of phenol by Trichosporom sp. LE3 cells immobilized in alginate. J. Basic Microbiol.,41:171-178. Sevgi E, Coral G, Gizir AM, Sangün MK. 2010. Investigation of heavy metal resistance in some bacterial strains isolated from industrial soils. Turk. J. Biol., 34: 423 - 431. Silva AAL, Pereira MP, Filho RGS, Hofer E. 2007. Utilization of phenol in the presence of heavy metals by metal-tolerant nonfermentative gram-negative bacteria isolated from wastewater. Rev. Latinoam Microbiol., 49: 68 - 73. Suleimanov RA. 1995. Conditions of waste fluid accumulation at petrochemical and processing enterprises and prevention of their harm to water bodies. Meditsina Trudai Promyshlennaia Ekologiia. 12: 31 - 36. Wei G, Yu J, Zhu Y, Chen W and Wang L. 2008. Characterization of phenol degradation by Rhizobium sp. CCNWTB 701 isolated from Astragalus chrysopteru in mining tailing region. Journal of Hazardous Materials 151(1): 111 - 117. Worden RM, Subramanian R, Bly MJ, Winter S, Aronson CL. 1991. Growth kinetics of Bacillus stearothermophilus BR219. Appl. Biochem. Biotechnol., 28/29: 267 - 275. Yang R and Humphrey AF. 1975. Dynamics and Steady Studies of Phenol Biodegradation in Pure and Mixed Cultures. Biotechnol.Bioeng.,17:1211 - 1235. Nwanyanwu et al., 2013 931 Journal of Research in Biology (2013) 3(3): 922-931
  • 86. JournalofResearchinBiology Effect of Chromolaena odorata leaf extract on haematological profiles in Salmonellae typhi infested Wistar rats Keywords: Salmonellae typhi, Chromolaena odorata, Blood cells, Anti-haematotoxic, Rats. ABSTRACT: Haematological indices provide an insight about the internal environment of a given organism. In this present study, the possible anti-haemototxic effect of Chromolaena odorata on Salmonellae typhi – induced haematotoxicity in rats were investigated. The experimental animals were divided into three groups. Group A received only food and water (control). Group B and C received in addition to food and water, single dose of stock Salmonellae typhi at a dose of 106 cfu/ml. The animals in group B and C were allowed to be infected with Salmonellae typhi for 7 days and confirmed by widal test, after which group C was treated with 750mg/kg body weight/ day ethanolic extract of Chromolaena odorata for 10 days. The result showed a significant (p < 0.05) decrease in Red Blood Cells (RBC) count, packed cell volume (PCV), haemoglobin (Hb), mean corpuscular haemoglobin (MCH), Mean Corpuscular haemoglobin Concentration (MCHC), neutrophil and increase in platelet, total White Blood Cell (WBC) and lymphocytes in animals infected with Salmonellae typhi when compared to the control non-infected group. Treatment of animals in group C with ethanolic extract of Chromolaena odorata showed a significant (P < 0.05) increase in mean values of RBC count, PCV, Hb, MCH, MCV, MCHC and decrease in platelets, WBC and lymphocytes when compared to the group infested with Salmonellae typhi only. The results above suggest the anti-haematotoxic potential of ethanolic extract of Chromolaena odorata in Salmonellae typhi infested rats. 932-939 | JRB | 2013 | Vol 3 | No 3 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.jresearchbiology.com Journal of Research in Biology An International Scientific Research Journal Authors: Nwankpa P1 , Ezekwe AS1 , Ibegbulem CO3 and Egwurugwu JN2 . Institution: 1. Department of Medical Biochemistry Imo State University, Owerri, Nigeria 2. Department of Physiology, Imo State University, Owerri, Nigeria 3. Department of Biochemistry, Federal University of Technology Owerri, Nigeria. Corresponding author: Promise Nwankpa Web Address: http://jresearchbiology.com/ documents/RA0337.pdf. Dates: Received: 15 Feb 2013 Accepted: 05 Mar 2013 Published: 11 May 2013 Article Citation: Nwankpa P, Ezekwe AS, Ibegbulem CO and Egwurugwu JN. Effect of Chromolaena odorata leaf extract on haematological profiles in Salmonellae typhi infested Wistar rats. Journal of Research in Biology (2013) 3(3): 932-939 Journal of Research in Biology An International Scientific Research Journal Original Research
  • 87. INTRODUCTION Enteric fever, also called typhoid fever caused by the bacterium Salmonellae typhi, is an acute life threatening febrile ailment (Kotton, 2007). Typhoid fever is distributed worldwide and prevalent throughout the tropics where it is the commonest cause of fever (Wilcocks and Manson-Bahr, 1972). Literature reports have shown that two million cases of typhoid and 200 thousand related deaths occur worldwide each year (Steinberg et al., 2004). One challenge of development in developing countries, is the provision of portable water for the populace as poor sanitary condition and hygiene has been reported to increase the prevalence of Salmonellae typhi infection with reduced incidence in developed countries (Kotton, 2007). Available reports indicate that typhoid infection is the leading cause of morbidity and mortality in a developing country like Nigeria where water carriage method of sewage disposal is inefficient (Crump et al., 2004). Salmonellae typhi infection causes gastroenteritis which symptoms include nausea, vomiting and diarrhea (Parry et al., 2002). The affected organs include spleen, liver and other tissues which habor the bacterium before entering the blood (Jones and Falkow, 1996). During metabolism, bacterial cells, release chemical toxins which interactions damage the tissue of the host organism. This tends to disrupt the blood components or blood forming tissues. Blood is one of the specialized body fluid responsible for the transportation of nutrients, oxygen, hormones and other metabolites to the body’s cell and metabolic waste products away from those cells to sites of elimination. It is known to be the most important body fluid that regulates various vital functions of the body such as excretion, respiration, circulation, osmotic and temperature balance etc. Mammalian circulation of blood transports specific nutrients, gases, metabolic products and hormones between different tissues and organs (Baynes and Dominiczak, 2005). Literature reports indicated that haematological profiles of different species of animals may be influenced adversely by diabetic condition (Edet et al., 2011), phenylhydrazine (Sanni et al., 2005), some anti-retroviral drugs (Kayode et al., 2011) and aqueous leaf extract of Ocimum gratissimum (Obianime et al., 2011). Chromolaena odorata (known as siam weed, independent weed, killer weed) is a perennial shrub which grow in rainforest, grassland and arid bushvelds (Timbilla and Braimah, 2002). The leaves of the plant has been reported to be widely used as herbal remedy for the treatment of various ailments. Available reports have shown a decotion of the leaf extract effective in the treatment of malaria and cough (Suksamran et al., 2004). Akah (1990) has reported the haemostatic and anti-inflammatory property of the leaf extract while Thang et al., (2005) has shown the stimulation of granular tissue and re-epithelization of the epithelial tissue during wound healing. Recently Nwankpa et al., (2012) reported the antioxidative effect of ethanolic leaf extract of Chromolaena odorata in rats. Other medicinal uses including anti-hypertensive, anti-diarrhoeal and diuretic has been reported (Iwu, 1993). In rural communities in Nigeria, the use of Chromolaena odorata for treating Salmonellae typhi infection is common but the effect of the plant on haematological indices in typhoid fever is not known. This study was therefore designed to assess the effect of Chromolaena odorata on haematological profiles in Salmonellae typhi infested rats. MATERIALS AND METHODS Plant Material: The Chromolaena odorata leaves were collected from a natural habitat in Owerri and authenticated by professor S.C. Okeke, a taxonomist at the department of Plant Science and Biotechnology, Imo State University Owerri, Nigeria. The voucher specimen was kept in the university herbarium for references. Preparation of Extract: Large quantities of fresh leaves of Chromolaena odorata, washed free of sand and Nwankpa et al., 2013 933 Journal of Research in Biology (2013) 3(3): 932-939
  • 88. debris, were dried under shade at room temperature at 27°C for 3 weeks. Electric blender was used to homogenize the dried leaves to a powder form. A 700g of the powder macerated in 1.1 litres of 80% (v/v) ethanol were allowed to stand for 24 hours. A chess clot was used to filter the mixture and the filtrate concentrated in vacuo at 37-40°C to 10% its original volume using a rotary evaporator. The concentrate was evaporated in a water bath at 40°C to a solid residue, the extract. The extract was dissolved in 100ml of 10% ethanol to an approximate concentration used for the experiment. Salmonellae typhi: The stock Salmonellae typhi was procured from Federal College of Veterinary and Medical Laboratory Technology of the National Veterinary Research Institute Vom, Jos, Plateau State, Nigeria. Nutrient agar plate, cesteine lactose electrolyte deficient plate (DCA) was used to sub-culture the micro- organism which was incubated at 37°C for 24 hours and examined for growth. The stock sample used for the experiment was prepared as culture slants using McCartney bottle and nutrient agar. Salmonellae typhi from the sub-cultured medium was aseptically incubated for 18 hours at 37°C. Animals: Albino Wistar rats of both sexes weighing between 150-200g were obtained from the animal house of Faculty of Medicine, Imo State University Owerri, Nigeria. They were maintained at room temperature and acclimatized for 12 days to daily handling. They were fed ad-libitum with commercial rat chow (Product of Pfizer Nigeria Ltd) and had free access to water. Induction of typhoid: Each rat was orally administered with 1ml of Salmonellae typhi at a dose of 106 cfu/ml to induce typhoid (Kirby, 1960). Experimental design: Twenty - four albino Wistar rats were used for the study. They were randomly assigned into 3 groups. Each group has 8 rats. Group A: The rats in this group were fed with rat chow and had free access to water. They were not administered with Salmonellae typhi and serve to monitor successful induction of typhoid. Group B: The rats in this group served as control. They were fed with rat chow and had free access to water. Single dose of Salmonellae typhi at106 cfu/ml was orally administered to rats in this group but were not treated with the plant extract. Group C: The rats in this group were fed with rat chow and had access to water. Single dose of Salmonellae tysphi at 106 cfu/ml were orally administered to the rats in this group. After 7 days of infection, 750 mg/kg ethanolic leaf extract of Chromolaena odorata were orally administered to the animals daily for 10 days. Collection and preparation of blood samples for analysis At the end of the treatment, the animals were fasted for 24 hours, re-weighed and sacrificed under chloroform anesthesia. By cardiac puncture, blood sample was collected from each animal with a sterile syringe and needle, in EDTA anti coagulated bottle. The anti-coagulated blood samples were used for haematological analyses which were carried out within 24 hours of sample collection. Haematological analysis Full blood counts such as packed cell volume (PCV), Haemoglobin (Hb), Red Blood Cell (RBC), Total White Blood Cells (TWBC), Platelet count, differential white blood cell (like lymphocytes, monocytes, eosinophils, neutrophils) and red cell indices including Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Cell Haemoglobin Concentration (MCHC) were estimated using the Sysmex® Automated Haematology Analyzer KX-2IN, Sysmex Corporation, Kobe, Japan. Statistical analysis Data generated were statistically analysed by one-way analysis of variance (ANOVA) of the SPSS statistical programme of Microsoft Excel. Values were Nwankpa et al., 2013 Journal of Research in Biology (2013) 3(3): 932-939 934
  • 89. declared significantly different at p<0.05. RESULTS AND DISCUSSION Table 1 and 2 shows the effect of Salmonellae typhi infection and subsequent treatment with ethanolic leaf extract of Chromolaena odorata on haematological parameters in rats. The results showed a significant (P < 0.05) decrease in Red Blood Cells (RBC) count, haemoglobin (Hb), Packed Cell Volume (PCV), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin Concentration (MCHC) and percentage nuetrophil levels in Salmonellae typhi infested rats compared to the non-infested group (Table 1 and 2). On the contrary, the total White Blood Cell (WBC), platelets and lymphocyte levels in rats infested with Salmonellae typhi showed a significant (P < 0.05) increase compared to the non-infested group (Table 2). Treatment of the rats in group C with ethanolic leaf extract of Chromolaena odorata showed a significant (P < 0.05) increase in RBC count, Hb, PCV, MCH, MCV, MCHC and percentage neutrophil levels compared to the Salmonellae typhi infested non-treated group (Table 1 and 2) while treatment of rats in group C with ethanolic leaf extract of Chromolaena odorata showed a significant (P < 0.05) decrease in platelets, WBC and lymphocyte levels compared to the non-treated Salmonellae typhi infested group (Table 2). However the results of this study showed no significant (P > 0.05) difference in RBC, Hb, PCV, MCV, MCH, MCHC, platelets, WBC, and lymphocytes in Salmonellae typhi infested rats treated with Chromolaena odorata compared to the non-infested rats (Table 1 and 2). Haematological indices provide relevant information regarding the internal milieu of an organism. Nutritional, environmental and microbial infection are among several other factors which have been reported to have adverse effects on the haematological profiles of most organisms. Vitamin B12 and folic acid deficiency (Jee et al., 2005, Murray et al., 2007) and exposure to environmental pollutants such as carbondisulphide, insecticide, hexane, gasoline vapour, nitrocellulose thinner has been reported (Dhembara and Pandhe, 2000; Uboh et al., 2007; 2009; 2012 and Savithri et al., 2010). Bacterial infection in living cells release toxins which metabolism results to increase in release of free radical species with attendant damage to the cells (Stipanuk, 2000). In this study, Salmonellae typhi infection significantly decreases the level of RBC, PCV, Hb, MCH, MCV, MCHC, neutrophils and increases the level of WBC and lymphocytes. The observation made in this study agrees with the report of Wilcocks and Manson- Bahr (1972) in Salmonellae typhi infection and Kumar and Kuttan (2005) on cyclophosphamide induced Nwankpa et al., 2013 935 Journal of Research in Biology (2013) 3(3): 932-939 Group Treatment RBC X1012 /L Hb (g/dL) PCV (%) MCV (fL) MCH (pg) MCHC (g/dL) A Negative control/water 3.69 ± 0.21 14.43 ± 0.65 44.33 ± 2.13 63.12 ± 1.60 17.19 ± 1.12 31.27 ± 1.20 B Salmonellae typhi (Positive control) 1.62 ± 0.03a 10.09 ± 0.71a 33.26 ± 2.14a 54.85 ± 1.55a 12.52 ± 1.30a 24.12 ± 1.23a C Salmonellae typhi + Chromolaena odorata 3.49 ± 0.05bc 14.15 ± 0.79bc 43.40 ± 2.34bc 61.95 ± 1.32bc 16.55 ± 1.02bc 30.12 ± 1.33bc Table 1: Effect of Chromolaena odorata on mean values of red blood cells, packed cell volume, hemoglobin and red cell indices in both experimental and control groups. Mean ± SD (n = 8) a Significantly different compared with negative control (P < 0.05). b Significantly different compared with Salmonellae typhi (positive control) (P < 0.05). c No significant difference compared with negative control (P > 0.05).
  • 90. toxicity. The haematotoxic effect of Salmonellae typhi infection may be explained by the interaction of the bacteria or its toxins with the blood forming tissues/ organs which may inhibit the rate at which some specific or generalized haemopoeitic committed stem cells are synthesized by the tissues. Some reports have shown that hexane, cyclophosphamide and benzene induced haematotoxic effect is associated with the interaction of their metabolites with the haematopoeitic tissues and cause depression in their haematopoeitic activities (Synder and Hedli, 1996; Kumar and Kuttan, 2005). Increase in total white blood cells and lymphocytes as well as decrease in neutrophils seen in this study is consistent with the reports on effect of insecticides and pesticides such as fenvalerate, lindane, aldrin among others, on total white blood cells and the differential counts in experimental animals (Synder and Hedli, 1996; Kumar et al., 1996; Savithri et al., 2010). This may be explained by increased lymphopoeisis and/or enhanced release of lymphocytes from lymph myeloid tissue (Das and Mukherjee, 2003). This response may be a direct stimulatory effect of toxic substance on lymphoid tissue/ pollutant induces tissue damage and disturbance of the non-specific immune system leading to increase in production of leukocytes. Neutrophils are known to be involved in the phagocytosis of foreign substances in the body during which some of them are ruptured. This may explain the decrease in neutrophil count on infection with Salmonellae typhi. Ethanolic extract of Chromolaena odorata significantly increased the level of RBC, Hb, PCV, MCV, MCH and MCHC thereby reducing and ameliorating the anaemic condition induced by Salmonellae typhi infection. The observed increase in RBC, Hb, and PCV may be explained by the role of Chromolaena odorata extract in reversing bone marrow depression with attendant improvement in erythrocyte membrane stability through the antioxidant potential of the plant extract, thus reducing haemolysis (Krause and Mahan, 1984; Naaz et al., 2007, Nwankpa et al., 2012). The improvement on the haematopoetic activities of the tissues and/or maintenance of red blood cell membrane integrity relieves the anaemic condition observed in Salmonellae typhi infection. Consequently, increase in RBC count on administration of Chromolaena odorata leaf extract translates to an increase in MCV while increase in Hb translates, to an increase in MCH and MCHC. Furthermore, inhibition of microbial growth by the plant extract has been reported. Okigbo and Ajalie (2005) and Alisi et al., (2011) showed that Chromolaena odorata leaf extract possess antibacterial activity which inhibit the growth of Salmonellae typhi in cells. Decrease in total white blood cell, lymphocytes and attendant increase in neutrophils on administration of the plant extract may be explained by the inhibition of growth of Nwankpa et al., 2013 Journal of Research in Biology (2013) 3(3): 932-939 936 Group Treatment Platelets X103 μL-1 TWBC X103 μL-1 Lymphocytes (%) Neutrophils (%) Eosinophils (%) Monocytes (%) A Negative control/ water 855.18 ± 2.11 16.24 ± 0.78 70.11 ± 2.01 20.19 ± 1.15 1.98 ± 0.6 2.51 ± 0.11 B Salmonellae typhi (Positive control) 880.13 ± 1.5a 25.85 ± 1.16a 82.14 ± 2.11a 11.56 ± 0.87a 3.20 ± 1.10 2.90 ± 0.55 C Salmonellae typhi + Chromolaena odorata 858.82 ± 1.46bc 17.14 ± 1.21bc 72.18 ± 1.88bc 19.26 ± 1.11bc 2.10 ± 0.80 2.6 ± 0.52 Table 2: Effect of CO on mean values of platelets, total white blood cells and differential cell counts in both experimental and control groups Mean ± SD (n = 8) a Significantly different compared with negative control (P < 0.05). b Significantly different compared with Salmonellae typhi (positive control) (P < 0.05). c No significant difference compared with negative control (P > 0.05).
  • 91. Salmonellae typhi in the cell. The inhibition of growth of the microorganism lead to the destruction of excess WBC and lymphyocytes released by the cell in response to bacterial infection (Nancy et al., 2005). Conversely, increase in neutrophil count on administration of the plant extract may be explained by reduced phagocytosis of the microbial cell consequent upon drastic reduction in the growth of microbial cell. CONCLUSION This study has established the anti-haematotoxic potential of ethanolic leaf extract of Chromolaena odorata against Salmonellae typhi induced haematotoxicity in rats. REFERENCES Akah PA. 1990. Mechanism of hemostatic activity of Eupatorium odoratum. International Journal of Crude Drug Research 28(40):253-256. Alisi CS, Nwaogu LA, Ibegbulem CO and Ujowund CU. 2011. Antimicrobial action of methanol extract of Chromolaena odorata-Linn is logistic and exerted by inhibition of dehydrogenase enzymes. Journal of Research in Biology 1(3):209-216. Baynes WJ and Dominiczak HM. 2005. Medical Biochemistry (2nd edn). Elseview Mosby Ltd, Philadelphia. Crump JA, Luby SP and Mintz ED. 2004. The global burden of typhoid fever. Bulletin of World Health Organization 82(5):346-355. Das BK and Mukherjee SC. 2003. Toxicity of cypermethrin in Labeo rohita fingerlings: Biochemical enzymatic and haematological consequence. Comp. Biochem. Physoil. Toxicol. Pharmacol., 134(1):109-121. Dhembara AJ and Pondhe GM. 2000. Haematological changes in fish, Punctivs sophore exposed to some insecticides. J. Exp. Zool., India. 3:41-44. Edet EE, Akpanabiatu MI, Uboh FE, Edet TE, Eno AE, Itam EH and Umoh B. 2011. Gongronema latifolium crude leaf extract reverses alterations in haemotological indices and weight-loss in diabetic rats. J. Pharmacol. Toxicol., 6(2):174-181. Iwu NM. 1993. Handbook of African Medical Plants. CRC Press London. Jee LH, Masroor F and Kang JC. 2005. Responses of cypermethrin-induced stress in haematological parameters of Korean rockfish, Sebastes schlegeli (Hilgendorf) Aquacult. Res., 36(9):898-905. Jones BD and Falkow S. 1996. Salmonellosis: Host Immune responses and bacterial virulence determination. Annual Review of Immunology 14:533-556. Kayode AAA, Kayode OT, Aroyeun OA and Stephen MC. 2011. Haematologic and hepatic enzyme alterations associated with acute administration of Antiretroviral drugs. J. Pharmacol., Toxicol., 6(3):293-302. Kirby B. 1960. Determination of anti-bacterial sensitivity. In (J. Ochie and A. Kochatkar, eds). Medical Laboratory Science, theory and practice (6th edn). McGraw Hill, New Delhi. 801-803. Kotton C. 2007. Typhoid fever medicine plus http:// www.n/m-nih.gov/medicineplys/cartride/001332.htm. Retrieved 04/05/2007. Krause M and Mahan LK. 1984. Food, nutrition and diet therapy (7th edn). W.B. Sanders, Philadelphia. Kumar DMHSA, Sushma NJ, Kumar DJS and Rao KJ. 1996. Haematological changes in albino rats under aldrin intoxication. Indian J. Compar. Anim. Physiol. 14:63-66. Kumar KBH and Kuttan R. 2005. Chemopreventive activity of an extract of Phyllanthus amarus against Nwankpa et al., 2013 937 Journal of Research in Biology (2013) 3(3): 932-939
  • 92. cyclophosphamide-induced toxicity in mice. Phytomedicine 7:494-500. Murray RK, Granner DK, Mayes PA and Rodwell VW. 2007. Harper’s illustrated Biochemistry (26th edn). McGraw-Hill Companies, Asia 46-47. Naaz F, Javed S and Abdinn MZ. 2007. Hepato- protective effect of ethanolic extract of Phyllanthus amarus shcum, Thonn on aflatoxin B1-induced-liver damage in mice. Journal of Ethnopharmacology 113 (3):503-509. Nancy E, Saab OD, Abdala LR and Castillo MC. 2005. Antibacterial activity of Satureja boliviana. International Journal of Molecular Medicine and Advance Sciences 1(1):29-33. Nwankpa P, Eteng MU, Oze G, Nwanjo HU and Ezekwe S. 2012. Effect of Chromolaena odorata on serum lipid profile and oxidative stress status in Salmonellae typhi infested wistar rats. Annals of Biological Research 3(100):4696-4700. Obianime AW, Aprioku JS and Esomonu C. 2011. The effects of aqueous Ocimium gratissimum leaf extract on some biochemical and hematological parameters in male mice. Asian J. Biol. Sci., 4:44-52. Okigbo RN and Ajalie AN. 2005. Inhibition of some human pathogens with tropical plant extracts – Chromolaena odorata and Citrus aurantifolia, and some antibiotics. International Journal of Molecular Medicine and Advance Sciences 1(1):34-40. Parry CM, Hein TTS, Dougan G, Ehite NJ and Farar JS. 2002. Typhoid fever. New England Journal of Medicine 347:1770-1782. Sanni FS, Ibrahim S, Esievo KAN and Sanni S. 2005. Effect of oral administration of aqueous extracts of Khaya Senegalensis stem bark on phenylhydrazine- induced anaemia in rats. Pak. J. Biol. Sci., 8:255-258. Savithri Y, Sekhar PR and Doss PJ. 2010. Changes in haematological profiles of albino rats under chlorpyrifos toxicity. Int. J. Pharma. Bio Sci. 1(3):1-7. Steinberg EB, Bishop RB and Dempsey AF. 2004. Typhoid fever intravellers: who should be targeted for prevention? Clinical Infectious Disease 39(2):186-191. Stipanuk MH. 2000. Biochemical and physiological aspects of human nutrition. W.B. Saunder, Philadelphia. Suksamrarn A, Chotipong A, Suavansri T, Boongira S, Timsukasai P, Vimuttipong S and Chuaynugul A. 2004. Antimycobacterial activity and cytotoxicity of flavonoids from the flowers of Chromolaena odorata. Archives of Pharmcaol. Research 27(95):507-511. Synder R and Hedli CC. 1996. An overview of benzene metabolism (Review). Environ Health Perspect. 104:1165-1171. Thang PT, Patrick S, Teik LS and Yung CS. 2005. Antioxidant effects of the extract from the leaves of Chromolaena odorata on human dermal fibroblast and epirdermal keratinocytes against hydrogen peroxide and hypoxanthine-Xanthine oxidase induced damage. Burns 27(4):319-327. Timbilla JA and Braimah C. 2002. Successful biological control of Chromolaena odorata (L) King and Ribonson in Ghana. The potential of a regional programme in Africa. Proceedings of the 5th international workshop in biological control ad management of Chromolaena odorata, Durban South Africa. Uboh FE, Akpanabiatu MI, Ebong PE and Umoh IB. 2007. Gender differences in the haematotoxicity and weight changes associated with exposure to gasoline vapours in wistar albino rats. Acta Toxicol., 15(2):125- 131. Uboh FE, Akpanabiatu MI, Alozie Y, Edet EE, Ndem JI and Ebong PE. 2009. Comparative effect of vitamin Nwankpa et al., 2013 Journal of Research in Biology (2013) 3(3): 932-939 938
  • 93. Submit your articles online at www.jresearchbiology.com Advantages Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php. A and E on gasoline vapours-induced haematotoxicity and weight-loss in male rats. Int. J. Pharmacol., 5(3):215- 221. Uboh FE, Usoh IF, Nwankpa P and Obochi GI. 2012. Effect of oral exposure to Nitrocellulose thinner on haematological profiles of male Albino rats. American Journal of Biochemistry and Molecular Biology 2(4): 227-234. Wilcock C and Manson-Bahr PEC. 1972. Manson’s tropical disease (17th edn). Macmillan, Tindall. Nwankpa et al., 2013 939 Journal of Research in Biology (2013) 3(3): 932-939
  • 94. Guidelines for Authors The article should be addressed to "The Editor". Submission of an article implies that it has never been published in any other journals and if accepted, it will not be published elsewhere. All papers are first reviewed by the editor. Papers found lacking will not be considered. Others will be sent for a detailed peer-review process. Journal Manuscript Format The manuscript should be typed in “Times new Roman” font with font size 11 and 1.5 line spacing. The page size should be strictly A4. All images should be in JPEG format. The article is to be submitted should accompany a covering letter with name and complete address (including Telephone Number and e-mail ID) of the author/s. The completed article should be sent to submit@jresearchbiology.com Title The title should briefly identify the subject and indicate the purpose of the document. The title should supply enough information for the reader to make a reliable decision on probable interest. Do not use all caps; instead use caps only at the first word of the title and/or at scientific names, abbreviations etc., Center the authors' initials and last names directly below the title. Abstract The abstract should include a hypothesis or rationale for the work, a brief description of the methods, a summary of the results, and a conclusion: The abstract should be less than 250 words. Do not include literature citations or references to tables, figures or equations. Keywords A short list of keywords or phrases should be included immediately after the abstract as index words. Choose keywords that reflect the content of your article. Note that words in the title are not searchable as keywords unless they are also included in the keyword list. Body of the Article The introductory section of the text should include a brief statement of why the research was conducted. It should also define the problem and present objectives along with a plan of development of the subject matter. The introductory section also usually includes a brief survey of the relevant literature on the topic. Materials and Methods Provide sufficient detail so that the work may be repeated. Do not give details of methods described in readily available sources. Instead, refer to the source and describe any modification. Figures that illustrate test apparatus and tables of treatment parameters or equipment specifications are appropriate here. Results and Discussion This section describes the solution to the problem stated in the introductory section. Use figures and tables to visually supplement the presentation of your results. The text must refer explicitly to all visuals, and you must interpret the visual elements to emphasize the evidence on which your conclusions are based. Do not omit important negative results. In addition, relate your findings to previous findings by identifying how and why there are differences and where there is agreement. Speculation is encouraged, but it must be identified. Conclusion This is a summary of your results. In this section, state any conclusions that can be drawn from your data. You may also include suggestions for future research. The conclusion may be a subsection of the Results and Discussion section, or it may be a separate section. Data or statements cited in your conclusion must have been stated previously in the article. Do not introduce new information in the conclusion. Acknowledgement Acknowledgements are optional. Use them to thank individuals or organizations that provided assistance in materials, expertise, or financing. The acknowledgements will appear at the end of the text and should be limited to one or two sentences. References All sources cited in the text must be listed in the References, and all documents listed in the References must be cited in the text. Accuracy of citation is the author's responsibility.
  • 95. Reference Style References should be cited in the text in the form (Author et al, 1987) and listed in alphabetical order at the end of the article as follows: Schernewski G, Neumann T. The trophic state of the Baltic Sea a century ago: a model simulation study. J Mar Sys., 2005;53:109– 124. Kaufman PD, Cseke LJ, Warber S, Duke JA and Brielman HL. Natural Products from plants. CRC press, Bocaralon, Florida. 1999; 15-16. Kala CP. Ecology and Conservation of alphine meadows in the valley of flowers national park, Garhwal Himalaya. Ph.D Thesis, Dehradun: Forest Research Institute, 1998; 75-76. http://www.ethnobiomed.com/content/pdf/1746-4269-1-11.pdf. Appendix Use an appendix for material that is too long to include in the text of the article. Manuscript Charges Journal of Research in Biology is an International Research Journal. This Journal provides immediate access to all published full-text articles to interested readers from all around the world. The availability of the author’s paper makes the scientific community to understand and develop an impact in the concerned research field. It also increases the chance of more citations of the published work, which in turn can be translated into more recognition of research. This journal also accelerates research and knowledge building worldwide. Publishing an article in Journal of Research in Biology requires payment of the manuscript processing charges, once the manuscript is accepted for publication. The payment is to be made by one of the authors, their university/organization, or funding entity. The manuscript processing charges are fixed so as to allow publishers to recover manuscript processing expenses and the cost of making the full-text available on the Internet to all interested researchers. For Indians The charges for submission of a Research article is Rs 2100, up to 8 pages and for more pages, each page costs Rs 250. For Foreign nationals The charges for submission of a Research article is USD 100, up to 8 pages and for more pages, each page costs 15 USD. Copyright Authors who publish in Journal of Research in Biology retain the copyright of their work which allows the unrestricted use, distribution, and reproduction of an article in any medium, provided that the original work is properly cited. If you have any queries kindly contact us at contact@jresearchbiology.com Hard copy subscription of the Journal is available. Kindly subscribe it for your laboratory. Subscription Rates For India: Other countries One Year : Rs 1000 One Year : $120 Six Months : Rs 600 Six Months : $65 Three Months : Rs 300 Three Months : $35 Eight Issues per year will be produced and sent to your perusal. All overseas address is served by Airmail. Send subscription to: Abiya Chelliah , No.2, Ret chakar street, Murugankurichi, Palayamkottai, Tirunelveli - 627002 Tamil Nadu, India Payment mode: You can take a Demand Draft/ Cheque in favour of Abiya Chelliah Payable at State Bank of India, St. Xavier’s College, Tirunelveli, Tamil Nadu, India-627002. Kindly inform us through contact@jresearchbiology.com on doing your payment for subscription.