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Research Project Report

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Contents
1. Tolerance response of American bollworm, Heliothes armigera (Hubner) to Cry
expressing BT cotton.
i. Introduction
ii. Research objectives
iii. Sowing of plants:
iv. Experimental procedure:
v. Statistical analysis:
2. Mosquito Vector Survey in Sargodha, Pakistan.
vi. Study area and survey of adult
vii. Results and discussion
3. Understanding the Rearing of mosquito in laboratory and investigation of repellents
efficacy against female of Culex sp.
i. Introduction:
ii. Research objectives:
iii. Mosquito rearing protocol
iv. Laboratory and rearing procedures
v. Material and methods used
vi. Precautions
vii. Origin and rearing of the mosquitoes.
viii. Repellent test
4. References

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1. Tolerance response of American bollworm, Heliothes
armigera (Hubner) to Cry expressing BT cotton.
Helicoverpa armigera (H. armigera) (Lepidoptera: Noctuidae) is a notorious lepidopterous
pest of cotton, Gossypium hirsutum L., in Asia. Transgenic cotton expressing the Cry gene from
Bacillus thuringiensis (Bt) has been widely planted for control of this pest. This pest is evolving
resistance to Bt toxin. The present research is aimed at determining the tolerance response of
American bollworm to cry expressing Bt cotton.
RESEARCH OBJECTIVES:
The main objectives of this research work was:
 to assess the tolerance response of H. armigera against Cry expressing Bt-cotton
 to check the differential response of H. armigera to Bt and non-Bt-cotton
 to determine the effect of magnesium chloride on the expression of Cry gene
 to understand the host-pest interaction
 to intimate the resistance level of H. armigera against prevailing germplasm of cotton
Sowing of plants:
Fourteen Bt- and non-Bt cotton varieties were sown two times during the January and February
2015 in pots of 14 inches diameter under plastic sheet in research area of University college of
Agriculture, University of Sargodha. First time sown seeds were not germinated or only nine to
ten were germinated due to over treatment with Sulfuric acid. But second time we sowed seeds
without treatment with Sulfuric acid and those were germinated efficiently more than 25 in both
the plastic as well as in clay pots.
Varieties
The transgenic Bt-cotton line CIM-178,CIM-602,MNH-886.CYTO-124,FH-LZ,CIM-598.CIM-
600,VH-305,FH-142,CIM-599,CIM-616,CYTO-177 containing Cry1Ac”.
Non-transgenic parent cultivar CIM-608, CIM-612 were grown for this study. The pots filled with
soil and well-rotted farmyard manure (5%). Each variety sown in six pots.
Experimental procedure:
The experiment involves Bt- and non-Bt-cotton varieties treated with MgCl2 for american
bollworm bioassays and the relative average development rate (RADR) of this bollworm to be
determined. All the developmental parameters such as time period of larval growth, time period of

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pupae, weight of larvae, rate of food consumption and number of eggs of American bollworm are
including in this study.
Statistical analysis:
The application of suitable statistical package and least significant difference test at 5% probability
level to test the significance of the treatments are involved.
2. Mosquito Vector Survey in Sargodha, Pakistan.
Study area and survey of adult
Mosquitoes of family Culicidae are distributed worldwide and comprise about 3500 species. The
diversity of mosquito’s species varies among different regions of the world. Sargodha is situated
in the sub-tropical region in the fluctuation of temperature in different season and heavy rainfall
occurs in a particular period. so, mosquito vector survey was conducted in our 8th
semester started
from September till the end of May in 2015.mosquitoes adult were collected during this period
from different breeding sites including 6 localities (back side of university college of agriculture,
services colony, Y block, Railway station, back side of Misali Ravian school for boys and Tires
bazar, the city using areal net our target species was culex and Aedes. The site selection for the
present survey is mainly based on population density and unplanned drainage with poor garbage
management site of the Sargodha city of Pakistan from the seven breeding sites a total of 1002
adult mosquitoes were collected. Among these mosquitoes the only genus that we found was culex
species.
Sampling consisted of monthly inspection of randomly selected breeding sites of the Sargodha
city. After collecting the total adults of each collection site these were placed in mosquito cages
for our rearing project. The breeding sites were variable including sewage water canal, ditches,
empty gutters, temporary water logged areas, plastic, earthen parts and discarded tires.most of the
study sites were enriched with rotten organic matter sewage green algae short herbs and upright
vegetation.
Results and discussion
Results reveal that of the highest population of culex species was in the Y block than decreased
in other localities like services colony, back side of university college of agriculture, back side of
Misali Ravian school for boys, railway station and tire bazar respectively. The highest population
recorded in y block was may be due to insufficient drainage system and human population density
and expansion of habitat for mosquito development as a result of climate change will therefor tend
to increase the vector density in relation to the human population, favoring disease transmission.

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Table 1. Abundance of Mosquito (Culex sp.) species in the six localities of Sargodha city.
Locality
Month of
study
Y block services
colony
Back side
of UCA
back side of
Misali
Ravian
railway
station
tire bazar
March 203 143 130 94 12 0
April 150 58 28 49 4 0
May 93 14 8 14 0 0
Values in each cell are the sum of adult collected three times with aerial net per locality and months
3. Understanding the Rearing of mosquito in laboratory
and investigation of repellents efficacy against female of
Culex sp.
Introduction:
Mosquito is very important insect in relation to public health because it transmits numerous
diseases including dengue, filariasis, malaria, yellow fever and Japanese encephalitis. Every year
more than 500 million people around word suffer from one of the other tropical diseases including
dangue, malaria and filariasis (Madhumathy et al 2007; Kumar et al., 2012). Culex quinquefasiatus
is an important vector of filariasis in tropical and subtropical region of the world. About 90 million
people suffer from these diseases.
The mosquito control and personal protection from mosquito bites are currently the most important
measures to control mosquito transmitted diseases. However the repellents based on chemical
insecticides are considered to be very useful in producing and preventing the mosquito vectors.
On the other hand chemical insecticides are not safe to human health especially the children
because they may cause skin irritation, hot sensation rashes and allergy (Das et al., 2003). In recent
years there is an increase in public concern on the safety of many chemical products that instigated
a renewed interest on the use of natural products prom plant origin for mosquito vector
management. In addition, plant essential oils in general have been recognized as an important
natural source of insecticides and insect repellents, various essential oils have also been
documented to exhibit acute toxic effects against insects, including mosquitoes (Pavel 2008). The
repellents based on plant essential oils are effective against mosquito control, environmental
friendly, easily biodegradable, and readily available in many areas of the world, have no ill effect
on non-target organisms (Govindrajan 2011).

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Research objectives:
o To understand the population as well as their potential habitats of mosquito in Sargodha
city.
o To completely understand the mosquito rearing protocol in the laboratory.
o To investigate the repellency of different mosquito repellents available in the market and
against female of Culex sp. and to campare them with Taramira oil.
Role of repellent to deter mosuitoes
Many researchers reported the repellent activity of many essential oils against female mosquito
vectors. Essential oil of Cymbopogon citratus, Cinnomomum zelanicum, Rosmarinus officinals,
Mentha piperita and zingiber officinalis showed repellent activity against Culex quinquefasciatus
The US Environmental Protection Agency (USEPA) has registered Citronella, lemon, Eucaliptus
oil as insect repellent ingredients for application on skin, these natural products have been
frequently used due to their relatively low toxicity, camparable efficacy and customer approval
(Soonwera and Phasomkusolsil 2014).
MOSQUITO REARING PROTOCOL
No method for rearing mosquitoes is guaranteed. Perhaps the most important requirement for
successful rearing of mosquitoes is attention to detail. Mosquito rearing to be successful requires
attention 24 hours a day, 7 days a week (Eugene 1970).
The basic rules: Avoid over-crowding mosquitoes, and overfeeding larvae; avoid pesticide
contamination, observe temperature and humidity requirements, standardize rearing methods and
avoid non-standardized food (Eugene 1970).
Temperature and Humidty equipments: Various improvised humidifying methods have been
described, including such methods as using wet towels, blowing a fan through layers of wet
excelsior or gauze; steam from boiling water or from radiators; wet sand; chemicals in water. Use
a recording hygrothermograph for a permanent, continuous record of temperature and humidity
conditions (Eugene 1970).
Lighting: The photoperiod and light intensity affect the development of the various stages in the
life cycle of the mosquito and are the subject of many papers. In the insectary, a cycle of 14 hours
of light and I0 hours of darkness appears to allow the best and most uniform development, without
interfering with normal working hours of the staff (Eugene 1970).

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LABORATORY AND REARING PROCEDURES
1. Eggs
o Eggs are deposited, primarily at night, in well-defined rafts containing I00 or more eggs.
o Oviposition containers are placed in the stock cages 48-76 hours after the females have
been provided with a blood meal. It is advisable to withdraw sucrose prior to egg laying
(de Meillon and Sebastien 1964). These containers, as a matter of convenience, are
usually: placed in the cages and left over night.
o Almost any container holding water to a depth of about 2.5 cm (1”) can be utilized for
oviposition.
o Hatching occurs in approximately 30 hours at 26-7’ C. (80” F.).
o A female may lay up to 5 egg rafts (de Meillon and Thomas, 1966).
o The number of larvae expected from the egg rafts should be estimated and the egg rafts
transferred to the rearing trays.
o Store eggs in a container in the rearing room.
o Treat surplus unhatched eggs with hot water or chemicals to kill them. If possible burn the
old egg papers.
2. Larvae
o In most areas tap water is entirely satisfactory.
o Larvae can be reared in a variety of containers, but enamel or glass containers facilitate
cleaning.
o The surface requirements for this species appear to be 3 larvae/cm2 of water surface.
o The temperature was maintained at 29’ C. They introduced 750 first instar larvae.
o Larvae can be fed 1 gram of a finely ground dog biscuit each day, until day 4 or 5. Each
day the water should be stirred.
o The larval period took 5-7 days at 29 C0
.
3. Pupae
o Pupae should be separated from larvae daily.
o All pupae should be placed in a container with fresh clean water and the container placed
in the desired cages for emergence.
o The pupal period is approximately 36 hours at 27’ C. (80” F.).
o Place pupae in special containers inside cages. Provide each container with a lid so that the
container can be closed after the pupae have emerged. It should be removed from the cage
through a sleeve.
o Treat unneeded larvae, pupae, and discarded rearing water by heating or with chemicals so
that only dead waste materials enter the sewage system.
4. Adults
o Adults are maintained in screen cages of various sizes as small as I ft3.
o Adults can be maintained on 10% sucrose-soaked cottton pads, raisins or honey.

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o A blood meal (chicken, guinea pig) should be provided for the females 3-4 days after
emergence.
o A single chicken can be used once or twice weekly over a long period of time if care is
used to prevent death by exsanguination,
o Prior to mosquito feeding the bird must be rendered immobile so that mosquitoes will not
be frightened away prior to or during feeding since partial blood meals result in small egg
rafts and can seriously affect production.
o Since most strains prefer to feed during the night the host bird is usually left in the cage
overnight.
o Best blood feeding is obtained when sucrose is removed 24 hours prior to blood meal.
5. Adult mating
1. To assure that female mosquitoes are virgin before mating for crosses, female and male
mosquitoes must be separated within 24 hours of eclosion.
2. A single male may mate with multiple females
3. The smallest volume of enclosure containment suitable for mating is the Solo 16 oz paper cup
soup container
4. Males and females need to be at least 2 days old post-eclosion before mating
5. Leave male and female mosquitoes together for 24-48 hours to be certain that mating has
occurred
6. Sexing mosquitoes
Sexing at pupal stage
This can be done by visual size separation. Female pupae are generally larger than male pupae.
Sexing at adult stage
Mosquito antennae are decorated with sensilla (fine sensory hairs). The male mosquito has
significantly more long sensilla than female antennae. The “feathery” appearance of the male
antennae will allow you to tell males apart from females by eye without a microscope.
7. Preservation of Whole Larvae and Pupae and Adult
o Whole larvae and pupae were preserved by first killing them with very hot water. The hot
water fixes proteins which prevents later darkening. Then they were preserved in 95%
formalin in small sized bottles.
o Adults were killed using ethyl acetate. Glass killing jars with the bottom 1/3 filled with
oven dried plaster of paris were used.
o To pin adults, attach pin points to No. 3 stainless steel insect pins. The preferred adhesive
is Ambroid cement. Ambroid cement is a glue commonly found in hobby shops. Some
adults were also preserved in 95% Formalin.

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8. Precautions:
o It is essential that larval and pupal exuviae not remain in the water longer than 8 hours. The
exuviae begin to deteriorate almost immediately. Therefore, all rearing vials should be
checked and exuviae removed a minimum of three times per day.
o Great care must be taken to avoid cross-contamination of different strains; even a single
contaminating egg from a different strain will ruin a stock.
o Always wear fresh gloves when handling eggs. Change gloves often, and always between
handling different strains.
o Larvae must always have ample food. However, care must be taken not to overfeed as this
will lead to bacterial growth and death. Water should remain relatively clear and odor-free.
o Put cages, nets, and pupal cups in the freezer morgue
o Keep the benches clear
o Label your cages
o Replace sugar feeders if you they become moldy
Material and methods
Origin and rearing of the mosquitoes:
o Mosquitoes used in this study were Culex spp. They
were collected on March 28, 2015 from different
localities of Sargodha, Punjab, Pakistan. Then they
were reared for one generation, in the Laboratory of
Entomology at the Department of Entomology, Faculty
of Agriculture, University College of Agriculture,
University of Sargodha, Pakistan under controlled
conditions at temperature of 27+-2C0
, relative humidity
70+_10% and 10-14 light-dark regime.
o Adult mosquitoes were kept in (39 × 20 × 17 cm)
mosquito net and daily provided with cotton pieces
soaked in 2% honey solution for a period of 3-4 days
after emergence. After this period the females were
allowed to take a blood meal from a chicken host, which
is necessary for laying eggs (anautogeny).

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o Plastic cup for oviposition containing tap water was placed in the cage. The obtained egg
rafts picked up from the plastic dish and transferred into large sized petri dish available in
our laboratory containing tap water left for 24 h.
o The hatching larvae were provided daily with fish
food as a diet. This diet was found to be the most
preferable food for the larval development and a well
female fecundity (Kasap and Demirhan 1992.).
Repellent Test
Before the application of repellent, the arms of two human volunteers were
washed and cleaned thoroughly with distilled water. The right arms was
used for treatment and left arms used for control. 2ml of test repellent was
applied to the treatment area of right forearm of each volunteers. After
applying the test repellent, the volunteer was instructed not to rub, touch
or wet treated forearm. The left forearm, which acted as control was not
treated and was exposed for up to 30 second to mosquito cage containing
250 newly parous female mosquito (5,7 days old) if at least two mosquito
landed on or bite the arm the repellence test was then continue. The test
continued until at least two bites occurred in a three minute period. If no
mosquitoes bite or landed during three minute period the arm was
withdrawn from the cage. The repellency test period was carried out every
thirty minutes until fewer than two mosquito bite or landed during three
minute study period and then repellency test was stopped. The between application of the
repellence was recorded as the protection time (Sritabutra et al., 2011)
References
EUGENE J.G. 1970. Manual for Mosquito Rearing and Experimental Techniques. American
Msquito Control Assosiation. INC. Publisher of MOSQUITO NEWS. P (79-83) Kasap,
M. and Demirhan L. 1992. The effect of various larval foods on the rate of adult emergence and
fecundity of mosquitoes. Turkiye Parazitologi Dergisi. 161: 87-97.
SOONWERA, M. AND PHASOMKUSOLSIL, S. 2014. Mosquito repellent from Thai
essential oils against dandue fever mosquito (Aedes aegyptii (L.)) and filarial mosquito
vestor (Culex quinquefasciatus (Says)). Australian Journal of Microbiology
Research, Vol.8 (17): 1819- 1824.
SRITABUTRA, D., SOONWERA, M., WALTANACHANOBON, S. AND POUNGJAI, S.
2011 Evaluation of herbal essential oil as repellent against Aedes aegypti (L) and
Anopheles dirus Peyton and Harrion. Asian Pacific Journel of Tropical Biomedicine,
S124-S128.