This document summarizes a study that analyzed the genetic differentiation between the native Artemia franciscana population from San Francisco Bay, USA and introduced populations in Kenyan coastal saltworks using molecular markers. Analysis of mitochondrial DNA and heat shock protein 70 gene sequences found evidence of genetic differentiation and haplotype diversity between populations, indicating evolutionary changes have occurred since introduction. Specifically, a private haplotype was found in samples from Fundisha saltworks, providing molecular evidence of genetic differentiation between populations, though not at a statistically significant level. The heat shock protein 70 gene sequences did not show unique signatures between Kenyan and source populations, suggesting other factors contribute to their increased thermotolerance. Further genetic study using larger DNA fragments was recommended
The document describes a meta-analysis of microbial community samples collected by the Earth Microbiome Project (EMP) that used coordinated protocols and analytical methods to explore patterns of diversity at an unprecedented scale. By tracking individual bacterial and archaeal ribosomal RNA gene sequences across multiple studies, the analysis resulted in both a reference database providing global context to DNA sequence data and an analytical framework for incorporating future study data to further characterize Earth's microbial diversity. The meta-analysis found that standardized environmental descriptors and new analytical methods, particularly using exact sequences instead of clustered operational taxonomic units, enabled comparisons across studies and exploration of large-scale ecological patterns.
This document summarizes a study that reconstructed 7,903 bacterial and archaeal genomes from over 1,500 public metagenomes. Key findings include:
- The genomes increase phylogenetic diversity of bacterial and archaeal trees by over 30% and provide first representatives for 17 bacterial and 3 archaeal candidate phyla.
- 245 genomes were recovered from the Patescibacteria superphylum.
- The genomes vary substantially in quality, with 43.5% considered near-complete, 43.8% medium quality, and 12.7% partial.
- The genomes expand representation of underrepresented phyla like Aminicenantes, Gemmatimonadetes, and Lentisphaera
This study examined the growth and survival of 18 strains of flagellated protists isolated from four deep-sea hydrothermal vents under conditions of high pressure and chemical exposure similar to vent environments. Some key findings:
- Deep-sea isolates of two species, Caecitellus parvulus and Rhynchomonas nasuta, grew faster at higher pressures than shallow-water strains. Both were able to grow at pressures matching their collection depths.
- All three species tested, C. parvulus, Cafeteria sp. and R. nasuta, showed high tolerance to sulfides and metals at concentrations far exceeding naturally occurring levels.
- C. parvulus and R. nas
This master's seminar presentation speaks about the role of bacteriophage in the management of different plant diseases.
It deals with the history and discovery of bacteriophages up to current research studies and usage.
Microsatellite and mt-DNA phylogenies of the chamois (genus Rupicapra) and ta...Trinidad Mendez
To elucidate the evolutionary history of chamois, we had analysed DNA sequences of four mitochondrial regions and 20 loci microsatellites including all subspecies along its entire distribution range
Technical Research Paper-04242013_DSN-1-finalDanielle Nisan
This study analyzed genetic diversity in Short-tailed, Black-footed, and Laysan albatross using ancient and historic mitochondrial DNA samples. The researchers sequenced two mitochondrial DNA regions, cytochrome b and the d-loop region, from museum specimens and ancient bones to identify haplotypes. They found low haplotype diversity in cytochrome b across all three species, and in d-loop regions in Short-tailed albatross, which experienced a population bottleneck. D-loop regions showed greater diversity in Black-footed and Laysan albatross. This suggests reduced genetic diversity in Short-tailed albatross following its near extinction due to overhunting in the early 1900s.
This study analyzed the occurrence and diversity of integrons in bacteria isolated from an urban wastewater treatment plant. A total of 697 isolates of Enterobacteriaceae and Aeromonas were screened for integrons. Three new gene cassettes were identified, including a novel aadA variant and genes involved in cell signaling and unknown functions. Thirteen different gene cassette arrays were detected, with four representing novel integrons. Approximately 80% of isolates were resistant to at least 3 antibiotic classes. The presence of novel integron structures in treated effluent suggests wastewater treatment plants may facilitate the formation and spread of antibiotic resistance genes.
The document describes a meta-analysis of microbial community samples collected by the Earth Microbiome Project (EMP) that used coordinated protocols and analytical methods to explore patterns of diversity at an unprecedented scale. By tracking individual bacterial and archaeal ribosomal RNA gene sequences across multiple studies, the analysis resulted in both a reference database providing global context to DNA sequence data and an analytical framework for incorporating future study data to further characterize Earth's microbial diversity. The meta-analysis found that standardized environmental descriptors and new analytical methods, particularly using exact sequences instead of clustered operational taxonomic units, enabled comparisons across studies and exploration of large-scale ecological patterns.
This document summarizes a study that reconstructed 7,903 bacterial and archaeal genomes from over 1,500 public metagenomes. Key findings include:
- The genomes increase phylogenetic diversity of bacterial and archaeal trees by over 30% and provide first representatives for 17 bacterial and 3 archaeal candidate phyla.
- 245 genomes were recovered from the Patescibacteria superphylum.
- The genomes vary substantially in quality, with 43.5% considered near-complete, 43.8% medium quality, and 12.7% partial.
- The genomes expand representation of underrepresented phyla like Aminicenantes, Gemmatimonadetes, and Lentisphaera
This study examined the growth and survival of 18 strains of flagellated protists isolated from four deep-sea hydrothermal vents under conditions of high pressure and chemical exposure similar to vent environments. Some key findings:
- Deep-sea isolates of two species, Caecitellus parvulus and Rhynchomonas nasuta, grew faster at higher pressures than shallow-water strains. Both were able to grow at pressures matching their collection depths.
- All three species tested, C. parvulus, Cafeteria sp. and R. nasuta, showed high tolerance to sulfides and metals at concentrations far exceeding naturally occurring levels.
- C. parvulus and R. nas
This master's seminar presentation speaks about the role of bacteriophage in the management of different plant diseases.
It deals with the history and discovery of bacteriophages up to current research studies and usage.
Microsatellite and mt-DNA phylogenies of the chamois (genus Rupicapra) and ta...Trinidad Mendez
To elucidate the evolutionary history of chamois, we had analysed DNA sequences of four mitochondrial regions and 20 loci microsatellites including all subspecies along its entire distribution range
Technical Research Paper-04242013_DSN-1-finalDanielle Nisan
This study analyzed genetic diversity in Short-tailed, Black-footed, and Laysan albatross using ancient and historic mitochondrial DNA samples. The researchers sequenced two mitochondrial DNA regions, cytochrome b and the d-loop region, from museum specimens and ancient bones to identify haplotypes. They found low haplotype diversity in cytochrome b across all three species, and in d-loop regions in Short-tailed albatross, which experienced a population bottleneck. D-loop regions showed greater diversity in Black-footed and Laysan albatross. This suggests reduced genetic diversity in Short-tailed albatross following its near extinction due to overhunting in the early 1900s.
This study analyzed the occurrence and diversity of integrons in bacteria isolated from an urban wastewater treatment plant. A total of 697 isolates of Enterobacteriaceae and Aeromonas were screened for integrons. Three new gene cassettes were identified, including a novel aadA variant and genes involved in cell signaling and unknown functions. Thirteen different gene cassette arrays were detected, with four representing novel integrons. Approximately 80% of isolates were resistant to at least 3 antibiotic classes. The presence of novel integron structures in treated effluent suggests wastewater treatment plants may facilitate the formation and spread of antibiotic resistance genes.
This document discusses evidence of lateral transfer of a group IE intron between fungal and red algal small subunit rRNA genes. It finds that a group IE intron inserted at position 989 in the nuclear SSU rRNA gene of the red alga Hildenbrandia rubra is closely related to similar fungal IE introns, providing evidence the intron was laterally transferred rather than vertically inherited. Phylogenetic analysis of intron sequences and comparisons of intron secondary structures support a relationship between the red algal intron and fungal introns, making lateral transfer the most likely explanation for the intron's presence in H. rubra.
The document summarizes the identification of six hemocyte cell types in Culex quinquefasciatus by light and transmission electron microscopy:
1) Prohemocytes, the smallest hemocytes, with a large central nucleus and few organelles. They represent 9.3% of hemocytes.
2) Spherulocytes, small hemocytes with numerous spherules containing a lamellar pattern and dense core. They are 1.6% of hemocytes.
3) Adipohemocytes, rare cells with a large nucleolated nucleus, cytoplasm containing organelles and lipid inclusions. They are 0.8% of hemocytes.
4) Oenocytoids
Models of Human Diseases Conference (2010) Tetrahymena model by Dr. R. Pearl...Medical Education Advising
The Ciliate Protozoan Tetrahymena thermophila as an important animal model organism
Dr. R.E. Pearlman, York University
Models of Human Diseases Conference
June 29, 2010
This document summarizes key points from a class on microbial phylogenomics taught by Jonathan Eisen. It discusses reading scientific papers, specifically beginning with the introduction rather than the abstract. It also provides guidance on identifying the big question a field is trying to answer, summarizing the background and limitations of prior work, stating the specific questions authors are addressing, and identifying their experimental approach. The document does not summarize any specific paper.
An overview of ranavirus diagnostics, treatment and managementmgray11
This document discusses diagnostics, treatment, and management of ranavirus. It outlines several diagnostic methods like virus isolation, serology, histopathology, transmission electron microscopy, and PCR. Real-time PCR is commonly used but has limitations. Proper sample selection and testing the right samples is important to accurately detect ranavirus infections, especially subclinical infections. Both mortality events and disease surveillance require considering potential co-infections and the ability to differentiate ranavirus strains. Control of ranavirus involves regulatory practices, vaccination for small populations, and treatment of individuals, which is debated for persistent infections. Reintroduction programs necessitate easy strain differentiation of detected viruses.
This document reports on a study of the chromosome numbers and nuclear types of 44 species from 20 genera of Cymbidioid orchids occurring in Brazil. Chromosome numbers ranged from 2n=12 in Psygmorchis pusilla to approximately 2n=168 in Oncidium aff. flexuosum. Interphase nuclear types varied widely. Chromosome numbers observed included 2n=54 for subtribe Eulophiinae, 2n=44, 46, 92 for subtribe Cyrtopodiinae, 2n=54, approximately 108 for subtribe Catasetinae, 2n=52, approximately 96 for subtribe Zygopetalinae, 2n=40, 80 for subtribe Lycast
The researchers analyzed the genetic structure of eastern mud snail populations from Fort Wadsworth and Plumb Beach in New York to determine if they are from the same or different populations. They extracted and sequenced DNA from the cytochrome c oxidase I gene of mud snails collected from both locations. Analysis showed very low genetic divergence between the two populations and a phylogenetic tree grouped the DNA sequences together rather than into separate clades. This supports the hypothesis that the mud snail populations are from the same population and do not need to be managed separately.
This document is a thesis by Jonas Danielson from Lund University in 2010 on the topic of plant Major Intrinsic Proteins (MIPs), also known as aquaporins. It provides an introduction to MIPs and their structure and function. It then focuses on plant MIPs, discussing the large family of MIP isoforms in plants, their evolution and diversity across plant species. It aims to expand knowledge of plant MIP diversity and the roles of different subfamilies and isoforms using both traditional molecular biology approaches and comparative genetics methods.
The complete genome of a foot-and-mouth disease virus (FMDV) type O isolate from Greece in 1994 was sequenced. This virus belongs to the Middle East-South Asia genetic group. Analysis found no differences in the protein coding sequence compared to other viruses in this group, but a 43 nucleotide deletion in the 5' untranslated region shared with only two other viruses. Seven amino acid substitutions were also found in the 3A protein, which has been associated with host specificity.
This document discusses a study that found molecular and morphological evidence suggesting that the flagellate Ancyromonas is closely related to the common ancestor of metazoans, fungi, and choanoflagellates. Analyses of 18S rRNA gene sequences from major eukaryotic lineages using maximum likelihood, minimum evolution, and maximum parsimony supported Ancyromonas forming its own lineage, called Ancyromonadida, that is more closely related to opisthokonts than its nearest protist relatives. However, low bootstrap support for deep nodes limits the ability of 18S rDNA to fully resolve this aspect of eukaryotic phylogeny.
Molecular and cytogenetic phylogeography of h. malabaricuscmvolcker
Claudio Michael Völcker
Jorge A. Dergam
Molecular and karyotypic phylogeography in the Neotropical Hoplias malabaricus (Erythrinidae) fish in eastern Brazil
This study examined three species of aquatic freshwater turtles in Costa Rica for haemogregarine infections. All turtles sampled were positive for intraerythrocytic haemogregarines, representing the first report of these parasites in turtles from Central America. Black river turtles had a significantly higher average parasitemia (0.34%) than white-lipped mud turtles (0.05%). Parasites in the single scorpion mud turtle examined were smaller and did not displace the host cell nucleus like those in the other two species. This is the first report of haemogregarines in the white-lipped mud turtle, scorpion mud turtle, and any Rhinoclemm
The study isolated and identified bacteria from fresh and smoked Clarias gariepinus (African catfish) samples collected from three markets in Minna, Nigeria. Bacterial analysis revealed six species of bacteria present: Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermis, Salmonella epidermis, Salmonella typhii, and Shigella sp. Samples from Chanchaga market had the highest bacterial load and number of identified species for both fresh and smoked fish. The mean bacterial load was 1.84 x 106 cfu/ml for fresh fish and 2.06 x 106 cfu/ml for smoked fish.
The document discusses the need for increased genomic sampling of bacterial and archaeal diversity based on the tree of life. It summarizes the Genomic Encyclopedia of Bacteria and Archaea (GEBA) pilot project, which selected 200 organisms from diverse phylogenetic lineages for genome sequencing. The results showed that sequencing genomes from underrepresented lineages led to novel gene, protein, and structural discoveries that improved genome annotation and metagenomic analysis. The document argues for expanding GEBA-style systematic sequencing to better represent microbial diversity.
Is host specificity the cause or the consequence of parasite diversification:...Jan Stefka
This document summarizes research on the relationship between host specificity and parasite diversification. It discusses how host specificity can arise as both a cause and consequence of speciation processes in parasites. The document presents case studies on lice and helminths that show examples of both host specificity developing in allopatry during speciation, and ongoing host-dependent speciation within parasite lineages. It concludes that while host specificity sometimes drives speciation in parasites, it can also be a product of speciation occurring in isolation between parasite populations.
This document describes the development of 21 new nuclear genetic markers for the wall lizard genus Podarcis. DNA from one individual of P. vaucheri was used to generate anonymous sequence fragments which were sequenced and screened for repetitive elements. Primers were designed for fragments over 300bp without repeats. These markers showed high cross-amplification among closely related P. vaucheri, P. bocagei and P. liolepis, but lower success in more distant P. muralis and P. tiliguerta. Nucleotide diversity across the 5 species ranged from 0.35-3.5%, demonstrating their utility for population genetics and phylogenetics in Podarcis.
Astroviruses are non-enveloped viruses with positive-sense RNA genomes that cause gastroenteritis. They have an icosahedral capsid containing three major proteins and a genome organized into three open reading frames. ORF1 encodes nonstructural proteins involved in replication, ORF2 encodes the structural capsid polyprotein which is processed by cellular proteases. Infection involves binding to an unknown receptor, translation of viral proteins, processing of polyproteins, replication through a negative-sense intermediate, and assembly of new virions which are released from the cell.
This document describes the sequencing and analysis of the small subunit ribosomal RNA gene from an unusual form of the protistan parasite Ichthyophonus found infecting yellowtail flounder off Nova Scotia. Phylogenetic analysis shows it is distinct from two isolates of I. hoferi, supporting it being a separate species named I. irregularis. This finding raises the possibility that ichthyophoniasis in fish could be caused by different but related pathogens, in some cases concurrently.
Talk by Jonathan Eisen for GSAC2000 on "Phylogenomics"Jonathan Eisen
This document discusses phylogenomics, which combines evolutionary reconstructions and genome analysis into a single composite approach. It provides examples of how phylogenomics can be used to infer functional predictions, identify gene duplications, and compare closely related genomes. The document outlines the key components of a phylogenomic analysis, including constructing gene and species trees, analyzing patterns of presence/absence and evolutionary distribution of genes, and making functional predictions based on the integrated analysis.
Paper to Upload, MOLECULAR PHYLOGENY OF CATFISHES.pdfOanhTrng13
This document presents a study on the molecular phylogeny of catfishes (order Siluriformes) in the Lower Mekong River Basin, based on analysis of mitochondrial DNA sequences. Thirty catfish species from nine families collected in the Lower Mekong Basin were analyzed using the 16S rRNA and COI gene regions. Phylogenetic trees were constructed using neighbor joining, Bayesian inference and maximum likelihood methods. The results showed strong support for monophyly of Siluriformes and seven of the nine studied families. However, the families Pangasiidae and Bagridae were found to be paraphyletic. Interfamilial relationships varied between analysis methods and datasets, showing inconsistencies with previous catfish phylogen
1) The study examined genetic variation between early and late feeding Atlantic salmon from the River Shin in Scotland using 6 allozyme loci and 8 microsatellite markers.
2) Early feeding fish had higher rates of smolting and fewer parr compared to late feeding fish, showing different life history strategies between the groups.
3) Early and late feeding fish showed different allelic and genotypic distributions at both allozyme and microsatellite loci, with early feeders being more heterozygous on average. Higher heterozygosity was positively associated with earlier smolting and precocious maturation in male parr.
This document discusses evidence of lateral transfer of a group IE intron between fungal and red algal small subunit rRNA genes. It finds that a group IE intron inserted at position 989 in the nuclear SSU rRNA gene of the red alga Hildenbrandia rubra is closely related to similar fungal IE introns, providing evidence the intron was laterally transferred rather than vertically inherited. Phylogenetic analysis of intron sequences and comparisons of intron secondary structures support a relationship between the red algal intron and fungal introns, making lateral transfer the most likely explanation for the intron's presence in H. rubra.
The document summarizes the identification of six hemocyte cell types in Culex quinquefasciatus by light and transmission electron microscopy:
1) Prohemocytes, the smallest hemocytes, with a large central nucleus and few organelles. They represent 9.3% of hemocytes.
2) Spherulocytes, small hemocytes with numerous spherules containing a lamellar pattern and dense core. They are 1.6% of hemocytes.
3) Adipohemocytes, rare cells with a large nucleolated nucleus, cytoplasm containing organelles and lipid inclusions. They are 0.8% of hemocytes.
4) Oenocytoids
Models of Human Diseases Conference (2010) Tetrahymena model by Dr. R. Pearl...Medical Education Advising
The Ciliate Protozoan Tetrahymena thermophila as an important animal model organism
Dr. R.E. Pearlman, York University
Models of Human Diseases Conference
June 29, 2010
This document summarizes key points from a class on microbial phylogenomics taught by Jonathan Eisen. It discusses reading scientific papers, specifically beginning with the introduction rather than the abstract. It also provides guidance on identifying the big question a field is trying to answer, summarizing the background and limitations of prior work, stating the specific questions authors are addressing, and identifying their experimental approach. The document does not summarize any specific paper.
An overview of ranavirus diagnostics, treatment and managementmgray11
This document discusses diagnostics, treatment, and management of ranavirus. It outlines several diagnostic methods like virus isolation, serology, histopathology, transmission electron microscopy, and PCR. Real-time PCR is commonly used but has limitations. Proper sample selection and testing the right samples is important to accurately detect ranavirus infections, especially subclinical infections. Both mortality events and disease surveillance require considering potential co-infections and the ability to differentiate ranavirus strains. Control of ranavirus involves regulatory practices, vaccination for small populations, and treatment of individuals, which is debated for persistent infections. Reintroduction programs necessitate easy strain differentiation of detected viruses.
This document reports on a study of the chromosome numbers and nuclear types of 44 species from 20 genera of Cymbidioid orchids occurring in Brazil. Chromosome numbers ranged from 2n=12 in Psygmorchis pusilla to approximately 2n=168 in Oncidium aff. flexuosum. Interphase nuclear types varied widely. Chromosome numbers observed included 2n=54 for subtribe Eulophiinae, 2n=44, 46, 92 for subtribe Cyrtopodiinae, 2n=54, approximately 108 for subtribe Catasetinae, 2n=52, approximately 96 for subtribe Zygopetalinae, 2n=40, 80 for subtribe Lycast
The researchers analyzed the genetic structure of eastern mud snail populations from Fort Wadsworth and Plumb Beach in New York to determine if they are from the same or different populations. They extracted and sequenced DNA from the cytochrome c oxidase I gene of mud snails collected from both locations. Analysis showed very low genetic divergence between the two populations and a phylogenetic tree grouped the DNA sequences together rather than into separate clades. This supports the hypothesis that the mud snail populations are from the same population and do not need to be managed separately.
This document is a thesis by Jonas Danielson from Lund University in 2010 on the topic of plant Major Intrinsic Proteins (MIPs), also known as aquaporins. It provides an introduction to MIPs and their structure and function. It then focuses on plant MIPs, discussing the large family of MIP isoforms in plants, their evolution and diversity across plant species. It aims to expand knowledge of plant MIP diversity and the roles of different subfamilies and isoforms using both traditional molecular biology approaches and comparative genetics methods.
The complete genome of a foot-and-mouth disease virus (FMDV) type O isolate from Greece in 1994 was sequenced. This virus belongs to the Middle East-South Asia genetic group. Analysis found no differences in the protein coding sequence compared to other viruses in this group, but a 43 nucleotide deletion in the 5' untranslated region shared with only two other viruses. Seven amino acid substitutions were also found in the 3A protein, which has been associated with host specificity.
This document discusses a study that found molecular and morphological evidence suggesting that the flagellate Ancyromonas is closely related to the common ancestor of metazoans, fungi, and choanoflagellates. Analyses of 18S rRNA gene sequences from major eukaryotic lineages using maximum likelihood, minimum evolution, and maximum parsimony supported Ancyromonas forming its own lineage, called Ancyromonadida, that is more closely related to opisthokonts than its nearest protist relatives. However, low bootstrap support for deep nodes limits the ability of 18S rDNA to fully resolve this aspect of eukaryotic phylogeny.
Molecular and cytogenetic phylogeography of h. malabaricuscmvolcker
Claudio Michael Völcker
Jorge A. Dergam
Molecular and karyotypic phylogeography in the Neotropical Hoplias malabaricus (Erythrinidae) fish in eastern Brazil
This study examined three species of aquatic freshwater turtles in Costa Rica for haemogregarine infections. All turtles sampled were positive for intraerythrocytic haemogregarines, representing the first report of these parasites in turtles from Central America. Black river turtles had a significantly higher average parasitemia (0.34%) than white-lipped mud turtles (0.05%). Parasites in the single scorpion mud turtle examined were smaller and did not displace the host cell nucleus like those in the other two species. This is the first report of haemogregarines in the white-lipped mud turtle, scorpion mud turtle, and any Rhinoclemm
The study isolated and identified bacteria from fresh and smoked Clarias gariepinus (African catfish) samples collected from three markets in Minna, Nigeria. Bacterial analysis revealed six species of bacteria present: Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermis, Salmonella epidermis, Salmonella typhii, and Shigella sp. Samples from Chanchaga market had the highest bacterial load and number of identified species for both fresh and smoked fish. The mean bacterial load was 1.84 x 106 cfu/ml for fresh fish and 2.06 x 106 cfu/ml for smoked fish.
The document discusses the need for increased genomic sampling of bacterial and archaeal diversity based on the tree of life. It summarizes the Genomic Encyclopedia of Bacteria and Archaea (GEBA) pilot project, which selected 200 organisms from diverse phylogenetic lineages for genome sequencing. The results showed that sequencing genomes from underrepresented lineages led to novel gene, protein, and structural discoveries that improved genome annotation and metagenomic analysis. The document argues for expanding GEBA-style systematic sequencing to better represent microbial diversity.
Is host specificity the cause or the consequence of parasite diversification:...Jan Stefka
This document summarizes research on the relationship between host specificity and parasite diversification. It discusses how host specificity can arise as both a cause and consequence of speciation processes in parasites. The document presents case studies on lice and helminths that show examples of both host specificity developing in allopatry during speciation, and ongoing host-dependent speciation within parasite lineages. It concludes that while host specificity sometimes drives speciation in parasites, it can also be a product of speciation occurring in isolation between parasite populations.
This document describes the development of 21 new nuclear genetic markers for the wall lizard genus Podarcis. DNA from one individual of P. vaucheri was used to generate anonymous sequence fragments which were sequenced and screened for repetitive elements. Primers were designed for fragments over 300bp without repeats. These markers showed high cross-amplification among closely related P. vaucheri, P. bocagei and P. liolepis, but lower success in more distant P. muralis and P. tiliguerta. Nucleotide diversity across the 5 species ranged from 0.35-3.5%, demonstrating their utility for population genetics and phylogenetics in Podarcis.
Astroviruses are non-enveloped viruses with positive-sense RNA genomes that cause gastroenteritis. They have an icosahedral capsid containing three major proteins and a genome organized into three open reading frames. ORF1 encodes nonstructural proteins involved in replication, ORF2 encodes the structural capsid polyprotein which is processed by cellular proteases. Infection involves binding to an unknown receptor, translation of viral proteins, processing of polyproteins, replication through a negative-sense intermediate, and assembly of new virions which are released from the cell.
This document describes the sequencing and analysis of the small subunit ribosomal RNA gene from an unusual form of the protistan parasite Ichthyophonus found infecting yellowtail flounder off Nova Scotia. Phylogenetic analysis shows it is distinct from two isolates of I. hoferi, supporting it being a separate species named I. irregularis. This finding raises the possibility that ichthyophoniasis in fish could be caused by different but related pathogens, in some cases concurrently.
Talk by Jonathan Eisen for GSAC2000 on "Phylogenomics"Jonathan Eisen
This document discusses phylogenomics, which combines evolutionary reconstructions and genome analysis into a single composite approach. It provides examples of how phylogenomics can be used to infer functional predictions, identify gene duplications, and compare closely related genomes. The document outlines the key components of a phylogenomic analysis, including constructing gene and species trees, analyzing patterns of presence/absence and evolutionary distribution of genes, and making functional predictions based on the integrated analysis.
Paper to Upload, MOLECULAR PHYLOGENY OF CATFISHES.pdfOanhTrng13
This document presents a study on the molecular phylogeny of catfishes (order Siluriformes) in the Lower Mekong River Basin, based on analysis of mitochondrial DNA sequences. Thirty catfish species from nine families collected in the Lower Mekong Basin were analyzed using the 16S rRNA and COI gene regions. Phylogenetic trees were constructed using neighbor joining, Bayesian inference and maximum likelihood methods. The results showed strong support for monophyly of Siluriformes and seven of the nine studied families. However, the families Pangasiidae and Bagridae were found to be paraphyletic. Interfamilial relationships varied between analysis methods and datasets, showing inconsistencies with previous catfish phylogen
1) The study examined genetic variation between early and late feeding Atlantic salmon from the River Shin in Scotland using 6 allozyme loci and 8 microsatellite markers.
2) Early feeding fish had higher rates of smolting and fewer parr compared to late feeding fish, showing different life history strategies between the groups.
3) Early and late feeding fish showed different allelic and genotypic distributions at both allozyme and microsatellite loci, with early feeders being more heterozygous on average. Higher heterozygosity was positively associated with earlier smolting and precocious maturation in male parr.
Short Communication: Molecular identification of White Sea Squirt Didemnum sp...anbiocore
This document summarizes a study that used DNA barcoding to identify white colonial ascidian (sea squirt) colonies observed growing over corals in Raja Ampat, Indonesia. Molecular analysis of the COI gene region from 22 samples identified 11 haplotypes belonging to 4 potential cryptic Didemnum species. Phylogenetic analysis revealed 4 distinct clades with high genetic diversity within clades, suggesting the species are likely native rather than introduced. This work represents the first study to investigate these species in Raja Ampat and raises awareness of introduced species issues in this important marine biodiversity area.
This study identified 62 species of gastropods belonging to 35 families that were associated with Sargassum seaweed beds in the São Sebastião Channel region of Brazil. A total of 13,945 individual gastropods were collected, with the three most abundant families being Cerithiidae, Phasianellidae, and Columbellidae. The dominant species included Bittiolum varium, Eulithidium affine, Mitrella dichroa, Anachis fenneli, and Costoanachis sertulariarium. Juvenile specimens of many species were also found, indicating that the seaweed beds serve as a nursery for gastropod development. The high diversity and
This document describes the megalopae of Paraxanthus barbiger crab for the first time based on field-collected samples from Chile over a year. It finds high seasonal variation in size but consistent morphological characteristics. Molecular sequencing of the 16S rRNA gene from megalopae and adults confirms identification at the species level despite morphological plasticity. This validation technique and description of a dominant crab species' larvae aids population dynamics studies in the region.
Induction of tetraploidy in an ornamental fish koicarp Cyprinus carpio L, usi...researchanimalsciences
Koicarp is potentially an important cultured ornamental fish in freshwater. Moreover there were reports existing on genetic manipulation of koicarp by application of the heat shock. Hence the present study was made to contribute a protocol for induction of tetraploidy by heat shock in the koicarp.Induction of tetraploidy was attempted in Cyprinus carpio L, Koicarp by heat shock. Eggs from five females and milt from five males ok Koicarp were pooled to ensure the required quantity and quality of gametes for fertilization. After insemination the eggs were divided into three batches each experiment based on the post fertilization viz., 25min, 27min and 30min after insemination. Batches of eggs held in plastic containers were exposed to hot water at 38° C, 39° C, 40° C & 41° C for durations of 2min and four min. One batch of the eggs without heat shock treatment was used as control. After treatments, eggs were immediately transferred to incubation troughs. Tetraploidy was ascertained by karyotyping as well as RBC nuclear micro measurements.Heat shock of 41°C for four min, imparted to eggs for 20 min after fertilization induced a maximum of 60± 2% tetraploidy and maximum hatchability of 10± 1.5%. A large proportion of the heat shocked embryos displayed morphological abnormalities such as short and curved tail, destroyed yolksac, deformed vertebral column and malformed cephalic region. A maximum of 60± 2% tetraploids (4n = 156) were obtained when the fertilized eggs (20 min old) were heat shocked at 41° C for four min duration. The tetraploid red blood cells (RBCs) nucleus volume was 2.1 times greater than those of the diploid RBC nucleus.Given that koicarp are such a useful model for other areas of research, perhaps further studies on the induction of tetraploidy in this species will lead to a better understanding of polyploidy induction and the establishment of tetraploid lines of koicarp and other species as well.
Article Citation:
Ananth Kumar and Mohamed Abdul Kadher Haniffa.
Induction of Tetraploidy in an Ornamental Fish Koicarp
Cyprinus carpio L, Using Heat Shock.
Journal of Research in Animal Sciences (2012) 1(1): 013-019.
Full Text:
http://janimalsciences.com/documents/AS0006.pdf
Induction of tetraploidy in an ornamental fish koicarp Cyprinus carpio L, us...researchanimalsciences
Koicarp is potentially an important cultured ornamental fish in freshwater.
Moreover there were reports existing on genetic manipulation of koicarp by
application of the heat shock. Hence the present study was made to contribute a
protocol for induction of tetraploidy by heat shock in the koicarp.Induction of
tetraploidy was attempted in
Cyprinus carpio
L, Koicarp by heat shock. Eggs from five
females and milt from five males ok Koicarp were pooled to ensure the required
quantity and quality of gametes for fertilization. After insemination the eggs were
divided into three batches each experiment based on the post fertilization viz., 25min,
27min and 30min after insemination. Batches of eggs held in plastic containers were
exposed to hot water at 38° C, 39° C, 40° C & 41° C for durations of 2min and four min.
One batch of the eggs without heat shock treatment was used as control. After
treatments, eggs were immediately transferred to incubation troughs. Tetraploidy
was ascertained by karyotyping as well as RBC nuclear micro measurements.Heat
shock of 41°C for four min, imparted to eggs for 20 min after fertilization induced a
maximum of 60± 2% tetraploidy and maximum hatchability of 10± 1.5%. A large
proportion of the heat shocked embryos displayed morphological abnormalities such
as short and curved tail, destroyed yolksac, deformed vertebral column and
malformed cephalic region. A maximum of 60± 2% tetraploids (4n = 156) were
obtained when the fertilized eggs (20 min old) were heat shocked at 41° C for four
min duration. The tetraploid red blood cells (RBCs) nucleus volume was 2.1 times
greater than those of the diploid RBC nucleus.Given that koicarp are such a useful
model for other areas of research, perhaps further studies on the induction of
tetraploidy in this species will lead to a better understanding of polyploidy induction
and the establishment of tetraploid lines of koicarp and other species as well.
This study examines two species of photosynthetic sea slugs, Plakobranchus ocellatus and Elysia timida, that are able to retain functional chloroplasts from consumed algae for extended periods. The researchers sequenced expressed mRNA from actively photosynthesizing individuals of both species and found no evidence that nuclear genes specific to photosynthesis had been transferred from the algal food source to the slugs, despite the long-term maintenance of plastid function. This suggests the molecular basis for plastid longevity in these species does not involve lateral transfer of algal nuclear genes.
This document summarizes a study that characterized the ecdysone receptor (EcR) gene in the salmon louse (Lepeophtheirus salmonis), an economically important parasite in salmon farming. The researchers isolated and sequenced cDNA of the predicted L. salmonis EcR gene, which encoded a protein highly similar to other arthropod EcRs. In situ analysis showed the EcR transcript is expressed in ovaries, sub-cuticle, and oocytes of adult female lice. Knockdown of EcR using RNA interference terminated egg production, indicating it plays an important role in reproduction and oocyte maturation. This suggests disrupting EcR signaling may provide a way to control louse reproduction and infestation.
A sudden and mass outburst of the epitoky polychaete worm Nereis (Neanthes) virens (Sars)/ Alitta virens was observed of the surface waters of Middle Strait, Baratang, South Andaman Island during July 2014. This polychaeta worm was studied for its morphology and structural characteristics. We have taken nine consecutive seasonal samplings from July 2011 to January 2015, this was the first appearance of these worms in such a huge mass. These epitoky worms were observed in the month of July 2014 during monsoonal season in Andaman Nicobar Islands. Even though detailed studies were carried out on this worm in the world oceans, the present observation was the first report on the tropical island ecosystem of Andaman and Nicobar Islands.
This document summarizes a study that analyzed the diversity of rRNA genes in the guts of adult and fingerling Mugil cephalus (flathead grey mullet) fish inhabiting an Egyptian Mediterranean estuary. Bulk DNA was extracted from the guts and the eukaryotic 18S rRNA gene, bacterial 16S rRNA gene, and archaeal 16S rRNA gene were amplified via PCR, cloned, and sequenced. Rarefaction analyses identified 11, 18, and 13 phylotype groups of rRNA genes for eukaryotes, bacteria, and archaea, respectively, in adult guts, and 6 and 11 phylotype groups for eukaryotes and bacteria in fingerling guts (archaea were not detected in
ARTICLE IN PRESShttpwww.elsevier.deprotisPublished onl.docxfredharris32
ARTICLE IN PRESS
http://www.elsevier.de/protis
Published online date 7 August 2006
1
Correspondin
fax +81 29 853
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& 2006 Elsev
doi:10.1016/j
157, 401—419, August 2006
Protist, Vol.
ORIGINAL PAPER
Hatena arenicola gen. et sp. nov., a Katablepharid
Undergoing Probable Plastid Acquisition
Noriko Okamoto2, and Isao Inouye1
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1, Tennodai, Tsukuba,
Ibaraki 305-8572, Japan
Submitted February 27, 2006; Accepted May 27, 2006
Monitoring Editor: Robert A. Andersen
Hatena arenicola gen. et sp. nov., an enigmatic flagellate of the katablepharids, is described. It shows
ultrastructural affinities to the katablepharids, including large and small ejectisomes, cell covering,
and a feeding apparatus. Although molecular phylogenies of the 18S ribosomal DNA support its
classification into the katablepharids, the cell is characterized by a dorsiventrally compressed cell
shape and a crawling motion, both of which are unusual within this group. The most distinctive feature
of Hatena arenicola is that it harbors a Nephroselmis symbiont. This symbiosis is distinct from
previously reported cases of ongoing symbiosis in that the symbiont plastid is selectively enlarged,
while other structures such as the mitochondria, Golgi body, cytoskeleton, and endomembrane
system are degraded; the host and symbiont have developed a morphological association, i.e., the
eyespot of the symbiont is always at the cell apex of Hatena arenicola; and only one daughter cell
inherits the symbiont during cell division, resulting in a symbiont-bearing green cell and a symbiont-
lacking colorless cell. Interestingly, the colorless cells have a feeding apparatus that corresponds to
the location of the eyespot in symbiont-bearing cells, and they are able to feed on prey cells. This
indicates that the morphology of the host depends on the presence or absence of the symbiont. These
observations suggest that Hatena arenicola has a unique ‘‘half-plant, half-predator’’ life cycle; one cell
divides into an autotrophic cell possessing a symbiotic Nephroselmis species, and a symbiont-lacking
colorless cell, which later develops a feeding apparatus de novo. The evolutionary implications of
Hatena arenicola as an intermediate step in plastid acquisition are discussed in the context of other
examples of ongoing endosymbioses in dinoflagellates.
& 2006 Elsevier GmbH. All rights reserved.
Key words: Hatena arenicola; Katablepharidophyta/Kathablepharida; Nephroselmis symbiont; plant
evolution; plastid acquisition via secondary endosymbiosis; ultrastructure.
Abbreviations: EM ¼ electron microscopy; ER ¼
endoplasmic reticulum; ICBN ¼ International Code
of Botanical Nomenclature; ICZN ¼ International
Code of Zoological Nomenclature; LM ¼ light mi-
croscopy; SEM ¼ scanning electron microscopy;
SSU rDNA ¼ small subunit ribosomal DNA; TEM ¼
transmission electron microscopy.
g author;
4533
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This document describes a study that generated DNA barcodes from fish species collected at a fishing harbor in Visakhapatnam, India. DNA was extracted from tissue samples of 50 fish individuals and a 658 base pair region of the COI gene was amplified and sequenced. The sequences were analyzed using tools like ORF finder, BLAST, and multiple sequence alignment. A phylogenetic tree was constructed to investigate relationships between sequences. The goals were to create a reference barcode library for the region and investigate species identification and potential cryptic species. The study focused on analyzing barcodes of Tripletail fish, which previous work on barcoding this species is limited.
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This document summarizes a symposium presentation about the effects of saxitoxins (STXs), which are toxins produced by harmful algal blooms, on the keystone predator sea star Pisaster ochraceus. The study examined how exposure to STXs through consumption of toxic mussels affected Pisaster's feeding behavior, ability to attach to substrates, and reproductive success. The results showed that while feeding behavior was unaffected, Pisaster had lower attachment strength after STX exposure and accumulated STXs in its tissues. Incidence of fertilization also tended to decrease at high but ecologically relevant STX concentrations, suggesting STXs can negatively impact Pisaster and the intertidal community structure it helps maintain
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The status.
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Solid waste generation – collection – dumping
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Good practices:
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Genetic differentiation of Artemia feanciscana in Kenyan coastal saltworks
1. ISSN 2320-5407 International Journal of Advanced Research (2014), Volume 2, Issue 4 ,1154-1164
1154
Journal homepage: http://www.journalijar.com INTERNATIONAL JOURNAL
OF ADVANCED RESEARCH
RESEARCH ARTICLE
Genetic differentiation of Artemia franciscana (Kellogg, 1906) in Kenyan coastal saltworks
Erick Ochieng Ogello1*
, Betty M. Nyonje2
and Gilbert Van Stappen3
1.1*
Kenya Marine and Fisheries Research Institute, Kegati Aquaculture Research Station P.O. Box 3259 – 40200,
Kisii, Kenya
2.2
Kenya Marine and Fisheries Research Institute, Mombasa Research Centre P. O. Box 81651 Mombasa, Kenya
3.3
Laboratory of Aquaculture & Artemia Reference Center (ARC), Ghent University, Rozier 44, B-9000 Gent,
Belgium
.
Manuscript Info Abstract
Manuscript History:
Received: 12 February 2014
Final Accepted: 22 March 2014
Published Online: April 2014
Key words:
mtDNA, RFLP, Heat shock protein,
Kenya, Artemia franciscana
*Corresponding Author
Erick Ochieng Ogello
The nature of genetic divergence between the Artemia population native to
San Francisco Bay, (SFB) USA and those from the introductions of SFB
material in the Kenyan coast two decades ago were investigated using the
mitochondrial DNA (mtDNA) and heat shock protein 70 (Hsp70) gene
molecular markers. The DNA was extracted from 80 single Artemia cysts
using the Chelex protocol. The 1,500 bp fragment of the 12S - 16S region of
the mtDNA and a 1,935 bp fragment of the Hsp70 gene were amplified
through Polymerase Chain Reaction (PCR) followed by Restriction
Fragment Length Polymorphism (RFLP) digestion using appropriate
endonucleases. The mtDNA analysis indicated higher haplotype diversity
(0.76 ± 0.07) in Artemia from Fundisha saltworks while the rest of the
samples were monomorphic. A private haplotype (AAABBA) in Fundisha
samples confirmed a molecular evidence of a systematic genetic
differentiation albeit in an insignificant manner (P > 0.05). There was
molecular evidence of coexistence of SFB and GSL Artemia strains in
Fundisha saltworks. The monomorphic DNA fingerprint in Kensalt Artemia
cysts was probably caused by non-sequential Artemia culture system and
limited mtDNA fragment size analysed. The Hsp70 gene RFLP fingerprint
did not show any unique gene signatures in the Kenyan Artemia samples
suggesting that other factors other than Hsp70 were involved in their superior
thermotolerance. Further genetical studies based on the larger mtDNA
fragment using robust genetic markers are recommended. Ecological studies
of the heat shock protein family and the stress response would be more
relevant than the qualitative RFLP technique.
Copy Right, IJAR, 2014,. All rights reserved
Introduction
The brine shrimps Artemia are small crustaceans adapted to live in stressful environmental conditions of hypersaline
habitats such as salt lakes, coastal lagoons and solar saltworks, where they feed primarily on phytoplankton and
bacteria (Persone and Sorgeloos, 1980; Toi et al., 2013). Being osmotolerant animals, Artemia can withstand
habitats whose salinity levels range between 10 - 340 g L-1
with fluctuating ionic composition and temperature
profiles (Van Stappen, 2002). Artemia adaptation to these conditions has occurred at molecular, cellular,
physiological and population level making Artemia fit to survive and reproduce effectively in such insulting
environments (Gajardo and Beardmore, 2012). Artemia has high genetic variability (Kappas et al., 2004) that makes
them model animals for studying evolutionary processes such as genetic differentiation, which indeed, is the focus
of this paper. The most discussed reproductive adaptation mechanism of the genus Artemia is the existence of two
distinctly short cycles of development (Clegg et al., 2004; Kappas et al., 2004). During favourable environmental
2. ISSN 2320-5407 International Journal of Advanced Research (2014), Volume 2, Issue 4 ,1137-1143
1155
conditions, an ovoviviparous reproduction cycle occurs where the adult females produce the free swimming naupli
(Anderson et al., 1970). However, during stressing environmental conditions, an oviparous reproductive cycle
prevails and the adult female Artemia produces metabolically inactive cysts as the parental animals dies (Dutrieu,
1960; Van Stappen, 1996). When conducive environmental conditions return, the cysts hatch into free swimming
nauplii in a process that lasts for about 20 hours, thus completing the cycle (Pearson and Sorgeloos, 1980; Van
Stappen, 1996).
The ability of the brine shrimp Artemia to inhabit hypersaline environments gives them a wide global geographical
representation (Persoone and Sorgeloos, 1980). In fact, as Triantaphyllidis et al. (1998) put it; the only place where
Artemia cannot be found is Antarctica. So far, discrete Artemia populations have been identified in about 600
natural salt lakes and saltworks and further survey efforts are still on course to identify more Artemia biotopes all
over the world (Van Stappen, 2002).
For a long time, Artemia morphometric features have been used to discriminate between different populations
despite many human errors (Naceur et al., 2010). Today, Artemia phylogeny can be easily verified and cyst samples
scientifically authenticated thanks to molecular techniques (Bossier et al., 2004; Van Stappen, 2008). According to
Avise (2004), molecular techniques provide full access to unlimited pool of organism’s genetic variability. The
extensive study of inter- and intra-specific diversity of Artemia has been made possible due to a variety of nuclear
and mitochondrial DNA markers for instance ITS-1, Hsp26, COI, 12S and 16S mtDNA (Perez et al., 1994; Hou et
al., 2006) and tools such as Restriction Fragment Length Polymorphism (RFLP) (Bossier et al., 2004; Gajardo et al.,
2004; Eimanifar et al., 2006), Random Amplified Polymorphic DNA (RAPD) (Sun et al.,1999a; Camargo et al.,
2002), Amplified Fragment Length Polymorphism (AFLP) (Sun et al., 1999b) using either single or pooled
individuals or cysts samples (Kappas et al., 2004). Other tools include microsatellites and Single Strand
Conformation Polymorphism (SSCP) (Blouin et al., 1996).
In the RFLP molecular technique, the targeted DNA genome size is first PCR amplified before digestion with
restriction enzymes. The digested product is then separated according to their size by agaorose gel electrophoresis
(Eimanifar et al., 2006). The mitochondrial genome of A. franciscana is estimated to be 15,822 nucleotides long
(Valverde et al., 1994). The mtDNA is highly conserved compared to nuclear DNA, making it a robust marker for
tracking animals’ ancestry (Krieg et al., 2000). Eimanifar et al. (2006) found genetic nucleotide divergence within
Artemia populations found in different ecological zones of Lake Urmia using the RFLP method. Agh et al. (2009)
showed that bisexual A. urmiana and parthenogenetic populations in Iran are genetically close based on RFLP of
their 1,500 bp mtDNA fragment. Manaffar (2012) also conducted an RFLP analysis of the 1,500 bp mtDNA
fragment on A. urmiana cysts and detected high polymorphism among cysts from different stations in Urmia Lake.
Through RFLP analysis of a 1,500 bp mitochondrial rDNA fragment, Bossier et al. (2004) developed a methodology
to authenticate Artemia cyst samples. Kappas et al. (2004) investigated how A. franciscana native to SFB colonised
unfamiliar Vietnam environments through RFLP technique based on the 2,963 bp long mtDNA target sequence.
Unique genetic signatures were observed in the mtDNA genome of the Vietnam Artemia strain suggesting a process
of strong selective pressure in them (Kappas et al., 2004).
There is much information regarding Artemia’s ability to synthesize heat shock proteins, such as Hsp26 and Hsp70
(Clegg et al., 2001; Crack et al., 2002; Willsie and Clegg, 2002). Artemia cysts contain substantial amounts of heat
shock proteins because they are the surviving agents in stressful environments (Clegg et al., 1999; Van Stappen,
2002). Scientific evidence has proven that the family of heat shock proteins are critical for thermal resistance
(Frankenberg et al., 2000), desiccation tolerance (Ma et al., 2005) and reduces osmotic stress (DuBeau et al., 1998;
Todgham et al., 2005). Therefore Hsp70 protects organisms against multidimensional environmental challenges.
Clegg et al. (2001) found that Artemia cysts produced in hotter environments contain higher amounts of heat shock
proteins such as artemin, p26 and Hsp70. Therefore, the stress proteins could be involved in the adaptation of A.
franciscana from SFB growing in the much hotter environments such as salt ponds in Vietnam (Clegg et al., 2001)
and probably Kenyan coastal areas.
Between 1984 and 1986, a non-native A. franciscana was introduced along the Kenyan coast (Fundisha and Kurawa
salt farms). Today, the A. franciscana has permanently colonised the Kenyan coast, where about eight saltworks
exist today. Since 2009, Fundisha saltworks has been re-inoculated using GSL Artemia strains suggesting
coexistence of GSL and SFB Artemia strains. This is a subject that can only be revealed through molecular studies.
The laboratory culture experiments of Mremi (2011) and Kapinga (2012) showed that Kenyan Artemia are superior
to their original SFB inoculants in terms of reproductivity and thermotolerance at elevated temperatures. However,
no information is available on their genetic architecture to support these phenotypic characteristics. To date, it is not
known whether SFB and GSL Artemia strains coexist in Fundisha saltworks. Neither do we know the genetic micro-
evolutionary divergences that have occurred in the Kenyan Artemia population. Artemia population have also been
discovered in Tanga region (Tanzania). However, no scientific information is available about them. The
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polymorphic analysis of the 1,935 bpHsp70 gene was considered to add more perspective on the much anticipated
genetic adaptation levels of the Kenyan Artemia populations. The hypothesis of this study was that the genetic
pattern of the Kenyan Artemia strains would be mutually polymorphic. The present laboratory based study aimed to
genetically characterize the Kenyan Artemia cysts based on the mitochondrial DNA and heat shock protein 70
(Hsp70) genes. The study also determined the purity of Artemia populations in Kensalt and Fundisha saltworks and
established the genetic relationships between the Kenyan and Tanga (Tanzania) Artemia cysts.
Materials and methods
Source of Artemia cyst samples and study area
A total of 80 individual Artemia cysts, 10 replicates from each of 8 samples were used in the study. The 8 samples
included 4 samples (Fundisha, Ken1, Ken2 and Ken3) harvested between 1996 and 2012 from selected salt farms at
the Kenyan coast, located at located at 3° 50' 0" South, 39° 46' 0" East (Study area Fig. 1). One sample from Great
Salt Lake, Utah state in USA (GSL), another sample from San Francisco Bay (SFB) USA and one sample from Vinh
Chau (VC), Vietnam, were used as controls. Artemia samples from Tanga (Tanzania) were also analysed as
additional study. All the Artemia cyst samples were available at the Laboratory of Aquaculture & Artemia
Reference Center (ARC), Ghent University, Belgium where they were stored at 4o
C in the cyst bank. The selection
criteria for the Kenyan Artemia cyst followed Nyonje (2011) report. Cysts from Kensalt farm (Ken1, Ken2, Ken3),
which were different batches of the same population, were considered because of their known reproductive
characteristics (Mremi, 2011; Kapinga 2012) while the much hypothesised coexistence of SFB and GSL Artemia
strains in Fundisha saltwork was the reason for considering cysts from there. The use of cysts for DNA extraction
for mtDNA and Hsp70 analysis were preferred to individual Artemia adults to prevent loss of genetic information
due to selective hatching (Van stappen 2008).
DNA extraction
DNA was extracted from single Artemia cysts using the Chelex method (Walsh et al., 1991). The cyst was isolated
using a sterile 10 μL pipette point and transferred to a sterile eppendorf (1.7 mL) where 30 μL of milliQ water (PCR
water) was added and left to hydrate for 1 hr. In each eppendorf tube, the cyst was crushed using a sterile pellet
pestle (Sigma-Aldrich Z35997-1EA) before adding 30 μL of well homogenized 10% Chelex slurry (Chelex-100 -
Biorad, Belgium). The samples were vortexed for 10 - 15 s before spinning for 1 min at 13,000 rpm in a micro-
centrifuge. The samples were incubated for 20 min at 95o
C, vortexed again for 10 - 15 s and further spanned at
13,000 rpm for 1 min. The quantity of the extracted DNA was measured using a NanoDrop® ND-1000 machine
while the quality of the DNA was verified through agarose gel electrophoresis (Lind et al., 2006).
PCR amplification of the 1,500 bp 12S - 16S mtDNA fragment
The double stranded DNA amplification was performed in 50 µL reaction volumes each containing a mixture of
36.375 µL PCR water, 5 µL 10 x Taq buffer + KCl-MgCl2, 5 µL MgCl2 (25 mM solution), 1 µL dNTP (10 mM
each), 1 µL of primer 1 and 2 (work-solution), 0.125 µL BSA, 0.5 µL Taq-polymerase and 2 µL of approximately 5
- 30 ng of template DNA extract, except for the negative control tube. The DNA samples were PCR amplified for
the 1,500 bp mtDNA fragment between the 12S - 16S region (Valverde et al., 1994). A combination of the forward
primer 12S - SP (5’-cta-gga-tta-gat-acc-cta-3’), and the reverse primer 16S - SP (5’- ccg-gtc-tga-act-cag-atc-3’) was
used according to Bossier et al. (2004). The BioRad PCR equipment was programmed such that the first cycle of
PCR reaction heated the mixture to 94o
C for 2 minutes to activate the Taq polymerase enzyme. This was followed
by 34 cycles of: 1) a denaturing step at 94o
C for 1 min; 2) an annealing phase at 52o
C for 45 seconds; 3) an
elongation phase at 72o
C for 2 min and the final elongation cycle step at 72o
C for 4 minutes.
PCR amplification of the Hsp70 gene
The PCR reaction mixture of 25 µL contained 16.32 µL PCR water, 4.8 µL 10 x GoTaq buffer + KCl-MgCl2, 1.2 µL
MgCl2 (25 mM solution), 0.48 µL dNTP (10 mM each), 0.48 µL of primer 1 and 2 (work-solution), 0.24 µL GoTaq-
polymerase and 1 µL of approximately 150 ng of template DNA extract except for the negative control tube. The
DNA samples were assayed for PCR amplification of the 1,935 bp Hsp 70 gene fragment (Baruah et al., 2010). A
combination of the forward primer Hsp70forward (5’-cac-cat-ggc-aaa-ggc-acc-agc-aat-agg-3’) and the reverse primer
Hsp70reverse (5’-ata-gtt-ggg-cca-ctg-cct-gtt-cca-g-3’) were used (Baruah et al., 2010). The PCR conditions were
modified from Baruah et al. (2010) as follows: denaturation step at 94o
C for 5 min followed by 35 cycles of 95o
C for
1 min, annealing at 63o
C for 1 min and elongation at 72o
C for 4 min followed by a final extension step for 10 min at
72o
C.
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Restriction digestion: RFLP procedure
The amplified 1,500 bp mtDNA fragments were screened for polymorphism using six restriction endonucleases
(AluI, HaeIII, HinfI, RsaI, XbaI and HpaII) (Bossier et al., 2004; Kappas et al., 2004). The reactions were done
according the manufacturer’s instructions (see Tab. 1). For each reaction tube, a total reaction volume of 23.5 µL
consisted of 16 µL PCR water, 2 µL Tango buffer, 0.5 µL of enzyme and 5 µL of PCR amplified DNA product.
Digested products were electrophoretically separated on 2 % agarose gel in a 1 X TAE buffer solution and stained
with 1 µL of GelRed. A voltage of 100 V was used to push the digested DNA fragments through the solidified
agarose gel for 1 h. A 100 bp promega DNA ladder was loaded as reference. A UV transilluminator was used to
visualise the fragments and photographed with a digital camera (Canon power shot G10).
For the Hsp 70 gene, restriction enzymes were selected based on the number of cleavage sites in the 1,935 bp
fragment of the Artemia franciscana nucleotide sequence (cDNA). Four restriction enzymes (Sau3A, Rsal, AluI and
HinfI) with recognition sequences GATC, GTAC, AGCT and GAATC respectively were used. In each reaction
tube, a total reaction volume of 23.5 µL contained 16 µL PCR water, 2 µL Tango buffer, 0.5 µL of enzyme and 5
µL of PCR amplified DNA product. The incubation temperature was 37o
C while inactivation temperature was 65o
C
for 20 min for Sau3A enzyme. Gel-electrophoresis was as explained above but 1kb promega DNA ladder was
loaded as reference. The homologies of fragment patterns were established through side by side visual comparisons
for both mtDNA and Hsp70 gene.
Data analysis
The RFLP restriction pattern fragments were manually scored. Fragments less than 100 bp were neglected because
of technical inconsistencies. Unique endonuclease restriction patterns were identified by using specific letters. Each
cyst replicate was assigned a multi-letter code that described its composite mtDNA genotype haplotype. For each
sample, the haplotype frequency (hf) was manually calculated by counting the identical haplotypes and dividing by
the total replicates per sample (Nei, 1978). The mean haplotype frequency was calculated by adding all the
haplotype frequency in each haplotype then dividing by the total number of samples (Nei, 1987). The haplotype
diversity within samples was calculated based on Nei and Tajima’s (1981) formula.
Where: H = haplotype diversity; N = Sample size; x = haplotype frequency
The non-parametric Wilcoxon signed rank one sample t-test of SPLUS (Sportifire 2 + 8.2) statistical programme
was used to test significant difference among the sample’s haplotype frequencies at P = 0.05 level of significance.
The cluster dendrogram for samples was drawn using PyElph 1.4 software (Pavel and Vasile, 2012) based on the
unweighted average pair group method (UPGMA). For the RFLP of the Hsp70 gene, the sizes of the fragments were
only estimated by comparison with a 1kb ladder. No further data processing was done whatsoever for the Hsp70
RFLP marker.
Results
Based on A 260 / 280 index, some samples showed high quality DNA (Tanga, SFB, GSL and VC) while others had low
quality (Fundisha, Ken1, Ken2 and Ken3) (Table 2). Values of A 260 / 280 indexes between 1.7 and 2.0 indicate the
presence of pure DNA (Glasel, 1995).
H =
N
N - 1
1 - ∑ x2
∑∑
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Fig. 1: Study area - map of the Kenyan coast showing the location of the salt belt and a more detailed
impression of the salt belt showing the individual salt producing companies in a North – South Orientation.
Fundisha saltwork is also called Crystalline
Table 1: The list and recognition sequences of the restriction enzymes used in the study including incubation
and activation temperatures as described by the manufacturer; N = C, G, T or A.
Enzyme Recognition sequence Incubation temperature Inactivation temperature
AluI 5’...A G C T...3’
3’...T C G A...5’
37O
C 65O
C / 20minutes
HaeIII 5’...G G C C...3’
3’...C C G G...5’
37O
C 80O
C / 20minutes
HinfI 5’...G A N T C...3’
3’...C T N A G...5’
37O
C 65O
C / 20minutes
RsalI 5’...G T A C...3’
3’...C A T G...5’
37O
C 80O
C / 20minutes
Xbal
HpaII
5’...T C T A G A...3’
3’...A G A T C T...5’
5’...C C G G...3’
3’...G G C C...5’
37O
C
37O
C
65O
C / 20minutes
80O
C / 20minutes
Indian Ocean
Fundisha/crystalline
Saltwork: 1,020 ha
Kensalt: 3,000 ha
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Table 2: Average quantity of DNA extracted from individual cysts from each sample including Artemia
Reference Centre (ARC) code. The DNA quantity was measured using a NanoDrop® ND-1000 machine.
Values are mean ± SE.
Sample ARC code DNA (ng /µL) A 260 / 280
Fundisha 1780 9.55 ± 0.69 2.12 ± 0.03
Ken1 1762 15.01 ± 0.43 1.59 ± 0.12
Ken2 1439 7.51 ± 0.45 3.04 ± 0.32
Ken3 1779 7.53 ± 0.63 2.99 ± 0.22
Tanga 1773 14.38 ± 0.70 1.99 ± 0.10
GSL 1768 27.99 ± 1.95 1.87 ± 0.02
SFB 1574 37.25 ± 1.26 1.92 ± 0.01
VC 1771 28.80 ± 1.75 2.01 ± 0.03
PCR amplification of the 1,500 bp 12S -16S mtDNA fragment
The primer combinations produced identical 1,500 bp fragments in all the 10 replicates in every sample analysed.
Only one replicate per sample is shown (Fig. 2)
Fig. 2: Example of agarose gel for PCR-amplified 1500 bp 12S – 16S mtDNA fragment for a single cyst per
sample. L: 1500 bp ladder; NC: Negative control.
RFLP analysis of the mtDNA
The enzymes HaeIII and HpaII detected polymorphism only in the Fundisha sample (Fig. 3). The enzymes AluI,
XbaI, HinfI and RsaI were monomorphic across all samples (results not shown). In total, approximately 1,216
fragments were surveyed in the 1,500 bp 12S - 16S mtDNA target sequence. A total of three composite haplotypes
were identified in the mtDNA target sequence. All the three haplotypes were present in Fundisha Artemia samples
while the rest of the samples were monomorphic (Table 3). The most common haplotype was AAAAAA, being
detected in all the sample populations except GSL. This haplotype attained the highest frequency (0.4286) within
Fundisha samples. A private haplotype (AAABBA) was discovered in Fundisha sample while the haplotype
(AAAABA) was only shared between Fundisha and GSL (Table 3). The highest haplotype diversity (h) was
recorded in Fundisha cyst samples (0.76 ± 0.07).
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Figure 3: Example of agarose restriction fragment profile for the polymorphic HaeIII and HpaII enzymes on
Fundisha individual cyst samples. PCR-amplified 1500 bp of 12S – 16S mtDNA fragment for 7 single cyst
replicates per sample.
Table 3: Haplotype genotype frequencies, mean haplotype frequency (mhf), sample size, number of
haplotypes (nh) and haplotype diversity (h) in samples. Haplotype genotypes are denoted with capital letters,
each one corresponding to the restriction pattern obtained by a restriction enzyme in the following order;
AluI, Xbal, HinfI, HpaII, HaeIII and RsaI
The dendogram showed two major groups (GSL and SFB) while the Fundisha cyst samples appeared to be
intermediate (Fig.4).
Haplotype Haplotype
genotype
Samples
Fundisha Ken1 Ken2 Ken3 Tanga GSL SFB VC mhf
H1 AAAAAA 0.4286 1.0000 1.0000 1.0000 1.0000 0 1.0000 1.0000 0.80
H2 AAAABA 0.2857 0 0 0 0 1.0000 0 0 0.16
H3 AAABBA 0.2857 0 0 0 0 0 0 0 0.04
S. size 7 7 7 7 7 7 7 7
nh 3 1 1 1 1 1 1 1
h ± SD 0.76 ± 0.07 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Hae
III
Hpa
II
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Figur 4: UPGMA dendrogram of Nei's genetic distance for 8 Artemia franciscana population samples. The
values on the horizontal lines stand for Neis genetic distances in percentage.
Molecular analysis of the Hsp70 gene fragment
The 1,935 bp Hsp70 gene fragment produced a non-polymorphic pattern in all the enzymes. Only the RFLP pattern
of enzyme Rsal is shown (Fig. 5). Interestingly, even restriction enzymes such as AluI and Sau3A with 7 and 6
cleavage sites respectively on the 1,935 bp Hsp70 gene fragment did not show any polymorphism in any of the
samples.
Figure 5: Agarose restriction fragment profile for the enzymes: Sau3A, AluI, HinfI and Rsal. The PCR
fragment was generated using DNA extracted from pooled Artemia cysts. L: 1Kb ladder, Lanes 1: Fundisha,
2: Ken1, 3: Ken2, 4: Ken3, 5: Tanga, 6: GSL, 7: SFB, 8: VC, 9: Undigested PCR product (2, 000 bp) control.
Discussion
The present study analysed mitochondrial DNA using the RFLP tool to detect polymorphism in the Kenyan A.
franciscana using DNA extracted from individual cysts. The monomorhic DNA fingerprints corresponded with zero
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genetic distance as shown by the UPGMA dendrogram (Fig. 4), indicating lack of genetic differentiation between
and among the Artemia samples. Lack of genetic diversity is risky in times of genetic bottleneck as the entire
population may perish. Ecological processes such as migration can cause high Artemia population heterogeneity in
the habitat but limited effective gene flow is observed (Hajirostamloo, 2009) because effective gene flow is much
slower compared to the process of dispersal (Naihong et al., 2000). The process of assortative mating can prevent
intercrossing even among coexisting Artemia species to reduce chances of speciation (Beristain et al., 2010). The
absence of genetic polymorphism within samples could have been due to the limited 1,500 bp fragment of mtDNA
analysed. Kappas et al. (2004) used a larger mtDNA fragment of 2,973 bp and detected significant genetic
polymorphism within the A. franciscana introduced in Vietnam almost 2 decades ago. Since Kenya and Vietnam
share similar Artemia inoculation history, one would have expected similar genetic evolutions. However, a larger
fragment has high chances of showing detailed microevolutionary changes that might not be detected in a limited
DNA fragment.
The environmental conditions are critical factors that may influence the Artemia population patterns and genetic
expressions (Evjemo and Olsen, 1999; Van Stappen, 2002). In Kenya, integrated salt - Artemia culture is a
continuous process where Artemia flourishes year round. In Vietnam, the saltworks are predictably sequential and
this favoured a faster evolution of VC Artemia strain (Kappas et al., 2004). This conforms to Manaffar’s (2012)
observation that genetic drift in the presence of limited gene flow facilitates the speciation process. Therefore, the
absence of periodical genetic bottlenecks in the Kenyan situation suggests that only natural selection process is
responsible for gene loss. Natural selection requires long time to cause meaningful genetic divergence (Gajardo and
Beardmore, 2012). Permanence and seasonality of the environment are key instruments driving considerable genetic
differentiation of Artemia leading to specific biota with definite genetic structures (Lenz, 1987). However, the
exclusive ovoviviparity of the New Zealand A. franciscana population (inoculated in 1950s) was due to genetic
differentiation caused by constant year-round salinity and temperature conditions (Wear and Haslett, 1986). The
mutations caused by high UV radiation have also been linked to genetic evolutionary changes in Artemia
populations (Hebert et al., 2002).
The current study might not have sufficiently assessed the samples intra-population diversity due to limited the
RFLP technique, which is inferior to detect intra-specific polymorphism (Bossier et al., 2004; Avise, 2004). The fact
that the single haplotype identified in Tanga samples was similar to Kensalt and Fundisha samples suggested they
are genetically close. This provides evidence that Artemia in tanga region was introduced by Kensalt management
who own saltworks there.
Based on the RFLP fingerprint pattern and the number of haplotype genotypes obtained in this study, only the
Artemia population in Fundisha saltworks was polymorphic albeit in an insignificant manner (P > 0.05). Therefore,
if indeed there was significant genetic differentiation between Kenyan (Kensalt) Artemia and their SFB ancestors,
then the tool used was not sufficiently adequate to detect this micro-evolutionary divergence. Nevertheless, the
private haplotype (AAABBA) in Fundisha cyst samples suggested a systematic genetic differentiation thus
molecular evidence of an existing subpopulation and genetic divergence from their SFB ancestors. The population-
specific haplotype identified in Fundisha saltwork may become useful in monitoring the geographic expansion of the
Artemia populations along the Kenyan coast. However, further studies using superior genetic tools like AFLP and
microsatellites are needed to authenticate this finding. There was molecular evidence of co-existence of both SFB
and GSL Artemia strains in Fundisha saltwork, conforming to Nyonje (2011) report. This finding is consistent with
the studies of Van Stappen (2002), who documented that coexistence of different Artemia strains or species within
the same site is a common scientific possibility.
The lack of genetic variation in the Hsp70 RFLP fingerprint pattern suggested that the samples analysed had the
same Hsp70 gene structure. Feder and Hofmann (1999) reported that little variation in the Hsp70 gene could be due
to the fact that it is evolutionary and functionally conserved. Based on Kapinga (2012) and Mremi (2011) findings, it
was hypothesised that the Kenyan Artemia posses unique Hsp70 gene signatures. Having rejected this hypothesis, it
means that factors other than the Hsp70 gene are responsible for the observed adaptations (thermotolerance). Future
studies should focus on more quantitative Hsp70 analysis such as western blot by chemiluminescence techniques
(Schutz-Geschwender et al., 2004).
Conclusions and recommendations
The mtDNA sequence analysis has provided some diagnostic power in comparing SFB, GSL and Kenyan Artemia
strains. Even though the genetic differentiation of the Kenyan Fundisha Artemia population from its SFB ancestors
is not statistically significant, the presence of a private haplotype genotype in Fundisha saltwork could be the
beginning of a long term micro-evolutionary process, which could lead to eventual geographic differentiation and
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progressive speciation of A. franciscana in the Kenyan environment. It may also help to explore and monitor future
expansion of the Artemia population. The Kenyan Kensalt Artemia population is not contaminated by other Artemia
strains while there is co-existence of SFB and GSL Artemia strains in Fundisha saltworks. Other factors other than
the Hsp70 family could be involved in the much cited thermotolerance superiority of the Kenyan Artemia
populations. More robust molecular markers targeting larger mtDNA fragment should be considered concurrently
with Hsp70 quantitative technique.
Acknowledgement
This study was funded by the Flemish Interuniversity Council, the Vlaamse Interuniversitaire Raad and the
University Development Cooperation (VLIR-UOS) through a joint project bringing together Kenya Marine &
Fisheries Research Institute (KMFRI) and Gent University, Laboratory of Aquaculture & Artemia Reference Center
(ARC).
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