Cágado-de-barbelas (Phrynops geoffroanus (Schweigger, 1812), Testudines: Chelidae) como modelo para ecotoxicologia evolutiva: relacionamento entre contaminação ambiental, condição e variabilidade genética

Pós-graduação em Genética - IBILCE

Geoffroy's side-necked turtle [Phrynops geoffroanus (Schweigger, 1812), Testudines: Chelidae] as a model for evolutionary ecotoxicology: relationship between environmental contamination, conditions and genetic variability

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Feeding ecology, density and biomass of the freshwater turtle, Phrynops geoffroanus, inhabiting a polluted urban river in south-eastern Brazil

This study examines the ecology of a population of Geoffroy's side-necked turtle Phrynops geoffroanus inhabiting a polluted urban river in Ribeirão Preto city, São Paulo state, south-eastern Brazil. Adult turtles fed mainly on Chironomidae larvae and pupae (Chironomus cf. plumosus, 100% of occurrence frequency) and domestic waste, but they also consumed terrestrial items (cockroach, snails) and carrion. Juvenile turtles showed more feeding diversity than the adults and exhibited a trend for predation on Chironomidae pupae, but this is not reflected in resource partitioning. The elevated number of turtles (170-230 turtles/ha of river) and biomass (255-345 kg/ha of river) inhabiting this urban river is probably the result of the abundance of sewage and organic waste produced by humans, the absence of predators, and increased availability of nesting areas. Such factors convert this area into an environment highly advantageous for the survival of Geoffroy's side-necked turtle.

Hemoglobin polymorphism and hematological profile of Geoffroy's side-necked turtle (Phrynops geoffroanus, Testudines) in the northwestern region of São Paulo State, Brazil

Complete blood counts and hemoglobin isoform data were gathered from 36 specimens of the turtle species Phrynops geoffroanus from the northwestern region of São Paulo State, Brazil. They were collected in an urban area. The hemoglobin profiles were obtained after red blood cell lysis and by electrophoretic migration in alkaline pH, acid pH, and neutral pH buffer. The hemoglobin components were confirmed using high-performance liquid chromatography (HPLC). Erythrogram analysis included hematocrit, total hemoglobin concentration, total red blood cell count, and red blood cell indices. The leukogram included a total white blood cell count and a calculation of the percent values of neutrophils, lymphocytes, monocytes, basophils, eosinophils, heterophils, and azurophils. HPLC analysis revealed three hemoglobin components; the first with a concentration of 5.5%, the second was a major component with an average concentration of 67.1%, and the third with a concentration of 28.5%. The hematological profile obtained for these specimens allowed us to establish a pattern for P. geoffroanus in São Paulo State Northwestern region. The average hematocrit values were 22.5% for females and 24.0% for males. For total hemoglobin, we found average values of 6.66 g/dL in females and 7.22 g/dL in males. The number of white blood cells was 2725 x 103/μL for females and 2775 x 103/μL for males. There was a predominance of heterophils, eosinophils, and monocytes in both sexes. No significant differences were found between males and females for hematological profile. The hematological results were compared to literature data for other Chelonia. They were similar to what is known for fresh water turtles. © FUNPEC-RP.

Influence of historical landscapes, drainage evolution and ecological traits on patterns of genetic diversity in Southeast Asian freshwater snakehead fishes

Snakehead fishes in the family Channidae are obligate freshwater fishes represented by two extant genera, the African Parachannna and the Asian Channa. These species prefer still or slow flowing water bodies, where they are top predators that exercise high levels of parental care, have the ability to breathe air, can tolerate poor water quality, and interestingly, can aestivate or traverse terrestrial habitat in response to seasonal changes in freshwater habitat availability. These attributes suggest that snakehead fishes may possess high dispersal potential, irrespective of the terrestrial barriers that would otherwise constrain the distribution of most freshwater fishes. A number of biogeographical hypotheses have been developed to account for the modern distributions of snakehead fishes across two continents, including ancient vicariance during Gondwanan break-up, or recent colonisation tracking the formation of suitable climatic conditions. Taxonomic uncertainty also surrounds some members of the Channa genus, as geographical distributions for some taxa across southern and Southeast (SE) Asia are very large, and in one case is highly disjunct. The current study adopted a molecular genetics approach to gain an understanding of the evolution of this group of fishes, and in particular how the phylogeography of two Asian species may have been influenced by contemporary versus historical levels of dispersal and vicariance. First, a molecular phylogeny was constructed based on multiple DNA loci and calibrated with fossil evidence to provide a dated chronology of divergence events among extant species, and also within species with widespread geographical distributions. The data provide strong evidence that trans-continental distribution of the Channidae arose as a result of dispersal out of Asia and into Africa in the mid–Eocene. Among Asian Channa, deep divergence among lineages indicates that the Oligocene-Miocene boundary was a time of significant species radiation, potentially associated with historical changes in climate and drainage geomorphology. Mid-Miocene divergence among lineages suggests that a taxonomic revision is warranted for two taxa. Deep intra-specific divergence (~8Mya) was also detected between C. striata lineages that occur sympatrically in the Mekong River Basin. The study then examined the phylogeography and population structure of two major taxa, Channa striata (the chevron snakehead) and the C. micropeltes (the giant snakehead), across SE Asia. Species specific microsatellite loci were developed and used in addition to a mitochondrial DNA marker (Cyt b) to screen neutral genetic variation within and among wild populations. C. striata individuals were sampled across SE Asia (n=988), with the major focus being the Mekong Basin, which is the largest drainage basin in the region. The distributions of two divergent lineages were identified and admixture analysis showed that where they co-occur they are interbreeding, indicating that after long periods of evolution in isolation, divergence has not resulted in reproductive isolation. One lineage is predominantly confined to upland areas of northern Lao PDR to the north of the Khorat Plateau, while the other, which is more closely related to individuals from southern India, has a widespread distribution across mainland SE Asian and Sumatra. The phylogeographical pattern recovered is associated with past river networks, and high diversity and divergence among all populations sampled reveal that contemporary dispersal is very low for this taxon, even where populations occur in contiguous freshwater habitats. C. micropeltes (n=280) were also sampled from across the Mekong River Basin, focusing on the lower basin where it constitutes an important wild fishery resource. In comparison with C. striata, allelic diversity and genetic divergence among populations were extremely low, suggesting very recent colonisation of the greater Mekong region. Populations were significantly structured into at least three discrete populations in the lower Mekong. Results of this study have implications for establishing effective conservation plans for managing both species, that represent economically important wild fishery resources for the region. For C. micropeltes, it is likely that a single fisheries stock in the Tonle Sap Great Lake is being exploited by multiple fisheries operations, and future management initiatives for this species in this region will need to account for this. For C. striata, conservation of natural levels of genetic variation will require management initiatives designed to promote population persistence at very localised spatial scales, as the high level of population structuring uncovered for this species indicates that significant unique diversity is present at this fine spatial scale.

Genetic Diversity of Rice Tungro Spherical Virus in Tungro-Endemic Provinces of the Philippines and Indonesia

The two adjacent genes of coat protein 1 and 2 of rice tungro spherical virus (RTSV) were amplified from total RNA extracts of serologically indistinguishable field isolates from the Philippines and Indonesia, using reverse transcriptase polymerase chain reaction (RT-PCR). Digestion with HindIII and BstYI restriction endonucleases differentiated the amplified DNA products into eight distinct coat protein genotypes. These genotypes were then used as indicators of virus diversity in the field. Inter- and intra-site diversities were determined over three cropping seasons. At each of the sites surveyed, one or two main genotypes prevailed together with other related minor or mixed genotypes that did not replace the main genotype over the sampling time. The cluster of genotypes found at the Philippines sites was significantly different from the one at the Indonesia sites, suggesting geographic isolation for virus populations. Phylogenetic studies based on the nucleotide sequences of 38 selected isolates confirm the spatial distribution of RTSV virus populations but show that gene flow may occur between populations. Under the present conditions, rice varieties do not seem to exert selective pressure on the virus populations. Based on the selective constraints in the coat protein amino acid sequences and the virus genetic composition per site, a negative selection model followed by random-sampling events due to vector transmissions is proposed to explain the inter-site diversity observed

Inter-and Intra-Site Genetic Diversity of Natural Field Populations of Rice Tungro Bacilliform Virus in the Philippines

The genetic structure of rice tungro bacilliform virus (RTBV) populations within and between growing sites was analyzed in a collection of natural field isolates from different rice varieties grown in eight tungro-endemic sites of the Philippines. Total DNA extracts from 345 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV, a procedure shown in preliminary experiments capable of revealing high levels of polymorphism in RTBV field isolates. In the total population, 17 distinct EcoRV-based genome profiles (genotypes) were identified and used as indicators for virus diversity. Distinct sets of genotypes occurred in Isabela and North Cotabato provinces suggesting a geographic isolation of virus populations. However, among the sites in each province, there were few significant differences in the genotype compositions of virus populations. The number of genotypes detected at a site varied from two to nine with a few genotypes dominating. In general the isolates at a site persisted from season to season indicating a genetic stability for the local virus population. Over the sampling time, IRRI rice varieties, which have green leafhopper resistance genes, supported similar virus populations to those supported by other varieties, indicating that the variety of the host exerted no apparent selection pressures. Insect transmission experiments on selected RTBV field isolates showed that dramatic shifts in genotype and phenotype distributions can occur in response to host /environmental shifts.

Assessing genetic diversity in cultured aquatic species : the Sydney Rock Oyster (Saccostrea glomerata) stock improvement program as a model

Genetic variation is the resource animal breeders exploit in stock improvement programs. Both the process of selection and husbandry practices employed in aquaculture will erode genetic variation levels overtime, hence the critical resource can be lost and this may compromise future genetic gains in breeding programs. The amount of genetic variation in five lines of Sydney Rock Oyster (SRO) that had been selected for QX (Queensland unknown) disease resistance were examined and compared with that in a wild reference population using seven specific SRO microsatellite loci. The five selected lines had significantly lower levels of genetic diversity than did the wild reference population with allelic diversity declining approximately 80%, but impacts on heterozygosity per locus were less severe. Significant deficiencies in heterozygotes were detected at six of the seven loci in both mass selected lines and the wild reference population. Against this trend however, a significant excess of heterozygotes was recorded at three loci Sgo9, Sgo14 and Sgo21 in three QX disease resistant lines (#2, #5 and #13). All populations were significantly genetic differentiated from each other based on pairwise FST values. A neighbour joining tree based on DA genetic distances showed a clear separation between all culture and wild populations. Results of this study show clearly, that the impacts of the stock improvement program for SRO has significantly eroded natural levels of genetic variation in the culture lines. This could compromise long-term genetic gains and affect sustainability of the SRO breeding program over the long-term.

Comparison of koala LPCoLN and human strains of Chlamydia pneumoniae highlights extended genetic diversity in the species

Background Chlamydia pneumoniae is a widespread pathogen causing upper and lower respiratory tract infections in addition to a range of other diseases in humans and animals. Previous whole genome analyses have focused on four essentially clonal (> 99% identity) C. pneumoniae human genomes (AR39, CWL029, J138 and TW183), providing relatively little insight into strain diversity and evolution of this species. Results We performed individual gene-by-gene comparisons of the recently sequenced C. pneumoniae koala genome and four C. pneumoniae human genomes to identify species-specific genes, and more importantly, to gain an insight into the genetic diversity and evolution of the species. We selected genes dispersed throughout the chromosome, representing genes that were specific to C. pneumoniae, genes with a demonstrated role in chlamydial biology and/or pathogenicity (n = 49), genes encoding nucleotide salvage or amino acid biosynthesis proteins (n = 6), and extrachromosomal elements (9 plasmid and 2 bacteriophage genes). Conclusions We have identified strain-specific differences and targets for detection of C. pneumoniae isolates from both human and animal origin. Such characterisation is necessary for an improved understanding of disease transmission and intervention.

Impacts of traditional husbandry practices on exploitable levels of genetic diversity in cultured 'Tra' catfish (Pangasianodon hypophthalmus) in the Mekong Delta, Vietnam

Sutchi catfish (Pangasianodon hypophthalmus) – known more universally by the Vietnamese name ‘Tra’ is an economically important freshwater fish in the Mekong Delta in Vietnam that constitutes an important food resource. Artificial propagation technology for Tra catfish has only recently been developed along the main branches of the Mekong River where more than 60% of the local human population participate in fishing or aquaculture. Extensive support for catfish culture in general, and that of Tra (P. hypophthalmus) in particular, has been provided by the Vietnamese government to increase both the scale of production and to develop international export markets. In 2006, total Vietnamese catfish exports reached approximately 286,602 metric tons (MT) and were valued at 736.87 $M with a number of large new export destinations being developed. Total value of production from catfish culture has been predicted to increase to approximately USD 1 billion by 2020. While freshwater catfish culture in Vietnam has a promising future, concerns have been raised about long-term quality of fry and the effectiveness of current brood stock management practices, issues that have been largely neglected to date. In this study, four DNA markers (microsatellite loci: CB4, CB7, CB12 and CB13) that were developed specifically for Tra (P. hypophthalmus) in an earlier study were applied to examine the genetic quality of artificially propagated Tra fry in the Mekong Delta in Vietnam. The goals of the study were to assess: (i) how well available levels of genetic variation in Tra brood stock used for artificial propagation in the Mekong Delta of Vietnam (breeders from three private hatcheries and Research Institute of Aquaculture No2 (RIA2) founders) has been conserved; and (ii) whether or not genetic diversity had declined significantly over time in a stock improvement program for Tra catfish at RIA2. A secondary issue addressed was how genetic markers could best be used to assist industry development. DNA was extracted from fins of catfish collected from the two main branches of the Mekong River inf Vietnam, three private hatcheries and samples from the Tra improvement program at RIA2. Study outcomes: i) Genetic diversity estimates for Tra brood stock samples were similar to, and slightly higher than, wild reference samples. In addition, the relative contribution by breeders to fry in commercial private hatcheries strongly suggest that the true Ne is likely to be significantly less than the breeder numbers used; ii) in a stock improvement program for Tra catfish at RIA2, no significant differences were detected in gene frequencies among generations (FST=0.021, P=0.036>0.002 after Bonferroni correction); and only small differences were observed in alleles frequencies among sample populations. To date, genetic markers have not been applied in the Tra catfish industry, but in the current project they were used to evaluate the levels of genetic variation in the Tra catfish selective breeding program at RIA2 and to undertake genetic correlations between genetic marker and trait variation. While no associations were detected using only four loci, they analysis provided training in the practical applications of the use of molecular markers in aquaculture in general, and in Tra culture, in particular.

Genetic diversity in three redclaw crayfish (Cherax quadricarinatus, von Martens) lines developed in culture in China

The redclaw crayfish Cherax quadricarinatus (von Martens) accounts for the entire commercial production of freshwater crayfish in Australia. Two forms have been recognized, an 'Eastern' form in northern Queensland and a 'Western' form in the Northern Territory and far northern Western Australia. To date, only the Eastern form has been exported overseas for culture (including to China). The genetic structure of three Chinese redclaw crayfish culture lines from three different geographical locations in China (Xiamen in Fujian Province, Guangzhou in Guangdong Province and Chongming in Shanghai) were investigated for their levels and patterns of genetic diversity using microsatellite markers. Twenty-eight SSR markers were isolated and used to analyse genetic diversity levels in three redclaw crayfish culture lines in China. This study set out to improve the current understanding of the molecular genetic characteristics of imported strains of redclaw crayfish reared in China. Microsatellite analysis revealed moderate allelic and high gene diversity in all three culture lines. Polymorphism information content estimates for polymorphic loci varied between 0.1168 and 0.8040, while pairwise F ST values among culture lines were moderate (0.0020-0.1244). The highest estimate of divergence was evident between the Xiamen and Guangzhou populations.