Novel microsatellite loci for a deep sea fish (Macrourus berglax) and their amplification in other grenadiers (Gadiformes: Macrouridae)

Over-exploitation of traditional coastal stocks and a rising demand for seafood have resulted in the shift of commercial fishing towards less-known, deep-sea species in many parts of the world. Yet, the lack of knowledge of the biology, ecology and life-history of these species represents a serious impediment for establishing sound stock management plans. With the aim of providing tools that will allow assessment of the population genetic structure of Macrourus berglax, we have isolated and characterised a suite of novel microsatellite loci for this deep sea grenadier. Eight of these markers showed between 4 and 11 alleles per locus in two distant North Atlantic populations, with observed and expected heterozygosities between 0.17-0.83 and 0.35-0.87, respectively. Importantly, eight of these loci also cross-amplify in other Macrourid species. 

Evaluating genetic traceability methods for captive-bred marine fish and their applications in fisheries management and wildlife forensics

Growing demands for marine fish products is leading to increased pressure on already depleted wild populations and a rise in aquaculture production. Consequently, more captive-bred fish are released into the wild through accidental escape or deliberate releases. The increased mixing of captive-bred and wild fish may affect the ecological and/or genetic integrity of wild fish populations. Unambiguous identification tools for captive-bred fish will be highly valuable to manage risks (fisheries management) and tracing of escapees and seafood products (wildlife forensics). Using single nucleotide polymorphism (SNP) data from captive-bred and wild populations of Atlantic cod Gadus morhua L. and sole Solea solea L., we explored the efficiency of population and parentage assignment techniques for the identification and tracing of captive-bred fish. Simulated and empirical data were used to correct for stochastic genetic effects. Overall, parentage assignment performed well when a large effective population size characterized the broodstock and escapees originated from early generations of captive breeding. Consequently, parentage assignments are particularly useful from a fisheries management perspective to monitor the effects of deliberate releases of captive-bred fish on wild populations. Population assignment proved to be more efficient after several generations of captive breeding, which makes it a useful method in forensic applications for well-established aquaculture species. We suggest the implementation of a case-by-case strategy when choosing the best method.

Development of Cell Culture Systems from Selected Species of Fish and Prawns

The present work deals with the development of primary cell culture and diploid cell lines from two fishes, such as Poecilia reticulata and Clarias gariepinus. The greatest difficulty experienced was the avoidance of bacterial and fungi contamination. Three types of cell cultures are commonly developed, primary cell culture, diploid cell lines and heteroploid cell lines. Primary cell culture obtained from the animal tissues that have been cultivated in vitro for the first time. They are characterized by the same chromosome number as parent tissue, cultivated in vitro for the first time, have wide range of virus susceptibility, usually not malignant, six chromatin retarded and do not grow as suspension cultures. Diploid cell lines arise from a primary cell culture at the time of subculturing. Diploid cell lines commercially used in virology are W1-38 (human embryonic lung), W1-26 (human embryonic lung) and HEX (Human embryonic kidney). Heteroploid cell lines have been subcultivated with less than 75% of the cells in the population having a diploid chromosome constitution. Tissue cultures have been extensively used in biomedical research. The main applications are in three areas, Karyological studies, Identification and study of hereditary metabolic disorders and Somatic cell genetics. Other applications are in virology and host-parasite relationships. In this study an attempt was made to preserve the ovarian tissue at low temperature in the presence of cryoprotectants so that the tissue can be retrieved at any time and a cell culture could be developed.

Studies on biochemical genetics of the grey mullet Mugil cephalus linnaeus

The thesis deals with the results of an investigation on the "BIOCHEMICAL GENETICS OF MUGIL CEPHALUS" from Cochin, Madras and Orissa. It is presented under the following major headings: Introduction, Review of Literature, Materials and Methods, Results, Discussions, Conclusions, Recommendations, Summary and References.The introduction gives a brief account of historical and modern back ground on the stock concept in fisheries research and management, followed by the importance and potential role of biochemical genetics in the identification of natural units of fisheries management. In the review of literature published reports relevant to biochemical genetics with special reference to that of general proteins and enzyme systems of fish populations were considered. A detailed account of the source of experimental specimens, mode of collection, transportation, sample extraction, gel preparation/gel electrophoresis, buffer systems, staining procedures of proteins/enzymes, standardization of experiments, interpretation of electrophoretic data using basic formulae etc. are given in the materials and methods section. Four important conclusions were drawn on the basis of the results of the present investigation. Three recommendations were also made on the basis of evaluation of the results.

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.

A broad-spectrum mer operon in a multi-drug resistant strain of the fish pathogen, Aeromonas salmonicida

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

Supernumerary chromosomes in the pufferfish Sphoeroides spengleri: first occurrence in marine Teleostean Tetraodontiformes fish

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Twelve loci from HSA10, HSA11 and HSA20 were comparatively FISH-mapped on river buffalo and sheep chromosomes

Ten type I loci from HSA 10 (IL2RA and VIM), HSA11 (HBB and FSHB) and HSA20 (THBD, AVP/OXT, GNAS1, HCK and TOP1) and two domestic cattle type II loci (CSSM30 and BL42) were FISH mapped to R-banded river buffalo (BBU) and sheep (OAR) chromosomes. IL2RA (HSA 10) maps on BBU 14q13 and OAR13q13, VIM (HSA 10) maps on BBU14q15 and OAR13q15, HBB (HSA11) maps on BBU16q25 and OAR15q23, FSHB (HSA11) maps on BBU16q28 and OAR15q26. THBD (HSA20) maps on BBU 14q15 and OAR13q15 while AVP/OXT. GNAS1, HCK, and TOP I (HSA20) as well as CSSM30 and BL42 map on the same large band of BBU 14q22 and OAR13q22. All loci were mapped on the same homologous chromosomes and chromosome bands of the two species, and these results agree with those earlier reported in cattle homologous chromosomes 15 and 13. respectively, confirming the high degree of both banding and physical map similarities among the bovid species. Indirect comparisons between physical maps achieved on bovid chromosomes and those reported on HSA10, HSA11 and HSA20 were performed. Copyright (C) 2001 S. Karger AG, Basel.

5S rDNA characterization in twelve Sciaenidae fish species (Teleostei, Perciformes): depicting gene diversity and molecular markers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biotechnology and fish culture

Biotechnology can currently be considered of importance in aquaculture. The increase in the production of aquatic organisms over the last two decades through the use of biotechnology indicates that in a few generations biotechnology may overtake conventional techniques, at least for the commercially more valuable species. In the last few years, genetics has contributed greatly to fish culture through the application of the more recent techniques developed in biotechnology and in genetic engineering. At present, the most commonly used methods in fish biotechnology are chromosome manipulation and hormonal treatments, which can be used to produce triploid, tetraploid, haploid, gynogenetic and androgenetic fish. These result in the production of individuals and lineages of sterile, monosex or highly endogamic fish. The use of such strategies in fish culture has as a practical objective the control of precocious sexual maturation in certain species; other uses are the production of larger specimens by control of the reproductive process and the attainment of monosex lines containing only those individuals of greater commercial value. The use of new technologies, such as those involved in gene transfer in many species, can result in modified individuals of great interest to aquaculturists and play important roles in specific programmes of fish production in the near future.

Localization of 5-methylcytosine in metaphase chromosomes of diploid and triploid pacu fish, Piaractus mesopotamicus (Pisces, Characiformes)

The distribution of 5-methylcytosine (5-MeC) was investigated in fish chromosomes by indirect immunofluorescence using a highly specific 5-MeC monoclonal antibody. Diploid and artificially produced triploid specimens of the pacu fish, Piaractus mesopotamicus, were analyzed. The strong immunofluorescent signals were coincident with the heterochromatic regions of both diploids and triploids in a pattern that matched the C-banding pattern. In the euchromatin, heterogeneous labeling was observed along the chromatids. The weakness of this labeling hindered comparison of the fluorescence labeling of homologous chromosomes from diploid and triploid individuals. However, no striking differences were observed. The possibility that the euchromatin labeling by the 5-MeC antibody is related to the occurrence of mildly repetitive sequences in the genome of Piaractus is discussed.