Characterization of α-actin isoforms in white and red skeletal muscle types of Leporinus macrocephalus (Characiformes, Anostomidae).

Two α-actin genes of the fish Leporinus macrocephalus, referring to white and red muscle tissues, were isolated. Actin isoforms, that mainly differed by a Ser/Ala155 substitution, can have a functional significance related to actin-ATP interaction. An Ala155 residue, as observed in the α-skeletal actin from red muscle, results in a decrease in actin's affinity for ATP, which may also be associated to the slow contractile performance of this tissue. Furthermore, a Phe/Ile262 substitution at the red muscle actin leads to a hydrophobicity variation at the D-plug of the protein, which could alter its stability. Data on qRT-PCR evidenced a significant higher actin mRNA level in white muscle when compared to red muscle (T=105 Mann Whitney; p<0.001). This finding could be related to the energetic demands of the white muscle tissue, with fast contraction fibers and glycolytic metabolism for energy supply. Available data on muscle actins lead to the proposal that white and red α-skeletal actins are genetically and functionally distinguishable in fish species, a feature that is not found in other vertebrate groups.

Development of microsatellite markers for Hyacinth macaw (Anodorhynchus hyacinthinus) and their cross-amplification in other parrot species.

BACKGROUND: Hyacinth macaw Anodorhynchus hyacinthinus is the largest parrot of the world and is considered vulnerable to extinction due to its habitat loss and illegal trade associated to the international pet market demand. Genetic studies on this species are still incipient to generate a consistent characterization of the population dynamics and to develop appropriate conservation strategies. In this sense, microsatellite markers may support the detection of a population genetic structure for this bird species. However, at this time, none Hyacinth macaw species-specific primers for microsatellite loci have been so far established. This study aimed to develop and characterize polymorphic microsatellite markers for A. hyacinthinus and to check for their cross-amplification in other parrot species. FINDINGS: Sequences containing repeated dinucleotide motifs were prospected and optimized from a genomic library that was enriched for microsatellites using magnetic beads. The analyses of 43-57 samples from wild individuals of three distinct Brazilian subpopulations led to the characterization of five polymorphic microsatellite loci. Allele richness per locus ranged from two to 12. Three loci exhibited observed heterozygosity values higher than 50%, but the overall average value among all loci was close to 45%. In addition, successful primer cross-amplification was verified in seven other investigated species of Neotropical parrots. CONCLUSIONS: The newly developed markers have shown to be potentially useful for in situ and ex situ population studies to support future conservation actions of Hyacinth macaw and other parrots.

Marshes as "Mountain Tops": Genetic Analyses of the Critically Endangered São Paulo Marsh Antwren (Aves: Thamnophilidae).

Small populations of endangered species can be impacted by genetic processes such as drift and inbreeding that reduce population viability. As such, conservation genetic analyses that assess population levels of genetic variation and levels of gene flow can provide important information for managing threatened species. The São Paulo Marsh Antwren (Formicivora paludicola) is a recently-described and critically endangered bird from São Paulo State (Brazil) whose total estimated population is around 250-300 individuals, distributed in only 15 isolated marshes around São Paulo metropolitan region. We used microsatellite DNA markers to estimate the population genetic characteristics of the three largest remaining populations of this species all within 60 km of each other. We detected a high and significant genetic structure between all populations (overall FST = 0.103) which is comparable to the highest levels of differentiation ever documented for birds, (e.g., endangered birds found in isolated populations on the tops of African mountains), but also evidence for first-generation immigrants, likely from small local unsampled populations. Effective population sizes were small (between 28.8-99.9 individuals) yet there are high levels of genetic variability within populations and no evidence for inbreeding. Conservation implications of this work are that the high levels of genetic structure suggests that translocations between populations need to be carefully considered in light of possible local adaptation and that remaining populations of these birds should be managed as conservation units that contain both main populations studied here but also small outlying populations which may be a source of immigrants.

A comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach.

The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ± 57.71; 224.10 ± 91.73) when compared to NT samples (249.75 ± 30.08; 342.95 ± 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes.

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

In order to extend the genetic data on the Sciaenidae fish family, the present study had the purpose to characterize PCR-generated 5S rDNA repeats of twelve species of this group through PAGE (Polyacrylamide Gel Electrophoresis) analysis. The results showed the occurrence of at least two different 5S rDNA size classes in all the species. Moreover, 5S rDNA repeats of one of the studied species - Isopisthus parvipinnis - were cloned and subjected to nucleotide sequencing and Southern blot membrane hybridization analyses, which permitted to confirm the existence of two major 5S rDNA classes. Phylogenetic analysis based on the nucleotide sequences of different 5S rDNA repeats of I. parvipinnis lead to their separation into two major clusters. These results may reflect the high dynamism that rules the evolution rate of 5S rDNA repeats. The obtained data suggest that 5S rDNA can be useful in genetic analyses to identify species-specific markers and determine relationships among species of the Sciaenidae group.

Discrimination of Shark species by simple PCR of 5S rDNA repeats

Sharks are suffering from intensive exploitation by worldwide fisheries leading to a severe decline in several populations in the last decades. The lack of biological data on a species-specific basis, associated with a k-strategist life history make it difficult to correctly manage and conserve these animals. The aim of the present study was to develop a DNA-based procedure to discriminate shark species by means of a rapid, low cost and easily applicable PCR analysis based on 5S rDNA repeat units amplification, in order to contribute conservation management of these animals. The generated agarose electrophoresis band patterns allowed to unequivocally distinguish eight shark species. The data showed for the first time that a simple PCR is able to discriminate elasmobranch species. The described 5S rDNA PCR approach generated species-specific genetic markers that should find broad application in fishery management and trade of sharks and their subproducts.

Identities among actin-encoding cDNAs of the Nile tilapia (Oreochromis niloticus) and other eukaryote species revealed by nucleotide and amino acid sequence analyses

Actin-encoding cDNAs of Nile tilapia (Oreochromis niloticus) were isolated by RT-PCR using total RNA samples of different tissues and further characterized by nucleotide sequencing and in silico amino acid (aa) sequence analysis. Comparisons among the actin gene sequences of O. niloticus and those of other species evidenced that the isolated genes present a high similarity to other fish and other vertebrate actin genes. The highest nucleotide resemblance was observed between O. niloticus and O. mossambicus a-actin and b-actin genes. Analysis of the predicted aa sequences revealed two distinct types of cytoplasmic actins, one cardiac muscle actin type and one skeletal muscle actin type that were expressed in different tissues of Nile tilapia. The evolutionary relationships between the Nile tilapia actin genes and diverse other organisms is discussed.

Partial molecular characterization of the Nile tilapia (Oreochromis niloticus) alpha-cardiac muscle actin gene and its relationship to actin isoforms of other fish species

An alpha actin gene segment, isolated from Nile tilapia (Oreochromis niloticus), was characterized by nucleotide sequencing, predicted amino acid sequence and Southern blot hybridization. Genomic DNA amplification resulted in a 1063-bp fragment corresponding to a partial alpha-cardiac muscle actin gene containing exons 3 to 6. Southern blot analysis of the restriction-digested DNA revealed that the Nile tilapia genome contains multiple muscle actin isoforms. Although comparison of the nucleotide sequence, amino acid residues and exon-intron organization of the isolated actin gene with those of other vertebrates showed a high level of identity, diagnostic amino acid residues can still be correlated to distinct actin genes in fish species.

Genetic conservation of brazilian fishes - present state and perspectives

Natural environments have been worldwide affected by the growing impact of anthropogenic actions that promote the reduction or the extinction of several vertebrate species. Aquatic ecosystems represent one of the most affected environments and many fish species and/or populations have been increasingly fragmented distributed due to habitat degradation, predatory fishing, introduction of exotic species, river sedimentation, deforestation, pollution, reduction of food resource, and construction of hydroelectric dams. Actually, more than 150 Brazilian fish species, including freshwater, estuary and coastal species, can be considered threatened. Information on the diversity, conservation biology and population analysis on threatened species or populations, with several DNA markers, can be extremely useful for the success of fish species-recovery and maintenance programs. Although DNA analysis in Neotropical fish species are just beginning, they tend to increase with the widespread attention to the use of molecular approaches to minimize problems related to the risk of extinction. The accumulation of information on biology and pattern of genetic variation of fish epecies, associated with ecological and demographic data, and also education and respect to the nature, constitutes a crucial task to develop efficient conservation strategies in order to preserve the genetic diversity in aquatic environments

Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales.

DNA-based studies have been one of the major interests in conservation biology of endangered species and in population genetics. As species and population genetic assessment requires a source of biological material, the sampling strategy can be overcome by non-destructive procedures for DNA isolation. An improved method for obtaining DNA from fish fins and scales with the use of an extraction buffer containing urea and further DNA purification with phenol-chloroform is described. The methodology combines the benefits of a non-destructive DNA sampling and its high efficiency. In addition, comparisons with other methodologies for isolating DNA from fish demonstrated that the present procedure also becomes a very attractive alternative to obtain large amounts of high-quality DNA for use in different molecular analyses. The DNA samples, isolated from different fish species, have been successfully used on random amplified polymorphic DNA (RAPD) experiments, as well as on amplification of specific ribosomal and mitochondrial DNA sequences. The present DNA extraction procedure represents an alternative for population approaches and genetic studies on rare or endangered taxa.

Mitochondrial DNA variation in wild populations of Leporinus elongatus from the Parana River basin

Leporinus elongatus, a fish species widely distributed throughout the Parana River basin in South America, is an important fishery resource and a valuable species in aquaculture programs. Despite its great economic importance, several wild populations have been suffering a drastic reduction. The comprehension of its population structure represents an important step for the conservation of these organisms in natural environments, and also for the selection of wild stocks to be used in hatchery programs. In order to understand the genetic-population structure of L. elongatus, D-loop mitochondrial DNA analyses were applied in six wild populations of the species. The results were used to estimate the levels of within and among population genetic variability. Although the D-loop variations could not be correlated to the geographic distribution of these organisms, it was possible to detect high levels of genetic variability within each population and the occurrence of exclusive population haplotypes, which suggests a partial genetic differentiation among them. The obtained data can be useful in selecting fish stocks that preserve a better genetic diversity of L. elongatus for use in conservation and/or hatchery programs