Genetic structure and evidence of anthropogenic effects on wild populations of two Neotropical catfishes: baselines for conservation.

Genetic diversity and structure of Pseudoplatystoma corruscans and P. reticulatum, large migratory South America catfishes, where overfishing and the construction of numerous dams in their feeding and reproducing areas are affecting their migratory processes negatively, were studied using microsatellites in samples from Paraguay (that comprises the Pantanal biome), and the upper and lower Paraná Basins. Genetic diversity was in accordance to that observed for other large migratory fishes, but the most geographically isolated populations of P. reticulatum and those P. corruscans subject to anthropogenic effects (stocking and dams) showed lower genetic diversity and evidences of bottlenecks compatible with low effective population size. Pseudoplatystoma reticulatum presented subtle genetic differentiation within the Paraguay area, especially between the edges of its distribution. Pseudoplatystoma corruscans, in this same area, presented a quite homogeneous but significant genetic break between the Paraguay and upper Paraná populations, apparently resulting from natural and historical isolation between the basins until recently. These data demonstrates that, although these Pseudoplatystoma spp. are abundant in the Pantanal area, anthropogenic events are leading to negative effects on their populations, particularly in the upper Paraná Basin. Genetic differentiation observed along each species distribution demands conservation actions to preserve each population's biodiversity. These results represent important genetic information using new microsatellite markers and the first genetic study of P. reticulatum covering this area of its native distribution. Data may also contribute to a better understanding of species migration patterns and to be used as a baseline for proper management.

First record of non-native hybrid catfish Pseudoplatystoma reticulatum × Leiarius marmoratus in the Upper Paraná River basin, Brazil.

The aim of this study is to report the presence of a three non-native hybrid long-whiskered catfishes (family Pimelodidae) in the Upper Paraná River basin, Brazil. Genetic analyses demonstrated that the three presumptive hybrids were a result of the crossbreeding of Pseudoplatystoma reticulatum (central Amazonas River basin and Lower Paraná River) and Leiarius marmoratus (Amazonas, Essequibo and Orinoco rivers), producing a hybrid commonly known in Brazil as cachandiá. The potential threat to biodiversity, due to possible genetic contamination, competition and predation of wild stocks, of such artificially produced hybrid fishes is discussed.

Evolution and conservation of Characidium sex chromosomes.

Fish species exhibit substantial variation in the degree of genetic differentiation between sex chromosome pairs, and therefore offer the opportunity to study the full range of sex chromosome evolution. We used restriction-site associated DNA sequencing (RAD-seq) to study the sex chromosomes of Characidium gomesi, a species with conspicuous heteromorphic ZW/ZZ sex chromosomes. We screened 9863 single-nucleotide polymorphisms (SNPs), corresponding to ~1 marker/100 kb distributed across the genome for sex-linked variation. With this data set, we identified 26 female-specific RAD loci, putatively located on the W chromosome, as well as 148 sex-associated SNPs showing significant differentiation (average FST=0.144) between males and females, and therefore in regions of more recent divergence between the Z and W chromosomes. In addition, we detected 25 RAD loci showing extreme heterozygote deficiency in females but which were in Hardy-Weinberg equilibrium in males, consistent with degeneration of the W chromosome and therefore female hemizygosity. We validated seven female-specific and two sex-associated markers in a larger sample of C. gomesi, of which three localised to the W chromosome, thereby providing useful markers for sexing wild samples. Validated markers were evaluated in other populations and species of the genus Characidium, this exploration suggesting a rapid turnover of W-specific repetitive elements. Together, our analyses point to a complex origin for the sex chromosome of C. gomesi and highlight the utility of RAD-seq for studying the composition and evolution of sex chromosomes systems in wild populations.

Phylogeny and biogeography of Triportheidae (Teleostei: Characiformes) based on molecular data.

Triportheidae represents a relatively small family of characiform fishes with highly modified morphology. The relationship among characiform families is still unclear, and a phylogenetic analysis for the family including a representative number of Triportheus species has never been performed. Here, we inferred a phylogeny for 19 of the 22 species recognized for this family and two possible new Triportheus species using two mitochondrial and three nuclear genes. Our results show that (1) Triportheidae is monophyletic and a sister group of the clade consisting of the families Bryconidae and Gasteropelecidae; (2) Triportheus is monophyletic, but some species need to be reviewed and described; (3) all genera in Triportheidae, except for Agoniates originated in the period between Early Oligocene and Early Miocene; and (4) speciation in Triportheidae coincides with important geological events in South America, reinforcing the importance of time-calibrated trees to study fish evolution.

[An intriguing model for 5S rDNA sequences dispersion in the genome of freshwater stingray Potamotrygon motoro (Chondrichthyes: Potamotrygonidae)].

5S rDNA genes of the stingray Potamotrygon motoro were PCR replicated, purified, cloned and sequenced. Two distinct classes of segments of different sizes were obtained. The smallest, with 342 bp units, was classified as class I, and the largest, with 1900 bp units, was designated as class II. Alignment with the consensus sequences for both classes showed changes in a few bases in the 5S rDNA genes. TATA-like sequences were detected in the nontranscribed spacer (NTS) regions of class I and a microsatellite (GCT) 10 sequence was detected in the NTS region of class II. The results obtained can help to understand the molecular organization of ribosomal genes and the mechanism of gene dispersion.

Genetic differentiation among distinct karyomorphs of the wolf fish Hoplias malabaricus species complex (Characiformes, Erythrinidae) and report of unusual hybridization with natural triploidy.

In this study, genetic differentiation between karyomorphs A (2n = 42) and D (2n = 39/40) of the wolf fish Hoplias malabaricus, which is comprised of several cryptic species that present a wide variety of diploid chromosome numbers and sex chromosome systems, resulting in the identification of seven distinct karyomorphs (A-G), was investigated using a combination of molecular and cytogenetic tools. Deep sequence divergences for both karyomorphs were observed and indicate a long period of reproductive isolation between karyomorphs A and D. Additionally, one individual with 61 chromosomes was identified, which, as far as is known, is the first case of natural triploidy resulting from the hybridization between these highly differentiated karyomorphs of H. malabaricus. Molecular and cytogenetic analyses revealed that this allotriploid specimen carries two sets of maternal chromosomes from karyomorph D (2n = 40) and one set of chromosomes from karyomorph A (n = 21). Moreover, ribosomal sites and active nucleolus organizer regions from both parental contributors were found in the triploid hybrid. Considering the significant genetic distances between karyomorphs A and D, one of the primary reasons for the lack of recurrent reports of hybridization in the H. malabaricus species complex may be due to post-zygotic barriers, such as hybrid sterility or unviability.

Cytogenetic mapping of H1 histone and ribosomal RNA genes in hybrids between catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum.

A physical chromosome mapping of the H1 histone and 5S and 18S ribosomal RNA (rRNA) genes was performed in interspecific hybrids of Pseudoplatystoma corruscans and P. reticulatum. The results showed that 5S rRNA clusters were located in the terminal region of 2 chromosomes. H1 histone and 18S ribosomal genes were co-localized in the terminal portion of 2 chromosomes (distinct from the chromosomes bearing 5S clusters). These results represent the first report of association between H1 histone and 18S genes in fish genomes. The chromosome clustering of ribosomal and histone genes was already reported for different organisms and suggests a possible selective pressure for the maintenance of this association.

Common descent of B chromosomes in two species of the fish genus Prochilodus (Characiformes, Prochilodontidae).

To ascertain the origin of B chromosomes in 2 fish species of the genus Prochilodus, i.e. P. lineatus and P. nigricans, we microdissected them and generated B-specific DNA probes. These probes were used to perform chromosome painting in both species and in 3 further ones belonging to the same genus (P. argenteus, P. brevis and P. costatus). Both probes hybridized with the B chromosomes in P. lineatus and P. nigricans, but with none of the chromosomes in the 5 species. This indicates that the B chromosomes have low similarity with DNAs located in the A chromosomes and suggests the possibility that the B chromosomes in the 2 species have a common origin. The most parsimonious explanation would imply intergeneric hybridization in an ancestor of P. lineatus and P. nigricans yielding the B chromosome as a byproduct, which remained in these 2 species after their phylogenetic origin, but was perhaps lost in other Prochilodus species. This hypothesis predicts that B chromosomes are old genomic elements in this genus, and this could be tested once a species from a relative genus would be found showing homology of its A chromosomes with the B-probes employed here, through a comparison of B chromosome DNA sequences with those in the A chromosomes of this other species.

Chromosomal mapping of repetitive DNA and cytochrome C oxidase I sequence analysis reveal differentiation among sympatric samples of Astyanax fasciatus (Characiformes, Characidae).

The genus Astyanax is a specious Neotropical fish group, occurring from the south area of the United States to Argentina. During the past few years, intensive studies on representatives of this group have been performed, broadening the genetic, taxonomic and biogeographical knowledge considerably. However, phylogenetic relationships among many species remain uncertain. Significant karyotypic variations occur in A. bimaculatus, A. scabripinnis and A. fasciatus, suggesting the existence of species complexes. Samples with different diploid numbers may be found in A. fasciatus and, in some cases, morphological differentiation among them is not easily verified due to the absence of clear diagnostic characteristics. In the present study, the organization of the A. fasciatus genome from the Tietê River, Mogi das Cruzes, SP, Brazil, was analyzed, describing the karyotype structure and the chromosomal mapping of 18S and 5S ribosomal DNA (rDNA), H1 and H3 histones genes, telomeric DNA, and Rex3 transposable element. Sequencing of the cytochrome c oxidase I (COI) gene was also performed. Chromosomal analyses highlighted the occurrence of 2 distinct karyomorphs in the referred population, one with 2n = 46 and the other with 2n = 48 chromosomes. Furthermore, physical mapping of the repetitive sequences and nucleotide analysis of the COI gene reinforce the genomic differentiation between those karyomorphs and corroborate the existence of 2 different lineages morphologically similar to A. fasciatus living in sympatry.

Specific and non-specific immunity of piglets from sows fed diets containing specific fatty acids in field conditions.

The transfer of passive immunity from sows to piglets is important and it is the first immune protection of the new born piglet. Improving sows immunity by adding immuno-stimulating product in sows diet can positively affect colostrum composition and transfer of immune molecules to piglets. The aim of the current study is to evaluate the benefit of a different solution, made of specific fatty acids from marine origin that have been used in human medicine for decades, for sows and piglets. Two trials were conducted in commercial farm, involving 240 sows at different periods of the year. Sows were divided in a control group, without supplementation, and a test group, supplemented with the feed additive from the 90th day of gestation to weaning. Sows body condition, piglets viability and growth were recorded. Milk immunoglobulin content was measured, as well as Aujeszky antibodies in sows and piglets blood as marker of specific immunity, and blood bactericidal activity, complement activity and lysozyme as markers of non specific immunity. No effect of the product was observed on piglets zootechnical criteria and specific immunity parameters but significant improvement of piglet non specific immunity, was observed. No difference was observed neither in the piglets blood PRRSV and PCV2 antibodies and viruses nor in Aujeszky antibodies. Blood complement activity seems to be an accurate indicator of immuno-stimulating additive efficiency. Giving alkyl-glycerol fatty acids to sows in late gestation and lactation can improve the passive immunity transfer to piglets.

Molecular characterization and physical mapping of two classes of 5S rDNA in the genomes of Gymnotus sylvius and G. inaequilabiatus (Gymnotiformes, Gymnotidae).

The nucleotide sequences of the 5S rRNA multigene family and their distribution across the karyotypes in 2 species of Gymnotiformes, genus Gymnotus (G. sylvius and G. inaequilabiatus) were investigated by means of fluorescence in situ hybridization (FISH). The results showed the existence of 2 distinct classes of 5S rDNA sequences in both species: class I and class II. A high conservative pattern of the codifying region of the 5S rRNA gene was identified, contrasting with significant alterations detected in the nontranscribed spacer (NTS). The presence of TATA-like sequences along the NTS of both species was an expected occurrence, since such sequences have been associated with the regulation of the gene expression. FISH using 5S rDNA class I and class II probes revealed that both gene classes were collocated in the same chromosome pair in the genome of G. sylvius, while in that of G. inaequilabiatus, class II appeared more disperse than class I.

Identification and characterization of polymorphic microsatellite loci in the blue shark Prionace glauca, and cross-amplification in other shark species.

Two to 14 alleles were found to be segregating per locus (mean 5·2), with observed and expected heterozygosities ranging from 0·08 to 0·78 and 0·08 to 0·94, respectively. Cross-amplification of six of these microsatellite loci indicated that they are also polymorphic in three species of Carcharhiniformes and two species of Lamniformes. The newly developed primers reported here constitute a useful tool for genetic population analyses on Prionace glauca and, potentially, other related species.

Chromosome mapping of retrotransposable elements Rex1 and Rex3 in three fish species in the subfamily Hypoptopomatinae (Teleostei, Siluriformes, Loricariidae).

Transposons and retrotransposons represent a great portion of the eukaryotic genome. These repeated elements can be defined as genetic entities capable of self-inserting into different positions within the genome modifying the functions of the genes with which they are associated. The purpose of the present work was to isolate, identify, and map the sequences of Rex1 and Rex3 transposable elements present in the genomes of fishes of the family Loricariidae. The retrotransposable elements were identified through amplification with Rex1 and Rex3 primers, and the nucleotide composition of the retrotransposons was determined by automatic sequencing. In all the species analyzed it was possible to isolate Rex1 partial sequences from 520 to 563 bp in size and Rex3 partial sequences from 407 to 454 bp in size. Comparisons with the available literature showed that Loricariidae retrotransposons Rex1 and Rex3 have a high sequence similarity to putative homologous genes in different fish orders. Fluorescence in situ hybridization experiments conducted in 3 species, Hisonotus leucofrenatus, Pseudotocinclus tietensis, and Parotocinclus maculicauda, using the retrotransposable elements isolated for each species as probes, revealed that Rex1 and Rex3 share a similar dispersion pattern and are both found on all chromosomes of the 3 species organized in small clusters and dispersed on all the chromosomes, including euchromatic and heterochromatic regions, although a larger concentration was observed in the heterochromatic regions of H. leucofrenatus. The data found in the present study contradict much of the literature that substantiates that the retrotranspon elements are preferentially accumulated in the heterochromatin regions in fishes.

Chromosome mapping of H1 histone and 5S rRNA gene clusters in three species of Astyanax (Teleostei, Characiformes).

We report here on the physical mapping of the H1 histone genes (hisDNA) and the 5S ribosomal DNA (rDNA) in 3 Neotropical fish species of the genus Astyanax(A. altiparanae, A. bockmanni and A. fasciatus) and the comparative analysis of the chromosomes bearing these genes. Nucleotide analyses by sequencing of both genes were also performed. The distribution of the H1 histone genes was more conserved than that of the rRNA genes, since these were always located in the pericentromeric regions of 2 chromosome pairs. 5S rDNA was found on one of the pairs that presented an H1 histone cluster; this seems to be a conserved chromosomal feature of the genus Astyanax. In addition, individuals of A. bockmanni and A. fasciatus showed clusters of 5S rDNA on 1 pair of acrocentric chromosomes, not found in A. altiparanae. The results obtained by chromosome mapping as well as by sequencing of both genes showed that A.bockmanni is more closely related to A. fasciatus than to A. altiparanae. The results allow the characterization of cytogenetic markers for improved elucidation of the processes involved in karyotype differentiation of fish genomes.

Chromosomal diversification in populations of Characidium cf. gomesi (Teleostei, Crenuchidae).

Comparative cytogenetic studies carried out in two populations of Characidium cf. gomesi from Botucatu region, SP, Brazil, showed a similar karyotypic structure in a diploid number of 50 chromosomes, 32 metacentric and 18 submetacentric chromosomes for males and 31 metacentric and 19 submetacentric chromosomes for females as well as a ZZ-ZW sex chromosome system. Differences between both populations, however, were found in relation to the occurrence of B chromosomes and the distribution of 18S and 5S ribosomal DNA (rDNA) sites. Characidium cf. gomesi from the Alambari Stream, a component of the Tietê River basin, revealed 18S rDNA on Z and W chromosomes, while this gene was located on autosomes in the sample from the Paranapanema River basin. The 5S rDNA sites were observed in a single chromosomal pair (number 25) in the populations from Paranapanema and in two pairs in the specimens from Tietê (numbers 20 and 25). Besides that, in the sample from Paranapanema, both inter and intra-individual variations were found due to the occurrence of up to four heterochromatic supernumerary chromosomes in the cells. The life mode of this fish, restricted to headwaters and subjected to frequent breakdown into sub-populations, may have contributed to the fixation of such chromosomal differences. The karyotypic similarities found in the analysed populations, however, suggest that all are descended from the same ancestor group whereas their differences indicate that they are already existing in reproductively isolated populations.

Repetitive DNA probe linked to sex chromosomes in hybrids between Neotropical fish Leporinus macrocephalus and Leporinus elongatus (Characiformes, Anostomidae).

Interspecific hybrids of fish can offer important advances in the zootechnical exploration of this animal group. However, hybrids can pose serious biological risks to the environment and natural populations, making genetic characterization and monitoring of hybrids produced in fish culture important concerns. In the present study, individuals of the hybrid 'Piaupara' were characterized by fluorescence in situ hybridization (FISH) using sex-specific probes to differentiate the respective parental contributions from female Leporinus macrocephalus (Piauçu) and male Leporinus elongatus (Piapara). The application of this technique demonstrated a difference in hybridization patterns between males and females of L. elongatus and L. macrocephalus, mainly due to the fact that the NOR-bearing chromosomes of L. elongatus contain some homology with the probe. This allowed us to detect a clear sex-specific distinction of the hybrid 'Piaupara', demonstrating that this technique is well-suited for the characterization of these hybrids. Moreover, this technique offers an important methodological advance for the study of diverse sex chromosome systems of Leporinus, supplying valuable information about the origin and establishment of a recently described sexual system in this genus.

Genetics of neotropical fish: from chromosomes to populations.

The Neotropical freshwater fish fauna is very rich--according to the most recent catalogue 71 families and 4,475 species have been described. However, only a small amount of general information is available on the composition of Neotropical marine fishes. In Brazil, 1,298 marine species have been recorded. General analysis of available cytogenetic and population genetic data clearly indicates research has been mainly concentrated on freshwater fishes. Thus, today, cytogenetic information is available for 475 species of Characiformes, 318 species of Siluriformes, 48 species of Gymnotiformes, 199 freshwater species that do not belong to the superorder Ostariophysi, and only 109 species of marine fishes. For the species studied, only about 6% have sex chromosomes and about 5% have supernumerary or B chromosomes. A review of the cytogenetic studies shows that these data have provided valuable information about the relationships between fish groups, the occurrence of cryptic species and species complexes, the mechanism of sex determination and sex chromosome evolution, the distribution of nucleolus organizer regions, the existence supernumerary chromosomes, and the relationship between polyploidy and evolution. In relation to populations in Neotropical marine waters, the studies have shown the presence of cryptic species, which has important implications for fishery management. Different levels of genetic structuring can be found among Neotropical freshwater migratory fish species. This raises important implications for fish population genetic diversity and consequently its sustainable utilization in inland fisheries and aquaculture, specifically for conservation of ichthyo-diversity and survival.

Identification of a new repetitive element in the sex chromosomes of Leporinus elongatus (Teleostei: Characiformes: Anostomidae): new insights into the sex chromosomes of Leporinus.

Leporinus elongatus represents an interesting model for studies on chromosome evolution since it possesses a conspicuous ZZ/ZW sex chromosome system that has been characterized mainly by basic cytogenetic techniques. In the present study we describe a dispersed repetitive element (named LeSpeI) related to the sex chromosomes of L.elongatus. Females revealed clusters of LeSpeI on the long arm of the W chromosome and in the acrocentric NOR-bearing chromosome pair. In males, the signal was restricted to the pericentromeric region of the NOR-bearing chromosomes. Considering the results obtained in the present study using FISH, NOR and C-banding, together with findings from previous studies, it can be inferred that the sex chromosome system of L. elongatus is still undergoing an evolutionary process. The data suggest novelties in relation to the sex chromosomes of the genus Leporinus with the description of a multiple sex chromosome system involving the NOR-bearing chromosomes. Therefore, it is hypothesized that the simple ZW chromosome system previously described for L. elongatus rather is a multiple Z1Z1Z2Z2/Z1W1Z2W2 system.

Relationships among growth and different NOR phenotypes in a specific stock of rainbow trout (Oncorhynchus mykiss).

Growth is one of the most important aspects in the genetic improvement of cultured fish species. Consequently, genetic parameters related to this feature and their response to selection have been the focus of most research in this area. Such research indicates that, in general, there is enough additive genetic variance related to growth, justifying the use of selection. Based on the usefulness of cytogenetic and molecular markers in the fish culture, the aim of the present work was to analyze the possible relationships among cytogenetic characteristics, specifically the NOR phenotypes, and the increase in length and weight in specimens of the rainbow trout (Oncorhynchus mykiss), resultant from directed mating between homozygous females and heterozygous males according to their NOR phenotypic patterns. The equations of the relationship between length and weight of the analyzed specimens followed the model Wt = a Lt(b), showing b values higher than 3, determinant of a positive allometric growth. The results showed that the different NOR phenotypes were not related with the growth values for length and weight in any statistical test.

Cytogenetic analyses of two Curimatidae species (Pisces; Characiformes) from the Paranapanema and Tietê Rivers.

Cytogenetic analyses were performed in two Curimatidae species (Steindachnerina insculpta and Cyphocharax modesta) from the Paranapanema and Tietê Rivers (São Paulo State, Brazil), showing a karyotype composed of 54 meta-submetacentric chromosomes in both species. Silver- and chromomycyn-staining and fluorescent in situ hybridization (FISH) using a 18S rDNA probe indicated that the nucleolar organizer regions (NORs) of both species are localized in the terminal region of the long arm of two metacentric chromosomes. Although a single NOR system was evidenced in both analyzed species, S. insculpta and C. modesta presented the nucleolar organizer regions in distinct chromosome pairs, indicating that these cistrons can be considered cytogenetic markers. Variation on the amount and distribution of the constitutive heterochromatin (C-bands) could also be detected between the two species - while S. insculpta presented few heterochromatic blocks, intensely stained C-bands were evidenced in C. modesta specially in the terminal region of the long arm of the NOR-bearing chromosomes. Although most Curimatidae species have been characterized by homogeneous karyotypes, isolated populations could be established under different environmental conditions leading to karyotype micro-structure variations specially related to the NORs localization and C-banding distribution. The obtained data were useful for the cytogenetic characterization and differentiation of S. insculpta and C. modesta and could be used in evolutionary inferences in the Curimatidae group.