Cross species amplification and characterization of microsatellite markers in Leporinus friderici (Characiformes, Anostomidae) / Amplificação cruzada e caracterização de marcadores microssatélites em Leporinus friderici (Characiformes, Anostomidae)

ABSTRACT: Leporinus friderici is a migratory neotropical fish with elevated ecological and economic importance in Brazil. Microsatellite markers are highly important in population genetic studies, management, and conservation programs; however, no markers are available for this species. In this study, seven microsatellite loci, previously developed for Megaleporinus obtusidens, were successfully cross-amplified in L. friderici. Among these loci, five presented moderate to high genetic variability levels, with four to seven alleles per loci and expected heterozygosities varying from ≥ 0.574 to 1.000. These markers represent a valuable tool for the future management and ecological studies involving this species and group of neotropical fishes.

RESUMO: Leporinus friderici é um peixe neotropical migratório com elevada importância ecológica e econômica no Brasil. Os marcadores microssatélites são conhecidos por sua importância em estudos genéticos populacionais, programas de manejo e conservação, no entanto, não existem marcadores disponíveis para esta espécie. Neste estudo, sete locos microssatélites, previamente desenvolvidos para Megaleporinus obtusidens foram amplificados com sucesso em L. friderici. Dentre esses loci, cinco apresentaram variabilidade genética moderada a alta, com quatro a sete alelos por loci e heterozigosidades esperadas variando de ≥ 0,574 a 1.000. Esses marcadores representam uma ferramenta valiosa para futuros manejos e estudos ecológicos envolvendo esta espécie e este grupo de peixes neotropicais.

Molecular identification of intergenus crosses involving catfish hybrids: risks for aquaculture production

Monitoring of the interspecific hybrid production and trade is essential for the appropriate management of these animals in fish farms. The identification of catfish hybrids by morphological analysis is unreliable, particularly of juveniles and post-F1 individuals. Therefore, in the present study, we used five molecular markers (four nuclear genes and one mitochondrial gene) to detect hybrids in the trade of pimelodid juvenile fish from different stocks purchased of five seed producers in Brazil. Samples commercialized as pintado (pure species Pseudoplatystoma corruscans ) from three fish farms were genetically identified as hybrid cachapinta ( P. reticulatum x P. corruscans ). In the stocks purchased as cachandiá (hybrid between P. reticulatum x Leiarius marmoratus ) and cachapira (hybrid between P. reticulatum x Phractocephalus hemioliopterus ), we suggested the occurrence of intergenus crosses involving the hybrid cachapinta, which was used instead of the pure species P. reticulatum . The problems involving the hybrid cachapinta production were discussed in the present study, especially because these animals have caused genetic contamination and threatened the genetic integrity of natural and cultivated populations. In order to improve the surveillance of the production and provide criteria for the correct management of catfish hybrids, genetic markers has become an excellent alternative to the morphological identification, including juveniles or post-F1 generations.

O monitoramento da produção e comércio de híbridos interespecíficos é essencial para o manejo adequado desses animais em pisciculturas. A identificação de híbridos de bagres por análise morfológica não é confiável, especialmente de juvenis e indivíduos pós-F1. Portanto, no presente estudo, cinco marcadores moleculares (quatro genes nucleares e um gene mitocondrial) foram utilizados para detectar híbridos no comércio de juvenis pimelodídeos de diferentes estoques, comprados de cinco produtores de alevinos no Brasil. As amostras comercializadas como pintado (espécie pura Pseudoplatystoma corruscans ) foram geneticamente identificadas como híbrido cachapinta ( P. reticulatum x P. corruscans ). Nos estoques comprados como cachandiá (híbrido entre P. reticulatum x Leiarius marmoratus ) e cachapira (híbrido entre P. reticulatum x Phractocephalus hemioliopterus ), sugere-se a ocorrência de cruzamentos intergêneros envolvendo o híbrido cachapinta, que foi usado ao invés da espécie pura P. reticulatum . Os problemas envolvendo a produção de cachapinta foram discutidos no presente estudo, especialmente porque estes animais têm causado contaminação genética e ameaçado a integridade genética das populações naturais e cultivadas. Com o intuito de melhorar a fiscalização da produção e fornecer critérios para o manejo correto dos híbridos de bagre, marcadores genéticos têm se tornado uma excelente alternativa para a identificação morfológica, incluindo juvenis ou gerações pós-F1.

Genetic identification of interspecific hybrid of Neotropical catfish species (Pseudoplatystoma corruscans vs. Pseudoplatystoma reticulatum) in rivers of Mato Grosso do Sul State, Brazil

Hybrids of the species Pseudoplatystoma corruscans vs. Pseudoplatystoma reticulatum are extensively traded because they are more docile and precocious compared to their parents. This study aimed to identify the occurrence of hybrid 'Surubim' in rivers of the State of Mato Grosso do Sul, applying molecular techniques. Pseudoplatystoma corruscans, Pseudoplatystoma reticulatum and hybrid 'Surubim' were detected using multiplex PCR and PCR-RFLP in RAG2, GLOBIN, EF1α, 18S rRNA nuclear genes, and mitochondrial 16S rRNA gene. The results revealed the presence of F1 hybrids in rivers and the occurrence of genetic introgression, evidenced by the Post-F1 hybrids. Furthermore, discrepancies between the morphological and genetic identification were observed, demonstrating the effectiveness of using different molecular markers for the correct identification of these hybrids. The necessity of implementation of management and conservation projects to maintain the genetic integrity of native species populations threatened by hybridization in the rivers of Mato Grosso do Sul State is evident....

Híbridos envolvendo as espécies Pseudoplatystoma corruscans vs. Pseudoplatystoma reticulatum são amplamente comercializados por apresentarem maior docilidade e precocidade quando comparados aos parentais. Neste trabalho objetivou-se identificar, por meio de técnicas moleculares, a existência de surubins híbridos em rios do estado do Mato Grosso do Sul. Pseudoplatystoma corruscans, Pseudoplatystoma reticulatum e 'Surubim' híbrido foram identificados utilizando as técnicas de PCR multiplex e PCR-RFLP dos genes nucleares RAG2, GLOBINA, EF1α, 18S rRNA e mitocondrial 16S rRNA. Os resultados demonstraram a presença de híbridos F1 em rios e a ocorrência da introgressão genética, evidenciada pelos híbridos Pós-F1. Além disso, foram observadas discrepâncias entre a identificação morfológica e genética, demonstrando a eficácia da utilização de diversos marcadores moleculares para identificação correta destes híbridos. Torna-se evidente a necessidade da implantação de projetos de manejo e conservação, a fim de manter a integridade genética de populações de espécies nativas em rios do estado do Mato Grosso do Sul, que encontram-se ameaçadas pela hibridação...

Cytogenetic characterization of F1, F2 and backcross hybrids of the Neotropical catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum (Pimelodidae, Siluriformes)

The cytogenetic characteristics of Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum and their F1, F2 and backcross hybrids were assessed by using chromosome banding techniques. The diploid number of 56 chromosomes was constant in all species and lineages, with a karyotypic formula containing 20 metacentric, 12 submetacentric, 12 subtelocentric and 12 acrocentric chromosomes. Nucleolar organizer regions (NORs) were identified in two subtelocentric chromosomes in the parents and hybrids, with partial nucleolar dominance in F1 and F2 specimens. Heterochromatic blocks were detected in the terminal and centromeric regions of some chromosomes in all individuals. For parental and hybrid lineages, 18S ribosomal clusters corresponding to NORs and 5S ribosomal genes were identified in distinct pairs of chromosomes. The striking conservation in the chromosomal macrostructure of the parental species may account for the fertility of their F1 hybrids. Similarly, the lack of marked alterations in the chromosomal structure of the F1 hybrids could account for the maintenance of these features in post-F1 lineages.

Intraspecific crosses resulting in the first occurrence of eight and nine B chromosomes in Prochilodus lineatus (Characiformes, Prochilodontidae)

B chromosomes are supernumerary elements present in about 15 percent of eukaryotic species and are most frequently heterochromatic, behave parasitically, show a transmission rate higher than standard (A) chromosomes, and can provoke harmful effects on carriers. In the current work, Prochilodus lineatus individuals carrying eight and nine B chromosomes were obtained by induced crossing performed involving breeders with different B chromosome numbers in their cells. The high B chromosome numbers found in the offspring were recorded for the first time in this species. The use of cytogenetic techniques applied in the present study revealed that regardless of the increase in number of B chromosomes in the genome of these individuals, those elements did not presented active genes, and showed their normal heterochromatic characteristic.

Chromosome polymorphism of heterochromatin and nucleolar regions in two populations of the fish Astyanax bockmanni (Teleostei: Characiformes)

Karyotype and other chromosomal markers as revealed by C-banding and silver (Ag) impregnation in two Astyanax bockmanni populations (Barra Seca Stream and Campo Novo River) were examined. The diploid chromosome number 2n = 50 and nearly identical karyotypes were documented. C-banding revealed heterochromatic blocks on the terminal regions of some chromosomes, with high frequencies of polymorphisms. The Ag-impregnation showed that the nucleolus organizer regions (NORs) varied in number, location and organization. Astyanax bockmanni revealed chromosome characteristics similar those of the species complex "A. scabripinnis". Mechanisms that may be responsible for the high degree of polymorphism are discussed.

Cariótipo e outros marcadores cromossômicos revelados pelo bandamento C e impregnação da prata (Ag) em duas populações de Astyanax bockmanni (córrego Barra Seca e rio Campo Novo) foram examinados. O número cromossômico diploide 2n = 50 e cariótipo quase idênticos são documentados. Bandamento C revelou blocos heterocromáticos na região terminal de alguns cromossomos, com um elevado nível de polimorfismos. A impregnação da prata mostrou variabilidade do número, posição e organização para as regiões organizadoras de nucléolo (RONs). Astyanax bockmanni revelou características cromossômicas similares ao complexo "A. scabripinnis" e os mecanismos responsáveis pelo alto nível do polimorfismo foram discutidos.

Cytogenetic markers as diagnoses in the identification of the hybrid between Piauçu (Leporinus macrocephalus) and Piapara (Leporinus elongatus)

The genetic monitoring of interspecific hybrids involves the application of methodologies able to provide an easy and indubitable genetic characterization of both parental and hybrid individuals. In the present work, cytogenetic techniques were used to identify a hybrid lineage of "Piaupara" in order to caracterize them in relation to the parental species, Leporinus macrocephalus (piauçu) and L. elongatus (piapara). The cytogenetic analysis revealed that L. macrocephalus presented 2n = 54 chromosomes and a nucleolar organizer regions (NOR) at the telomere of the long arm of the submetacentric chromosome pair 2. Analysis of constitutive heterochromatin (C-banding) revealed a conspicuous block at the pericentromeric region on the long arm of a submetacentric chromosome pair. L. elongatus presented the same diploid number, 2n = 54, and a karyotypic formula similar to that of L. macrocephalus. The NORs were also at the telomere of the long arm of the submetacentric pair 2, which was morphologically different from that of L. macrocephalus. Heterochromatic blocks were observed at both telomeres of a submetacentric chromosome pair. The hybrid "Piaupara" presented the same diploid number (2n = 54) and karyotypic formula as the parental species and there were no visible differences between parental and hybrid individuals. Differently from the Giemsa staining, NOR- and C-banding analysis showed marked differences which allowed the identification of the hybrids by the different morphology and/or size of the chromosomes carrying the NORs and patterns of heterochromatin distribution in their chromosomes. Such genetic studies are important for fish culture since they can provide tools for monitoring natural and artificial hybridization. They are also useful in biological conservation programmes and in the proper management of natural and reared fish stocks.

Cytogenetic characterization of the strongly electric Amazonian eel, Electrophorus electricus (Teleostei, Gymnotiformes), from the Brazilian rivers Amazon and Araguaia

A karyotype analysis of the electric eel, Electrophorus electricus (Teleostei, Gymnotiformes), a strongly electric fish from northern South America, is presented. Two female specimens were analyzed, one from the Amazon River and one from the Araguaia River. The specimens had a chromosomal number of 2n = 52 (42M-SM + 10A). C-bands were present in a centromeric and pericentromeric position on part of the chromosomes; some interstitial C-bands were also present. Heteromorphic nucleolus organizer regions (NORs) were detected in two chromosome pairs of the specimen from the Amazon River. The chromosome number and karyotype characteristics are similar to those of other Gymnotidae species. The genera Electrophorus and Gymnotus are positioned as the basal lineages in the Gymnotiformes phylogeny.

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

Cytogenetic analyses carried out in eight specimens of Sphoeroides spengleri revealed the presence of 2n = 46 chromosomes (20 M/SM and 26 ST/A). Besides the standard karyotypical set, the presence of B microchromosomes was observed in two individuals, ranging from 0 to 2 microchromosomes per cell. A karyotype composed by 2n = 46 chromosomes with occurrence of M and SM chromosomes is considered basal for the species from the clade comprising the families Tetraodontidae, Balistidae, and Diodontidae, although it represents a derived condition for the order Tetraodontiformes, whose basal karyotype would be composed by 2n = 48 acrocentric chromosomes. The occurrence of B microchromosomes in marine Tetraodontiformes fish was not known, and this represents the first report of such a chromosomal type.

First report of a B chromosome in a natural population of Astyanax altiparanae (Characiformes, Characidae)

Several species of the genus Astyanax have already been genetically studied, and B-chromosomes have been considered to be an interesting feature in some species of this group. In the present paper we report, for the first time, the occurrence of a B microchromosome in a natural population of A. altiparanae. This additional genomic element was identified as an acrocentric chromosome, similar in size to the smallest chromosomal pairs of the standard karyotype. Analysis of the constitutive heterochromatin pattern by C-banding evidenced heterochromatic blocks located on centromeric, pericentromeric, and interstitial regions of some chromosomes, and also positive marks in a subtelocentric chromosomal pair that presented the short arms entirely heterochromatic. The application of this methodology also revealed a heterochromatic pattern in the extra chromosome, a typical feature of supernumerary chromosomes.

A lethal effect associated with polymorphism of the NOR-bearing chromosomes in rainbow trout (Oncorhynchus mykiss)

Cytogenetic analysis of a rainbow trout stock showed that the nucleolar organizing regions were located subterminally on the long arm of a submetacentric chromosome pair and occurred as a single chromosomal segment (phenotype N1) or as two chromosomal segments separated by a short euchromatic segment (phenotype N2). Cytogenetic analysis also showed that there were N1N1 and N1N2 individuals but no N2N2 individuals. Analysis of the different includedphenotypes incluted that the population was not in Hardy-Weinberg equilibrium (chi² = 19.333; p < 0.01), and that a higher frequency of individuals had the N1N2 phenotype. Experimental crosses involving four males (two N1N1 and two N1N2) and four females (one N1N1 and three N1N2) yielded eight broods. There were no significant differences between the expected and observed frequencies of offspring resulting from crosses involving N1N1 x N1N2 individuals. However, significant differences were seen in crosses involving N1N2 x N1N2 parents because of the a high incidence of N1N2 fishes and the absense of N2N2. The lack of N2N2 individuals in the parental sample and their absence among the offspring of the experimental crosses suggested that this genetic combination may be lethal in rainbow trout. The survival rates of embryonic eyed egg and fry stage individuals were not different, indicating that the possible lethal effect may occur during more advanced ontogenetic phases.

Analysis of NOR distribution in cultivated and naturalized stocks of rainbow trout (Oncorhynchus mykiss)

In salmonid species, nucleolar organizing regions (NORs) have frequently been found on a single chromosome pair. However, some other, smaller yet active NORs have also been described. In the present study, NORs were found to be located in different positions on the chromosomes of 12 rainbow trout samples (11 cultivated and one naturalized stocks). Three phenotypes were found: 1) NORs located on two submetacentric chromosomes, in a subterminal position on the short arm (AA); 2) NORs located on two submetacentric chromosomes, in a subterminal position on the long arm (BB); and 3) NORs located on two submetacentric chromosomes, one in a subterminal position on the short arm, and the other in a subterminal position on the long arm (AB). Phenotype AA was identified in individuals from ten cultivated stocks, phenotype AB was identified in individuals from three cultivated stocks, and phenotype BB was found in one cultivated and one wild stock. Some aspects of the origin and the distribution of the different phenotypes are discussed