Abstract Raphidascarid nematodes have been the focus of several studies, mainly due to the zoonotic potential of some species, even though the cases are underreported. Due to the difficulty in identifying their larvae, the use of diagnostic techniques involving morphological and molecular analyses has grown in the last 20 years. The present study had as objective the morphological and molecular characterization of the L3 larval types of Hysterothylacium collected in Pomatomus saltatrix and Pagrus pagrus from the Brazilian coast, close to the municipality of Santos, State of São Paulo. Twenty specimens of P. saltatrix were necropsied and Hysterothylacium type V (n = 257) and Hysterothylacium type X (n = 5) larvae were found. Five specimens of P. pagrus were necropsied and all were parasitized by Hysterothylacium type V larvae. The analyses showed a genetic proximity relationship between Hysterothylacium types V with other Hysterothylacium V and with H. deardorffoverstreetorum, although this is a species inquirenda. Haplotypes for Hysterothylacium type X found in the present study formed a monophyletic group with other Hysterothylacium X, H. amoyense, and H. zhoushanense. Through this study, new hosts and localities were registered for Hysterothylacium type V and Hysterothylacium type X.

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.

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.

Abstract The main objective of this study was to estimate the genetic diversity levels and haplotype traceability in pacu Piaractus mesopotamicus from the breeding program located in Brazil by analyses of the mitochondrial DNA control region (mtDNA). Moreover, broodstocks from eight commercial fish farms were used for comparative evaluation, four from Brazil (Br1-Br4) and four from Argentina (Ar1-Ar4). The descriptive results revealed 47 polymorphic sites and 51 mutations, which evidenced 34 haplotypes. Ten haplotypes were shared among fish farms and 24 were exclusive. The nucleotide diversity (π) ranged from 0.00031 to 0.01462 and haplotype diversity (Hd) from 0.125 to 0.868. The analysis of molecular variance (AMOVA) indicated high structure present in the analyzed stocks (F ST = 0.13356 and ФST = 0.52707). The genetic diversity was high in most of the commercial broodstocks, especially those from Brazil. We observed seven haplotypes in the genetic breeding population, of which four were exclusive and three shared among the commercial fish farms. The genetic diversity was moderate (π = 0.00265 and Hd = 0.424) and considered appropriated for this breeding population of pacu. Our results provide support for the genetic diversity maintenance and mtDNA traceability of pacu commercial broodstocks.

Abstract Supernumerary, or B, chromosomes are present in several eukaryotes, including characid fish of the genus Psalidodon. Notably, Psalidodon paranae carries the most studied B chromosome variant, a macro-B chromosome. The origin of this element was determined to be an isochromosome; however, data regarding its inheritance remain unavailable due to methodological barriers such as the lack of an efficient, non-invasive, and rapid protocol for identifying B-carrying individuals that would enable the design of efficient crossing experiments. Thus, in this study, we primarily aimed was to develop two non-invasive and fast (approximately 2 h) methods to identify the presence of B chromosomes in live specimens of P. paranae based on satellite DNA (satDNA) sequences known to be present in this element. The methods include fluorescence in situ hybridization in interphase nuclei and relative gene quantification of satDNAs using quantitative polymerase chain reaction. Our results reveal the efficiency of quick-fluorescence in situ hybridization and quantitative polymerase chain reaction for identifying B-carrying individuals using the proposed satDNA sequences and open up new possibilities to study B chromosomes.

Abstract B chromosomes are non-essential additional genomic elements present in several animal and plant species. In fishes, species of the genus Psalidodon (Characiformes, Characidae) harbor great karyotype diversity, and multiple populations carry different types of non-essential B chromosomes. This study analyzed how the dispensable supernumerary B chromosome of Psalidodon paranae behaves during meiosis to overcome checkpoints and express its own meiosis-specific genes. We visualized the synaptonemal complexes of P. paranae individuals with zero, one, or two B chromosomes using immunodetection with anti-medaka SYCP3 antibody and fluorescence in situ hybridization with a (CA)15 microsatellite probe. Our results showed that B chromosomes self-pair in cells containing only one B chromosome. In cells with two identical B chromosomes, these elements remain as separate synaptonemal complexes or close self-paired elements in the nucleus territory. Overall, we reveal that B chromosomes can escape meiotic silencing of unsynapsed chromatin through a self-pairing process, allowing expression of their own genes to facilitate regular meiosis resulting in fertile individuals. This behavior, also seen in other congeneric species, might be related to their maintenance throughout the evolutionary history of Psalidodon.

RESUMO A hibridação é uma questão amplamente discutida em vários estudos com espécies de peixes. Para alguns autores, hibridações podem estar relacionadas à diversificação e especiação de muitos grupos, ou à extinção de populações ou espécies. Dificuldades para diferenciar espécies e híbridos podem ser um problema para aplicar corretamente o manejo de espécies selvagens, porque linhagens híbridas, especialmente as mais avançadas, podem assemelhar-se aos parentais. O gênero Cichla Bloch & Schneider, 1801 constitui um interessante modelo experimental, considerando que a hibridação e as incertezas taxonômicas dificultam a correta identificação. Considerando estes problemas, neste estudo foram desenvolvidas metodologias genéticas e aplicadas abordagens merísticas e morfométricas em amostras selvagens para identificar espécies e para testar uma possível hibridação entre Cichla kelberi Kullander & Ferreira, 2006 e Cichla piquiti Kullander & Ferreira, 2006. Para isto, C. kelberi, C. piquiti e indivíduos do híbrido em potencial ( carijó) foram coletados nos rios Paraná e Tietê (SP, Brasil). Para os métodos merístico e morfométrico, os indivíduos foram analisados, utilizando-se o software estatístico Pcord 5:31, enquanto que para os métodos moleculares, primers para PCR-multiplex foram desenhados e enzimas para PCR-RFLP foram selecionadas, sob nucleotídeos espécie-específicos. Todos os resultados indicaram que o carijó não é um híbrido interespecífico, porque apresentou padrão genético idêntico e morfologia próxima à C. piquiti. Assim, propomos que o carijó é um morfotipo de C. piquiti. Além disso, este estudo promove uma nova ferramenta molecular que poderá ser utilizada em futuras pesquisas, monitoramento e manejo do gênero Cichla.

ABSTRACT The hybridization is a widely-discussed issue in several studies with fish species. For some authors, hybridization may be related with diversification and speciation of several groups, or also with the extinction of populations or species. Difficulties to differentiate species and hybrids may be a problem to correctly apply a management of wild species, because hybrid lineages, especially the advanced ones, may resemble the parental species. The genus Cichla Bloch & Schneider, 1801 constitutes an interesting experimental model, considering that hybridization and taxonomic uncertainties hinder a correct identification. Considering these problems, in this study, we developed genetic methodologies and applied meristic and morphometric approaches in wild samples in order to identify species and for test a possible hybridization between Cichla kelberi Kullander & Ferreira, 2006 and Cichla piquiti Kullander & Ferreira, 2006. For this, C. kelberi, C. piquiti and potential hybrid ( carijó) individuals were collected in Paraná and Tietê rivers (SP, Brazil). For meristic and morphometric methods, the individuals were analyzed using the statistical software Pcord 5:31, while for molecular methods, primers for PCR-multiplex were designed and enzyme for PCR-RFLP were selected, under the species-specific nucleotide. All results indicated that the carijó is not an interspecific hybrid, because it presented identical genetic pattern and morphology closed to C. piquiti. Thus, we propose that carijó is a C. piquiti morphotype. In addition, this study promotes a new molecular tool that could be used in future research, monitoring and management programs of the genus Cichla.

RESUMO 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.

ABSTRACT 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.

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.

Hybrids of the species Pseudoplatystoma corruscansvs.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.

Estudos citogenéticos foram desenvolvidos em Prochilodus lineatus (Valenciennes 1836), os quais descrevem um interessante sistema de pequenos cromossomos supranumerários. O objetivo deste trabalho foi estudar a frequência e morfologia dos cromossomos B em indivíduos a partir da linhagem parental e os padrões de herança destes elementos em indivíduos obtidos a partir de cruzamentos controlados na espécie P. lineatus. A taxa de transmissão dos cromossomos B revelaram um kB=0,388 para o tipo acrocêntrico, kB=0,507 para o tipo metacêntrico e kB=0,526 submetacêntrico. Os resultados obtidos levantam hipóteses de que os cromossomos B do tipo acrocêntrico se encontram em fase de extinção, enquanto que os supranumerários do tipo metacêntrico e submetacêntrico encontram-se em fase de neutralização, seguindo uma taxa de transmissão Mendeliana.

Cytogenetic studies were developed in Prochilodus lineatus (Valenciennes 1836), describing an interesting system of small supernumerary chromosomes. The purpose of this work is to study the frequency and morphology of B chromosomes in individuals from the parental line and the inheritance patterns of these elements in individuals obtained from controlled crosses in the species P. lineatus. The transmission rate of B chromosomes revealed a kB=0.388 for the acrocentric type, a kB=0.507 for the metacentric type and a kB=0.526 for the submetacentric type. The obtained results raise hypothesis that B-acrocentric chromosomes are involved in an extinction process in this species, while the metacentric and submetacentric supernumerary elements comprises a neutral mechanism and follows a Mendelian transmission rate.

The family Prochilodontidae is considered a group with well conserved chromosomes characterized by their number, morphology and banding patterns. Thence, our study aimed at accomplishing a cytogenetic analysis with conventional methods (Giemsa staining, silver staining of the nucleolus organizer regions-AgNOR, and C-banding) and fluorescence in situ hybridization (FISH) with 18S and 5S ribosomal DNA probes in five species of the Prochilodus genus (Prochilodus argenteus, Prochilodus brevis, Prochilodus costatus, Prochilodus lineatus and Prochilodus nigricans) collected from different Brazilian hydrographic basins. The results revealed conservatism in chromosome number, morphology, AgNORs 18S and 5S rDNAs location and constitutive heterochromatin distribution patterns. The minor differences observed in this work, such as an Ag-NOR on a P. argenteus chromosome and a distinct C-banding pattern in P. lineatus, are not sufficient to question the conservatism described for this group. Future work using repetitive DNA sequences as probes for FISH will be interesting to further test the cytogenetic conservatism in Prochilodus.

O Tambaqui (Colossoma macropomum) é a espécie de peixe mais comumente cultivada em pisciculturas no Brasil. A espécie é altamente adaptada às condições de cativeiro, apresentando rápido crescimento e alta fecundidade. Nos últimos anos tem ocorrido o cruzamento artificial entre espécies de Characiformes, produzindo os híbridos "Tambacu" e "Tambatinga". A identificação de híbridos é uma tarefa difícil, em virtude da grande similaridade morfológica entre as espécies parentais. O presente estudo apresenta uma abordagem molecular inovadora para identificação de híbridos com base em PCR Multiplex de um gene nuclear (α-Tropomiosina), em que foram testados 93 espécimes obtidos em pisciculturas da região norte do Brasil. O sequenciamento de um fragmento de 505 pares de bases da Região Controle permitiu a identificação da linhagem materna de todos os espécimes como sendo de C. macropomum. Inesperadamente foram encontrados apenas dois haplótipos nas 93 amostras, um baixíssimo índice de diversidade para populações cultivadas de Tambaqui. A PCR multiplex identificou 42 híbridos, em contraste aos 23 híbridos citados pelos fornecedores dos peixes, em uma identificação realizada com base na morfologia. Esta ferramenta inovadora possui um grande potencial para o desenvolvimento da aquicultura brasileira dada a possibilidade de identificação sistemática das características genéticas tanto das matrizes produtoras de alevinos quanto dos alevinos e juvenis criados em fazendas.

Tambaqui (Colossoma macropomum) is the fish species most commonly raised in the Brazilian fish farms. The species is highly adaptable to captive conditions, and is both fast-growing and relatively fecund. In recent years, artificial breeding has produced hybrids with Characiform species, known as "Tambacu" and "Tambatinga". Identifying hybrids is a difficult process, given their morphological similarities with the parent species. This study presents an innovative molecular approach to the identification of hybrids based primarily on Multiplex PCR of a nuclear gene (α-Tropomyosin), which was tested on 93 specimens obtained from fish farms in northern Brazil. The sequencing of a 505-bp fragment of the Control Region (CR) permitted the identification of the maternal lineage of the specimen, all of which corresponded to C. macropomum. Unexpectedly, only two CR haplotype were found in 93 samples, a very low genetic diversity for the pisciculture of Tambaqui. Multiplex PCR identified 42 hybrids, in contrast with 23 identified by the supplier on the basis of external morphology. This innovative tool has considerable potential for the development of the Brazilian aquaculture, given the possibility of the systematic identification of the genetic traits of both fry-producing stocks, and the fry and juveniles raised in farms.

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.

B chromosomes are supernumerary elements present in about 15% 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.

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.

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.

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.