Use of chromosome microdissection in fish molecular cytogenetics

Chromosome microdissection is a technique in which whole chromosomes or chromosomal segments are dissected under an inverted microscope yielding chromosome-specific sequences. Several protocol modifications introduced during the past 15 years reduced the number of chromosomes required for most applications. This is of particular interest to fish molecular cytogenetics, since most species present highly uniform karyotypes which make impossible the collection of multiple copies of the same chromosome. Probes developed in this manner can be used to investigate chromosome homologies in closely related species. Here we describe a protocol recently used in the gymnotiform species group Eigenmannia and review the major steps involved in the generation of these markers focusing on protocol modifications aiming to reduce the number of required chromosomes.

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.

Isolation and characterization of Flavobacterium columnare (Bernardet et al. 2002) from four tropical fish species in Brazil

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish, implicated in skin and gill disease, often causing high mortality. The aim of this study was the isolation and characterization of Flavobacterium columnare in tropical fish in Brazil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) and cascudo (Hypostomus plecostomus) were examined for external lesions showing signs of colunmaris disease such as greyish white spots, especially on the head, dorsal part and caudal fin of the fish. The sampling comprised 50 samples representing four different fish species selected for study. Samples for culture were obtained by skin and kidney scrapes with a sterile cotton swabs of columnaris disease fish and streaked onto Carlson and Pacha (1968) artificial culture medium (broth and solid) which were used for isolation. The strains in the liquid medium were Gram negative, long, filamentous, exhibited flexing movements (gliding motility), contained a large number of long slender bacteria and gathered into ‘columns'. Strains on the agar produced yellow-pale colonies, rather small, flat that had rhizoid edges. A total of four Flavobacterium columnare were isolated: 01 Brycon orbignyanus strain, 01 Piaractus mesopotamicus strain, 01 Colossoma macropomum strain, and 01 Hypostomus plecostomus strain. Biochemical characterization, with its absorption of Congo red dye, production of flexirubin-type pigments, H2S production and reduction of nitrates proved that the isolate could be classified as Flavobacterium columnare.

Elaboration of sausage using minced fish of Nile tilapia filleting waste

The objective of this study was to evaluate the inclusion of minced fish (MF) (0, 20, 40, 60, 80 and 100%), obtained from Nile tilapia filleting waste, in sausage and determine their physicochemical, nutritional and sensory properties. The sausages showed a decrease in protein and increase in fat content with increasing inclusion of MF. The nutritional quality of the products was high, with digestibility over 85%. The parameters of texture instrumental and yellow color (b*) decreased with the increasing inclusion of MF. The sensory evaluation of the color showed that the maximum level of inclusion of MF was not well accepted by the panelists. The sausage with the best acceptance for the flavor attribute was those with 60% of MF. The results showed good nutritional quality of sausages utilizing MF of Nile tilapia filleting waste and according to the sensory evaluation, the maximum level of inclusion should not exceed 60%.

Karyological study of Amphisbaena ridleyi (Squamata, Amphisbaenidae), an endemic species of the Archipelago of Fernando de Noronha, Pernambuco, Brazil

The karyotype of Amphisbaena ridleyi, an endemic species of the archipelago of Fernando de Noronha, in State of Pernambuco, Brazil, is described after conventional staining, Ag-NOR impregnation and fluorescence in situ hybridization (FISH) with a telomeric probe. The diploid number is 46, with nine pairs of macrochromosomes (three metacentrics, four subtelocentrics and two acrocentrics) and 14 pairs of microchromosomes. The Ag-NOR is located in the telomeric region of the long arm of metacentric chromosome 2 and FISH revealed signals only in the telomeric region of all chromosomes. Further cytogenetic data on other amphisbaenians as well as a robust phylogenetic hypothesis of this clade is needed in order to understand the evolutionary changes on amphisbaenian karyotypes.

Astyanax hastatus Myers, 1928 (Teleostei, Characidae): a new species complex within the genus Astyanax?

Four populations of Astyanax hastatus Myers 1928 from the Guapimirim River basin (Rio de Janeiro State) were analyzed and three distinct cytotypes identified. These cytotypes presented 2n = 50 chromosomes, with 4M+8SM+10ST+28A (Cytotype A), 8M+10SM+14ST+18A (Cytotype B), 6M+8SM+4ST+32A (Cytotype C) and scanty heterochromatin, mainly located throughout pericentromeric regions of several chromosomal pairs. No homologies with the As-51 satellite DNA were observed in the three cytotypes, although all of them presented multiple 18S rDNA sites, as detected by both silver nitrate staining and FISH (fluorescent in situ hybridization). The application of the term "species complex" in Astyanax is discussed from a cytotaxonomic viewpoint.

Localization of ribosomal genes in three Pimelodus species (Siluriformes, Pimelodidae) of the São Francisco River: 5S genes as species markers and conservation of the 18S rDNA sites

Pimelodidae is one of the most representative of Neotropical catfish families. However, these fish are still poorly studied in terms of cytogenetics, especially regarding the application of more accurate techniques such as the chromosomal localization of ribosomal genes. In the present work, fluorescent in situ hybridization with 5S and 18S rDNA probes was employed for rDNA site mapping in Pimelodus sp., P. fur and P. maculatus from the São Francisco River in the Três Marias municipality - MG. The results from the application of the 18S probe confirmed the previous data obtained by silver nitrate staining, identifying a simple nucleolar organizing region system for these species. However, the labeling results from the 5S rDNA probe demonstrated a difference in the number and localization of these sites between the analyzed species. The obtained data allowed inferences on the possible processes involved in the karyotypic evolution of this genus.

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.

Systematics of the neotropical fish subfamily Glandulocaudinae (Teleostei: Characiformes: Characidae)

The systematics of the Glandulocaudinae is reviewed in detail and justification for the recognition of the group as a subfamily is discussed. The subfamily Glandulocaudinae consists of three genera: Lophiobrycon with one species plesiomorphic in some anatomical features but some others exclusively derived relative to the species in the other genera; Glandulocauda with two species intermediate in phylogenetic derivation; and Mimagoniates with seven species (one new), all more phylogenetically derived concerning their pheromone producing caudal-fin organs and with other anatomical characters presumably more derived than in the species of the other genera. Glandulocauda melanogenys Eigenmann, 1911, is considered a junior synonym of Hyphessobrycon melanopleurus Ellis, 1911. A replacement name, Glandulocauda caerulea Menezes & Weitzman, is proposed for G. melanopleura Eigenmann, 1911. Gland cells found in the caudal-fin organs of all species are histologically indistinguishable from club cells and probably secrete a pheromone during courtship. The club cells are associated with somewhat modified to highly derived caudal scales forming a pheromone pumping organ in the more derived genera and species. This subfamily is distributed in freshwaters of eastern and southern Brazil, Paraguay, and northeastern Uruguay.

Population genetic structuring in pacu (Piaractus mesopotamicus) across the Paraná-Paraguay basin: evidence from microsatellites

The Paraná-Paraguay basin encompasses central western Brazil, northeastern Paraguay, eastern Bolivia and northern Argentina. The Pantanal is a flooded plain with marked dry and rainy seasons that, due to its soil characteristics and low declivity, has a great water holding capacity supporting abundant fish fauna. Piaractus mesopotamicus, or pacu, endemic of the Paraná-Paraguay basin, is a migratory species economically important in fisheries and ecologically as a potential seed disperser. In this paper we employ eight microsatellite loci to assess the population structure of 120 pacu sampled inside and outside the Pantanal of Mato Grosso. Our main objective was to test the null hypothesis of panmixia and to verify if there was a different structuring pattern between the Pantanal were there were no physical barriers to fish movement and the heavily impounded Paraná and Paranapanema rivers. All loci had moderate to high levels of polymorphism, the number of alleles varied from three to 18. The average observed heterozygosity varied from 0.068 to 0.911. After the Bonferroni correction three loci remained significant for deviations from Hardy-Weinberg, and for those the frequency of null alleles was estimated. F ST and R ST pairwise comparisons detected low divergence among sampling sites, and differentiation was significant only between Paranapanema and Cuiabá and Paranapanema and Taquari. No correlation between genetic distance and the natural logarithm of the geographic distance was detected. Results indicate that for conservation purposes and for restoration programs small genetic differences detected in the Cuiabá and Paranapanema rivers should be taken in consideration.

A phylogenetic analysis of Brycon and Henochilus (Characiformes, Characidae, Bryconinae) based on the mitochondrial gene 16S rRNA

The genus Brycon, the largest subunit of the Bryconinae, has 42 valid species distributed from southern Mexico to the La Plata River in Argentina. Henochilus is a monotypic genus, comprising a single species (H. wheatlandii) found in the upper Rio Doce basin. In the present study, partial sequences of the mitochondrial gene 16S were obtained for fifteen species of Brycon and for Henochilus wheatlandii. The results showed that the genus Brycon is paraphyletic, since Henochilus is the sister-group of B. ferox and B. insignis. The most basal species analyzed were the trans-Andean species B. henni, B. petrosus, and B. chagrensis.

Spatial and temporal distribution of fish in Palmas bay, Ubatuba, Brazil

The aim of this study was to analyze the distribution and abundance of the fish fauna of Palmas bay on Anchieta Island in southeastern Brazil. Specimens were caught in the summer and winter of 1992, using an otter trawl at three locations in the bay. The specimens were caught in both the nighttime and daytime. Data on the water temperature and salinity were recorded for the characterization of the predominant water mass in the region, and sediment samples were taken for granulometric analysis. A total of 7 656 specimens (79 species), with a total weight of approximately 300 kg, were recorded. The most abundant species were Eucinostomus argenteus, Ctenosciaena gracilicirrhus, Haemulon steindachneri, Eucinostomus gula and Diapterus rhombeus, which together accounted for more than 73% of the sample. In general, the ecological indices showed no differences in the composition of species for the abiotic variables analyzed. The multivariate analysis showed that the variations in the distribution of the fish fauna were mainly associated with intra-annual differences in temperature and salinity, resulting from the presence of South Atlantic Central Water (SACW) in the area during the summer. The analysis also showed an association with the type of bottom and a lesser association with respect to the night/day periods.

The ichthyofauna of upper rio Capivari: defining conservation strategies based on the composition and distribution of fish species

Although the rio Capivari basin is recognized as an area of great importance for the ichthyofauna, it lacks virtually every basic requirement for the definition of appropriate conservation strategies, since not even its species composition is known. The objective of this work is to determine the composition and distribution of fish species in the upper rio Capivari basin, relating them to the local physical features, and to evaluate the effectiveness of proposed conservations units, delimited based on areas of native vegetation remains, on the conservation of local ichthyofauna. During 2007, 50 different watercourses were sampled with gillnets, cast nets and kick nets. A total of 1308 individuals belonging to 41 species were captured. Degree of conservation, altitude and width were the parameters that best explained fish species richness. Considering the recently proposed boundaries for potential conservation units in the region only 15 or 20 out of 41 species would be found in the State Park and Environmental Protection Area respectively. In practice, the proposed conservation units would not be effective tools for fish conservation, since it would be located in mountainous areas of high altitude, of headwaters streams and where few species are found. In such context, the conservation of specific stretches of larger rivers is critical.

On the name of the lepidophagous characid fish Roeboexodon guyanensis (Puyo) (Teleostei: Characiformes: Characidae)

The arguments for the usage of the names Gnathoplax, Roeboexodon, R. geryi, and R. guyanensis are revised. Based on the literature and museum specimens, we recommend that the neotype of Exodon guyanensis should be considered valid; that the genus Gnathoplax should be considered an objective junior synonym of Roeboexodon; and that Roeboexodon geryi should be considered an objective junior synonym of Exodonguyanensis.