Investigations into the perplexing interrelationship of the Genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships within the genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae) remain unresolved. Because of the use of Takifugu as model organisms, the resolution of these relationships is crucial for the interpretation of evolutionary trends in biology. Pufferfishes of this genus are comprised of a comparatively small number of species and are mainly distributed along the coastal region of the western part of the Sea of Japan and the coastline of China. Mitochondrial gene sequences were employed to test the phylogenetic hypotheses within the genus. Seventeen species of the genus were examined. Molecular phylogenetic trees were constructed using the maximum parsimony, neighbor-joining, maximum likelihood and Bayesian methods. Our hypothesis of internal relationships within the genus differs from previous hypotheses. Our results indicate that (1) the genus Takifugu is a monophyletic assemblage; (2) the genus is divided into 6 subgroups based on the molecular data; and (3) there is low genetic diversity among the species within this genus. In addition, speciation within Takifugu appears to be driven by hybridization and isolation by distribution. Our results also suggested that the taxonomy in the genus should be clarified based on both molecular and morphological data.

Molecular phylogeny and species identification of pufferfish of the genus Takifugu (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships and species identification of pufferfishes of the genus Takifugu were examined by use of randomly amplified polymorphic DNA (RAPD) and sequencing of the amplified partial mitochondrial 16S ribosomal RNA genes. Amplifications with 200 ten-base primers under predetermined optimal reaction conditions yielded 1962 reproducible amplified fragments ranging from 200 to 3000 bp. Genetic distances between 5 species of Takifugu and Lagocephalus spadiceus as the outgroup were calculated from the presence or absence of the amplified fragments. Approximately 572 bp of the 16S ribosonial RNA gene was amplified, using universal primers, and used to determine the genetic distance values. Topological phylogenic trees for the 5 species of Takifugu and outgroup were generated from neighbor-joining analysis based on the data set of RAPD analysis and sequences of mitochondrial 16S rDNA. The genetic distance between Takifugu rubripes and Takifugu pseudommus was almost the same as that between individuals within cacti species, but much smaller than that between T. rubripes, T. pseudommus, and the other species. The molecular data gathered from both analysis of mitochondria and nuclear DNA strongly indicated that T. rubripes and T. pseudommus should be regarded as the same species. A fragment of approximately 900 bp was amplified from the genome of all 26 T. pseudommus individuals examined and 4 individuals of intermediate varieties between T. rubripes and T. pseudommus. Of the 32 T. rubripes individuals, only 3 had the amplified fragment. These results suggest that this fragment may be useful in distinguishing between T. rubripes and T. pseudommus.

Processos carioevolutivos na ordem tetraodontiformes: uma visão através de suas diferentes linhagens

Given the great diversity of fishes, the Order Tetraodontiformes stands to show genetic and morphological characteristics enough singular. The fishes of this order have a compact DNA which favors molecular studies, as well as comparisons with more basal species. Model of genome evolution, there are still many gaps in knowledge about their chromosomal patterns and how evolutionary rearrangements influence the marked variation in DNA content of this order. In view of this, we present cytogenetic analyzes of the species Acanthostracion quadricornis (Ostraciidae), A. polygonius (Ostraciidae) Melichthys niger (Balistidae) Cantherhines macrocerus (Monacanthidae) and C. pullus (Monacanthidae), Lagocephalus laevigatus, Colomesus psittacus and Canthigaster figueiredoi (Tetraodontidae), to contribute with cytogenetic data for this group. The analysis was performed by C-banding, Ag-RONs, coloring with base-specific fluorochromes DAPI-CMA3, restriction enzymes AluI, EcoRI, TaqI, PstI and HinfI and in situ hybridization with probes for ribosomal DNA 18S and 5S. The heterochromatic ultrastructure of A. quadricornis and A. polygonius revealed a outstanding heterochromatin content, which may indicate that the accumulation or loss of extensive heterochromatin content could be responsible for large variations in genomic content displayed in different Tetraodontiformes families. The species Cantherhines macrocerus, C. pullus (Monacanthidae) and Melichthys niger (Balistidae) shows a huge karyotypic similarity both numerically and structural. L. laevigatus showed similar cytogenetic features (2n = 44 and single RONs) to the species of the genus Takifugu, which reinforces the idea of their phylogenetic relationships. C. psittacus presented the highest diploid number described for the family (2n = 56) and large amount of HC, features that related with its sister family Diodontidae. Cytogenetic analysis in C. figueiredoi revealed heterochromatic polymorphisms, RONs multiple and Bs chromosomes. These events are rare in marine fishes, and are possibly associated with the strong restructuring and genomic reduction that this family has been suffered. These features, plus the morphological and molecular data suggests that these species share the same ancestral branch, with a possible monophyletic origin. In this study, new contributions to the knowledge of evolutionary patterns facing by Tetraodontiformes are provided and discussed under cytotaxonomyc, genomic and evolutionary perspectives.

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Padrões cariotípicos em tetraodontiformes (Osteichthypes) :redução genômica e mecanismos de diversificação

The Tetraodontiformes order is composed for about 400 species of fish, distributed in ten families, with circuntropical distribution. The morphologic diversity of each family reflects in, part, the different levels of specialization. This group represents an ancestry after-Perciformes and constitutes the last branch of the diffusion of the Teleósteos, occupying a position of prominence. The phylogenetics relationships of the Tetraodontiformes exist diverse works examining and, in all, these families are recognized as groups brothers, being Diodontidae next to Tetraodontidae and Balistidae next to Monacanthidae. Although it possesss a representative number of species, the works involving of the families Balistidae and Monacanthidae are few exemplary, especially species of oceanic islands. In this work cytogenetic studies in five species had been analyzed Cantherhines macrocerus, Cantherhines pullus (Monacanthidae), Melichthys niger (Balistidae), Sphoeroides testudíneus (Tetraodontidae) and Chilomycterus antennatus (Diodontidae); through conventional coloration, Ag-NORs and C banding. Ahead of the different karyological trends of evolution presented by the Tetraodontiformes, the present work also searched to verify the relation existence enters the total size of the chromosomes with the amount of DNA in these groups of Tetraodontiformes. For such, they had been correlated the total size of the chromosomes of these species, with values of content of available DNA in literature. The cytogenetics analyses for the species C.macrocerusJ C.pullus (Monacanthidae) and M.niger (Balistidae), had disclosed 40 chromosomes, all acrocentrics. All possess only one pair of NORs and pericentromeric heterochromatin. For S.testudíneus the found dyploid number was equal 2n=46, with NF=78 (16m+18sm+8st+4a), while that for C.antennatus it possesss 2n=50, with NF=76 (4m+22st+24a). Both species possess simple NORs and pericentromeric heterochromatin blocks. In M.niger, the presence of positive marking (heterochromatin and NOR) in the secondary constriction in the second chromosomic pair suggesting the occurrence of a rearrangement, possibly a fusing involving these homologous ones, indicating that these events had been important for the establishment of the karyological history of this group. A maintenance of the chromosomic constancy found in the populations of C.macrocerus (Monacanthidae) and S.testudineus (Tetraodontidae) perhaps if must for the aiding of the gene flow through oceanic chains. These data contrast with the differentiated kinds of chromosomes of C.antennatus between the Northeast coast and Southeastern, suggesting that the ecological standards of each species, added to the conditions of the marine environment, can be responsible for the karyological delineation of each species. The found characteristics for the species C.macrocerus, C.pullus, M.niger, S.testudineus and C.antennatus add it the available data for other species of Tetraodontiformes. From the data gotten in the present study, it can be inferred that the DNA content possesss direct relation with the total length of the chromosomes

Population biology and diet of the puffer fish Lagocephalus laevigatus (Tetraodontiformes: Tetraodontidae) in Caraguatatuba Bay, south-eastern Brazil

This study describes the spatio-temporal distribution, population biology, and diet of the puffer fish Lagocephalus laevigatus in Caraguatatuba Bay, south-eastern Brazil. Monthly samples were taken between August 2003 and October 2004 by trawls in two areas, south and north, at depths of 1 to 4 m. The fish were measured and their sex and reproductive stage determined. The abundance of this species was compared between areas and among months, and the items in the diet were identified and quantified. Lagocephalus laevigatus was rare in Caraguatatuba Bay, where only 199 small individuals (4.8 to 15.4 cm) were obtained in the entire study period, suggesting that this species uses the estuary as a nursery. None of the specimens of L. laevigatus captured in Caraguatatuba Bay were sexually mature. Higher densities of L. laevigatus in the bay were recorded in the south area and between October and December 2003, i.e. in the spring, suggesting that spawning may occur from late winter to spring (August through to November). The diet items consumed by L. laevigatus in Caraguatatuba Bay were, as expected from the current literature, crustaceans, mainly amphipods, and fish. However, the most-consumed item was the sea whip Leptogorgia setacea (Cnidaria). This feeding habit may be related to the presence of toxins (tetrodotoxin and saxitoxin) that are frequently found in the skin and viscera of L. laevigatus, which may be sequestered from the sea whip, which possibility still needs to be specifically evaluated.

Branchial vascular pathways in two species of Tetraodontiformes and the concept of secondary vessels and nutrient arteries

The vascular organisation of the branchial basket was examined in two Tetraodontiform fishes; the three-barred porcupinefish, Dicotylichthys punctulatus and the banded toadfish, Marylina pleurosticta by scanning electron microscopy of vascular casts and standard histological approaches. In D. punctulatus, interarterial anastomoses (iaas) originated at high densities from the efferent filamental and branchial arteries, subsequently re-anastomosing to form progressively larger secondary vessels. Small branches of this system entered the filament body, where it was interspersed between the intrafilamental vessels. Large-bore secondary vessels ran parallel with the efferent branchial arteries, and were found to constitute an additional arterio-arterial pathway, in that these vessels exited the branchial basket in company with the mandibular, the carotid and the afferent and efferent branchial arteries, from where they gave rise to capillary beds after exit. Secondary vessels were not found to supply filament muscle; rather these tissues were supplied by single specialised vessels running in parallel between the efferent and afferent branchial arteries in both species examined. Although the branchial vascular anatomy was generally fairly similar for the two species examined, iaas were not found to originate from any branchial component in the banded toadfish, M. pleurosticta, which instead showed a moderate frequency of iaas on other vessels in the cephalic region. It is proposed that four independent vascular pathways may be present within the teleostean gill filament, the conventional arterio-arterial pathway across the respiratory lamellae; an arterio-arterial system of secondary vessels supplying the filament and non-branchial tissues; a system of vessels supplying the filament musculature; and the intrafilamental vessels (central venous sinus). The present study demonstrates that phylogenetic differences in the arrangement of the branchial vascular system occur between species of the same taxon.

Comparison of sample preparation methods, validation of an UPLC-MS/MS procedure for the quantification of tetrodotoxin present in marine gastropods and analysis of pufferfish

Tetrodotoxin (TTX) is one of the most potent marine neurotoxins reported. The global distribution of this toxin is spreading with the European Atlantic coastline now being affected. Climate change and increasing pollution have been suggested as underlying causes for this. In the present study, two different sample preparation techniques were used to extract TTX from Trumpet shells and pufferfish samples. Both extraction procedures (accelerated solvent extraction (ASE) and a simple solvent extraction) were shown to provide good recoveries (80-92%). A UPLC-MS/MS method was developed for the analysis of TTX and validated following the guidelines contained in the Commission Decision 2002/657/EC for chemical contaminant analysis. The performance of this procedure was demonstrated to be fit for purpose. This study is the first report on the use of ASE as a mean for TTX extraction, the use of UPLC-MS/MS for TTX analysis, and the validation of this method for TTX in gastropods.

Ecomorfologia de dez espécies de peixes marinhos mais abundantes de Galinhos/RN

Ecomorphology is a science based on the idea that morphological differences among species could be associated with distinct biological and environmental pressures suffered by them. These differences can be studied employing morphological and biometric indexes denominated Ecomorphological attributes , representing standards that express characteristics of the individual in relation to its environment, and can be interpreted as indicators of life habits or adaptations suffered due its occupation of different habitats. This work aims to contribute for the knowledge of the ecomorphology of the Brazilian marine ichthyofauna, specifically from Galinhos, located at Rio Grande do Norte state. 10 different species of fish were studied, belonging the families Gerreidae (Eucinostomus argenteus), Haemulidae (Orthopristis ruber,Pomadasyscorvinaeformis,Haemulonaurolineatum,Haemulonplumieri,Haemulonsteindachneri), Lutjanidae (Lutjanus synagris), Paralichthyidae (Syaciummicrurum), Bothidae (Bothus ocellatus) and Tetraodontidae (Sphoeroidestestudineus), which were obtained during five collections, in the period time of September/2004 to April/2005, utilizing three special nets. The ecomorphological study was performed at the laboratory. Eight to ten samples of each fish specie were measured. Fifteen morphological aspects were considered to calculate twelve ecomorphological attributes. Multivariate statistical analysis methods such as Principal Component Analysis (PCA) and Cluster Analysis were done to identify ecmorphological patterns to describe the data set obtained. As results, H.aurolineatumwas the most abundant specie found (23,03%) and S.testudineusthe less one with 0,23%. The 1st Principal component showed variation of 60,03% with influence of the ecomorphological attribute related to body morphology, while the 2nd PC with 23,25% variation had influence of the ecomorphological attribute related to oral morphology. The Cluster Analiysis promoted the identification of three distinct groups Perciformes, Pleuronectiformes and Tetraodontiformes. Based on the obtained data, considering morphological characters differences among the species studied, we suggest that all of them live at the medium (E.argenteus,O.rubber, P.corvinaeformis,H.aurolineatum,H.plumieri,H.steindachneri,L.synagris) and bottom (S.micrurum,B.ocellatus,S.testudineus) region of column water.

Estudios citogenéticos en teleosteos marinos y dulceacuícolas de venezuela

Pós-graduação em Ciências Biológicas (Zoologia) - IBB

Systematic Variation in the Pattern of Gene Paralog Retention between the Teleost Superorders Ostariophysi and Acanthopterygii

Teleost fish underwent whole-genome duplication around 450 Ma followed by diploidization and loss of 80-85% of the duplicated genes. To identify a deep signature of this teleost-specific whole-genome duplication (TSGD), we searched for duplicated genes that were systematically and uniquely retained in one or other of the superorders Ostariophysi and Acanthopterygii. TSGD paralogs comprised 17-21% of total gene content. Some 2.6% (510) of TSGD paralogs were present as pairs in the Ostariophysi genomes of Danio rerio (Cypriniformes) and Astyanax mexicanus (Characiformes) but not in species from four orders of Acanthopterygii (Gasterosteiformes, Gasterosteus aculeatus; Tetraodontiformes, Tetraodon nigroviridis; Perciformes, Oreochromis niloticus; and Beloniformes, Oryzias latipes) where a single copy was identified. Similarly, 1.3% (418) of total gene number represented cases where TSGD paralogs pairs were systematically retained in the Acanthopterygian but conserved as a single copy in Ostariophysi genomes. We confirmed the generality of these results by phylogenetic and synteny analysis of 40 randomly selected linage-specific paralogs (LSPs) from each superorder and completed with the transcriptomes of three additional Ostariophysi species (Ictalurus punctatus [Siluriformes], Sinocyclocheilus species [Cypriniformes], and Piaractus mesopotamicus [Characiformes]). No chromosome bias was detected in TSGD paralog retention. Gene ontology (GO) analysis revealed significant enrichment of GO terms relative to the human GO SLIM database for growth, Cell differentiation, and Embryo development in Ostariophysi and for Transport, Signal Transduction, and Vesicle mediated transport in Acanthopterygii. The observed patterns of paralog retention are consistent with different diploidization outcomes having contributed to the evolution/diversification of each superorder.

Evolution and genomic organization of muscle microRNAs in fish genomes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Moravecia australiensis n. g., n. sp (Dracunculoidea : Guyanemidae) from the gills of the green porcupine fish Tragulichthys jaculiferus (Cuvier) in Australia

A new dracunculoid genus and species, Moravecia australiensis, is described from gill-filaments of the green porcupine fish Tragulichthys jaculiferus (Cuvier) (Tetraodontiformes: Diodontidae) from Moreton Bay, Queensland, Australia. Abundant mobile larvae and a few adult males with females occurred in the gill-filament between the epithelial basement membrane and efferent artery. Gills of all 69 fish examined contained larvae. Eleven harboured adult nematodes of a previously undescribed species belonging to the family Guyanemidae. The new species is placed within a newly proposed genus because it differs from the four existing genera in the family in possessing fine cuticular transverse striations, two forward protruding cephalic elevations, a circumoral elevation, a small triangular mouth surrounded by six cephalic papillae arranged in two lateral clusters of three each and a pair of large lateral amphids. Males have two pairs of pedunculate caudal papillae supporting the caudal alae. A key to the genera of the Guyanemidae is presented.

Ecomorfologia de dez espécies de peixes marinhos mais abundantes de Galinhos/RN

Ecomorphology is a science based on the idea that morphological differences among species could be associated with distinct biological and environmental pressures suffered by them. These differences can be studied employing morphological and biometric indexes denominated Ecomorphological attributes , representing standards that express characteristics of the individual in relation to its environment, and can be interpreted as indicators of life habits or adaptations suffered due its occupation of different habitats. This work aims to contribute for the knowledge of the ecomorphology of the Brazilian marine ichthyofauna, specifically from Galinhos, located at Rio Grande do Norte state. 10 different species of fish were studied, belonging the families Gerreidae (Eucinostomus argenteus), Haemulidae (Orthopristis ruber,Pomadasyscorvinaeformis,Haemulonaurolineatum,Haemulonplumieri,Haemulonsteindachneri), Lutjanidae (Lutjanus synagris), Paralichthyidae (Syaciummicrurum), Bothidae (Bothus ocellatus) and Tetraodontidae (Sphoeroidestestudineus), which were obtained during five collections, in the period time of September/2004 to April/2005, utilizing three special nets. The ecomorphological study was performed at the laboratory. Eight to ten samples of each fish specie were measured. Fifteen morphological aspects were considered to calculate twelve ecomorphological attributes. Multivariate statistical analysis methods such as Principal Component Analysis (PCA) and Cluster Analysis were done to identify ecmorphological patterns to describe the data set obtained. As results, H.aurolineatumwas the most abundant specie found (23,03%) and S.testudineusthe less one with 0,23%. The 1st Principal component showed variation of 60,03% with influence of the ecomorphological attribute related to body morphology, while the 2nd PC with 23,25% variation had influence of the ecomorphological attribute related to oral morphology. The Cluster Analiysis promoted the identification of three distinct groups Perciformes, Pleuronectiformes and Tetraodontiformes. Based on the obtained data, considering morphological characters differences among the species studied, we suggest that all of them live at the medium (E.argenteus,O.rubber, P.corvinaeformis,H.aurolineatum,H.plumieri,H.steindachneri,L.synagris) and bottom (S.micrurum,B.ocellatus,S.testudineus) region of column water.