Genome-wide characterization of microsatellites in cobia Rachycentron canadum (Linnaeus, 1766): Survey and analysis of their abundance and diversity.

The cobia Rachycentron canadum, mainly distributed in the warm waters of tropical and subtropical regions around the world, remains a fish of considerable economic importance. Detailed diversity and the number of microsatellite sequences in the cobia genome are still unintelligible. The primary aim of this work was to identify and quantify the miscellaneous SSR sequences in the cobia genome. More than 280,000 sequences were sequenced and screened using next-generation sequencing technology and microsatellite identification. Perfect mononucleotide repeats, dinucleotide microsatellites, and trinucleotide microsatellites contain (A)10 /(T)10 , (AC)6 /(TG)6 , and (AAT)5-32 as the largest number of motifs in each type of microsatellite, respectively. The tetranucleotide and pentanucleotide microsatellites (TTM and PTM) consist of the largest number of motifs of both (ATCT)5-32 and (TCAT)5-31 in TTMs, and (CTCTC)5-9 in PTMs, whereas the hexanucleotide microsatellites are rarely observed in the cobia genome. All c. 38000 sequences of composite microsatellites are extremely diverse, including compound (11.71%), interrupted compound (71.77%), complex (0.45%), and interrupted complex (16.07%). In this study, we developed a convenient and useful recording system for writing down and categorizing diverse composite microsatellite types. This system will provide great support for exploring repeat origins, evolutionary mechanisms, and the application of polymorphic microsatellites.

Comparative Studies of 5S rDNA Profiles and Cyt b Sequences in two Onychostoma Species (Cyprinidae).

Onychostoma barbatulum and O. alticorpus, two primarily freshwater cyprinid fish, have similar morphological characters and partially overlapping ecological habitats. In order to explore the genetic differences between these two species, chromosomal characteristics and genetic variations were examined by fluorescence in situ hybridization (FISH) of 5S rDNA and cytochrome (Cyt) b gene analysis. Ten specimens of O. barbatulum and O. alticorpus were collected from the Nanzihsian Stream in southern Taiwan. FISH revealed that the 5S rDNA loci of O. barbatulum and O. alticorpus were found at a pericentromeric and subtelomeric position, respectively, in a pair of submetacentric chromosomes. Cyt b genes were amplified and sequenced from five individuals of each species. Intraspecific genetic distances ranged from 0.001-0.004 in O. barbatulum and from 0.001-0.006 in O. alticorpus. Genetic distances between these two species ranged from 0.132-0.142. The phylogenetic tree showed these two species are not sister species. In conclusion, FISH cytogenetic information and Cyt b gene analyses indicated that these two species have significantly different genetic characteristics; nevertheless, their morphological similarities may be due to environmental adaptation.

Evolution of microsatellite Loci of tropical and temperate anguilla eels.

Anguilla eels are divided into temperate and tropical eels, based on their major distributions. The present study collected two temperate eels, Anguilla japonica and Anguilla anguilla, and two tropical eels, Anguilla marmorata and Anguilla bicolor pacifica, to examine two questions: do temperate and tropical Anguilla eels have different genetic polymorphic patterns?; and do temperate Anguilla japonica and Anguilla anguilla have a closer relationship to each other than to tropical eels? In total, 274 sequences were cloned and sequenced from six conserved microsatellite loci to examine polymorphic patterns of these four catadromous eels. Different mutational events, including substitutions, and repeat-unit deletions and insertions, appeared in major regions, while different point mutations were observed in flanking regions. The results implied that parallel patterns of microsatellite sequences occurred within both tropical and temperate freshwater eels. Consensus flanking sequences of six homologous loci from each of the four species were constructed. Genetic distances ranged from 0.044 (Anguilla bicolor pacifica vs. Anguilla marmorata) to 0.061 (Anguilla marmorata vs. Anguilla anguilla). The tree topology suggests the hypothesis of Anguilla japonica and Anguilla anguilla being a sister group must be rejected.

Screening of new microsatellite DNA markers from the genome of Platyeriocheir formosa.

The catadromous Platyeriocheir formosa is a crab endemic in Taiwan. To conserve P. formosa population diversity and ensure the sustainable use of this natural resource, we have developed new genetic markers, 17 polymorphic microsatellite loci, to promote the study of its population genetics in the future. In this study, more than 70 microsatellite sequences were found. Among these, 18 loci were selected to analyze the genetic diversity of P. formosa. With the exception of the Pfo15 locus, all of the remaining loci were polymorphic with allelic numbers ranging from 3-14. Heterozygosity within all 17 polymorphic loci ranged from 0.2-0.95 with an average of 0.55, which suggested that these loci are proper markers for studying population genetics. After we tested cross-specific amplification, eight and six primer sets could be successfully used for the amplification of microsatellite loci in morphologically similar Eriocheir sinensis and E. japonica, respectively; this suggests that they are useful markers for closely related species.

Variation in the Karyotype, Cytochrome b Gene, and 5S rDNA of Four Thunnus (Perciformes, Scombridae) Tunas.

Yan-Horn Lee, Tsair-Bor Yen, Chiu-Fen Chen, and Mei-Chen Tseng (2018) Thunnus tunas in Scombridae are divided into the temperate subgenus Thunnus (bluefin group) and tropical subgenus Neothunnus (yellowfin group) species based on anatomic traits and distributions. The main purpose of this study was to examine the systematic status of T. obesus based on karyotype, cytochrome (Cyt) b gene, and 5S ribosomal DNA sequences. All T. obesus, T. albacares, T. alalunga, and T. orientalis specimens were caught in southeastern coastal waters off the main island of Taiwan. The karyotypical formula of T. obesus was 2 m + 2 st + 44 t, that of T. albacares was 2 m + 2 sm + 2 st + 42 t, that of T. alalunga was 2 m + 2 sm + 2 st + 42 t, and that of T. orientalis was 2 m + 2 sm + 44 t (m: metacentric; sm: submetacentric; st: subtelocentric; t: telocentric chromosome). According to a molecular genetics analysis for these species using Cyt b gene sequences (1141 bp), interspecific genetic distances ranged from 0.004 (T. orientalis vs. T. alalunga) to 0.038 (T. alalunga vs. T. obesus). The genealogy tree exhibited these 4 species as being categorized into 4 monophyletic groups with high bootstrapping values; T. alalunga and T. orientalis are sister species. This result suggests that the species currently allocated in Thunnus and Neothunnus might need new taxonomic characters to redefine the monophyly of the two subgenera. The sequence lengths of all cloned 5S genes from the 4 species ranged from 327-342 bp. Interspecific genetic distances ranged from 0.016 (T. orientalis vs. T. alalunga) to 0.111 (T. orientalis vs. T. albacares). The phylogenetic tree based on 5S rDNA shows T. obesus divided into 2 groups: one similar to T. albacares and the other close to T. orientalis. These results imply that Thunnus tunas have a common synapomorphic character with Scombridae fish (2n = 48) and high numbers of telocentric chromosomes (42-44). Thunnus orientalis and T. alalunga are sister based on molecular data. Thunnus obesus may have been derived from a more-complicated speciation processes.

Comparative Study on Hatching Rate, Survival Rate, and Feminization of Onychostoma barbatulum (Pellegrin, 1908) at Different Temperatures and Examining Sex Change by Gonad and Karyotype Analyses.

Mei-Chen Tseng, Dian-Hao Yang, and Tsair-Bor Yen (2017) Onychostoma barbatulum has become an aquaculture species with high economic value in Taiwan. It was observed that females can grow faster to a larger size than males on aquaculture farms. Therefore, development of feminized fry can increase farm profits in the future. The purpose of this study was to establish the optimal incubation, feed, and feminization temperatures to produce a high feminization ratio and better survival rates for O. barbatulum fry. The performance mode of sex-determination by temperature was also explored in the study. Adults were collected from Nanzixian Stream in southwestern Taiwan and artificially propagated in tanks. Fertilized eggs were placed in environments of different temperatures to determine the hatching rate (n = 3000) and the survival rate (n = 3000) of fry. After 6 months, the sex ratio was established from gonad tableting (n = 360). A karyotype analysis (n = 86) was also conducted to verify the existence of gender-reversed individuals. The results showed that hatching rates at 17.5, 19.5, 21.5, 23.5, and 25.5°C were 70.7%, 67.3%, 73.3%, 33.7%, and 34.7%, respectively. Survival ratios from low to high temperatures were 34.7%, 47.7%, 33.7%, 12.3%, and 23.3%, respectively. These results indicated that both the hatching rates and survival ratios of fry performed poorly at temperatures higher than 21.5°C. The performance mode of sex-determination by temperature of O. barbatulum revealed that female ratios significantly decreased at the two extremes of the temperature range, while the female ratio was highest at an intermediate temperature. The best feminized ratio (83.4%) was observed at 21.5°C among all tested temperatures (p < 0.05). Meanwhile according to a karyotype analysis, sex-reversed individuals were found in each group, indicating that temperature is a critical phenotypic sex-determining factor. Therefore, rearing O. barbatulum fry at a proper water temperature can effectively increase the female sex ratio and maintain high hatchability and survival ratios. These results can potentially be applied to produce a high proportion of female fry on aquaculture farms.

Cytogenetics of Two Onychostoma Species in Taiwan by Ag-NOR and 18S rDNA Profiles.

Chiao-Chuan Han, Tsair-Bor Yen, Nian-Cih Chen, and Mei-Chen Tseng (2017) Both Onychostoma barbatulum and O. alticorpus are primary freshwater fish in Taiwan. The former has been developed as an aquaculture species with high economic value, while the latter is a native endemic species in Taiwan. Understanding the cytogenetic information of these two species is necessary for their selected breeding, recovery, and management. In this study, Giemsa staining, silver-binding nucleolar organizer region (Ag-NOR), C-banding, and fluorescence in situ hybridization (FISH) with 18S ribosomal (r)DNA probes were used to analyze the cytogenetic characteristics. Results of Giemsa staining showed that the two Onychostoma species shared the same number of chromosomes, 2n = 50. Respective karyotype formulas of the female and male were 10 m + 22 sm + 10 st + 8 t and 11 m + 22 sm + 10 st + 7 t in O. barbatulum, and 14 m + 18 sm + 8 st + 10 t and 15 m + 18 sm + 8 st + 9 t in O. alticorpus. Karyotypes of both species showed a pair of heteromorphic chromosomes in male fish. Their sex determination should be the XX/XY system. Two pairs of Ag-NORs were found in O. barbatulum, but only one pair occurred in O. alticorpus. C-banding areas were observed on centromeres or telomeres of some chromosomes. FISH revealed different cytogenetic characters between these two species. The above cytogenetic information will contribute to species identification, population recovery, and advantages for breeding and management in the future.

Serotype-specific detection of enterovirus 71 in clinical specimens by DNA microchip array.

Enterovirus 71 is an important pathogen that causes high morbidity and mortality in children in Taiwan. Virus isolation in cell cultures has been the standard method for enterovirus 71 identification in Clinical Virology Laboratories. However, virus isolation takes 5-10 days when using cell culture. A microchip for enterovirus 71 detection was developed as an alternative diagnostic method. The novel approach is based on hybridization of amplified DNA specimens with oligonucleotide DNA probes immobilized on a microchip. Two oligonucleotides were used as detection probes, the pan-enterovirus sequence located in the 5'-noncoding region (5'-NCR) and the enterovirus 71-specific sequence located in the VP2 region. The diagnostic procedure takes 6 h. One hundred specimens identified as enteroviruses by viral cultures were tested using this microchip, including 67 enterovirus 71 specimens. The sensitivity of the novel method is 89.6% and its specificity is 90.9%. The enterovirus 71-microchip can detect the amplicon derived from viral RNA corresponding to 1-10 virions in a clinical specimen. Microchip array is a potential diagnostic method for identification of enterovirus in the future.

Behavioral effects of titanium dioxide nanoparticles on larval zebrafish (Danio rerio).

In this study, zebrafish embryos were exposed to titanium dioxide nanoparticles (TiO2 NPs at 0.1, 0.5, 1, 5, 10 mg/L or control) from fertilization to free swimming stage. Hatchability, survival, and malformation rate were not affected by TiO2 NPs at these exposure levels. However, larval swimming parameters, including average and maximum velocity and activity level were significantly affected by TiO2 NPs. Co-exposure to either the glutathione precursor, N-acetylcysteine (NAC), or the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), did not significantly alter the behavioral effects resulting from TiO2 NPs, suggesting that other factor(s) besides oxidative stress may contribute to the behavioral toxicity of TiO2 NPs. Our study also demonstrated that the behavioral endpoints were more sensitive than the others (e.g., hatchability and survival) to detect toxicity of TiO2 NPs on developing fish.

Identification and cloning of the antioxidant enzyme, glutathione peroxidase, of white shrimp, Litopenaeus vannamei, and its expression following Vibrio alginolyticus infection.

cDNA encoding glutathione peroxidase (GPx) mRNA of the white shrimp Litopenaeus vannamei was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA (RACE) using oligonucleotide primers based on the GPx sequence of Homo sapiens (NM002083), Mus musculus (NM008160), Arabidopsis thaliana (U94495), Bos taurus (NM174770), and Capsicum chinense (AJ973135). The 727-bp cDNA contained an open reading frame (ORF) of 567 bp, a 101-bp 5'-untranslated region, and a 59-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid (aa) sequence (189 aa) was 19.25 kDa long with an estimated pI of 8.39. It contains a putative selenocysteine residue which is encoded by the unusual stop codon, TGA, and forms the active site with residues Glu(75) and Trp(153). Comparison of amino acid sequences showed that white shrimp GPx is more closely related to GPx1 and GPx2 than to GPx3 and GPx4 of various animals. The GPx cDNA was synthesized in haemocytes, gills, the hepatopancreas, intestines, and muscles. The respiratory bursts of shrimp increased significantly after a Vibrio alginolyticus injection in order to kill the pathogen, and then induced increases in the activities of SOD and GPx to protect cells against damage from oxidation. However, GPx activity increased as a result of upregulated expression of GPx mRNA which was induced by the increase in H(2)O(2).