Assessing Genetic Diversity of Wild Populations and Different Color Varieties for Genetic Improvement of Siamese Fighting Fish Betta splendens in Thailand.

To establish sustainable resources and founder populations for genetic improvement of the Siamese fighting fish Betta splendens, genetic diversity in wild and hatchery stocks was examined using mitochondrial (mt) DNA genes cytochrome b (cytb), 16S ribosomal DNA (16S rDNA), and cytochrome oxidase subunit I (COI), and eight microsatellite loci. Based on mtDNA sequences, restrictive levels of polymorphism (0, 3, and 1 substitutions) were observed in this study. For analysis of microsatellites, fluorescent multiplex PCR was developed, and subsequently identifying moderate levels of observed (Ho = 0.4488) and expected (He = 0.6627) heterozygosities and a high number of alleles per locus (15.125 alleles) for overall samples. Comparison of Siamese fighting fish from different sources revealed large genetic differences between pairs of farmed fish (eight groups) and between wild (three geographic locations) and farmed fish (P < 0.0031 following Bonferroni correction). This suggested limited exchanges of genetic resources between commercial farms. When different color varieties of B. splendens were compared, large genetic distances and significant FST estimates and genetic heterogeneity were found (P < 0.0031). Effective population sizes (Ne) were estimated and two farms (NP2-2BS and BK1-4BS) showed Ne greater than 10. Among color varieties, Multi-colors and Blue revealed reasonable Ne (large and 27.9), but lower Ne values (3.6-8.4) were found for the remaining color varieties. These results indicate an urgent need for the establishment of gene pool resources of B. splendens for effective genetic improvement of Siamese fighting fish in Thailand.

Development of polymorphic microsatellites for genetic studies of white scar oyster (Crassostrea belcheri) using paired-end shotgun sequencing.

White scar oyster Crassostrea belcheri is a commercially important bivalve species in Thailand. Appropriate genetic markers are needed for effective management to elevate its production efficiency. Type II microsatellites of C. belcheri were identified and characterized using an Illumina paired-end shotgun sequencing. A total of 14,743,710 reads were generated for which 198,849 reads containing microsatellites and 217,998 microsatellite loci were found. Twenty out of 60 microsatellite loci (33.33%) were polymorphic and these microsatellites were further tested against DNA bulks (N = 10 each) originating from 7 different geographic locations in Thai waters. Results indicated that newly developed microsatellites can be used for genetic diversity analysis of C. belcheri. Genotyping of C. belcheri collected from Surat Thani (Gulf of Thailand; N = 50) were performed. The number of alleles per locus ranged from 2 to 12 (average = 4.95). Observed and expected heterozygosities ranged from 0.0000 to 0.9400 (average = 0.3419) and 0.1139 to 0.8190 (average = 0.5844), respectively. Genome information and 20 newly isolated microsatellites will facilitate further studies in population genetics, stock management, and genetic improvement of C. belcheri in Thailand.

Identification of a Growth-Associated Single Nucleotide Polymorphism (SNP) in Cyclin C of the Giant Tiger Shrimp Penaeus monodon.

The full-length cDNA of cyclin C of the giant tiger shrimp Penaeus monodon (PmCyC) was isolated by RACE-PCR. It was 1443 bp in length containing an open reading frame (ORF) of 804 bp and 267 deduced amino acids. Tissue distribution analysis indicated that PmCyC was more abundantly expressed in ovaries and testes than other tissues of female and male juveniles (P < 0.05). A pair of primers was designed, and an amplification product of 403 bp containing an intron of 123 bp was obtained. Polymorphism of amplified PmCyC gene segments of the 5th (3-month-old G5, N = 30) and 7th (5-month-old G7, N = 18) generations of domesticated juveniles was analyzed. Four conserved SNPs (T>C134, T>C188, G>A379, and T>C382) were found within the examined sequences. A TaqMan genotyping assay was developed for detection of a T>C134 SNP. Association analysis indicated that this SNP displayed significant association with body weight (P < 4.2e-10) and total length (P < 2e-09) of the examined G7 P. monodon (N = 419) with an allele substitution effect of 5.02 ± 0.78 g and 1.41 ± 0.19 cm, respectively. Juveniles with C/C134 (22.80 ± 2.51 g and 12.97 ± 0.53 cm, N = 19) and T/C134 (20.41 ± 0.93 g and 12.77 ± 0.21 cm, N = 129) genotypes exhibited a significantly greater average body weight and total length than those with a T/T134 genotype (14.72 ± 0.53 g and 11.37 ± 0.13 cm, N = 271) (P < 0.05).

A SNP of the hemocyanin gene (LvHc) is a marker for high growth and ammonia-tolerance in Pacific white shrimp Litopenaeus vannamei.

Expression levels of hemocyanin (LvHc), activating transcription factor 4 (LvAtf4), glutathione S-transferase (LvGst), caspase 2 (LvCasp2) and anti-lipopolysaccharide factor (LvAlf) were examined in the hepatopancreas of Pacific white shrimp Litopenaeus vannamei juveniles exposed to a lethal concentration of ammonia-N (32.15 mg/l). The expression levels of all transcripts except LvAlf were significantly greater (P < 0.05) in tolerant shrimp (Lv-AT; N = 30) that survived up to 72 h post treatment (hpt) than in susceptible shrimp (Lv-AS24 and Lv-AS72; N = 45 and 15), that died within 24 h or between 24 and 72 hpt, respectively. Subsequently, effects of non-lethal concentrations of ammonia-N (control, 10 and 20 mg/l) on the expression of LvHc in juvenile shrimp were examined. Compared to the control, expression levels of LvHc transcripts in hemocytes and the hepatopancreas of tested shrimp changed after exposure to ammonia-N. One SNP (C > T545) was found in the LvHc322 gene segment. Real-time PCR amplification of specific alleles (real-time PASA) was developed for detection of C > T545 genotypes. Juveniles in the lethal exposure test that carried a C/T545 genotype showed a greater average body weight and total length (8.46 ± 0.36 g and 10.05 ± 0.16 cm) than those with a C/C545 genotype (7.48 ± 0.31 g and 9.60 ± 0.13 cm) (P < 0.05). Similar results were found in the second generation (G2) of a growth-improved stock (3 and 4 families of BIOTEC-G2-L1 and BIOTEC-G2-L2) and in commercially farmed shrimp (2 groups). Accordingly, expression levels and SNP of LvHc can serve as markers for selection high growth performance in ammonia-tolerant L. vannamei.

The development of species-specific AFLP-derived SCAR and SSCP markers to identify mantis shrimp species.

Mantis shrimp has become commercially valuable in many countries, while the commercially aquaculture still unsuccessful. The stable supply of the species-specific markers for precise identification can play a key role of foods authentication as well as restoring/enhancing mantis shrimp stocks in future. The aim of this research was to identify species-specific markers for Squillid and Harpiosquillid mantis shrimp taxa using Amplified fragment length polymorphism-Single strand conformation polymorphism (AFLP-SSCP) approaches. Selective amplification would be substituted as a total of 40 primer combinations was performed using either three-base (i.e., EcoRI+3 and MseI+3 in 20 primer combinations) or two-base (i.e., EcoRI+2 and MseI+2 in 20 primer combinations) selective primers. These had been size-fractionated via 6% denaturing polyacrylamide gel electrophoresis, ten AFLP fragments exhibiting species or genus-specific characteristics were cloned, sequenced, and GenBank interrogated. A primer pair was designed and their specificity was tested versus the genomic DNA of various species. Results show that the primer E+2-13/M+2-13Hr158 generated PCR products for just H. harpax, while E+3-14/M+3-2HhHr151 and E+2-13/M+2-13Hh150 generated PCR products for both H. harpax and H. raphidea and not others (i.e., M. nepa, O. oratoria, and E. woodmasoni). SSCP was then applied in order to differentiate between H. harpax and H. raphidea. These SSCP results indicate that species can be differentiated based on polymorphic fragment nucleotides. Indeed, primers E+2-13/M+2-13Hr158, E+3-14/M+3-2HhHr151, and E+2-13/M+2-13Hh150 were all successfully confirmed as present in processed mantis shrimp samples (i.e., saline-preserved and heat-dried). These results provide new species-specific markers for mantis shrimp identification.

Cloning, expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris.

cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35-37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg2+, Mn2+, Li+, Ca2+, Ni2+, CHAPS, DTT, and EDTA and inhibited by Hg2+, Ag+, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.

Identification of pigmentation genes in skin, muscle and tail of a Thai-flag variety of Siamese fighting fish Betta splendens.

Pigmentation genes expressed in skin, body muscle and tail of Thai-flag compared with Blue, White and Red varieties of Siamese fighting fish Betta splendens were identified. In total, 22,919 new unigenes were found. Pearson correlation and PCA analysis revealed that expression profiles of genes in muscle, skin and tail across solid color variety were similar. In contrast, those in skin and red tail part of Thai-flag were closely related but they showed different expression profiles with the white tail part. Moreover, 21,347-64,965 SNPs were identified in exonic regions of identified genes. In total, 28,899 genes were differentially expressed between paired comparisons of libraries where 13,907 genes (48.12 %) were upregulated and 14,992 genes (51.88 %) were downregulated. DEGs between paired libraries were 106-5775 genes relative to the compared libraries (56-2982 and 50-2782 for upregulated and downregulated DEGs). Interestingly, 432 pigmentation genes of B. splendens were found. Of these, 297 DEGs showed differential expression between varieties. Many DEGs in melanogenesis (Bsmcr1r, Bsmcr5r, and Bsslc2a15b), tyrosine metabolism (Bstyr, Bstyrp1b and Bsdct), stripe repressor (BsAsip1 and BsAsip2b), pteridine (Bsgch2) and carotenoid (BsBco2) biosynthesis were downregulated in the Thai-flag compared with solid color varieties. Expression of Bsbco1l, Bsfrem2b, Bskcnj13, Bszic2a and Bspah in skin, muscle and tail of Thai-flag, Blue, Red and White varieties was analyzed by qRT-PCR and revealed differential expression between fish varieties and showed anatomical tissue-preferred expression patterns in the same fish variety. The information could be applied to assist genetic-based development of new B. splendens varieties in the future.

Characterization and expression of cell division cycle 2 (Cdc2) mRNA and protein during ovarian development of the giant tiger shrimp Penaeus monodon.

The meiotic maturation of oocytes is regulated by the maturation-promoting factor (MPF), a complex of Cdc2 (Cdk1) and Cyclin B. Here, the complete open reading frame (ORF) of Cdc2 in Penaeus monodon was characterized. PmCdc2 were 900bp in length corresponding to a polypeptide of 299 amino acids with the conserved Thr14, Tyr15 and Thr161 residues. Quantitative real-time PCR indicated that the expression level of PmCdc2 in wild intact broodstock was significantly increased in stages II (vitellogenic) and III (early cortical rod) ovaries relative to stage I (previtellogenic) ovaries and peaked in stage IV (mature) ovaries (P<0.05). The expression level of PmCdc2 in stages I-IV ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian developmental stages in intact broodstock (P<0.05). Expression levels of PmCdc2 in ovaries of 18-month-old P. monodon upon 5-HT injection (50µg/g body weight) were significantly increased at 1hour post injection (hpi, P<0.05). Recombinant PmCdc2 protein and its polyclonal antibody were successfully produced. Western blot analysis revealed the expected 34kDa band (PmCdc2) along with a smaller band of 23kDa (ribosomal protein S3) in ovaries of juveniles and various ovarian stages of broodstock. Using phospho-Cdc2 (Thr161) polyclonal antibody, the positive signal of 34kDa was observed in all ovarian stages but the most intense signal was found in stage IV ovaries. Results in the present study indicated that PmCdc2 gene/protein plays an important role in the development and maturation of oocytes/ovaries in P. monodon.

Transcriptome comparison for identification of pigmentation-related genes in different color varieties of Siamese fighting fish Betta splendens.

Transcriptome comparison was performed to identify genes expressed in skin, muscle and tails of mono-color (Red, Blue, Black, White and Yellow), bi-color (Cambodian) and multi-color (Marble) varieties of Siamese fighting fish Betta splendens. In total, 163,140 unigenes covering 26.348 Gb were found. Of these, 93,899 (57.55 %) unigenes significantly matched at least one database. In total, 5039 differentially expressed genes (DEGs) were found where 2415 genes (47.93 %) showed higher expression and 2624 genes (52.07 %) showed lower expression for all pairwise comparisons. DEGs between paired color varieties were 133-443. Of these, 38-220 genes were more highly expressed while 37-280 genes were more lowly expressed relative to the compared varieties. A total of 897 sequences (148 genes) significantly matched pigmentation-related genes of Danio rerio (E-value < 1e-06). Of these, 19 DEGs were identified. Examples are tyrosinase-related protein 1a (BsTyrp1a), epidermal growth factor receptor (BsEgfr) and neurofibronin 1a (BsNf1a). Moreover, 711,123 SNPs were identified and 1365 of these were located in pigmentation-related genes. Interestingly, an A > C474 SNP in the gene BsTrpm7 and an indel (position 3571) in the BsItgb1a gene were found only in Cambodian. A C > T2520 SNP in BsFzd4 and 10 of 11 SNPs in BsTyrp1a were found only in Black. Different expression levels (P < 0.05) were found for tyrosinase (BsTyr), BsTyrp1a, BsNf1a and BsEgf1 among skin, body muscle and tails of the same variety and among the same tissues of different varieties (Red, Green, Blue, Black, Cambodian and Multi-colors, N = 5 each).

A cDNA microarray, UniShrimpChip, for identification of genes relevant to testicular development in the black tiger shrimp (Penaeus monodon).

BACKGROUND: Poor reproductive maturation in captive male broodstock of the black tiger shrimp (Penaeus monodon) is one of the serious problems to the farming industries. Without genome sequence, EST libraries of P. monodon were previously constructed to identify transcripts with important biological functions. In this study, a new version of cDNA microarray, UniShrimpChip, was constructed from the Peneaus monodon EST libraries of 12 tissues, containing 5,568 non-redundant cDNA clones from 10,536 unique cDNA in the P. monodon EST database. UniShrimpChip was used to study testicular development by comparing gene expression levels of wild brooders from the West and East coasts of Thailand and domesticated brooders with different ages (10-, 14-, 18-month-old). RESULTS: The overall gene expression patterns from the microarray experiments revealed distinct transcriptomic patterns between the wild and domesticated groups. Moreover, differentially expressed genes from the microarray comparisons were identified, and the expression patterns of eight selected transcripts were subsequently confirmed by reverse-transcriptase quantitative PCR (RT-qPCR). Among these, expression levels of six subunits (CSN2, 4, 5, 6, 7a, and 8) of the COP9 signalosome (CSN) gene family in wild and different ages of domesticated brooders were examined by RT-qPCR. Among the six subunits, CSN5 and CSN6 were most highly expressed in wild brooders and least expressed in the 18-month-old domesticated group; therefore, their full-length cDNA sequences were characterized. CONCLUSIONS: This study is the first report to employ cDNA microarray to study testicular development in the black tiger shrimp. We show that there are obvious differences between the wild and domesticated shrimp at the transcriptomic level. Furthermore, our study is the first to investigate the feasibility that the CSN gene family might have involved in reproduction and development of this economically important species.

Molecular cloning and expression analysis of the Mitogen-activating protein kinase 1 (MAPK1) gene and protein during ovarian development of the giant tiger shrimp Penaeus monodon.

Isolation and characterization of genes and/or proteins differentially expressed in ovaries are necessary for understanding ovarian development in the giant tiger shrimp (Penaeus monodon). In this study, the full-length cDNA of P. monodon mitogen-activating protein kinase 1 (PmMAPK1) was characterized. PmMAPK1 was 1,398 bp in length containing an open reading frame of 1,098 bp that corresponded to a polypeptide of 365 amino acids. PmMAPK1 was more abundantly expressed in ovaries than in testes of P. monodon. Quantitative real-time PCR revealed differential expression levels of PmMAPK1 mRNA during ovarian development of intact broodstock, where it peaked in early cortical rod (stage III) ovaries (P < 0.05) and slightly decreased afterwards (P > 0.05). Likewise, the expression level of PmMAPK1 in early cortical rod and mature (IV) ovaries was significantly greater than that in previtellogenic (I) and vitellogenic (II) ovaries of eyestalk-ablated broodstock (P < 0.05). The PmMAPK1 transcript was localized in ooplasm of previtellogenic oocytes. In intact broodstock, the expression of the PmMAPK1 protein was clearly increased from previtellogenic ovaries in subsequent stages of ovarian development (P < 0.05). In contrast, the level of ovarian PmMAPK1 protein was comparable during oogenesis in eyestalk-ablated broodstock (P > 0.05). The PmMAPK1 protein was localized in ooplasm of previtellogenic and vitellogenic oocytes. It was also detected around the nuclear membrane of early cortical rod oocytes in both intact and eyestalk-ablated broodstock. Results indicated that PmMAPK1 gene products seem to play functional roles in the development and maturation of oocytes/ovaries in P. monodon.

Genetic differentiation of the stingless bee Tetragonula pagdeni in Thailand using SSCP analysis of a large subunit of mitochondrial ribosomal DNA.

Genetic diversity and population differentiation of the stingless bee Tetragonula pagdeni (Schwarz) was assessed using single-strand conformational polymorphism (SSCP) analysis of a large subunit of the ribosomal RNA gene (16S rRNA). High levels of genetic variation among individuals within each population (North, Northeast, Central, Prachuap Khiri Khan, Chumphon, and Peninsular Thailand) of T. pagdeni were observed. Analysis of molecular variance indicated significant genetic differentiation among the six geographic populations (Φ (PT) = 0.28, P < 0.001) and between samples collected from north and south of the Isthmus of Kra (Φ (PT) = 0.18, P < 0.001). In addition, Φ (PT) values between all pairwise comparisons were statistically significant (P < 0.01), indicating strong degrees of intraspecific population differentiation. Therefore, PCR-SSCP is a simple and cost-effective technique applicable for routine population genetic analyses in T. pagdeni and other stingless bees. The results also provide an important baseline for the conservation and management of this ecologically important species.

Identification of testis-relevant genes using in silico analysis from testis ESTs and cDNA microarray in the black tiger shrimp (Penaeus monodon).

BACKGROUND: Poor reproductive maturation of the black tiger shrimp (Penaeus monodon) in captivity is one of the serious threats to sustainability of the shrimp farming industry. Understanding molecular mechanisms governing reproductive maturation processes requires the fundamental knowledge of integrated expression profiles in gonads of this economically important species. In P. monodon, a non-model species for which the genome sequence is not available, expressed sequence tag (EST) and cDNA microarray analyses can help reveal important transcripts relevant to reproduction and facilitate functional characterization of transcripts with important roles in male reproductive development and maturation. RESULTS: In this study, a conventional testis EST library was exploited to reveal novel transcripts. A total of 4,803 ESTs were unidirectionally sequenced and analyzed in silico using a customizable data analysis package, ESTplus. After sequence assembly, 2,702 unique sequences comprised of 424 contigs and 2,278 singletons were identified; of these, 1,133 sequences are homologous to genes with known functions. The sequences were further characterized according to gene ontology categories (41% biological process, 24% molecular function, 35% cellular component). Through comparison with EST libraries of other tissues of P. monodon, 1,579 transcripts found only in the testis cDNA library were identified. A total of 621 ESTs have not been identified in penaeid shrimp. Furthermore, cDNA microarray analysis revealed several ESTs homologous to testis-relevant genes were more preferentially expressed in testis than in ovary. Representatives of these transcripts, homologs of saposin (PmSap) and Dmc1 (PmDmc1), were further characterized by RACE-PCR. The more abundant expression levels in testis than ovary of PmSap and PmDmc1 were verified by quantitative real-time PCR in juveniles and wild broodstock of P. monodon. CONCLUSIONS: Without a genome sequence, a combination of EST analysis and high-throughput cDNA microarray technology can be a useful integrated tool as an initial step towards the identification of transcripts with important biological functions. Identification and expression analysis of saposin and Dmc1 homologs demonstrate the power of these methods for characterizing functionally important genes in P. monodon.

Cloning and characterization of a melanization inhibition protein (PmMIP) of the black tiger shrimp, Penaeus monodon.

Melanization is an important component of the innate immune responses in invertebrates and it is essential for defense against invading microorganism. Melanin formation, which is a result of activation of the so called prophenoloxidase activating system, needs to be controlled due to the dangerous effects of quinones and melanin which are produced during the process of melanization. Here, a cDNA for a melanization inhibition protein (MIP), named PmMIP, was identified from the black tiger shrimp, Penaeus monodon by RT-PCR using degenerated oligonucleotide primers and RACE-PCR. The complete sequence significantly matched MIP of the freshwater crayfish Pacifastacus leniusculus (PlMIP). PmMIP contains an N-terminal signal peptide and a fibrinogen related domain (FReD). RT-PCR was applied to examine the expression profiles of PmMIP in various tissues of juvenile P. monodon. PmMIP was expressed in all examined tissues except hemocytes and at very low levels in hepatopancreas and ovaries. The expression of this gene was very low during the larval stages and hardly present in egg and at the nauplius stage. A time-course expression analysis of PmMIP upon Vibrio harveyi challenge at protein levels in plasma was determined. The result shows that MIP protein in plasma was induced at 6 h and disappeared at 12 and 24 h and then the protein reappeared at 48 and 72 h post injection. These results suggest that upon bacterial infection the PmMIP protein is first released from tissues into hemolymph and then degraded to allow melanization to occur for fighting against bacteria.

Molecular cloning and expression of progestin membrane receptor component 1 (Pgmrc1) of the giant tiger shrimp Penaeus monodon.

Knowledge on molecular mechanisms of steroid hormonal induction on oocyte development may lead to the possible ways to effectively induce ovarian maturation in shrimp. In this study, progestin membrane receptor component 1 (Pgmrc1) of the giant tiger shrimp (Penaeus monodon) initially identified by EST analysis was further characterized. The full-length cDNA of Pgmrc1 was 2015bp in length containing an ORF of 573bp corresponding to a polypeptide of 190 amino acids. Northern blot analysis revealed a single form of Pgmrc1 in ovaries of P. monodon. Quantitative real-time PCR indicated that the expression level of Pgmrc1 mRNA in ovaries of both intact and eyestalk-ablated broodstock was greater than that of juveniles (P<0.05). Pgmrc1 was up-regulated in mature (stage IV) ovaries of intact broodstock (P<0.05). Unilateral eyestalk ablation resulted in an earlier up-regulation of Pgmrc1 since the vitellogenic (II) ovarian stage. Moreover, the expression level of Pgmrc1 in vitellogenic, early cortical rod and mature (II-IV) ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian stages in intact broodstock (P<0.05). Pgmrc1 mRNA was clearly localized in the cytoplasm of follicular cells, previtellogenic and early vitellogenic oocytes. Immunohistochemistry revealed the positive signals of the Pgmrc1 protein in the follicular layers and cell membrane of follicular cells and various stages of oocytes. Taken the information together, Pgmrc1 gene products seem to play the important role on ovarian development and may be used as the bioindicator for monitoring progression of oocyte maturation of P. monodon.

Development of a species-diagnostic marker for identification of the stingless bee Trigona pagdeni in Thailand.

A species-diagnostic SCAR marker for identification of the stingless bee (Trigona pagdeni Schwarz) was successfully developed. Initially, amplified fragment length polymorphism analysis was carried out across representatives of 12 stingless bee species using 64 primer combinations. A 284 bp band restrictively found in T. pagdeni was cloned and sequenced. A primer pair (CUTP1-F/R) was designed and tested for species-specificity in 15 stingless bees. The expected 163 bp fragment was successfully amplified in all examined individuals of T. pagdeni (129/129). Nevertheless, cross-species amplification was also observed in T. fimbriata (1/3), T. collina (11/112), T. laeviceps (1/12), and T. fuscobalteata (15/15), but not in other species. SSCP analysis of CUTP1 further differentiated T. fuscobalteata and T. collina from T. pagdeni. Although T. laeviceps, T. fimbriata, and T. pagdeni shared an identical SSCP genotype, they are not taxonomically problematic species.

Molecular cloning and expression analysis of ovary-specific transcript 1 (Pm-OSTI) of the giant tiger shrimp, Penaeus monodon.

Isolation and characterization of genes specifically expressed in ovaries are necessary for understanding sex differentiation and ovarian development processes in the giant tiger shrimp, Penaeus monodon. In this study, a transcript that significantly matched the polehole precursor was further characterized by RACE-PCR. The sequence obtained was 5151 bp in length and contained a coding region of 5031 bp corresponding to 1677 amino acids. This transcript was only expressed in ovaries but not in testes of Juveniles (N = 10) and broodstock (N = 22) of P. monodon. A tissue distribution analysis further confirmed ovary-specific expression of this transcript (called P. monodon ovary-specific transcript 1, Pm-OST1) in female broodstock. Expression levels of Pm-OST 1 in ovaries of juvenile P. monodon upon 5-HT Injection (33.9+/-6.40 g; 50 microg/g body weight) were significantly higher at 12-72 hours post Injection (P<0.05). Quantitative real-time PCR Indicated that Pm-OST1 was comparably expressed throughout ovarian development in normal P. monodon broodstock (P>0.05). However, the expression level of Pm-OST1 was significantly higher in stage-III ovaries in eyestalk-ablated broodstock (P<0.05). Pm-OST1 was clearly localized in the ooplasm of previtellogenic and vitellogenic oocytes. Our results suggest that Pm-OST1 plays a functionally Important role in promoting the development of female germ cells and oocytes in P. monodon.

Identification, characterization, and expression of the genes TektinA1 and Axonemal protein 66.0 in the tropical abalone Haliotis asinina.

The genes Tektin A1 and axonemal protein 66.0 were successfully Isolated and characterized in the tropical abalone Haliotis asinina. The full-length cDNAs of Ha-TekA1 and Ha-Axp66.0 were 2166 and 2038 bp long, with ORFs of 1350 and 1683 bp, respectively. Both Ha-TekA1 and Ha-Axp66.0 were expressed in the testes but not in the ovaries or hemocytes of H. asinina adults. In addition, HaAxp66.0 was not expressed in H. asinina juveniles (2, 3, and 5 months old). A tissue expression analysis showed Ha-Axp66.0 to be expressed specifically in the testes, whereas Ha-TekA1 was expressed abundantly in the testes but weakly in the foot, gill, digestive gland, left hypobranchial gland, and mantle. The relative expression levels of Ha-TekA1 and Ha-Axp66.0 were significantly lower in undeveloped testes (stage I) than in developed testes (stages II, III, and IV) of H. asinina (P < 0.05).

Molecular characterization and expression profiles of cyclin A and cyclin B during ovarian development of the giant tiger shrimp Penaeus monodon.

The meiotic maturation of oocytes is regulated by maturation promoting factor (MPF), a complex of cdc2 (Cdk1) and cyclin B and other Cdk/cyclin complexes. To better understand molecular aspects governing reproductive maturation of the giant tiger shrimp (Penaeus monodon), the full length cDNAs and genomic organization of cyclins A and B (PMCyA and PMCyB) were characterized. A single form of PMCyA contained an open reading frame (ORF) of 1326 bp corresponding to a deduced protein of 441 amino acids. Its genomic sequence contained 5 exons, 4 introns and untranslated regions (UTRs) spanning 2586 bp in length. In contrast, PMCyB possessed three isoforms with an identical ORF of 1206 bp (401 amino acids) but three different 3' UTR lengths of 416, 543 and 1117 bp, respectively. Their respective genomic sequences were composed of 8 exons, 7 introns and UTRs covering 4181, 4307 and 4940 bp. Expression levels of both PMCyA and PMCyB in ovaries of broodstock were much greater than those of juveniles (P<0.05). During ovarian development and after spawning of normal shrimp broodstock, PMCyA was not differentially expressed (P>0.05) whereas the level of PMCyB in stage IV was greater than that of stage I ovaries (P<0.05). Unilateral eyestalk ablation, a technique commonly used to induce spawning in P. monodon female brooders, had no effects on transcription of PMCyB (P>0.05) but resulted in a lower expression of PMCyA at stage IV of ovarian development of this economically important species (P<0.05).

Genetic diversity and geographic differentiation of the giant tiger shrimp (Penaeus monodon) in Thailand analyzed by mitochondrial COI sequences.

Genetic diversity and geographic differentiation of the giant tiger shrimp, Penaeus monodon, in Thai waters (Satun, Trang, Phangnga, and Ranong in the Andaman Sea and Chumphon and Trat in the Gulf of Thailand) were examined by COI polymorphism (N = 128). We observed 28 COI mitotypes across all investigated individuals. The sequence divergence between pairs of mitotypes was 0.00-20.76%. A neighbor-joining tree clearly indicated lineage separation of Thai P. monodon and large nucleotide divergence between interlineage mitotypes but limited divergence between intralineage mitotypes. High genetic diversity was found (mean sequence divergence = 6.604%, haplotype diversity = 0.716-0.927, pi = 2.936-8.532%). F-statistics (F(ST)) and an analysis of molecular variance (AMOVA) indicated that the gene pool of Thai P. monodon was not homogeneous but genetically differentiated intraspecifically (P < 0.05). Six samples of P. monodon could be allocated into three different genetic populations: Trat (A), Chumphon (B), and the Andaman samples Satun, Trang, Phangnga, and Ranong (C). Contradictory results regarding patterns of geographic differentiation previously reported by various molecular approaches were clarified by this study.