Bavornlak Khamnamtong

Molecular Taxonomy of Cupped Oysters (Crassostrea, Saccostrea, and Striostrea) in Thailand Based on COI, 16S, and 18S rDNA Polymorphism

Genetic diversity of oysters Crassostrea belcheri (Sowerby, 1871), C. iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), S. forskali (Gmelin, 1791), and Striostrea (Parastriostrea) mytiloides (Lamarck, 1819) (Ostreoida, Mollusca) was analyzed by polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) of 16S ribosomal DNA with AcsI, AluI, DdeI, DraI, RsaI, and TaqI, 18S ribosomal DNA with HinfI, and cytochrome oxidase subunit I with AcsI, DdeI and MboI. A total of 54 composite haplotypes were observed. Species-diagnostic markers were specifically found in C. belcheri, C. iredalei, and S. cucullata, but not in S. forskali and Striostrea mytiloides, which shared common composite haplotypes. Neighbor-joining trees constructed from genetic distances between pairs of composite haplotypes and species indicated large genetic differences between Crassostrea and Saccostrea (including Striostrea mytiloides), but closer relationships were observed within each genus. Four groups of unidentified oysters (Crassostrea sp. and Saccostrea sp. groups 1, 2, and 3) were also genetically analyzed. Fixed RFLP markers were found in Crassostrea sp. and Saccostrea sp. group 2, but not in Saccostrea sp. groups 1 and 3. Phylogenetic and genetic heterogeneity analyses indicated that Crassostrea sp. and Saccostrea sp. group 2 should be considered as newly unidentified oyster species in Thailand.

Genetic Heterogeneity of the Blue Swimming Crab (Portunus pelagicus) in Thailand Determined by AFLP Analysis

Genetic diversity and population differentiation of the blue swimming crab (Portunus pelagicus) in Thailand, originating from Ranong and Krabi located in the Andaman Sea (west) and Chanthaburi, Prachuap Khiri Khan, and Suratthani located in the Gulf of Thailand (east), were examined by AFLP analysis. High genetic diversity of P.xa0pelagicus in Thai waters was found (Nxa0=xa072). The four primer combinations generated 227 AFLP fragments, and the percentage of polymorphic bands in each geographic sample was 66.19–94.38%. The mean genetic distance between pairs of samples was 0.1151–0.2440. Geographic heterogeneity analyses using the exact test and FST-based statistics between all pairwise comparisons were statistically significant (Pxa0<xa00.01), indicating a fine-scale level of intraspecific population differentiation of Thai P.xa0pelagicus. The estimated number of migrants per generation (Nem) was 0.26–0.76, suggesting restricted gene flow levels of P.xa0pelagicus in Thai waters.

Characterization and expression analysis of the Broad-complex (Br-c) gene of the giant tiger shrimp Penaeus monodon

Broad-complex (Br-c) is the early ecdysone responsive gene encoding a family of zinc-finger transcription factors. In this study, the full-length cDNA of the Br-c gene of the giant tiger shrimp (Penaeus monodon) was identified. PmBr-c was 1897 bp in length containing an ORF of 1329 bp deducing to a polypeptide of 442 amino acids. PmBr-c was more abundantly expressed in ovaries than testes of P. monodon broodstock. The expression levels of PmBr-c mRNA in ovaries of juveniles was significantly greater than that in stages II (vitellogenic), IV (mature) and V (post-spawning) ovaries of intact broodstock. The expression level of PmBr-c was significantly increased in stage III (nearly mature) ovaries of intact wild broodstock and in stage IV ovaries of eyestalk-ablated broodstock (P<0.05). In domesticated broodstock, ovarian PmBr-c was expressed lower in 18-month-old shrimp compared to 6-month-old shrimp (P<0.05). In situ hybridization revealed that PmBr-c mRNA was localized in ooplasm of previtellogenic oocytes in various ovarian stages of P. monodon broodstock. Serotonin (5-HT, 50 μg/g body mass; 18-month-old shrimp) and progesterone (0.1 μg/g body mass; 14-month-old shrimp) injection significantly promoted the expression level of PmBr-c in ovaries of domesticated broodstock at 24 and 48-72 h post injection (hpi, P<0.05). The expression levels of PmBr-c in ovaries of juvenile P. monodon was significantly increased following 20-hydroxyecdysone treatment (1 μg/g body mass; 4-month-old shrimp) for 168 hpi (P<0.05). Taken together, PmBr-c seems to play an important role during ovarian development of P. monodon.

Isolation and Characterization of Testis-Specific DMRT1 in the Tropical Abalone (Haliotis asinina)

The Doublesex Male abnormal-3 Related Transcription factor-1 (DMRT1) gene encodes a protein containing the DNA-binding motif called the DM domain, involved in the sexual development of various species. To gain insight into its implications for gonadal differentiation in the tropical abalone (Haliotisxa0asinina), a DMRT1 homolog was identified and characterized. The full length cDNA of HADMRT1 (1,740xa0bp with an ORF of 732xa0bp corresponding to a putative polypeptide of 243 amino acids) and its DM domain-less variant (HADMRT1-like, 1,430xa0bp with an ORF of 312xa0bp, 103 amino acids) were successfully isolated and reported for the first time in molluscs. HADMRT1 was specifically expressed in the testes of adult H.xa0asinina (Nxa0=xa016) but not in whole juveniles (2, 3, 5xa0months old, Nxa0=xa06 for each group) and ovaries (Nxa0=xa016), and pooled hemocytes (from 50 individuals) of adults. Tissue distribution analysis further revealed testis-specific expression of HADMRT1. Semiquantitative RT-PCR illustrated that the relative expression level of HADMRT1 in developed testes (stages II, III, and IV) was significantly greater than that in undeveloped testes (stage I) of abalone broodstock (Pxa0<xa00.05).

Genetic population differentiation of the blue swimming crab Portunus pelagicus (Portunidae) in Thai waters revealed by RAPD analysis

Genetic diversity and population differentiation of the blue swimming crab, Portunus pelagicus, in Thailand were analyzed by RAPD analysis. One hundred and twelve RAPD fragments were generated from 109 individuals of P. pelagicus using OPA02, OPA14, OPB10, UBC122, and UBC158 primers. The percentage of polymorphic bands in each geographic sample and that of each primer across overall samples were 72.7-85.0 and 92.0-100%, respectively. Large numbers of polymorphic bands found in the RAPD analysis suggested high genetic diversity of Thai P. pelagicus. The mean genetic distance between samples across all primers was 0.0929-0.2471. Significant geographic heterogeneity was observed across samples overall and between all pairs of geographic samples (P < 0.01 for theta and P < 0.0001 for the exact test), indicating strong genetic differentiation of P. pelagicus in Thai waters, despite its high potential of dispersal. Limited gene flow levels (0.44-1.19 individuals per generation) of Thai P. pelagicus were also observed. A fine scale level of differentiation suggested that P. pelagicus from each geographic sample in Thai waters should be regarded as a separate genetic population and treated as a different exploited stock.

Identification of reproduction-related proteins and characterization of the protein disulfide isomerase A6 cDNA in ovaries of the giant tiger shrimp Penaeus monodon.

Proteomic analysis was carried out for identification of proteins functionally involved in ovarian development of the giant tiger shrimp (Penaeus monodon). A total of 335 protein spots including 183 spots from vitellogenic (stage II) and 152 spots from mature (stage IV) ovaries of intact P. monodon broodstock were examined. Of these, 75 (40.98%) and 59 (38.82%) spots significantly matched known proteins in the databases, respectively. In addition, 270 protein spots including 167 and 103 spots from respective ovarian stages of eyestalk-ablated broodstock were also characterized. A total of 95 (56.89%) and 62 (60.19%) spots matched known proteins, respectively. Among differentially expressed reproduction-related proteins, the full-length cDNA of protein disulfide isomerase A6 (PmPDIA6) was further characterized by RACE-PCR. PmPDIA6 was 1946bp in length containing an open reading frame (ORF) of 1293bp corresponding to a polypeptide of 430 amino acids. PmPDIA6 was up-regulated at stage III ovaries in intact shrimp (P<0.05). Interestingly, eyestalk ablation resulted in a lower expression level of PmPDIA6 in each stage of ovarian development compared to that of intact broodstock (P<0.05). Results in this study clearly indicated the potential of cellular proteomic studies and gene expression analysis for identification of proteins/genes differentially expressed during ovarian development of P. monodon.

Proteomic analysis of ovarian proteins and characterization of thymosin-β and RAC-GTPase activating protein 1 of the giant tiger shrimp Penaeus monodon

Cellular proteomics of total proteins in ovaries of domesticated and wild giant tiger shrimp (Penaeus monodon) were examined using GeLC-MS/MS. In total, 1638 proteins matched those previously deposited in databases and 1253 (76.50%) of these significantly matched known proteins. Several reproduction-related proteins (e.g. Cdc2, Cyclin B, Cdc25, 14-3-3, thymosin-β and Rac-GTPase activating protein 1) were identified. In addition, the full-length cDNA of P. monodon thymosin-β (PmTmsb; 1084 bp with an ORF of 387 bp and 128 deduced aa) and Rac-GTPase activating protein 1 (PmRacgap1; an ORF of 1881 bp and 626 deduced aa) were further characterized. PmTmsb was constitutively expressed in all tissues. In contrast, PmRacgap1 was more abundantly expressed in gonads than in several non-reproductive tissues (e.g. subcuticular epithelium, hepatopancreas, intestine, pleopods, stomach and thoracic ganglion). The expression levels of PmTmsb and PmRacgap1 in ovaries of wild adult broodstock were significantly greater than those in ovaries of juveniles (P<0.05). However, their expression levels did not vary significantly during ovarian development stages in intact broodstock. However, eyestalk ablation resulted in a significant reduction in PmTmsb expression at stages I and III ovaries (P<0.05), although it did not affect PmRacgap1 transcription significantly at these stages. On the other hand, use of polyclonal antibodies derived from recombinant PmTmsb and PmRacgap1 revealed that levels of both proteins decreased at the late stage (IV) of ovarian development. Our results suggested that PmTmsb and PmRacgap1 may act as negative effectors during ovarian development in P. monodon.

Identification of reproduction-related proteins and characterization of proteasome alpha 3 and proteasome beta 6 cDNAs in testes of the giant tiger shrimp Penaeus monodon

Cellular proteomic analysis was carried out to identify reproduction-related proteins in testes of wild and domesticated broodstock of Penaeus monodon. In total, 642 protein spots were characterized and 287 spots (44.70%) significantly matched protein sequences in the databases (P<0.05). To examine a role of the proteasome system in testicular development of P. monodon, the expression profiles of proteasome alpha 3 subunit (PmPsma3) and proteasome beta 6 (PmPsmb6) mRNA in different groups of domesticated shrimp and in wild broodstock were examined. The expression levels of these transcripts in testes of 18-month-old domesticated shrimp were significantly lower than those of wild broodstock (P<0.05). Interestingly, the expression levels of testicular PmPsma3 and PmPsmb6 in 18-month-old shrimp were significantly increased at 24 h following serotonin injection (50 μg/g body weight). Results suggested that reduced degrees of maturation in captive P. monodon males may be partially resolved by exogenous 5-HT administration.

Characterization, expression and localization of valosin-containing protein in ovaries of the giant tiger shrimp Penaeus monodon

Valosin-containing protein (VCP), a member of the ATPase-associated with diverse cellular activity (AAA) family, was identified from the giant tiger shrimp (Penaeus monodon). The full-length cDNA of the PmVCP mRNA consisted of 2,724 bp containing an ORF of 2,367 bp corresponding to a deduced polypeptide of 788 amino acids. The deduced PmVCP protein contained two putative Cdc48 domains (positions 17-103, E-value=2.00e-36 and 120-186, E-value=3.60e-11) and two putative AAA domains (positions 232-368, E-value=3.67e-24 and 505-644, E-value=3.73e-25). PmVCP mRNA expression in ovaries was greater than that in testes in both juveniles and broodstock. PmVCP was significantly up-regulated in stages II and IV ovaries in intact wild broodstock (P<0.05) but it was not differentially expressed during ovarian development in eyestalk-ablated broodstock (P>0.05). The expression level of PmVCP mRNA in ovaries of 14-month-old shrimp was not affected by progesterone injection (0.1μg/g body weight, P>0.05). In contrast, exogenous 5-HT administration (50μg/g body weight) resulted in an increase of PmVCP mRNA in ovaries of 18-month-old shrimp at 6 and 24h post-injection (hpi) (P<0.05). The rPmCdc48-VCP protein and its polyclonal antibody were successfully produced. Cellular localization revealed that PmVCP was localized in the ooplasm of previtellogenic oocytes. Subsequently, it was translocated into the germinal vesicle of vitellogenic oocytes. Interestingly, PmVCP was found in nucleo-cytoplasmic compartments, in the cytoskeletal architecture and in the plasma membrane of mature oocytes in both intact and eyestalk-ablated broodstock.

Development of a Species-diagnostic SCAR Marker of the Blue Swimming Crab (Portunus pelagicus)

The blue swimming crab, Portunus pelagicus, is distributed from the intertidal zone to approximately 50 m depth along the coastlines (Kangas 2000). Commercial fishing of P. pelagicus has rapidly expanded in the last few years, resulting in overexploitation of natural P. pelagicus in Thailand. Species-specific markers play important roles in quality control of products of economically important species (Klinbunga et al. 2000). They can be used to verify species origins of various forms of the blue swimming crab products from Thailand to prevent intentional use of the wrong species in canning. Amplified fragment length polymorphism (AFLP) is a relatively simple method for the identification of genetic markers at different taxonomic levels without the need for knowledge of sequences of the genome under investigation (Vos et al. 1995; Lui and Cordes 2004). Recently, Zhang et al. (2004) applied AFLP to identify and characterize 11 Lutjanus species captured in Nansha coral reefs. The similarity