Microsatellite and mitochondrial haplotype diversity reveals population differentiation in the tiger shrimp (Penaeus monodon) in the Indo-Pacific region.

The black tiger shrimp (Penaeus monodon) is an ecologically and economically important penaeid species and is widely distributed in the Indo-Pacific region. Here we investigated the genetic diversity of P. monodon (n = 355) from eight geographical regions by genotyping at 10 microsatellite loci. The average observed heterozygosity at various loci ranged from 0.638 to 0.743, indicating a high level of genetic variability in this region. Significant departures from Hardy-Weinberg equilibrium caused by heterozygote deficiency were recorded for most loci and populations. Pairwise F(ST) and R(ST) values revealed genetic differentiation among the populations. Evidence from the assignment test showed that the populations in the West Indian Ocean were unique, whereas other populations examined were partially admixed. In addition, the non-metric multidimensional scaling analysis indicated the presence of three geographic groups in the Indo-Pacific region, i.e. the African populations, a population from western Thailand and the remaining populations as a whole. We also sequenced and analysed the mitochondrial control region (mtCR) in these shrimp stocks to determine whether the nuclear and mitochondrial genomes show a similar pattern of genetic differentiation. A total of 262 haplotypes were identified, and nucleotide divergence among haplotypes ranged from 0.2% to 16.3%. Haplotype diversity was high in all populations, with a range from 0.969 to 1. Phylogenetic analysis using the mtCR data revealed that the West Indian Ocean populations were genetically differentiated from the West Pacific populations, consistent with the microsatellite data. These results should have implications for aquaculture management and conservation of aquatic diversity.

Development of polymorphic expressed sequence tag-derived microsatellites for the extension of the genetic linkage map of the black tiger shrimp (Penaeus monodon).

In this study, microsatellite markers were developed for the genetic linkage mapping and breeding program of the black tiger shrimp Penaeus monodon. A total of 997 unique microsatellite-containing expressed sequence tags (ESTs) were identified from 10 100 EST sequences in the P. monodon EST database. AT-rich microsatellite types were predominant in the EST sequences. Homology searching by the blastn and blastx programs revealed that these 997 ESTs represented 8.6% known gene products, 27.8% hypothetical proteins and 63.6% unknown gene products. Characterization of 50 markers on a panel of 35-48 unrelated shrimp indicated an average number of alleles of 12.6 and an average polymorphic information content of 0.723. These EST microsatellite markers along with 208 other markers (185 amplified fragment length polymorphisms, one exon-primed intron-crossing, six single strand conformation polymorphisms, one single nucleotide polymorphism, 13 non-EST-associated microsatellites and two EST-associated microsatellites) were analysed across the international P. monodon mapping family. A total of 144 new markers were added to the P. monodon maps, including 36 of the microsatellite-containing ESTs. The current P. monodon male and female linkage maps have 47 and 36 linkage groups respectively with coverage across half the P. monodon genome.

Population structure of tropical abalone (Haliotis asinina) in coastal waters of Thailand determined using microsatellite markers.

Three partial genomic libraries were constructed from genomic DNA of the tropical abalone (Haliotis asinina) that was digested with AluI, vortexed/sonicated, and digested with mixed enzyme (AluI, HincII, and RsaI). The libraries yielded 0.02%, 0.42%, and 1.46% positive microsatellite-containing clones, respectively. Eleven clones each of perfect, imperfect, and compound microsatellites were isolated. Ten primer pairs (CUHas1-CUHas10) were analyzed to evaluate their polymorphic level. The numbers of alleles per locus, observed heterozygosity (H0), and expected heterozygosity (He) ranged from 3 to 26 alleles, and varied between 0.27 and 0.85 and between 0.24 and 0.93, respectively. Three microsatellite loci (CUHas2, CUHas3, and CUHas8) were further used for examination of genetic diversity and differentiation of natural H. asinina in coastal waters of Thailand. Genetic variabilities in terms of the effective number of alleles (n(e)), H0, and He were higher in 2 samples from the Gulf of Thailand (n(e)=9.37, 7.66; H0=0.62, 0.78; and He=0.87, 0.86) than those of one sample (n(e)=6.04; H0=0.58; and He=0.62) derived from the Andaman Sea. Assessment of genetic heterogeneity, including allele frequency comparison and pairwise F(ST) analysis, indicated interpopulational differentiation, between natural H. asinina from the Gulf of Thailand and that from the Andaman Sea (P<0.0001).

Genetic diversity and molecular markers of the tropical abalone (Haliotis asinina) in Thailand.

Genetic diversity of abalone in Thailand, Haliotis asinina, H. ovina, and H. varia, was analyzed by polymerase chain reaction (PCR) of 18S and 16S rDNAs, with randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). Species-specific RAPD markers were found in each abalone species. Restriction analysis of 18S (nuclear) ribosomal DNA with Alu I, Taq I, and Hae III and 16S (mitochondrial) rDNA with Bam HI, Eco RI, Hae III, and Alu I gave 12 and 13 digestion patterns, respectively. A total of 49 composite haplotypes were found. A dendogram obtained by the unweighted pair-group method with arithmetic mean, constructed from divergence between pairs of composite haplotypes, revealed reproductively isolated gene pools of these abalone and indicated that H. asinina and H. ovina are genetically closer than H. varia. When H. varia was discovered owing to small sample sizes, geographic heterogeneity analysis and FST estimate indicated clear genetic differentiation between H. ovina originating from the Andaman Sea (west) and the Gulf of Thailand (east, P<0.0001), whereas partial differentiation was observed between the Philippines and the remaining H. asinina samples (P<0.0021). The amplified 16S rDNAs of individuals representing composite haplotypes found in this study were cloned and sequenced. A neighbor-joining tree constructed from sequence divergence of 16S rDNA accurately allocated those sequences according to species origins of abalone. Species-specific PCR based on 16S rDNA polymorphism was successfully developed in H. asinina and H. varia but not in H. ovina.

Molecular genetic identification tools for three commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand.

Molecular genetic keys for identification of 3 commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand were developed based on restriction analysis of 18S ribosomal DNA and cytochrome oxidase subunit I (COI). Digestion of the amplified 18S rDNA with Hinf I unambiguously differentiated Crassostrea oysters from Saccostrea oysters and Striostrea (Parastriostrea) mytiloides. In addition, species-specific restriction fragment length polymorphism patterns of C. belcheri, C. iredalei, and S. cucullata were consistently observed when the gel-eluted COI was digested with Mbo I and Dde I. Thirty composite haplotypes were observed across all individuals. Species-specific composite haplotypes were found in C. belcheri (AAAA and AAAB), C. iredalei (AABC and AABU), and S. cucullata (BBCD and BBCE), respectively. The most common composite haplotype of COI in C. belcheri (AAAA), C. iredalei (AABC), and S. cucullata (BBCD) was amplified, cloned, and sequenced. Detection of C. belcheri and C. iredalei based on polymerase chain reaction was further developed using more specific primers (HCO2198 and R372) followed by digestion of a 372-bp product with Mbo I.

Genetic heterogeneity of the giant tiger shrimp (Penaeus monodon) in Thailand revealed by RAPD and mitochondrial DNA RFLP analyses.

Genetic diversity of the giant tiger shrimp (Penaeus monodon) collected from 5 areas, Chumphon and Trat (Gulf of Thailand), and Phangnga, Satun, and Trang (Andaman Sea), was examined by randomly amplified polymorphic DNA (RAPD) and mitochondrial DNA (16S ribosomal DNA and an intergenic COI-COII) polymorphism. A total of 53 polymorphic fragments from UBC299, UBC273, and UBC268 was consistently scored across all samples. From the respective primers 26, 32, and 30 genotypes were generated. A 260-bp RAPD fragment generated by the primer UBC268 was specifically observed in 95.8% of Trat P. monodon, suggesting that this RAPD could be used as a marker for comparing phenotypic performance of P. monodon from Trat and other geographic samples. In addition, 37 mtDNA composite haplotypes were observed from restriction analysis of the same P. monodon samples. High haplotype diversity (0.855) and nucleotide diversity (3.328%) of Thai P. monodon were observed. Population differentiation of P. monodon between the Andaman Sea and Gulf of Thailand was clearly illustrated by both techniques (P <.0001). Nevertheless, contradictory results on patterns of differentiation were observed between P. monodon within the Gulf of Thailand. Analysis of nuclear DNA polymorphism (RAPD) indicated a genetically significant difference between Chumphon and Trat ( P <.0001), whereas mtDNA polymorphism did not show differentiation between these samples (P =.0497). Under the presumption of selective neutrality of these markers, biased female gene flow between Trat and Chumphon P. monodon may exist and be responsible for an anomalous differentiation pattern between these geographic samples.

Genetic diversity and molecular markers of cupped oysters (genera Crassostrea, Caccostrea, and Striostrea) in Thailand revealed by randomly amplified polymorphic DNA analysis.

Genetic diversity and species-diagnostic markers of 5 oysters in Thailand, Crassostrea belcheri (Sowerby, 1871), Crassostrea iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), Saccostrea forskali (Gmelin, 1791), and Striostrea Parastriostrea) mytiloides (Lamarck, 1819), were investigated by randomly amplified polymorphic DNA (RAPD) analysis. In a total, 135, 127, and 108 genotypes were observed from primers OPA09, OPB01, and OPB08 (Operon Technologies Inc., kits A and B), and 131 and 122 genotypes from primers UBC210 and UBC220 (University of British Columbia), respectively. Two hundred fifty-four reproducible and polymorphic fragments (200-2500 bp in length) were generated across the 5 investigated species. The average number of bands per primer varied between 12.4 and 32.2. The percentage of polymorphic bands within Crassostrea (53.23%-77.67%) was lower than that within Saccostrea and Striostrea oysters (86.21%-99.36%). Nine, species-specific markers were found in C. belcheri, 4 in C. iredalei, and 2 in S. cucullata. The mean of a ratio between the number of genotypes generated by each primer and the number of investigated specimens of C. belcheri (0.58) was lower than that of the remaining species (0.90-1.00). Genetic distances between pairs of oyster samples were between 0.105 and 0.811. A neighbor-joining tree indicated distant relationships between Crassostrea and Saccostrea oysters, but closer relationships were observed between the latter and Striostrea mytiloides.

A non-stop, single-tube, semi-nested PCR technique for grading the severity of white spot syndrome virus infections in Penaeus monodon.

A single-tube, non-stop, semi-nested polymerase chain reaction (PCR) technique was developed for simultaneous detection and severity grading of white spot syndrome virus (WSSV) infections in the black tiger shrimp Penaeus monodon. The test uses 1 sense primer and 3 antisense primers that produce up to 3 PCR products (1100, 526 and 250 base pairs [bp]) depending upon the severity of infection. Specifically, heavy infections (> or = 2 x 10(4) viral particles) of WSSV produce all 3 fragments, while moderate infections (around 2 x 10(3) viral particles) produce 2 (526 and 250 bp) and light infections (20 to 200 viral particles) produce 1 (250 bp). In addition, the technique uses internal control primers that yield a shrimp characteristic fragment for non-infected samples and samples with a low quantity of viral target in order to assure integrity and reproducibility of the PCR assays. The non-stop, single-tube, semi-nested PCR technique is simple and convenient and can detect as little as 5 fg WSSV DNA (20 viral particles) in crude extracts of postlarval samples or extracts of pleopods and haemolymph from larger shrimp.

Microsatellite Polymorphism and the Population Structure of the Black Tiger Shrimp (Penaeus monodon) in Thailand.

Genetic variation and differentiation of Thai Penaeus monodon from five geographic locations (Chumphon, Trad, Phangnga, Satun, and Trang) were investigated using five microsatellite loci (CUPmo18, Di25, Di27, CSCUPmo1, and CSCUPmo2). The number of alleles across the five loci ranged from 19 to 30, and heterozygosities ranged from 0.49 to 0.95. The mean number of alleles and effective number of alleles per locus were 21.0 to 26.6 and 13.1 to 20.4, respectively. The average heterozygosity across all investigated samples was 0.78, indicating high genetic diversity in this species. Geographic heterogeneity analysis of the results from two of the loci, CUPmo18 and Di25, showed significant differences among the Gulf of Thailand (Trad and Chumphon) but not the Andaman samples. Comparison between regions revealed significant heterogeneity of the Andaman and Trad P. monodon (P <.001), whereas those from Chumphon and the Andaman were genetically similar (P >.05). Significant genetic differentiation was consistently observed between the Andaman-Trad samples (F(ST) = 0.0101, P <.0001) and the Chumphon-Trad samples (F(ST) = 0.0101, P <.0001). On the basis of our analyses, the investigated samples from five geographic locations were allocated to three distinct populations composed of the Andaman Sea (A), Chumphon (B), and Trad (C).

Development of Species-Specific Markers of the Tropical Oyster (Crassostrea belcheri) in Thailand.

Randomly amplified polymorphic DNA (RAPD) analysis was used to identify species-specific markers of 5 oyster species in Thailand: Crassostrea belcheri, Crassostrea iredalei, Saccostrea cucullata, Saccostrea forskali, and Striostrea (Parastriostrea) mytiloides. Species-specific markers were found in C. belcheri, C. iredalei, and S. cucullata but not in S. forskali and S. mytiloides. Three C. belcheri-specific RAPD fragments were cloned and sequenced. A primer set was designed from each of the recombinant clones (pPACB1, pPACB2, and pPACB3). The polymerase chain reaction products showed expected sizes of 536, 600, and 500 bp, respectively, with the sensitivity of detection approximately 30 pg of C. belcheri total DNA template. The specificity of pPACB1 was examined against 135 individuals of indigenous oyster species in Thailand and against outgroup references S. commercialis (N = 12) and Perna viridis (N = 12). Results indicated the species-specific nature of primers developed from pPACB1. This primer set can be used for broodstock selection and determination of C. belcheri larvae to assist the selective breeding program for this commercially important species.

Mitochondrial DNA Diversity in Three Populations of the Giant Tiger Shrimp Penaeus monodon.

: Mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) was utilized for determination of genetic variation and population structure in Penaeus monodon collected from Satun (the Andaman Sea) and Surat and Trat (the Gulf of Thailand). Twenty-eight composite haplotypes were generated from 52 restriction profiles of P. monodon mtDNA digested with 11 restriction endonucleases. The size of the entire P. monodon mitochondrial genome was estimated to be 15.913 +/- 0.177 kb. The average haplotype diversity in P. monodon was 0.864, whereas the mean nucleotide diversity within populations was 2.51%, 2.22%, and 1.91% for Satun, Trat, and Surat, respectively. Geographic heterogeneity analysis indicated population differentiation between P. monodon from the Andaman Sea and P. monodon from the Gulf of Thailand (p <.0001). On the basis of the high genetic diversity level of P. monodon in Thailand, the Satun and Trat P. monodon populations from the west and east of the pennisula were selected to be founder stocks in our selective breeding program.

Experimental transmission of white spot syndrome virus (WSSV) from crabs to shrimp Penaeus monodon.

White spot syndrome virus (WSSV) of the black tiger prawn Penaeus monodon is a recently discovered baculo-like virus disease which is currently the cause of very serious and widespread losses in the shrimp industry in Thailand and elsewhere in Asia. Three suspected crab carriers of this virus commonly found in shrimp-rearing areas were investigated. These were Sesarma sp., Scylla serrata and Uca pugilator. All these crabs could be infected with WSSV by injection and they sustained heavy viral infections for up to 45 d (confirmed by normal histology, specific in situ DNA hybridization and PCR amplification) without visible signs of disease or mortality. All of them also transferred the disease to P. monodon via water while physically separated in aquarium cohabitation tests. Transfer of the virus to the shrimp was monitored using in situ DNA hybridization and PCR assay at 12 h intervals after cohabitation began. With U. pugilator, WSSV could be detected in the shrimp cohabitants after 24 h using PCR amplification and after 60 h using in situ hybridization. With S. serrata, the shrimp were positive for WSSV after 36 h using PCR and after 60 h using DNA in situ hybridization. With Sesarma sp. they were positive after 48 h using PCR and 72 h using in situ hybridization. These laboratory studies demonstrated that crab carriers of WSSV may pose a real threat to cultivated shrimp. However, the studies were carried out in containers with a small volume and with relatively clean sea water as compared to shrimp cultivation ponds. Pond-based studies are now needed to determine whether factors such as pond volume, pond water quality and shrimp and crab behavior can influence the rate and success of transfer.

Genetic structure in wild populations of black tiger shrimp (Penaeus monodon) using randomly amplified polymorphic DNA analysis.

Randomly amplified polymorphic DNA (RAPD) analysis was used to examine genetic variation in wild black tiger shrimp, Penaeus monodon. Specimens were collected from five geographically separated locations (Satun-Trang, Phangnga, and Medan in the Andaman Sea and Chumphon and Trad in the Gulf of Thailand). A total of 100 P. monodon individuals were investigated using seven arbitrarily selected primers. Fifty-eight (72.5%) of eighty reproducible RAPD fragments ranging in size from 200 to 2200 bp were polymorphic. The percentages of polymorphic bands of the five geographic populations investigated varied from 51.5 to 57.7%. The genetic distance between populations and UPGMA dendrograms indicated that the Medan population was genetically different from Thai P. monodon (Dij = 14.976%). Within Thailand, the Satun-Trang P. monodon was separated from the remaining geographic populations with a genetic distance of 2.632%. RAPD analysis in the present study yielded a total of 252 genotypes. A Monte Carlo analysis illustrated geographic heterogeneity in genotype frequencies within this species, suggesting that genetic population structure does exist in this taxon (P < 0.001 for all primers). Signficant differences in genotype frequencies between Thai and Indonesian (Medan) P. monodon were observed (P < 0.0001). Within Thailand, the Andaman Sea P. monodon was significantly different from that of the Gulf of Thailand (P values between 0.0000 and 0.0387), indicating population differentiation between P. monodon from these two main fishery regions of Thailand.

Random amplified polymorphic DNA (RAPD) markers for determination of genetic variation in wild populations of the black tiger prawn (Penaeus monodon) in Thailand.

Random amplified polymorphic DNA (RAPD) analysis was used to amplify the genome of black tiger prawns (Penaeus monodon) to detect DNA markers and assess the utility of the RAPD method for investigating genetic variation in wild P. monodon in Thailand. A total of 200 ten-base primers were screened, and 84 primers yielded amplification products. Six positive primers that gave highly reproducible RAPD patterns were selected for the analysis of three geographically different samples of Thai P. monodon. A total of 70 reproducible RAPD fragments ranging in size from 200 to 2000 bp were scored, and 40 fragments (57%) were polymorphic. The RAPD analysis of broodstocks from three different locales, Satun-Trang, Trat, and Angsila, revealed different levels of genetic variability among the samples. The percentages of polymorphic bands were 48% and 45% in Satun-Trang and Trat, respectively, suggesting a high genetic variability of the two samples to be used in selective breeding programs. Only 25% polymorphic bands were found in the Angsila sample, indicating the lowest polymorphic level among the three samples examined. Primer 428 detected a RAPD marker that was found only in P. monodon originating from Satun-Trang, suggesting the potential use of this marker as a population-specific marker in this species.

A simple, rapid and sensitive detection of salmonella in food by polymerase chain reaction.

A polymerase chain reaction (PCR) method was developed for detection of salmonella in food. A set of PCR primers was designed to amplify a 199 bp salmonella-specific DNA fragment derived from a repetitive DNA of Salmonella Weltevreden. The assay detected all 52 most prevalent serovars found in contaminated food in Thailand and no cross-reaction was observed with other non-salmonella organisms. The limit of detection was 1 fg of purified target DNA or five bacteria from pure culture. The detection of artificially contaminated food performed following a 6 h enrichment step was three bacteria per gram and the result was obtained within 4 h.

Simple nonradioactive DNA hybridization method for identification of sibling species of Anopheles dirus (Diptera: Culicidae) complex.

A simple method was developed for species identification of mosquitoes in the Anopheles dirus complex found in Thailand using horseradish peroxidase-labeled DNA probes and a chemiluminescent detection system. Species-specific DNA probes for Anopheles dirus, species B, C, and D detected 1-5 ng of target DNA, a sensitivity that was comparable with the radioisotopic detection system. Identification of individual mosquitoes was performed by dot-blot analysis of crude mosquito DNA. The method allowed identification using third or fourth instars as well as the adult head, thorax, or abdomen. This technique successfully identified the sibling species of the A. dirus complex in field collections.

Sensitive detection of Plasmodium falciparum in blood and mosquito by DNA amplification.

By means of enzymatic amplification of Plasmodium falciparum DNA using the polymerase chain reaction (PCR), we have been able to detect as little as 20 parasites in 20 ml infected human blood based on the visualization of a 206 bp fragment in ethidium bromide-stained agarose gel. Comparison, through microscopic examination of 2030 blood samples collected from various endemic areas in Thailand, indicates that the PCR-based detection system is 96% specific and 81% sensitive, with a disagreement of 6.6%. The same protocol can be applied to detect a minimum of 10 sporozoites or a single oocyst dissected from P. falciparum-infected mosquito.

Polymerase chain reaction detection of Plasmodium falciparum in mosquitoes.

The polymerase chain reaction (PCR) procedure using a primer set derived from a repetitive deoxyribonucleic acid (DNA) sequence specific to Plasmodium falciparum was used to detect parasite DNA in mosquitoes. In laboratory-infected mosquitoes, PCR could detect as few as 10 sporozoites in a dissected salivary gland and a single oocyst in a dissected midgut. The ability to detect P. falciparum DNA in wild-caught mosquitoes indicated an advantage of the PCR over enzyme-linked immunosorbent assay for the detection of Plasmodium sporozoites in mosquitoes with low-grade parasite infections.

Improved resolution and sensitivity of human DNA fingerprinting by specific-primed labelling of M13 DNA.

A method to label M13 DNA probe by primer extension using a specific oligonucleotide primer is described. The method specifically labels the two 15-bp repeats in M13 DNA which hybridize to target DNA giving rise to DNA fingerprinting patterns. The M13 probe labelled by this method gave superior DNA fingerprinting patterns that that labelled by random primers. As little as 0.25 microgram of target DNA was sufficient for DNA fingerprinting. Non-isotopic labelling by the specific primer also showed improved DNA fingerprinting pattern. The results demonstrate the methodology to improve DNA fingerprinting based on M13 DNA probe.