Genetic Diversity and Molecular Markers of Cupped Oysters (Genera Crassostrea, Saccostrea, 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. Received June 6, 2000; accepted September 12, 2000     

Molecular Genetic Identification Tools for Three Commercially Cultured Oysters (<Emphasis Type="Italic">Crassostrea belcheri</Emphasis>, <Emphasis Type="Italic">Crassostrea iredalei</Emphasis>, and <Emphasis Type="Italic">Saccostrea cucullata</Emphasis>) in Thailand

Abstract 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.     

Stone-tool usage by Thai long-tailed macaques (Macaca fascicularis)

In January and March of 2005, we conducted surveys of long-tailed macaques at Piak Nam Yai Island, Laem Son National Park (9 degrees N 34-35', 98 degrees E 28'), Ranong Province, situated in southern Thailand. Two of the three troops of long-tailed macaques found on the island were observed using axe-shaped stones to crack rock oysters, detached gastropods (Thais tissoti, Petit, 1852), bivalves (Gafrarium divaricatum, Gmelin, 1791), and swimming crabs (Thalamita danae, Stimpson, 1858). They smashed the shells with stones that were held in either the left or right hand, while using the opposite hand to gather the oyster meat. Some monkeys used both hands to handle the stones. According to Matsuzawa's 1996 hierarchical classification of tool usage (levels 0-3), the tool usage by Thai long-tailed macaques could be characterized as either level 1 (cracking rock oysters with stones) or level 2 (cracking drifting mollusks and crabs with stones by placing them on a rock). Our discovery of stone-tool usage by Thai long-tailed macaques provides a new point of reference for discussions regarding the evolution of tool usage and the material culture of primates.     

Prevalence of Perkinsus marinus (dermo), Haplosporidium nelsoni (MSX), and QPX in bivalves of Delaware's inland bays and quantitative, high-throughput diagnosis of dermo by QPCR

Restoration of oyster reef habitat in the Inland Bays of Delaware was accompanied by an effort to detect and determine relative abundance of the bivalve pathogens Perkinsus marinus, Haplosporidium nelsoni, and QPX. Both the oyster Crassostrea virginica and the clam Mercenaria mercenaria were sampled from the bays. In addition, oysters were deployed at eight sites around the bays as sentinels for the three parasites. Perkinsus marinus prevalence was measured with a real-time, quantitative polymerase chain reaction (PCR) methodology that enabled high-throughput detection of as few as 31 copies of the ribosomal non-transcribed spacer region in 500 ng oyster DNA. The other pathogens were assayed using PCR with species-specific primers. Perkinsus marinus was identified in Indian River Bay at moderate prevalence ( approximately 40%) in both an artificial reef and a wild oyster population whereas sentinel oysters were PCR-negative after 3-months exposure during summer and early fall. Haplosporidium nelsoni was restricted to one oyster deployed in Little Assawoman Bay. QPX and P. marinus were not detected among wild clams. While oysters in these bays have historically been under the greatest threat by MSX, it is apparent that P. marinus currently poses a greater threat to recovery of oyster aquaculture in Delaware's Inland Bays.     

Oyster reefs as processors of estuarine materials

Oyster reefs are dense concentrations of filter-feeding animals in estuarine ecosystems. A flow-through plastic tunnel is a feasible method of determining significant changes in material concentrations in tidal waters passing over an oyster reef. The oyster reef reduces the amplitude of the particulate organic carbon and chlorophyll a signals while increasing the amplitude, of the ammonia signal. The observations suggest that oyster reefs have one of the highest reported release rates of ammonia (1680–7250 μg at.·m^?2·h^?1), and thus are probably important in material cycles in marsh-estuarine ecosystems. The magnitude of particulate organic carbon removal by the oyster reef is many times greater than that expected from biofiltration alone, suggesting that removal due to physical factors may be important.     

二倍体和三倍体太平洋牡蛎鳃扫描电镜的比较

利用扫描电子显微镜技术对二倍体和三倍体太平洋牡蛎(Crassostrea gigas)鳃的表面结构进行了观察和比较。结果显示：三倍体牡蛎鳃丝的宽度、鳃丝间的距离较二倍体大;鳃丝的微细结构比二倍体更致密;鳃丝间通过丝问连接形成的孔洞大于二倍体。这些不同表明二倍体和三倍体呼吸及摄食可能存在差异。     

New anonymous nuclear DNA markers for the pearl oyster Pinctada margaritifera and other Pinctada species

The black‐lipped oyster Pinctada margaritifera is highly exploited in French Polynesia where the pearl industry relies mostly on spat collection, and therefore on resources available in the wild. Little is known of these resources, and population genetic studies would be useful to improve management. We used two methods, direct amplification of length polymorphism (DALP) and exon primed intron crossing (EPIC) to develop five new nuclear markers presenting length polymorphism. Although these markers remained anonymous after using blast on GenBank, they are codominant and follow Hardy–Weinberg equilibrium. Tests on related species or subspecies of Pinctada gave encouraging results.     

Metabolic adjustments in the oyster Crassostrea gigas according to oxygen level and temperature

The purpose of this study was to examine the responses of the oyster Crassostrea gigas to oxygen levels at subcellular and whole organism levels. Two experiments were carried out. The first experiment was designed to measure the clearance and oxygen consumption rates of oysters exposed at different concentrations of oxygen at 15, 20 and 25°C for 20 h. The goal of this first part was to estimate the hypoxic threshold for oysters below which their metabolism shifts towards anaerobiosis, by estimating the oxygen critical point (PcO₂) at 15, 20 and 25°C. The second experiment was carried out to evaluate the metabolic adaptations to hypoxia for 20 days at three temperatures: 12, 15 and 20°C. The metabolic pathways were characterized by the measurement of the enzymes pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK), the alanine and succinate content and the adenylate energy charge. Respiratory chain functioning was estimated by the measurement of the activity of the electron transport system (ETS). The values of PcO₂ were 3.02±0.15, 3.43±0.20 and 3.28±0.24 mg O₂ l^-1 at 15, 20 and 25°C, respectively. In whole oysters, hypoxia involved the inhibition of PK whatever the temperature, but PEPCK was not stimulated. Succinate accumulated significantly only at 12°C and alanine at 12 and 15°C. A negative relationship between the PK activity and the alanine content was only found in hypoxic oysters. Finally, hypoxia increased significantly the activity of ETS. With high PcO₂ values, the metabolic depression occurred quickly, showing that oysters had a low capacity to regulate their respiration when oxygen availability is reduced, particularly in the summer.     

Coastal acidification impacts on shell mineral structure of bivalve mollusks

Ocean acidification is occurring globally through increasing CO₂ absorption into the oceans creating particular concern for calcifying species. In addition to ocean acidification, near shore marine habitats are exposed to the deleterious effects of runoff from acid sulfate soils which also decreases environmental pH. This coastal acidification is being exacerbated by climate change‐driven sea‐level rise and catchment‐driven flooding. In response to reduction in habitat pH by ocean and coastal acidification, mollusks are predicted to produce thinner shells of lower structural integrity and reduced mechanical properties threatening mollusk aquaculture. Here, we present the first study to examine oyster biomineralization under acid sulfate soil acidification in a region where growth of commercial bivalve species has declined in recent decades. Examination of the crystallography of the shells of the Sydney rock oyster, Saccostrea glomerata, by electron back scatter diffraction analyses revealed that the signal of environmental acidification is evident in the structure of the biomineral. Saccostrea glomerata, shows phenotypic plasticity, as evident in the disruption of crystallographic control over biomineralization in populations living in coastal acidification sites. Our results indicate that reduced sizes of these oysters for commercial sale may be due to the limited capacity of oysters to biomineralize under acidification conditions. As the impact of this catchment source acidification will continue to be exacerbated by climate change with likely effects on coastal aquaculture in many places across the globe, management strategies will be required to maintain the sustainable culture of these key resources.