Manila Clam Populations Research Articles

Co-infection of two eukaryotic pathogens within clam populations in Arcachon Bay.

The parasitic species Perkinsus olseni (= atlanticus) (Perkinsea, Alveolata) infects a wide range of mollusc species and is responsible for mortality events and economic losses in the aquaculture industry and fisheries worldwide. Thus far, most studies conducted in this field have approached the problem from a "one parasite-one disease" perspective, notably with regards to commercially relevant clam species, while the impact of other Perkinsus species should also be considered as it could play a key role in the disease phenotype and dynamics. Co-infection of P. olseni and P. chesapeaki has already been sporadically described in Manila clam populations in Europe. Here, we describe for the first time the parasitic distribution of two Perkinsus species, P. olseni and P. chesapeaki, in individual clam organs and in five different locations across Arcachon Bay (France), using simultaneous in situ detection by quantitative PCR (qPCR) duplex methodology. We show that P. olseni single-infection largely dominated prevalence (46-84%) with high intensities of infection (7.2 to 8.5 log-nb of copies. g-1of wet tissue of Manila clam) depending on location, suggesting that infection is driven by the abiotic characteristics of stations and physiological states of the host. Conversely, single P. chesapeaki infections were observed in only two sampling stations, Ile aux Oiseaux and Gujan, with low prevalences 2 and 14%, respectively. Interestingly, the co-infection by both Perkinsus spp., ranging in prevalence from 12 to 34%, was distributed across four stations of Arcachon Bay, and was detected in one or two organs maximum. Within these co-infected organs, P. olseni largely dominated the global parasitic load. Hence, the co-infection dynamics between P. olseni and P. chesapeaki may rely on a facilitating role of P. olseni in developing a primary infection which in turn may help P. chesapeaki infect R. philippinarum as a reservoir for a preferred host. This ecological study demonstrates that the detection and quantification of both parasitic species, P. olseni and P. chesapeaki, is essential and timely in resolving cryptic infections and their consequences on individual hosts and clam populations.

Numerical study of hydrodynamic effects on Manila clam population distribution and transport in the Southwest Laizhou Bay, China

Pelagic larvae of marine organisms are abundant on many continental shelves. Due to their importance as a biological resource and contribution to marine biodiversity, the dynamics of pelagic larvae have received wide attention. Numerical models are an efficient approach to assessing pelagic larval transport driven by various ocean dynamics. However, few studies have focused on the pelagic larval distribution of Manila clam populations and related dynamic factors in the southwestern Laizhou Bay (SLB) during late spring. A hydrodynamic model including realistic meteorologic and oceanic boundary conditions was coupled with a larval transport model to describe the pelagic larval distribution and transport processes. The hydrodynamic model was validated with observational and satellite-derived data. Effects of hydrodynamics and biological behavior on pelagic dynamics were further simulated with numerical experiments. During the early planktonic period, most clam larvae in the SLB were transported eastward or northeastward. Two potential larval settlement areas (LSAs) were recognized: the east side of the dike outside Guangli port and the Lao River estuary. The dominant controlling factor was southerly wind, and larvae's response to river plume or estuarine circulation played a secondary role. Larval transport depended on bottom subtidal currents when late-stage larvae settled near the bottom layer. The hydrodynamic and larval transport model established in this study can be applied to other bays and coastal waters. Understanding the clam larval distribution and transport dynamics in the SLB can provide a reference for predicting LSAs and biological resource management in coastal aquaculture areas.

Molecular and microscopic identification of Eomarteilia granula infection in Manila clam Ruditapes philippinarum off the south coast of Korea.

A report on the new species Eomarteilia (=Marteilia) granula infecting Manila clam Ruditapes philippinarum from Japan in 2014 suggests the possibility of E. granula infecting other Manila clam populations in the Northwest Pacific region, including Korea. In this study, we report the first infections by E. granula in Manila clams off the south coast of Korea. Histology revealed Marteilia-like plasmodia in the digestive tubule epithelia. Tissue imprints demonstrated that each parasite sporangium enclosed 4 spores and transmission electron microscopy (TEM) revealed ultrastructure of primary cells enclosing secondary cells, which contained spores. Mature spores consisted of 3 sporoplasms: outermost, intermediate, and innermost. The outermost sporoplasm showed a peripheral electron-dense monolayer characteristic of E. granula. The 18S rDNA amplified from the Marteilia-like parasite yielded 1784-bp PCR amplicon sequences which were 99.8% similar to that of E. granula previously reported (as M. granula) from Japan. In the molecular phylogenetic analysis, the novel Marteilia-like organism formed a well-supported clade with E. granula. Accordingly, we concluded that the novel Marteilia-like parasite that we found infecting some Korean Manila clams is Eomarteilia granula. Field surveys revealed that the infection was limited to clams of the south coast of Korea, with the prevalence ranging from 3.3 to 5.0%.

Population genetics of the Manila clam (Ruditapes philippinarum) in East Asia

The Manila clam Ruditapes philippinarum is the world’s second most important bivalve mollusk commercially farmed, whose indigenous populations are mainly distributed in the coastal areas of East Asia. However, with the development of commercialization, mixture of populations and loss of local germplasm have become prominent problems. Here, genetic differentiation of seven Manila clam populations from East Asia was investigated through analyzing the polymorphism of the mitochondrial cytochrome C oxidase subunit I (COI) gene as well as 20 simple sequence repeat (SSR) molecular loci. In total, 40 haplotypes were identified, among which 31 were unique. Moreover, two main haplotypes were detected with several radiating derived haplotypes. Populations in Japan-North Korea shared haplotype Hap_31, and populations in China shared haplotype Hap_7, suggesting that the natural geographical isolation of the Yangtze River and the Yalu River might have divided the East Asian indigenous populations into three groups, which were located in South China, North China, and Japan-North Korea, respectively. The Aquaculture breeding activities from South to North in China might have promoted gene exchange among Manila clam populations. Population in Laizhou had the highest genetic diversity and therefore could be an excellent germplasm source.

Molecular detection of protists associated with the Manila clam Ruditapes philippinarum inhabiting the Wajiro tidal flat in Hakata Bay, Fukuoka Prefecture

Tidal flats are ecologically important as they support a large community of animals (e.g., crabs, mollusks, and polychaetes) and restore water quality. However, information regarding microbial eukaryotes, most of which are protists, associated with animals inhabiting these environments is limited. In the present study, utilizing polymerase chain reaction techniques, we detected fragments of the small subunit ribosomal RNA (SSU rRNA) gene of protists from the gill tissues of the Manila clam Ruditapes philippinarum in the Wajiro tidal flat, Hakata Bay, Japan. Based on the phylogeny of the SSU rRNA gene, the detected organisms were shown to belong to not only Perkinsozoa but also to Ciliophora, Kinetoplastida, and Haplosporidia. For the protist affiliated with Perkinsozoa, the internal transcribed spacer (ITS) of the rRNA genes was retrieved from the same gill tissue samples for precise identification at the species level. The ITS sequence obtained was identical to that of Perkinsus olseni, which is highly prevalent in Manila clam populations in Japan. Our findings suggest that protists potentially parasitic on this representative mollusk in tidal flats are phylogenetically diverse, and that the organisms detected may affect the population dynamics of the host clams.

Euclidean Genetic Distances of Four Manila Clam (Ruditapes philippinarum) Populations analyzed by PCR Research.

ABSTRACTThe PCR analysis was performed on DNA samples extracted from a total of 20 individuals using six oligonucleotides primers. The author accomplished clustering analyses to reveal the Euclidean genetic distances among four clam populations from Gochang, Seocheon, Taean and Anmyeon of the Korean peninsula. The oligonucleotides primer OPA-08 generated 5 unique loci to each population, approximately 550 bp and 600 bp, respectively, in the MCS population. Especially, the primer OPA-20 generated 15 unique loci to each population, which were identifying each population, approximately 400 bp, 750 bp and 800 bp, in the MCT population. Individuals from MCG clam population (0.637±0.227) exhibited higher bandsharing values than did individuals from MCG clam population (0.402±0.115) (P<0.05). The dendrogram obtained by the six oligonucleotides primers indicates four genetic clusters: cluster 1 (MCG 01, 02, 04 and 05), cluster 2 (MCS 06, 07, 08, 09 and 10), cluster 3 (MCT 11, 12, 13, 14 and 15) and cluster 4 (MCA 16, 17, 18, 19, 20 and MCG 03). Among the twenty clam individuals, the shortest genetic distance that displayed significant molecular differences was between individuals 14 and 15 from the MCT population (genetic distance = 0.094), while the longest genetic distance among the twenty individuals that displayed significant molecular differences was between individuals MCG no. 01 and MCG no. 02 (genetic distance = 0.687). Comparatively, individuals of MCS clam population were fairly closely related to that of MCT clam population, as shown in the hierarchical dendrogram of Euclidean genetic distances.

Survey on &lt;i&gt;Perkinsus&lt;/i&gt; Species in Manila Clam &lt;i&gt;Ruditapes philippinarum&lt;/i&gt; in Korean Waters Using Species-Specific PCR

We investigated possible infection with Perkinsus olseni and/or P. honshuensis in Manila clams in Korean waters using Perkinsus species-specific PCR. Ray’s fluid thioglycollate medium assay revealed that 97.3% of the clams examined were infected with Perkinsus sp., with the mean intensity ranging from 2.2 × 104 to 6.8 × 106 cells/g gill. In the Perkinsus species-specific PCR assay, all the amplified Perkinsus-positive bands were identified as P. olseni, while none of P. honshuensis-specific band was amplified. Our survey results indicate that P. olseni is the main causative agent of perkinsosis in Manila clam populations in Korean waters, while more extensive survey must be carried out.

A history of invasion: COI phylogeny of Manila clam Ruditapes philippinarum in Europe

The Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) is now one of the top 5 most commercially valuable bivalve species worldwide. Originally from the Indo-Pacific region, it has been introduced in many countries for fisheries and aquaculture, including estuarine environments along Atlantic and Mediterranean European coasts. Yet despite its commercial value and widespread distribution, the precise origins of stocks remain speculative and the genetic diversity of introduced populations is poorly known. Thus, the aim of this work was to collect mtDNA COI (Cytochrome oxidase I) gene sequences from 5 European countries with Manila clam stocks and compare them with native Asian populations to evaluate their genetic diversity and identify possible routes of invasion. The COI gene sequencing supported a strong founder effect in the European populations with 3 main haplotypes occurring at high frequencies, derived from Japan. However, high haplotype diversity was also observed due to the occurrence of 10 rare haplotypes. This supports hypotheses (i) there have been additional, previous unrecorded, introductions as previously hypothesized by analysis of 16S rDNA, and (ii) there has been a limited loss of genetic diversity in introduced populations, as previously suggested by microsatellite data. This is the first genetic comparison of Manila clam populations introduced in to Europe with native clams. Genetic data herein presented are fundamentally important for the traceability of clam products and stock management programmes and will also inform discussion on the potential resilience of exploited Manila clam populations.

Genetic diversity and structure of Manila clam (Ruditapes philippinarum) populations from Liaodong peninsula revealed by SSR markers

Manila clam (Ruditapes philippinarum) is an important commercially marine bivalve, and its wild populations have been severely declining in the coast of China during the last decade. In this study, a set of 7 genomic simple sequence repeat (SSR), and 5 expressed sequences tag (EST)-derived SSR markers were analyzed on eight wild R. philippinarum populations to assess the genetic diversity and population differentiation. A total of 114 alleles were detected on 12 loci, and the number of alleles per locus in each population ranged from 2 to 11, and allelic richness per locus varied from 2.00 to 10.88 for each sample. The average of observed and expected heterozygosity ranged from 0.386 to 0.550, and from 0.533 to 0.707, respectively. Pairwise FST values indicated that all population pairs had significant genetic differentiation (overall FST = 0.242, P < 0.01). Cluster analysis using unweighted pair group method with the arithmetic mean (UPGMA) separated the eight populations into two groups. This study will shed light on the domestication and cultivation on population genetic diversity of R. philippinarum, and also provide the foundation for conservation of R. philippinarum germplasm resources in clam breeding practices.

Morphological Trends of Four Manila Clam Populations (Venerupis philippinarum) on the French Atlantic Coast: Identified Spatial Patterns and Their Relationship to Environmental Variability

For species dynamics highly influenced by environmental conditions, sustainable management for conservation and/or exploitation purposes requires the ability to segregate natural processes from anthropogenic impacts. In this context, identification of phenotypic variations in morphological patterns, and clarification of the mechanisms involved, constitute useful tools to identify adequate management measures from Europe-wide to local conditions. Although such approaches are already used effectively in the management of fish stocks, they are more recent for bivalve stocks, which are also good candidates for such management tools. The current study examines morphological patterns in the Manila clam (Venerupis philippinarum) through morphometric studies of 4 populations on the French Atlantic coast (Banc du Guer, Morbihan Gulf, Bellevue, and Arcachon Bay). The methodology relies mainly on conventional shape analysis using 8 morphometric ratios involving metrics and weight. In addition, an outline description was created for selected individuals that revealed different forms of the anterior-dorsal valve margin identified for future research. Using supervised classification methods, morphometric-latitudinal trends were revealed between northern and southern populations of V. philippinarum. Relationships between discriminatory morphometric ratios (describing elongation, valve density, and weight related to length) and available environmental conditions (temperature, salinity, and chlorophyll a concentrations) were considered using linear multilevel models. They revealed significant relationships with chlorophyll a concentrations and with 1 ratio with seawater temperature ranging from 12-20C. Four potential causes were hypothesized to explain the unexpected relationship identified between morphometric characteristics and trophic conditions: (1) a negative effect resulting from high chlorophyll a concentrations, (2) a masked effect of other food resources affecting valve growth, (3) interspecific competition for food among filter-feeding populations, (4) and effects of other environmental parameters such as sediment type. Observed differences in shape may result from a combination of these 4 causes with varying degrees, depending on site-specific environmental conditions. Last, the consequences of morphometric variation on population regulation are addressed.

Genetic diversity of introduced Manila clam Ruditapes philippinarum populations inferred by 16S rDNA

Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) – is one of the most successful marine invaders, being introduced worldwide for aquaculture and fisheries. Genetic diversity and structure of its populations are largely unknown, especially in the invaded environments. Herein we present molecular genetic data on 12 introduced Manila clam populations in Italy, Spain, and Portugal from both Mediterranean (Adriatic) and Atlantic sampling sites. The phylogenetic information was investigated by the direct sequencing of 16S mitochondrial DNA. Results of mtDNA analyses showed the occurrence of 11 haplotypes for European introduced populations, as a consequence of multiple founder effects from different source populations. Rp hap1 was the most frequent, shared among all populations. The other 10 haplotypes were rare and distributed at local scale, in agreement with what was observed in other invasive bivalves. Biogeographic and phylogenetic analyses based on 16S rDNA of introduced Manila clam populations showed a complex scenario, dominated by multiple pools of individuals coming from different sources, as a consequence of multiple introductions.

New polymorphic microsatellite markers for the Korean manila clam (Ruditapes philippinarum) and their application to wild populations.

Manila clam (Ruditapes philippinarum) is a valuable and intensively exploited shellfish species in Korea. Despite its importance, information on its genetic background is scarce. For the genetic characterization of R. philippinarum, expressed sequence tag-derived microsatellite markers were developed using next-generation sequencing. A total of 5879 tandem repeats containing di- to hexanucleotide repeat motifs were obtained from 236,746 reads (mean = 413 bp). Of the 62 loci screened, 24 (38.7%) were successfully amplified, and 10 were polymorphic in 144 individuals from 2 manila clam populations (Incheon and Geoje, Korea). The number of alleles ranged from 2 to 17 in the Incheon population and from 3 to 13 in the Geoje population (overall AR = 7.21). The mean observed and expected heterozygosities were estimated to be 0.402 and 0.555, respectively. Hence, there is less genetic variability in the Geoje population than in the Incheon population, although no significant reductions of genetic diversity were found between the populations (P > 0.05). However, significant genetic differentiation was detected between the populations (FST = 0.064, P < 0.001). Significant deviations from Hardy-Weinberg equilibrium and high inbreeding coefficients (mean FIS = 0.22-0.26) were detected in both populations. The 10 novel polymorphic microsatellite loci used in this study will be useful for future genetic mapping studies and for characterizing population structures, monitoring genetic diversity for successful aquaculture management, and developing conservation strategies for manila clam populations in Korea.

都市圏の小規模な造成干潟におけるアサリの生産構造

To clarify capability of small scale artificial tidal flat (SAT) in urban area, we focused on manila clam production and investigated some SATs in Tokyo Bay. Manila clam production rate on a main SAT was comparable or higher than natural tidal flat in Tokyo Bay. But, manila clam was damaged by hypoxia seriously in late summer to autumn in one SAT which was main research area. Another one SAT, manila clam wasn't damaged hypoxia, because of the shallow area was widely than main SAT. Further, benthic microalgae was main food source of Manila clam in all SATs, and manila clam populations related to benthic microalgae abundance. It suggested manila clam production was affected directly benthic microalgae production, and benthic microalgae production on SAT was changed by design or maintenance condition of each SAT.

Experimental challenges of juvenile and adult Manila clams with the protozoan Perkinsus olseni at different temperatures

Infection with Perkinsus species, primarily P. olseni, is thought to be a major cause of the decline of Manila clam populations in Japan since the 1980s. However, the pathogenicity of the infection has not been sufficiently evaluated to estimate the impact of infection on wild Manila clam populations. We experimentally challenged juvenile (3- to 6-mm shell length) and adult (18- to 22-mm shell length) Manila clams with P. olseni at 18, 23, 28, and 30 °C. Mortality was significantly higher in challenged groups than in control groups. The difference in mean mortality between the challenged and control groups (all life stages and temperatures) was only significant above a threshold of infection intensity ~106 cells/g soft wet tissue (SWT). As temperature increased, the onset of mortality occurred more rapidly. The increase in mortality occurred earlier in juveniles than adults at 28 °C and lower. Our results suggest that the pathogenicity of P. olseni is higher in juveniles than in adults and at higher water temperatures. Given the infection intensities (ca. 106 cells/g SWT) previously reported in wild Manila clams, the parasite likely has considerable impact on wild Manila clam populations, particularly juveniles during periods of high temperature.

Clam culture in the Venice lagoon: stock assessment of Manila clam (Venerupis philippinarum) populations at a nursery site and management proposals to increase clam farming sustainability

The Venice lagoon is the main Manila clam (Venerupis philippinarum ) production site in Europe. Following the unsustainable exploitation of this site (free-access fishery) local authorities have been striving to regulate fishing activities and most clams are produced using mollusc culture techniques based on wild spat harvesting and on its seeding in appropriate, designated culture areas. Therefore, the quality and abundance of wild clam stocks are critical factors that could limit the full development of this activity. The present study aims at assessing Manila clam stocks (spat, adults per square meter) using ordinary kriging, in an area of the Venice lagoon located near the Porto Marghera industrial zone. Previous studies have identified this area as one of the most important natural clam settlement sites in the Venice lagoon. The study area was approximately 1085 ha; 46 sampling stations were examined. Stock assessment (animals were grouped by size and age group), biomass and correlation with sediment texture were evaluated. The results obtained and comparisons with studies from the literature enabled an estimation of clam recruitment potential and an analysis of the conservation status of clam stocks. Thereafter, in view of a full reclamation and an ecological restoration of the study area, different measures are proposed for the sustainable use of this area that include the establishment of a biological protection area and a shared and rational management of the nursery area; these measures could potentially increase the sustainability of clam culture in the Venice lagoon.

The invasive Manila clam Ruditapes philippinarum (Adams and Reeve, 1850) in Northern Adriatic Sea: Population genetics assessed by an integrated molecular approach

The coastal lagoons of the Northern Adriatic Sea are among the most worldwide productive locations of Manila clam Ruditapes philippinarum. Although introduced in Italy in 1983 from the Indo-Pacific, fishing and exploitation of Manila clam improved during the years as Italy became the leading country in Europe for production of this shellfish. Despite its commercial importance, genetic structure of R. philippinarum in Northern Adriatic Sea has not been previously investigated. Here we present the first genetic study on Manila clam populations inhabiting a Mediterranean area, assessed by both mitochondrial ( 16S rDNA) and nuclear DNA (microsatellite loci). Our study showed that this species has a limited genetic differentiation at the mitochondrial level, but a higher rate of genetic diversity can be identified by polymorphic markers as microsatellites. In particular, out of 28 alleles, 7 private ones were recorded for the Venice Lagoon populations, 2 for those of Scardovari and one for the Po River Delta populations. These molecular markers suggest the occurrence of at least two different introduction events from different recruitment stocks, representing a powerful tool not only to assess genetic diversity of an introduced species, but also helpful information to manage aquaculture and fishery stocks, and to warrant food quality, safety and for the authentication of shellfish products, and traceabilty path.

Bivalve population health: Multistress to identify hot spots

This study investigated some stress (metals, parasites) and response (immunity, metallothionein) factors in two cockle and two Manila clam populations. Data from eight seasons were averaged to obtain global baseline values. Stress/response characteristics of each population were compared to population health status that was determined through population dynamics parameters. Four different scenarios were discussed: (1) a lightly stressed cockle population with correct population health but with a risk of deterioration (hot spot); (2) a lightly stressed introduced cockle population threatened of extinction. In this case ecological factors were suspected; (3) a moderately stressed clam population with moderate adaptative response. The population was sustainable but the level of stress should not increase (hotspot); and (4) a stressed clam population and unfavourable ecological conditions preventing clam settlement. This monitoring highlighted that the discrepancy between population health and stress levels could be due to insufficient response by bivalves and/or by unfavourable ecological factors.

Influence of Perkinsus Infection on the Physiology and Behavior of Adult Manila Clam Ruditapes philippinarumi

Annual catch landings of the Manila clam Ruditapes philippinarum have decreased in Japan since the early 1980s because of the decreased size of many Manila clam populations. It is hypothesized that Perkinsus olseni infection contributed to these decreases. However, the effect of Perkinsus infection on the survival and physiology of Manila clam is poorly understood. We examined the influence of Perkinsus infection on clearance rate, tolerance against high water temperature, and burrowing activity in a captive adult clam population. Infection intensity did not affect clearance rates, burrowing activity, or tolerance against high water temperature. In addition, we monitored infection intensity in wild, adult clams from a tidal flat lacated in western Seto Inland Sea over a one-year period. Infection levels were relatively stable in the wild population between June 2004 and May 2005, but we noted a significant decrease in the infection intensity at the end of the spawning period in November 2004. However, in the present study, clear evidence showing the negative impact of Perkinsus on clam physiology and survival was not obtained.

Effect of tributyltin on veliger larvae of the Manila clam, Ruditapes philippinarum

We investigated the effects of waterborne and maternal exposure to tributyltin (TBT) on veliger larvae of the Manila clam, Ruditapes philippinarum. In a waterborne exposure test, veliger larvae (D-larvae stage: 24 h after fertilization) were exposed to TBT at measured concentrations of <0.01 (control), 0.055, 0.130, 0.340, and 0.600 μg/l for 13 d. The percentage of normal veliger larvae (the ratio of normal veliger larvae to all larvae) decreased significantly in all TBT treatment groups compared with that in the control group. In a maternal exposure test, 100 clams were exposed to TBT at measured concentrations of <0.01 (control), 0.061, and 0.310 μg/l at 20–22 °C for 3 weeks, and the percentage of normal veliger larvae assessed for 13 d. No maternal effects on veliger larvae from TBT were observed in TBT treatment groups as compared with the control group. These results demonstrate that waterborne TBT affects Manila clam veliger larvae, and indicates that TBT may have reduced Manila clam populations by preventing the development and survival of veliger larvae.