Reproduction of an alien Ruditapes clam (Bivalvia: Veneridae) on recreational clam-gathering grounds in Tokyo Bay,Japan

The Manila clam Ruditapes philippinarum is an important fisheries resource and popular recreational native species in Japan. To counter the domestic shortage of Manila clams caused by depleted catches, similar clams were introduced into Japan from China and the Korean peninsula. The introduced clam is an unnamed morph closely related to R. philippinarum that originates from southern China. Its shell is morphologically different from that of the typical Manila clam and there are small but distinct genetic differences. There is evidence to suggest that the Chinese morph may hybridize with the Manila clam in the Ariake Sea, but not in Tokyo Bay. We carried out studies to determine the degree of overlap of the breeding seasons of both taxa by collecting samples of the Manila clam and the alien clam released onto recreational clam-gathering grounds in Tokyo Bay and their gonadal maturity was examined histologically. Spawning-stage individuals of the Manila clam appeared from April to August and those of the southern Chinese morph from July to August, indicating overlapping spawning periods and thus the potential for hybridization. We discussed the forming condition of the hybridized clam population in terms of reproduction and recruitment process of the clams.     

Development of microsatellite markers for the Manila clam Ruditapes philippinarum

We isolated nine polymorphic microsatellites from the Manila clam Ruditapes philippinarum. These loci provide one class of highly variable genetic marker as the number of alleles ranged from six to 22, and the observed and expected heterozygosity ranged from 0.136 to 0.909 and from 0.553 to 0.954, respectively. We consider that these loci are potentially useful for detailing the genetic structure and gene flow among R. philippinarum 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 F_ST values indicated that all population pairs had significant genetic differentiation (overall F_ST = 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.     

Molecular cloning and characterization of Y-box gene (Rpybx) from Manila clam and its expression analysis in different strains under low-temperature stress

Manila clam, Ruditapes philippinarum, is an economically important marine bivalve species. Y-box proteins are members of the cold shock proteins family and highly conserved from bacteria to humans. In this study, a novel Y-box gene (Rpybx) was cloned from the Manila clam and gene expression profiling was performed on three shell color strains (white, zebra and white zebra) and two wild populations (Southern and Northern) of R. philippinarum. The complete ORF length of Rpybx is 1367 bp, encoding 253 amino acids residues. Based on the amino acid sequence analysis and phylogenetic analysis, the Rpybx gene was identified as a member of the invertebrate Y-box proteins family. Rpybx has a similar tertiary structure to human Y-box protein YB-1. The Rpybx mRNA levels were analyzed by qPCR under acute and gradually varied cold stress. Under acute low-temperature stress, the expression of Rpybx mRNA in gills and hepatopancreas was significantly increased in all selected strains and populations (P < 0.05). The Northern population showed the lowest relative expression level of Rpybx. The expressions of Rpybx were greatly upregulated in gills and hepatopancreas of different stains and populations at 5 or −2°C under gradually varied temperature stress (P < 0.05). The results shed light on the biological function of the Rpybx gene in defending against low-temperature challenge and further exploring the molecular mechanism of cold tolerance and resistance in R. philippinarum.     

An environmentally induced tidal periodicity of microgrowth increment formation in subtidal populations of the clam Ruditapes philippinarum

The periodicity of increment formation in the shell of the Manila clam Ruditapes philippinarum was investigated in the subtidal zone of the Auray River estuary (South Brittany, France). Calcein markings were performed at different periods between May and October 2007 using in situ benthic chambers tented by scuba divers. This study shows that shell microgrowth increments were well-defined and deposited with a tidal periodicity in the subtidal zone, providing the calendar base for high-resolution ecological studies and environmental reconstruction from these R. philippinarum shells. Endogenous rhythmicity in shell microgrowth increment formation and oxygen consumption was previously documented in this species from intertidal flats. Our study suggests that, in the subtidal zone, Manila clams' rhythmic activity may be controlled by such an endogenous process, synchronized by tidal cues. As in other bivalves, R. philippinarum is an osmoconformer euryhaline bivalve. The tidal rhythmicity of shell microgrowth increments in subtidal specimens of this species could be explained by a behavioral adaptation of valve closure at low tide to protect the clam from low salinities and/or to synchronize with food availability. Finally, large inter-individual variability in tidally associated growth rates and asynchronous growth breaks were observed, and could be due to genetic variability between individuals, asynchronous partial spawning events or predation.     

Short-term hypoxia modulates Rapana venosa (Muricidae) prey preference in Adriatic lagoons

We studied the effect of short-term hypoxia on predation by the non-native rapa whelk (Rapana venosa) on the three most common bivalve prey in northern Adriatic lagoons: the non-native ark shell (Scapharca inaequivalvis) and Manila clam (Tapes philippinarum), and the native cockle Cerastoderma glaucum. In mesocosm experiments, bivalves were offered to predator as single prey, and as multiple choice of prey. Bivalve prey showed behavioral responses when exposed to short-term hypoxia. Under normoxia, prey were consumed in almost equal numbers when offered separately, but when choice was offered to the predator R. venosa showed a marked preference for S. inaequivalvis. Short-term hypoxia increased the vulnerability of T. philippinarum, and, consequently, rapa whelks modified their predatory behavior and switched their preference to T. philippinarum. We hypothesize this to be a case of environmentally-mediated, non-reciprocal indirect interaction between the two non-native prey species, whose causes are based upon differences in specific traits of prey. Hypoxia may facilitate the coexistence of the two non-native bivalves via predator switching. The native cockle seems the net loser of the game.     

Mislabeled: eco-labeling an invasive alien shellfish fishery

The invasive alien Manila clam fishery from Ria Arousa, Spain, was recommended for a Marine Stewardship Council certificate which implies the fishery maintains the integrity of the ecosystem. By certifying an environmentally harmful invasive alien clam fishery, the MSC harms the environment and risks its credibility, at the time when environmental responsibility and social responsibility are increasingly important to policymakers, industry, and consumers, particularly in Europe. We call on the MSC to reassess their evaluation of the Ruditapes philippinarum fishery in Ria Arousa, Spain.     

Insights into the Establishment of the Manila Clam on a Tidal Flat at the Southern End of an Introduced Range in Southern California,USA

Coastal ecosystem modifications have contributed to the spread of introduced species through alterations of historic disturbance regimes and resource availability, and increased propagule pressure. Frequency of occurrence of the Manila clam (Venerupis phillipinarum, Veneridae) in Southern California estuaries has increased from absent or sparse to common since the mid-1990s. Potential invasion vectors include seafood sales and aquaculture, and spread from established northern populations over decades. The clam’s post-settlement habitat preferences are, however, uncertain in this region. Our project aimed to identify factors associated with established patches of the clam within a bay toward the southern end of this introduced range. During summer 2013, we sampled 10 tidal flat sites in Mission Bay, San Diego; each containing an area with and without hard structure (e.g., riprap, boulders). We measured likely environmental influences (e.g., sediment variables, distance to ocean). Manila clam densities across the bay were most strongly associated with site, where highest densities were located in the northern and/or back halves of the bay; and weakly correlated with lower porewater salinities. Within sites, Manila clam density was enhanced in the presence of hard structure in most sites. Prevailing currents and salinity regimes likely contribute to bay wide distributions, while hard structures may provide suitable microhabitats (refuge from predators and physical stress) and larval entrapment within sites. Results provide insights into decisions about future shoreline management efforts. Finally, we identify directions for future study to better understand and therefore predict patterns of establishment of the Manila clam in the southern portion of its introduced range.     

Gymnophallid digenean Parvatrema duboisi uses Manila clam as the first and second intermediate host

To identify the metacercariae of a gymnophallid trematode in the Manila clam Ruditapes philippinarum from the Ariake Sea, experimental infection and molecular analysis were conducted. Based on the morphology of adult worms obtained from experimentally infected mice, the parasite was identified as Parvatrema duboisi. Comparison of internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) sequences for metacercariae and sporocysts from Manila clams and adult worms collected from wild wigeon Anas penelope showed minor differences ranging from 0 to 0.8%. These data strongly suggest that in the Ariake Sea, the parasite has a lifecycle using the Manila clam as the first and second intermediate hosts and wigeon as the definitive host.     

Impact of the infaunal Manila clam, <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>, on sediment stability

The aim of this study was to examine the impact of bioturbation by the Manila clam, Ruditapes philippinarum, on sediment stability. A laboratory benthic annular flume system (AFS) was deployed to evaluate the relationship between sediment stability of a subtidal mudflat and density of the infaunal clam under the influence of different current velocities. There was a significant correlation between mean erosion rate and current velocities in all treatments with clams (p < 0.001). There was also a significant correlation between mean erosion rate and R. philippinarum density (p < 0.001), reflecting bioturbation-enhanced sediment erosion. The effects of clam density on sediment erodability were more marked at the lower current velocities. In the control, the critical erosion velocity (Ū_crit) was about 32 cm s⁻¹. With increasing R. philippinarum density, Ū_crit decreased down to the minimum value of about 20 cm s⁻¹ at a density of 206 clams m⁻². This study demonstrated that the burrowing activity of R. philippinarum reduces sediment stability, particularly at relatively low current velocities (25 cm s⁻¹) and at densities below those found in the clam cultivation areas within the Sacca di Goro lagoon.     

Molecular and morphological evidence of hybridization between native Ruditapes philippinarum and the introduced Ruditapes form in Japan

Marine aquaculture and stock enhancement are major causes of the introduction of alien species. A good example of such an introduction is the Japanese shortneck clam Ruditapes philippinarum, one of the most important fishery resources in the world. To meet the domestic shortage of R. philippinarum caused by depleted catches, clams were imported to Japan from China and the Korean peninsula. The imported clam is an alien species that has a very similar morphology, and was misidentified as R. philippinarum (hereafter, Ruditapes form). We genotyped 1,186 clams of R. philippinarum and R. form at four microsatellite loci, sequenced mitochondrial DNA (COI gene fragment) of 485 clams, 34 of which were R. variegatus, and measured morphometric and meristic characters of 754 clams from 12 populations in Japan and China, including the Ariake Sea and Tokyo Bay, where large numbers of R. form were released. Our analyses confirmed that R. form was from the genus Ruditapes, and the genetic differentiation between R. philippinarum and R. form was distinct, but small, compared with five bivalve outgroups. However, R. form had distinct shell morphology, especially larger numbers of radial ribs on the shell surface, suggesting that R. form might be a new Ruditapes species or a variation of R. philippinarum that originated from southern China. A genetic affinity of the sample from the Ariake Sea to R. form was found with the intermediate shell morphology and number of radial ribs, and the hybrid proportion was estimated at 51.3 ± 4.6 % in the Ariake Sea.     

FIRST RECORD OF GRACILARIA VERMICULOPHYLLA (GRACILARIALES,RHODOPHYTA) IN THE PO DELTA LAGOONS,MEDITERRANEAN SEA (ITALY)1

Molecular outcomes led us to report the first occurrence of the invasive alien species Gracilaria vermiculophylla (Ohmi) Papenf. in the Mediterranean Sea. This species was recorded for the first time in the Po Delta lagoons in May and October 2008, probably introduced by the importation of the Manila clam Tapes philippinarum. At present, G. vermiculophylla is spread only near some clam‐farming areas, but its diffusion is expected to increase with the colonization of other lagoons where aquaculture is spread, as already observed for other alien species such as Agardhiella subulata and Solieria filiformis. The present study supplies further information on the morphology of this species, the ecological characteristics of the colonized areas, and the most probable introduction vector, confirming that the species spreading occurs in eutrophic and turbid coastal systems.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2 kDa. Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni-associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1-2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes.     

Assessing the geographic scale of genetic population management with microsatellites and introns in the clam Ruditapes decussatus

The clam Ruditapes decussatus is commercially important in southwestern Europe, suffering from population decline and hybridization with exotic Manila clam (R. philippinarum). Previous studies with intronic markers showed a genetic subdivision of the species in three races (Atlantic, West Mediterranean, and Adriatic‐Aegean). However, detailed population genetic studies to help management of the main production areas in the southwest of Europe are missing. We have analyzed eight Atlantic and two Mediterranean populations from the Spanish coasts using 14 microsatellites and six intronic markers. Microsatellites confirmed the Atlantic and West Mediterranean races detected with introns and showed that genetic variability was higher in Mediterranean than in Atlantic populations. Both marker types showed that genetic differentiation of Atlantic populations was low and indicated that populations could be managed at the regional level in the case of Cantabrian and Gulf of Cadiz areas, but not in the case of Rias Baixas and the Mediterranean. This study shows the interest of including different types of markers in studies of genetic population structure of marine organisms.     

Brown muscle disease: Impact on Manila clam Venerupis (=Ruditapes) philippinarum biology

This study assessed the effect of Brown Muscle Disease (BMD) on Manila clam Venerupis philippinarum fitness. BMD was discovered in 2005. It affects the posterior adductor muscle and leads to clam gaping and eventually death. Three statuses of clams were compared: buried individuals with no signs of BMD (BUR); clams at the surface of the sediment with no signs of BMD (SURF) and clams at the surface of the sediment exhibiting signs of brown muscle disease (BMD). Physiological (condition index), immune (hemocyte parameters) and molecular (gene expressions) parameters collected seasonally were analyzed and compared.Results demonstrated a seasonal pattern in condition index (CI) with peaks in spring/summer and decreases in autumn/winter. At each season, the highest CI was observed in BUR and the lowest CI was observed in BMD.In terms of immune response, phagocytosis rate and capacity were higher in clams with BMD whereas the health status of the clams did not influence the total hemocyte count. Genes involved in the immune system (comp, tnf, inter) were upregulated in clams with BMD. The molecular analysis of gill and posterior muscle showed higher mitochondrial metabolism (cox-1, 16S) in cells of infected clams, suggesting a stronger energetic demand by these cells. Finally, genes involved in oxidative stress response (cat, sod), detoxification (mt) and DNA repair (gadd45) were also overexpressed due to reactive oxygen species production.Most of the studied parameters underlined a cause–effect correlation between Manila clam health status (BUR, SUR, BMD) and physiological parameters. An important stress response was observed in BMD-infected clams at different scales, i.e. condition index, immune parameters and stress-related gene expression.     

Molecular phylogeography of Ruditapes philippinarum in the Northwestern Pacific Ocean based on COI gene

To examine the pattern of phylogeography of Ruditapes philippinarum, a length of 644-bp gene fragment of the mtDNA COI was sequenced. A total of 170 individuals from 19 locations were analyzed yielding 74 haplotypes, most of which were unique. The levels of haplotype diversity and nucleotide diversity for the species ranged from 0.80 ± 0.16 (Notsuke Bay) to 1.00 ± 0.13 (Nanao Bay, Miyazu Bay, Dalian 1 and Ariake), and from 0.002 ± 0.001 (Notsuke Bay) to 0.011 ± 0.006 (Qingdao), respectively. Both the phylogenetic (NJ tree) and minimum spanning trees (MST) showed three significant genealogical clusters corresponding to sampling localities, a genetic differentiation speculated to be caused by the isolation of the marginal seas of the Northwestern Pacific during Pleistocene low sea-level stands. Both AMOVA and pairwise Fst analyses revealed significant genetic differentiation between the populations from Japan and China. The pattern of isolation by distance was also detected in this species (r = 0.50, P < 0.001). Both mismatch distribution analysis and the neutrality tests showed that R. philippinarum had undergone a recent population expansion. The estimate of population expansion time was about 425 kya-1580 kya.