Impact of preparation method on gonad domoic acid levels in the scallop, Pecten maximus (L.)

The king scallop, Pecten maximus (L.), fishery is a valuable economic resource in the UK, and is reliant on supplying premium “roe-on” processed scallops to the continental market. A considerable degree of variability is observed in domoic acid (DA) levels among individual P. maximus and their body components, which complicates the management of the fishery during amnesic shellfish poisoning (ASP) events. This study examined the impact of professional processing and three differing laboratory preparation techniques on final gonadal DA levels. DA analysis was conducted using a LC–MS/MS procedure. The results demonstrate that different methods of preparation can significantly alter gonadal toxicities in scallops from the same site, and the extent to which DA within the digestive loop, which passes through the gonad, contributes to total gonadal DA. Mean gonadal toxicity attributed to the digestive loop contents was estimated at 4.7–24.7 μg DA g⁻¹. Despite large individual variations in toxin levels; in scallops with elevated gonadal toxicities resulting from higher digestive loop content toxicity, the effect of flushing out the contents of the digestive loop significantly reduced the DA content of the tissue and lowered the frequency of individuals harbouring levels above the 20 μg DA g⁻¹ statutory safety limit. Removal of the digestive loop contents can potentially result in an 87% decrease in gonadal DA burden. Furthermore, the method applied by professional processors effectively removed the contents of the digestive loop and reduced gonadal DA to levels comparable with the laboratory techniques. Deliberate contamination with scallop mucus did not increase gonadal DA levels. The extent of toxin variation resulting from differing gonad preparations demonstrates the need to standardize scallop tissue preparation techniques during ASP events. Consequently, detailed protocols aimed at minimizing the contamination of edible components should be developed and adhered to by both processing facilities and monitoring bodies.     

Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile

The morphology and toxicity of the four ubiquitous species belonging to the genus Pseudo-nitzschia found in mixed blooms of phytoplankton from northern Chilean waters were studied. The phytoplankton samples and cultures obtained were identified by scanning electron microscopy, revealing the presence of Pseudo-nitzschia australis, P. calliantha, P. pseudodelicatissima and P. subfraudulenta. This is the first report of P. calliantha in northern Chile. Toxin analyses using the LC–MS method confirmed the presence of domoic acid in P. australis and P. calliantha. Domoic acid was not detected in cultures of P. subfraudulenta. This study therefore confirms P. australis and P. calliantha as an unequivocal source of domoic acid in Chilean waters. P. australis is probably the most important producer of amnesic shellfish toxin in view of its domoic acid content. However, more research is needed to evaluate the potential for toxin production in P. pseudodelicatissima.     

Effect of storage on amnesic shellfish poisoning (ASP) toxins in king scallops (Pecten maximus)

Since 1998, king scallops (Pecten maximus) obtained from Scottish offshore sites have been monitored for domoic acid (DA) and epi-domoic acid (epi-DA), the principal toxic compounds associated with amnesic shellfish poisoning (ASP). The presence of these toxins in king scallops harvested from Scottish waters at concentrations exceeding the current regulatory limit (20 μg g⁻¹ shellfish flesh) is a recurrent event. However, little information was available to determine the effects that different storage conditions experienced during sample transportation to the monitoring laboratory may have on the toxin concentrations, which are subsequently detected. Furthermore, the stability of DA and epi-DA in the solvents (methanol:water (1:1, v/v) and citric acid buffer (0.5 M, pH 3.2)) routinely used for their extraction from shellfish has not previously been assessed. Results from this study demonstrate that when king scallop samples were stored for 2–3 days at 12 °C, a significantly higher toxin concentration was detected in the gonad than when samples were stored at 4 °C and analysed within 48 h. This implies that monitoring programmes must consider transport and storage conditions between harvest and analysis. Stability studies showed rapid decomposition of DA and epi-DA in aqueous methanol extracts while DA and epi-DA seem acceptably stable when stored refrigerated in citrate buffer.     

Karyotype and Chromosomal Location of 18S–28S and 5S Ribosomal DNA in the Scallops Pecten maximus and Mimachlamys varia (Bivalvia: Pectinidae)

This work describes the karyotype and chromosomal location of the ribosomal DNA (rDNA) of Pecten maximus and Mimachlamys varia, two commercial scallop species from Europe. According to the chromosome centromeric index values found, the karyotype of P. maximus is composed of 1 metacentric, 2 metacentric–submetacentric, 1 telocentric–subtelocentric and 15 telocentric pairs, and that of M. varia of 4 metacentric, 2 subtelocentric–submetacentric, 9 subtelocentric, 3 subtelocentric–telocentric and 1 telocentric–subtelocentric pairs. In P. maximus, 18S-28S rDNA was located by FISH on a metacentric–submetacentric pair, and in M. varia on a subtelocentric–submetacentric pair using both silver staining and FISH. PCR amplification of the 5S rDNA unit yielded a single product of about 460 bp (P. maximus) and 450 bp (M. varia), that used as probe revealed a 5S rDNA site on a telocentric pair in P. maximus and a subtelocentric pair in M. varia. Two-color FISH or sequential silver staining of 5S rDNA-FISH-metaphases corroborated that the two gene families are located on different chromosomes in both species. A comparative analysis of the data allowed the inference of karyotypic relationships within scallops.     

Margolisiella islandica sp. nov. (Apicomplexa: Eimeridae) infecting Iceland scallop Chlamys islandica (Müller, 1776) in Icelandic waters

Wild Iceland scallops Chlamys islandica from an Icelandic bay were examined for parasites. Queen scallops Aequipecten opercularis from the Faroe Islands and king scallops Pecten maximus and queen scallops from Scottish waters were also examined. Observations revealed heavy infections of eimeriorine parasites in 95–100% of C. islandica but not the other scallop species. All life stages in the apicomplexan reproduction phases, i.e. merogony, gametogony and sporogony, were present. Trophozoites and meronts were common within endothelial cells of the heart’s auricle and two generations of free merozoites were frequently seen in great numbers in the haemolymph. Gamonts at various developmental stages were also abundant, most frequently free in the haemolymph. Macrogamonts were much more numerous than microgamonts. Oocysts were exclusively in the haemolymph; live mature oocysts contained numerous (>500) densely packed pairs of sporozoites forming sporocysts.Analysis of the 18S ribosomal DNA revealed that the parasite from C. islandica is most similar (97.7% identity) to an unidentified apicomplexan isolated from the haemolymph of the giant clam, Tridacna crocea, from Japan. Phylogenetic analyses showed that the novel sequence consistently grouped with the Tridacna sequence which formed a robust sister clade to the rhytidocystid group.We propose the name Margolisiella islandica sp. nov., referring to both type host and type locality.     

Previously unknown apicomplexan species infecting Iceland scallop, Chlamys islandica (Müller, 1776), queen scallop, Aequipecten opercularis L., and king scallop, Pecten maximus L

Examination of three scallop species from three separate locations: Iceland scallop from Icelandic waters, king scallop from Scottish waters and queen scallop from Faroese and Scottish waters, revealed infections of a previously unknown apicomplexan parasite in all three scallop species. Developmental forms observed in the shells appeared to include both sexual and asexual stages of the parasite, i.e. merogony, gametogony and sporogony, which suggests a monoxenous life cycle. Meronts, gamonts, zygotes and mature oocysts were solely found in the muscular tissue. Zoites, which could be sporozoites and/or merozoites, were observed in great numbers, most frequently in muscles, both intracellular and free in the extracellular space. Zoites were also common inside haemocytes. Examination of the ultrastructure showed that the zoites contained all the major structures characterizing apicomplexans. This apicomplexan parasite is morphologically different from other apicomplexan species previously described from bivalves. Presently, its systematic position within the phylum Apicomplexa cannot be ascertained.     

Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR

The toxic dinoflagellate Alexandrium fundyense is widespread in the northeastern part of North America, including the Gulf of Maine, and is responsible for seasonal harmful algal blooms in these regions. Even at low cell densities, A. fundyense toxins can accumulate in shellfish and result in paralytic shellfish poisoning (PSP). PSP can be debilitating or lethal to humans and other shellfish consumers and is a public health concern. As a result, accurate measurements of A. fundyense distributions, particularly at low cell density, are critical to continued PSP monitoring and mitigation efforts. Towards this end we have developed a real-time quantitative PCR (qPCR) method to monitor A. fundyense. Laboratory validation indicates that the qPCR assay is sensitive enough to detect 10 cells per sample, and that it does not detect co-occurring dinoflagellates such as Alexandrium ostenfeldii. The qPCR methodology was used to quantify A. fundyense cell densities in samples collected during a spring 2003 transect in the Gulf of Maine, and the data were compared to those obtained in parallel from light microscope and DNA hybridization-based methods. Results show that A. fundyense cell density was low during this period relative to typical cell densities required for PSP contamination of local shellfish, and that qPCR values were comparable to numbers determined by independent methods.     

Response on scallop culture to enhanced nutrient supply by experimental fertilisation of a landlocked bay

The effect of an enhanced nutrient supply to coastal waters of a landlocked bay, Hopavågen in Central Norway, on the phytoplankton production and biomass, and on growth of scallops (Pecten maximus) was studied in 1997–1999. Nitrogen, silicon and phosphorous (N:Si:P = 16:8:1, atomic) were added daily between May and October in 1998 at a level of 0.4 mg P m^–3 day^–1. The concentration of nutrient addition was doubled in 1999 during the same period. High addition of nutrients (1999) resulted in a significantly higher phytoplankton biomass in the summer period, expressed as chlorophyll a content, than without nutrient (1997) and low nutrient (1998). The respective mean chlorophyll a levels were 2.4 in 1999, 1.6 in 1998 and 1.2 g l^–1 in 1997. The mean primary production during the summers generally increased with the addition of nutrients from an average level of 320 mg carbon m^–2 day^–1 in 1997 to 1200 mg carbon m^–2 day^–1 in 1999. Scallops placed at 10 m depth in Hopavågen showed an increase in growth rate of the outer scallop shell in the period July–September from 0.16% day^–1 in 1997 to 0.53% day^–1 in 1998. Scallops grown in an unfertilised control station in the fjord outside Hopavågen had a significantly lower growth rate than those grown in the fertilised water of Hopavågen. The results showed decreased growth rate with increasing shell sizes. However, for all size groups studied a higher growth rate of the scallops was observed when nutrients were added to the bay. The tissue dry weight content of scallops grown in Hopavågen was 2–4 times higher than in the control scallops.     

A study of spatial variability of domoic acid in razor clams: recommendations for resource management on the Washington coast

Domoic acid (DA) levels in Washington State razor clams (Siliqua patula) have been extremely variable and unpredictable, resulting in emergency closures of harvest areas in 1991, 1998, and 1999. Information concerning toxin variability relative to sampling location is important in developing a more reliable plan for managing DA outbreaks. In November 1998, record levels of DA in razor clams (up to 295 ppm) were reported at Kalaloch beach, Washington. A razor clam resource survey conducted there during the summer of 1999, along with the long retention time of DA in the clams, permitted a study of DA levels as a function of tidal elevation and north-south beach location. During the summer low tides (28–31 July 1999) razor clams were collected from six east-west transects, approximately 1.6 km apart. Each clam was individually analyzed for DA to determine the distribution of toxin between (interspecific variability) and within (intraspecific variability) transects. While average DA levels were similar between transects, toxin levels varied substantially among individual clams. The coefficient of variation among all samples (n=445) was 122%, indicating that harvest closures based upon composite analyses of only six clams could be in error. Here we recommend a sampling strategy of razor clams for regulatory purposes that will result in DA measurements more representative of total population toxin values.     

The effect of scallop dredging on Irish Sea benthos: experiments using a closed area

A 2 km² area off the southwest coast of the Isle of Man (Irish Sea) has been closed to commercial fishing with mobile gear since March 1989. This area was heavily fished for Pecten maximus (Linnaeus, 1758) prior to closure, and the seabed immediately surrounding the closed area is still one of the most heavily dredged in the Irish Sea. Two methods have been used to study the effect of scallop dredging on the benthos in this closed area and adjacent fished areas. Firstly, twice-yearly grab sampling of experimental plots inside and outside the closed area since 1995 has enabled comparisons of the benthic infauna and epifauna of experimentally dredged plots, undredged control plots and plots exposed to commercial dredging. Secondly, divers have carried out visual transect surveys of P. maximus numbers regularly since closure. Communities of experimentally disturbed plots have become less similar to adjacent undisturbed control areas and more similar to commercially dredged areas. At each sampling date, similarity between dredged samples was greater than between undredged samples. Since 1989, there have been increases in the mean numbers of P. maximus in the closed area. The age structure of the closed area P. maximus population is also different to that outside, with a higher mean age due to the presence of large, old individuals.These results present strong evidence that scallop dredging alters benthic communities and suggest that the closure of areas to commercial dredging may allow the development of more heterogeneous communities and permit the populations of some species to increase. A common problem with studying fishing disturbance is the lack of good control sites and this work also demonstrates the value of closed areas to scientific studies of demersal fishing.     

Note on the occurrence of Pseudo-nitzschia australis and domoic acid in squid from Monterey Bay, CA (USA)

Over the past decade diatom blooms of domoic acid (DA)-producing Pseudo-nitzschia spp. have been responsible for numerous marine mammal and bird mortalities in Monterey Bay, CA. One possible toxin vector is the market squid, Loligo opalescens, a small pelagic mollusk that plays an important role in the near-shore food web of the California Current ecosystem as a favored vertebrate prey species. This study examined the trophic link between toxic Pseudo-nitzschia and L. opalescens using toxin and stomach content analyses of animals collected from Monterey Bay, CA in 2000. Receptor binding assay data (confirmed by tandem mass spectrometry), demonstrated the presence of DA in squid during a toxic Pseudo-nitzschia event, with P. australis frustules observed in stomach samples. Though DA levels were low (<0.5 μg DA g⁻¹ tissue) in L. opalescens during the study period, it is now clear that this potent neurotoxin can occur in squid and is likely delivered through its krill prey species, which are known to retain DA after feeding on toxic Pseudo-nitzschia. Our findings suggest that further study of the relationship between Pseudo-nitzschia blooms and DA contamination of squid is warranted to better evaluate the potential health risk to humans and wildlife associated with this major commercial seafood species and important prey item.     

Ionic factors affecting motility,respiration and fertilization rate of the sperm of the bivalvePecten maximus (L.)

Fertilization of the scallopPecten maximus occurs after gametes were naturally released in sea water by the bivalve which has undergone stimulation. The motility of the spermatozoa requires their dilution in sea water (1/40). Dilution triggers an immediate increase of oxygen consumption by sperm, reflecting an activation of a cyanide-sensitive respiration of a cellular origin. When scallops were stimulated by thermal shocks or by serotonin injection, sperm sampled at the urogenital pore output duct shows a respiration-motility activation after sea water dilution which is not seen in sperm scarified from the gonad. Dilution of kidney-sampled sperm into acidic (pH 5) or Na⁺-free artificial sea water reversibly inhibits both respiration and motility. In all cases fertilization rate of sperm is correlated to the increase of respiratory rate and motility measured after dilution in different media. Whether the scallop was stimulated or not, the pH of haemolymph and pericardic fluids were one pH unit below the value of sea water, the pH of the gonad and of the kidney tissues being more acidic (6.5 in average). Our results suggest that the acidic pH of the genital tract maintains the spermatozoa in a quiescent state and that capacitation occurs when male gametes move from the gonad to the kidney from where it is naturally released.Abbreviations ASW artificial sea water - SW sea water - TRIS trishydroxymethyl-aminomethane     

Paralytic shellfish toxin profile in strains of the dinoflagellate Gymnodinium catenatum Graham and the scallop Argopecten ventricosus G.B. Sowerby II from Bahía Concepción, Gulf of California, Mexico

Gymnodinium catenatum Graham is a paralytic shellfish poison (PSP) producer that was described for the first time from the Gulf of California in 1943. During the last decade, its distribution along the Mexican Pacific coastline has increased. In Bahía Concepción, a coastal lagoon on the western side of the Gulf of California, G. catenatum has been linked to significant PSP concentrations found in mollusks. In this study, we describe the saxitoxin profile of 16 strains of G. catenatum, and catarina scallops (Argopecten ventricosus) from Bahía Concepción. Toxins were analyzed by HPLC with post-column oxidation and fluorescence detection. The average toxicity of the G. catenatum strains was 26.0±6.0 pg and 28.0±18.0 pg STX eq/cell after 17 and 22 days of growth, respectively. Ten toxins were recorded, but only dcSTX, dcGTX2, dcGTX3, C1, and C2 were always present in all strains at both growth stages. Since toxin profiles in scallops were similar to the cultures, biotransformations are not significant in catarina scallop. NeoSTX, GTX2, GTX3, and B2 were present in some G. catenatum strains and their presence varied with the age of the culture. In scallop samples, dcSTX, dcGTX2, and dcGTX3 were the most abundant toxins, and from the C-toxin group, only C2 was found. This unique toxin profile can be used as a biomarker for this population, when compared with strains of G. catenatum from other geographic regions.