Does uptake of Alexandrium fundyense cysts contribute to the levels of PSP toxin found in the sea scallop, Placopecten magellanicus?

Atlantic sea scallops, Placopecten magellanicus, in most areas of the Bay of Fundy, New Brunswick, Canada, have year-round concentrations of paralytic shellfish posioning (PSP) toxins greater than the regulatory concentration of 80 μg STX eq. 100 g⁻¹ wet weight. Scallops (mean shell height of 10.7 cm, age 3–5 years) were collected by SCUBA and individually tagged near Parker Island, Bay of Fundy. Half were hung 2 m below the low tide water level and the remainder were placed on the bottom (11 m depth at low tide) under the scallops held at 2 m. Scallop, water and sediment samples were collected monthly for determination of concentrations of PSP toxins and Alexandrium fundyense.In October, 1993, mean concentrations of PSP toxins in digestive gland, and mantle were 3205 and 1018 μg STX eq. 100 g⁻¹ wet weight, respectively. Eight months later (June 1994), PSP concentrations in digestive glands from the surface and bottom had declined to 504 and 682 μg STX eq. 100 g⁻¹ wet weight, respectively, whereas those in the mantle had declined to 802 and 681 μg STX eq. 100 g⁻¹ wet weight. During July 1994, A. fundyense concentrations observed at Parker Island and offshore were 320 cells l⁻¹ and 14,200 cells l⁻¹, respectively. Subsequently, toxin concentrations in surface and bottom scallop digestive glands increased to 12,720 and 11,408 μg STX eq. 100 g⁻¹ wet weight, whereas concentrations in mantles increased to 2126 and 1748 μg STX eq. 100 g⁻¹ wet weight, respectively. Concentrations of PSP toxins in these tissues in October 1994 were similar to those measured in October 1993. Concentrations of PSP toxin were less than the regulatory concentration in the gonads and non-detectable in adductor muscles of all scallops sampled.There were no statistically significant differences in profiles for uptake and depuration of PSP toxins in scallops held at the surface compared to those from bottom, suggesting that A. fundyense cysts at the concentrations found in the sediment (45 cysts cm⁻³) did not contribute significantly to the year-round presence of PSP toxins within scallop tissues. The year-round occurrence of PSP toxin is probably due to accumulation during summer blooms followed by a very slow rate of depuration.     

PRODUCTION OF PARALYTIC SHELLFISH TOXINS BY APHANIZOMENON SP. LMECYA 31 (CYANOBACTERIA)1

We examined intracellular and extracellular paralytic shellfish toxins (PST) in a strain of Aphanizomenon sp. (LMECYA31) isolated from a Portuguese freshwater reservoir throughout the growth cycle and under different conditions affected by temperature and nitrate and phosphate availability. PST concentrations and compositions were greatly influenced by cell density, growth stage, and temperature and nutrients conditions. On a per‐cell basis results showed (1) the enhancement of PST cell quota after the end of exponential growth phase in nutrient replete batch cultures, (2) the absence of a PST increment at late growth stages under phosphate limitation, (3) a rise in PST maximum cell quota under nitrate depletion, and (4) the enhancement of toxin production at higher temperatures. The relative proportion of the four toxins detected, neoSTX, dcSTX, STX and GTX5, also changed within and between culture settings. While growing under phosphate rich media cells produced mainly GTX5 and neoSTX, whereas under phosphate limitation the proportion of STX and dcSTX increased substantially with culture age. Large amounts of extracellular toxins were found in the culture medium, increasing during culture time. Extracellular toxin composition in each culture was fairly constant and always similar to the intracellular composition found at late stages of growth. This further supported other research that indicates that PSTs are released to the water through cell lysis, and a significant concentration of PST may be expected to remain in the water upon the collapse of a toxic bloom or after cells removal by water treatment.     

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.     

Characterization of paralytic shellfish toxins in seawater and sardines (<Emphasis Type="Italic">Sardina pilchardus</Emphasis>) during blooms of <Emphasis Type="Italic">Gymnodinium catenatum</Emphasis>

The re-emergence of Gymnodinum catenatum blooms after a 10 year hiatus of absence initiated the present investigation. This study aims to evaluate the exposure of small pelagic fishes to paralytic shellfish toxins (PST) during blooms of G. catenatum. Sardines (Sardina pilchardus) were selected as a representative fish species. In order to assess toxin availability to fish, both intracellular PSTs (toxin retained within the algal cells) and extracellular PSTs (toxin found in seawater outside algal cells) were quantified, as well as toxin levels within three fish tissue matrices (viscera, muscle and brain). During the study period, the highest cell densities of G. catenatum reached 2.5 × 10⁴ cells l⁻¹ and intracellular PST levels ranged from 3.4 to 398 ng STXeq l⁻¹ as detected via an enzyme linked immunosorbent assay (ELISA). Measurable extracellular PSTs were also detected in seawater (0.2–1.1 μg STXeq l⁻¹) for the first time in Atlantic waters. The PST profile in G. catenatum was determined via high performance liquid chromatography with fluorescence detection (HPLC-FLD) and consisted mostly of sulfocarbamoyl (C1+2, B1) and decarbamoyl (dcSTX, dcGTX2+3, dcNEO) toxins. The observed profile was similar to that reported previously in G. catenatum blooms in this region before the 10-year hiatus. Sardines, planktivorous fish that ingest a large number of phytoplankton cells, were found to contain PSTs in the viscera, reaching a maximum of 531 μg STXeq kg⁻¹. PSTs were not detected in corresponding muscle or brain tissues. The PST profile characterized in sardine samples consisted of the same sulfocarbamoyl and decarbamoyl toxins found in the algal prey with minor differences in relative abundance of each toxin. Overall, the data suggest that significant biotransformation of PSTs does not occur in sardines. Therefore, planktivorous fish may be a good tracer for the occurrence of offshore G. catenatum blooms and the associated PSTs produced by these algae.     

Paralytic shellfish poisoning (PSP) in Margarita Island, Venezuela

A severe outbreak of Paralytic Shellfish Poisoning (PSP) occurred in Manzanillo and Guayacán, northwestern coast of Margarita Island, Venezuela, between August and October 1991. A bloom of dinoflagellates including Prorocentrum gracile, Gymnodinium catenatum and Alexandrium tamarense seemed to be responsible for this outbreak. Levels of PSP toxins in mussels (Perna perna) exceeded the international safety limit of saxitoxin, 80 microg STX/100 microg meat. PSP toxin values varied between 2548 and 115 microg STX/100 g meat in Manzanillo, and between 1422 and 86 microg STX/100 g meat in Guayacán. At both locations, the highest levels were detected in August, when 24 patients exhibited typical symptoms of PSP toxicity after consuming cooked mussels (16 required hospitalization). A high pressure liquid chromatographic (HPLC) procedure was recently used on the 1991 samples. The major toxin detected in samples of both locations was decarbamoyl saxitoxin (dcSTX), but low concentrations of saxitoxin were also found in Manzanillo samples. Gonyautoxins GTX1, GTX2 and GTX3 were detected only at Guayacán, while in both locations, decarbamoylgonyatouxin (dcGTX2,3) toxins were detected. These findings represent the first time that causative toxins of PSP in Venezuela have been chemically identified, and confirm the presence of dcSTX and dcGTX in mussels from the Caribbean Sea. The presence of dcSTX and dcGTX in shellfish is indicative that Gymnodinium catenatum was a causative organism for outbreak of PSP.     

Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China

The composition of the paralytic shellfish toxins (PSTs) of five Alexandrium tamarense strains isolated from the coastal waters of southern China and one Alexandrium minutum strain from Taiwan Island were investigated. A. tamarense CI01 and A. tamarense Dapeng predominantly produced C2 toxin (over 90%) with trace amounts of C1 toxin (C1), gonyautoxin-2 (GTX2) and GTX3; two strains of A. tamarense HK9301 maintained in different locations produced C1-4 toxins and GTX1, 4, 5 and 6; no PSTs were found in A. tamarense NEW, while A. minutum TW produced only GTX1-4. The toxin compositions of cultured A. tamarense strains did not vary as much during different growth phases as did the toxin composition of A. minutum TW. The toxin compositions of A. tamarense HK9301-1 did not change significantly under different salinity, light intensity, and nitrate and phosphate levels in the culture medium, although the toxin productivity varied expectably. Another strain HK9301-2 maintained in a different location produced much less toxins with a considerably different toxin composition. Under similar culture maintenance conditions for 3 years, the toxin profiles of A. tamarense HK9301-1 did not change as much as did A. tamarense CI01. Our results indicate that toxin compositions of the dinoflagellate strains are strain-specific and are subject to influence by nutritional and environmental conditions but not as much by the growth phase. Use of toxin composition in identifying a toxigenic strain requires special caution.     

The toxigenic marine dinoflagellate Alexandrium tamarense as the probable cause of mortality of caged salmon in Nova Scotia

The toxins associated with paralytic shellfish poisoning (PSP) are potent neurotoxins produced by natural populations of the marine dinoflagellate Alexandrium tamarense. In early June 2000, a massive bloom (>7×10⁵ cells l⁻¹) of this dinoflagellate coincided with an unusually high mortality of farmed salmon in sea cages in southeastern Nova Scotia. Conditions in the water column in the harbour were characterised by the establishment of a sharp pycnocline after salinity stratification due to abundant freshwater runoff. In situ fluorescence revealed a high sub-surface (2–4 m depth) chlorophyll peak related to the plankton bloom. The intense bloom was virtually monospecific and toxicity was clearly related to the concentration of Alexandrium cells in plankton size fractions. Cultured clonal isolates of A. tamarense from the aquaculture sites were very toxic on a per cell basis and yielded a diversity of PSP toxin profiles, some of which were similar to those from plankton concentrates from the natural bloom population. The toxin profile of plankton concentrates from the 21–56 μm size fraction was complex, dominated by the N-sulfocarbamoyl derivative C2, with levels of other PSP toxins GTX4, NEO, GTX5 (=B1), GTX3, GTX1, STX, C1, and GTX2, in decreasing order of relative abundance. Although no PSP toxin was found systemically in the fish tissues (liver, digestive tract) from this salmon kill event, the detection of Alexandrium cells and low levels of PSP toxins in salmon gills provide evidence that the enhanced mortalities were caused by direct exposure to toxic Alexandrium cells and/or to soluble toxins released during the bloom.     

A proteomics assay to detect eight CBRN‐relevant toxins in food

A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero‐hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody‐free sample preparation followed by bottom‐up LC‐MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public‐health domain, it opens the way for multiplex investigation of food‐borne toxins using targeted LC‐MS/MS.     

The effect of size and age on depuration rates of diarrhetic shellfish toxins (DST) in mussels (Mytilus edulis L.)

Depuration or elimination of diarrhetic shellfish toxins (DST) was followed for 73 days in 1- and 2-year-old mussels. The age groups also differed in size, providing a broad approach to studying the effect of the differences in physiology accompanying the differences in size. Content of DST was analysed both on groups and individual mussels. Environmental variables were measured to evaluate their effect on depuration.We found no significant differences in elimination rate of DST between 1- and 2-year-old mussels under natural conditions. This suggests that size and age do not affect the elimination rate of the DST. The present study is the first study on the effect of age and size on the elimination rate of algal toxins in bivalves. The natural variations in food levels and temperature were not found to affect the elimination rate of DST.The digestive gland weights in the 1-year-old mussels increased four times while the DST content per individual decreased eight times. This demonstrated that dilution of toxins due to tissue growth could have an important contribution to declines in toxin concentrations. Changes in tissue mass are affected by environmental variables via growth or starvation, and when such changes lead to concentration or dilution of toxins this does not reflect the accumulation or removal of toxins from the tissues. We hence suggest that when evaluating the actual elimination capacity of the mussels, as in the present study, the total content of toxins per individual should be used, rather than toxin concentrations.The 1-year-old mussels had faster growth compared to the 2-year-old mussels in both total soft tissue and digestive glands. The mechanism of DST elimination is still unknown. If this process involves metabolism of the toxins, one could expect the rates of elimination to follow overall metabolic rates. However, the results from the present study suggest that large differences in growth rates, which also include difference in feeding and metabolic rates, do not affect the elimination rate of DST. Our results support the assumption that the depuration rates cannot be accelerated, even in artificial systems, as a cost-effective way to solve the problem with toxic mussels for the industry.     

In vitro transformation of paralytic shellfish toxins in the clams Mya arenaria and Protothaca staminea

Dissected tissues of two clam species, the Pacific littleneck, Protothaca staminea, and soft-shell, Mya arenaria, were evaluated for in vitro conversion of paralytic shellfish poisoning (PSP) toxins. Tissue homogenates were incubated with purified PSP toxins to determine the time-course of toxin conversion. The effects of boiling and addition of a natural reductant (glutathione) on toxin conversion were also assessed. For P. staminea, the digestive gland showed the greatest capacity for biotransformation, followed by gill, but mantle, adductor muscle, and siphon tissues exhibited very low conversion. In this species, the production of decarbamoyl derivatives was much greater from low potency N-sulfocarbamoyl toxins than from carbamate analogues. Decarbamolyation exhibited apparent specificity for α-epimers of all toxin substrates and this reaction was inhibited by boiling. Glutathione-mediated desulfation was tissue specific and had apparent specificity for β-epimers. These observations on P. staminea suggest that the above reactions are enzyme-mediated. In contrast, there was little toxin conversion in M. arenaria homogenates, but even this low activity was heat-labile and thus likely enzyme-mediated.     

Does the trophic habitat influence the biochemical quality of the gonad of Octopus vulgaris? Stable isotopes and lipid class contents as bio-indicators of different life-cycle strategies

This study aims to test whether environmental conditions including the trophic habitat and diet impact the biochemical composition of storage organs and affect the nutritional quality of eggs of Octopus vulgaris. Trophic habitat and gonad quality of neighbouring populations off the Portuguese coast, subject to different oceanographic regimes, were compared using the digestive gland and beaks as recorders of trophic and habitat preferences, and gonads as indicators of egg quality. Cholesterol, phospholipids and triacylglycerol content, essential fatty acid (EFA) profile of the digestive gland and stable isotopes, δ¹⁵N and δ¹³C, in the buccal mass flesh and beaks were indicators of the differences in the trophic habitat between populations. For gonad quality, the same bio-indicators were used to identify differences with maturation. The study shows that, although diet influences the EFA profile of the gonads to a certain degree, the main lipid content, phospholipids and cholesterol content in the gonads are not influenced by habitat conditions. This, therefore, suggests that O. vulgaris is able to influence the quality of egg content independent of diet. The species is believed to be an income breeder which attains maturity upon reaching a sufficient condition level, then channelling energy directly from food to gonad development.     

New saxitoxin analogues in the marine environment: developments in toxin chemistry,detection and biotransformation during the 2000s

Scientific study of paralytic shellfish poisoning toxins (PSTs) started in the early XXth century. In the 1920s it was understood the link between the toxicity observed in mussels with certain microalgae species. The poison was eventually purified from the clam Saxidomus giganteus, taking its name from it: saxitoxin (STX). Along the 1970s and 1980s it was understood that other STX analogues existed, both in dinoflagellates and bivalves. These were grouped into three major occurring families: the carbamate, N-sulfocarbamoyl and decarbamoyl, depending on the variation of the side chain of the tetrahydropurine core. The deoxydecarbamoyl family was additionally recognised in the dinoflagellate Gymnodinium catenatum. Chemical research into these STX analogues was conducted worldwide during the 1990s mainly by HPLC with pre- or post-column oxidation and fluorescence detection. Implementation of fluorescence detection with spectral capabilities and mass spectrometry detection during the 2000s led to the recognition of new analogues. Metabolites originated by single or double hydroxylation at C11 position were found in mussels, and later suspected in other bivalves. Designated M1-M4, these present very low fluorescence, and can only be studied resorting to HILIC-MS. Three hydroxybenzoate analogues were characterised as an important toxin fraction of the dinoflagellate Gymnodinium catenatum, and named GC1-GC3. Later, many more analogues were suspected: the corresponding N1-hydroxyl variants of GC1-GC3 (GC4-GC6), di-hydroxybenzoate variants (GC1a-GC6a), and sulphate-benzoate variants (GC1b-GC6b). In bivalves, carbamoylase activity renders these analogues into decarbamoyl analogues. Other compounds with PST-like characteristics have been detected in bivalves from Angola, Argentina and Vietnam. Today, the range of naturally occurring STX derivatives, both in marine and freshwater environments, accounts to more than fifty structural variants. This poses a problem for carrying out food safety analysis based solely in chemical methods. Fortunately, most modifications to the side chain of the tetrahydropurine core result in diminished toxicity.     

Examination of the seasonal dynamics of the toxic dinoflagellate Alexandrium catenella at Redondo Beach, California, by quantitative PCR

The presence of neurotoxic species within the genus Alexandrium along the U.S. coastline has raised concern of potential poisoning through the consumption of contaminated seafood. Paralytic shellfish toxins (PSTs) detected in shellfish provide evidence that these harmful events have increased in frequency and severity along the California coast during the past 25 years, but the timing and location of these occurrences have been highly variable. We conducted a 4-year survey in King Harbor, CA, to investigate the seasonal dynamics of Alexandrium catenella and the presence of a particulate saxitoxin (STX), the parent compound of the PSTs. A quantitative PCR (qPCR) assay was developed for quantifying A. catenella in environmental microbial assemblages. This approach allowed for the detection of abundances as low as 12 cells liter⁻¹, 2 orders of magnitude below threshold abundances that can impact food webs. A. catenella was found repeatedly during the study, particularly in spring, when cells were detected in 38% of the samples (27 to 5,680 cells liter⁻¹). This peak in cell abundances was observed in 2006 and corresponded to a particulate STX concentration of 12 ng liter⁻¹, whereas the maximum STX concentration of 26 ng liter⁻¹ occurred in April 2008. Total cell abundances and toxin levels varied strongly throughout each year, but A. catenella was less abundant during summer, fall, and winter, when only 2 to 11% of the samples yielded positive qPCR results. The qPCR method developed here provides a useful tool for investigating the ecology of A. catenella at subbloom and bloom abundances.     

Metallothioneins and trace elements in digestive gland,gills, kidney and gonads of Octopus vulgaris

Metallothionein-like proteins (MT) and V, Cr, Co, Ni, Zn, Cu, As and Cd were determined in digestive gland, gills, kidney and gonads of Octopus vulgaris, from the Portuguese coast. To our knowledge these are the first data on MT in octopus. High concentrations (µg g^− 1, dry mass) of Zn (48050) and Cd (555) were found in digestive gland, and MT reached levels one order of magnitude above the ones registered in wild bivalves. Significantly higher levels of MT in digestive gland and gills of specimens from A and B were in line with elevated Cd concentrations. Principal component analyses (PCA) point to MT-Cd and MT-Cr associations in digestive gland and gills. Despite the high levels of Zn in specimens from B, association with Zn was not obtained. Due to the affinity of MT to various elements, it should not be excluded the possibility of Cd replacing Zn in Zn-MT. Kidney presented higher levels of Cd, Co, Ni and As than gills and gonads, and in the case of As surpassing the levels in digestive gland, but PCA showed no relation with MT. Likewise the MT levels in gonads had no correspondence to the metal concentration variation.     

Pseudoalteromonas Bacteria Are Capable of Degrading Paralytic Shellfish Toxins

Marine bacterial isolates cultured from the digestive tracts of blue mussels (Mytilus edulis) contaminated with paralytic shellfish toxins (PSTs) were screened for the ability to reduce the toxicity of a PST mixture. Seven isolates reduced the overall toxicity of the algal extract by ≥90% within 3 days. These isolates shared at least 99% 16S rRNA gene sequence similarity with five Pseudoalteromonas spp. Phenotypic tests suggested that all are novel strains of Pseudoalteromonas haloplanktis.Among the marine algal biotoxins identified to date; paralytic shellfish toxins (PSTs) constitute the most serious threat to the safety of the food supply, mainly due to their high acute toxicities and the absence of antidotes or effective medical treatments (8). Paralytic shellfish poisoning is caused by ingestion of one or more of the chemically related PSTs (see Fig. S1 in the supplemental material). PSTs are mainly produced by marine dinoflagellates, including Alexandrium spp., Gymnodinium catenatum, and Pyrodinium bahamense var. compresssum (16). Since bivalve molluscs filter-feed on marine algae, they tend to concentrate PSTs largely, but not exclusively, in their digestive organs (7, 9, 10, 29). Not affected by commercial sterilization (14, 18) or cooking, PSTs present significant risks to the food supply, particularly during periods of toxic algal blooms. Practical methods for PST detoxification of living shellfish do not exist (5).Transformations of PSTs by bacteria have been reported in the literature (23-25, 31, 35, 36, 38); early studies focused on the conversion of hydroxysulfate carbamate derivatives (gonyautoxins 1 and 4) to the more highly toxic saxitoxin (STX) (23-25). In addition, several reports have noted the high capacity of the digestive gland for PST transformation (12, 28, 32, 39), suggesting the presence of toxin-transforming enzymes and/or microorganisms in bivalve molluscs. The partial degradation of gonyautoxins 1 and 4 and C1/C2 by marine bacteria has also been reported (38). In addition, Stewart et al. (37) discovered the bacterial degradation of domoic acid (another marine toxin that causes amnesic shellfish poisoning), collectively suggesting that bacteria might play a role in the elimination of marine toxins from toxic bivalve molluscs. The capacity to catabolize domoic acid is greater in cultures isolated from blue mussels that rapidly eliminate domoic acid than in bacterial isolates from bivalves known to retain the toxin for longer time periods (e.g., scallops), suggesting these bacteria play a role in the elimination of marine toxins.Recently, we reported the kinetics of PST destruction for a group of marine bacteria isolated from toxic blue mussels (11). Here we report the phenotypic and taxonomic characterization of these unique marine bacteria.     

Paralytic shellfish toxins or spirolides? The role of environmental and genetic factors in toxin production of the Alexandrium ostenfeldii complex

Dinoflagellates of the Alexandrium ostenfeldii complex (A. ostenfeldii, A. peruvianum) are capable of producing different types of neurotoxins: paralytic shellfish toxins (PSTs), spirolides and gymnodimines, depending on the strain and its geographic origin. While Atlantic and Mediterranean strains have been reported to produce spirolides, strains originating from the brackish Baltic Sea produce PSTs. Some North Sea, USA and New Zealand strains contain both toxins. Causes for such intraspecific variability in toxin production are unknown. We investigated whether salinity affects toxin production and growth rate of 5 A. ostenfeldii/peruvianum strains with brackish water (Baltic Sea) or oceanic (NE Atlantic) origin. The strains were grown until stationary phase at 7 salinities (6–35), and their growth and toxin production was monitored. Presence of saxitoxin (STX) genes (sxtA1 and sxtA4 motifs) in each strain was also analyzed. Salinity significantly affected both growth rate and toxicity of the individual strains but did not change their major toxin profile. The two Baltic Sea strains exhibited growth at salinities 6–25 and consistently produced gonyautoxin (GTX) 2, GTX3 and STX. The two North Sea strains grew at salinities 20–35 and produced mainly 20-methyl spirolide G (20mG), whereas the strain originating from the northern coast of Ireland was able to grow at salinities 15–35, only producing 13-desmethyl spirolide C (13dmC). The effects of salinity on total cellular toxin concentration and distribution of toxin analogs were strain-specific. Both saxitoxin gene motifs were present in the Baltic Sea strains, whereas the 2 North Sea strains lacked sxtA4, and the Irish strain lacked both motifs. Thus sxtA4 only seems to be specific for PST producing strains. The results show that toxin profiles of A. ostenfeldii/peruvianum strains are predetermined and the production of either spirolides or PSTs cannot be induced by salinity changes. However, changes in salinity may lead to changed growth rates, total cellular toxin concentrations as well as relative distribution of the different PST and spirolide analogs, thus affecting the actual toxicity of A. ostenfeldii/peruvianum populations.     

Anatomical and histochemical features of the digestive system of Octopus vulgaris Cuvier, 1797 with a special focus on secretory cells

This study reports a detailed anatomical and histological study of the digestive system of Octopus vulgaris. Emphasis was placed on characterising the glands and glandular cells and their distribution throughout the digestive tract. The use of classic histological and histochemical techniques revealed two morphological types of glandular cells: granular and mucous. Moreover, the histochemical analysis indicated specialisation of mucous glandular cells in the buccal mass, the submandibular gland and the caecum for secreting acid and neutral glycoconjugates. The cells of the anterior salivary glands are specialised for secreting neutral glycoproteins, and those of the posterior salivary glands are specialised for granular and mucous secretion. The oesophagus, crop and stomach lack glandular cells, but both granular and mucous glandular cells are found in the intestine. An unusual structure resembling the typhlosole of bivalves is described for the first time in the intestine of O. vulgaris. The highly ciliated epithelium and location of the structure in the anterior part of the intestine suggest a possible role in bypassing the caecum, stomach and intestine. We discuss how these cells and organs contribute to the process of digestion in the light of the present histological and histochemical data and of previously published information on the morphology and physiology of digestion in the octopus.     

Fine structure and absorption of ferritin in the digestive organs of Loligo vulgaris and L. Forbesi (Cephalopoda,Teuthoidea)

The digestive organs possibly involved in food absorption in Loligo vulgaris and L. forbesi are the caecum, the intestine, the digestive gland, and the digestive duct appendages. The histology and the fine structure showed that the ciliated organ, the caecal sac, and the intestine are lined with a ciliated epithelium. The ciliary rootlets are particularly well developed in the ciliated organ, apparently in relation to its function of particle collection. Mucous cells are present in the ciliated organ and the intestine. Histologically, the digestive gland appears rather different from that of other cephalopods. However, the fine structure of individual types of squid digestive cell is actually similar to that of comparable organs in other species, and the squid cells undergo the same stages of activity. Digestive cells have a brush border of microvilli, and numerous vacuoles, which sometimes contain “brown bodies.” However, no “boules” (conspicuous protein inclusions of digestive cells in other species) could be identified in their cytoplasm; instead only secretory granules are present. In the digestive duct appendages, numerous membrane infoldings associated with mitochondria are characteristic features of the epithelial cells in all cephalopods. Two unusual features were observed in Loligo: first, the large size of the lipid inclusions in the digestive gland, in the caecal sac, and in the digestive duct appendages; and second, the large number of conspicuous mitochondria with well-developed tubular cristae. When injected into the caecal sac, ferritin molecules can reach the digestive gland and the digestive duct appendages via the digestive ducts, and they are taken up by endocytosis in the digestive cells. Thus, it appears that the digestive gland of Loligo can act as an absorptive organ as it does in other cephalopods.     

Mineral congregations, “spherites” in the midgut gland ofCoelotes terrestris (Araneae): Structure,composition and function

Summary Spherites in the digestive and secretory cells of the midgut gland of the agelenid spiderCoelotes terrestris were studied by electron microscopy and histochemical methods. Spherites measured 1–6 m in diameter and were characterized by alternating layers of electron dense and electron lucent material. The main-components of spherites were calcium phosphates and calcium carbonates. Guanine and barium, as well as aminopeptidase and alkaline phosphatase were also present. The matrix consisted of proteins and carbohydrates. Numerous spherites were found together with excretory products within the excretory vacuoles of the digestive cells.Spiders fed with food containing lead, showed deposition of the metall in the spherites. It is then proposed that spherites, aside from their role in storing calcium and other ions, may function in detoxification of heavy metals.     

Paralytic shellfish toxin profiles of different geographic populations of Gymnodinium catenatum (Dinophyceae) in Korean coastal waters

Paralytic shellfish poisoning toxin profiles of dinoflagellate cultures of Gymnodinium catenatum Graham from the Yellow and South Seas in Korea were investigated by high performance liquid chromatography fluorometric detection. Strains from the Yellow Sea had predominantly carbamate toxins, while strains from Sujeongri and Chindong in the South Sea contained the N‐sulfocarbamoyl toxins, Cl,2, as major components including the presence of GTX5 and dcSTX in some strains. Toxin profiles from St. Deukryang Bay strains (South Sea) showed both characteristics of those in the South Sea and those in the Yellow Sea. Thirty strains could be divided into three groups based on cluster analysis of toxin compositions. Group I (Yellow Sea strains) was distinguished from Group II (Sujeongri and Chindong strains) by the absence of GTX5 and dcSTX. Group III comprised Deukryang Bay strains. In conclusion, the Yellow Sea and the South Sea were found to have different dinoflagellate populations with different toxin compositions.