A non-lethal method for detection of Bonamia ostreae in flat oyster (Ostrea edulis) using environmental DNA.

Surveillance and diagnosis of parasitic Bonamia ostreae infections in flat oysters (Ostrea edulis) are prerequisites for protection and management of wild populations. In addition, reliable and non-lethal detection methods are required for selection of healthy brood oysters in aquaculture productions. Here we present a non-lethal diagnostic technique based on environmental DNA (eDNA) from water samples and demonstrate applications in laboratory trials. Forty oysters originating from Limfjorden, Denmark were kept in 30 ppt sea water in individual tanks. Water was sampled 6 days later, after which all oysters were euthanized and examined for infection, applying PCR. Four oysters (10%) were found to be infected with B. ostreae in gill and mantle tissue. eDNA purified from the water surrounding these oysters contained parasite DNA. A subsequent sampling from the field encompassed 20 oysters and 15 water samples from 5 different locations. Only one oyster turned out positive and all water samples proved negative for B. ostreae eDNA. With this new method B. ostreae may be detected by only sampling water from the environment of isolated oysters or isolated oyster populations. This non-lethal diagnostic eDNA method could have potential for future surveys and oyster breeding programs aiming at producing disease-free oysters.

Effects of the DSP-toxic dinoflagellate Dinophysis acuta on clearance and respiration rate of the blue mussel, Mytilus edulis.

Diarrheic Shellfish Poisoning toxins (DST) are a severe health risk to shellfish consumers and can be a major problem for the shellfish industry. Bivalve molluscs can accumulate DST via ingestion of toxic dinoflagellates like Dinophysis spp., which are the most prominent producers of DST. The effects of DST-containing dinoflagellate Dinophysis acuta on bivalve clearance and respiration rate were investigated in the blue mussel (Mytilus edulis) exposed to different algal densities in a controlled laboratory study. Results showed that M. edulis exposed to D. acuta displayed a reduced clearance rate compared to M. edulis exposed to equivalent bio-volumes of the non-toxic cryptophyte Rhodomonas salina. Furthermore, M. edulis ceased to feed on D. acuta after 1 to 4 h, depending on D. acuta densities. The quickest response was observed at the highest densities of D. acuta. The estimated total amount of DST accumulated in the M. edulis exceeded the regulatory limit for human consumption and furthermore, intoxication of the M. edulis seemed to occur faster at high cell toxicity rather than at high cell density. However, respiration rates were, similar, irrespective of whether M. edulis were fed single diets of R. salina, D. acuta or a mixed diet of both algal species. In conclusion, the DST-containing D. acuta had a severe negative effect on the clearance of M. edulis, which can affect the conditions of the M. edulis negatively. Hence, DST may cause low quality M. edulis, due to reduced feeding when exposed to DST-containing D. acuta.

Impact of temperature and pyrene exposure on the functional response of males and females of the copepod Calanus finmarchicus.

We know very little about the effects of two global stressors, elevated temperature and contaminants, on the grazing of marine copepods. To address this issue, we tested the hypotheses that the individual and combined effects of these two stressors may reduce grazing rates and may depend on food availability and gender. We exposed male and female Calanus finmarchicus copepods to pyrene at two temperatures (10 and 14 °C) and six food concentrations (25-800 µg C Rhodomonas baltica L-1) and measured fecal pellet size, and grazing rate (GR) from pellet production. Males had smaller fecal pellets and lower GR than did females. Temperature and pyrene exposure had no effect on pellet size. Temperature alone had no effect on GR of males, but females had lower GR at elevated temperature. Pyrene-exposed males and females had lower GR only at the food concentrations of 200-800 µg C R. baltica L-1 and those patterns were independent of temperature. Pyrene-induced reduction in GR was stronger in females than in males. The negative effects of both elevated temperature and pyrene may reduce the abundance and trophic success of C. finmarchicus in a warmer, more polluted future.

The blue mussel Mytilus edulis is vulnerable to the toxic dinoflagellate Karlodinium armiger-Adult filtration is inhibited and several life stages killed.

Blooms of the toxic dinoflagellates Karlodinium armiger and K. veneficum are frequently observed in Alfacs Bay, Spain, causing mass mortality to wild and farmed mussels. An isolate of K. armiger from Alfacs Bay was grown in the laboratory and exposed to adults, embryos and trochophore larvae of the blue mussel, Mytilus edulis. Adult mussels rejected to filter K. armiger at cell concentrations >1.5·103 cells ml-1. Exposure of adult mussels (23-33 mm shell length) to a range of K. armiger cell concentrations led to mussel mortality with LC50 values of 9.4·103 and 6.1·103 cells ml-1 after 24 and 48 h exposure to ~3.6·104 K. armiger cells ml-1, respectively. Karlodinium armiger also affected mussel embryos and trochophore larvae and feeding by K. armiger on both embryos and larvae was observed under the microscope. Embryos exposed to low K. armiger cell concentrations suffered no measurable mortality. However, at higher K. armiger cell concentrations the mortality of the embryos increased significantly with cell concentration and reached 97% at 1.8·103 K. armiger cells ml-1 after 29 h of exposure. Natural K. armiger blooms may not only have serious direct effects on benthic communities, but may also affect the recruitment of mussels in affected areas.

Accumulation, transformation and breakdown of DSP toxins from the toxic dinoflagellate Dinophysis acuta in blue mussels, Mytilus edulis.

Okadaic acid (OA), dinophysistoxins (DTX) and pectenotoxins (PTX) produced by the dinoflagellates Dinophysis spp. can accumulate in shellfish and cause diarrhetic shellfish poisoning upon human consumption. Shellfish toxicity is a result of algal abundance and toxicity as well as accumulation and depuration kinetics in mussels. We mass-cultured Dinophysis acuta containing OA, DTX-1b and PTX-2 and fed it to the blue mussel, Mytilus edulis under controlled laboratory conditions for a week to study toxin accumulation and transformation. Contents of OA and DTX-1b in mussels increased linearly with incubation time, and the net toxin accumulation was 66% and 71% for OA and DTX-1b, respectively. Large proportions (≈50%) of both these toxins were transformed to fatty acid esters. Most PTX-2 was transformed to PTX-2 seco-acid and net accumulation was initially high, but decreased progressively throughout the experiment, likely due to esterification and loss of detectability. We also quantified depuration during the subsequent four days and found half-life times of 5-6 days for OA and DTX-1b. Measurements of dissolved toxins revealed that depuration was achieved through excreting rather than metabolizing toxins. This is the first study to construct a full mass balance of DSP toxins during both accumulation and depuration, and we demonstrate rapid toxin accumulation in mussels at realistic in situ levels of Dinophysis. Applying the observed accumulation and depuration kinetics, we model mussel toxicity, and demonstrate that a concentration of only 75 Dinophysis cells l(-1) is enough to make 60 mm long mussels exceed the regulatory threshold for OA equivalents.

The potent respiratory system of Osedax mucofloris (Siboglinidae, Annelida)--a prerequisite for the origin of bone-eating Osedax?

Members of the conspicuous bone-eating genus, Osedax, are widely distributed on whale falls in the Pacific and Atlantic Oceans. These gutless annelids contain endosymbiotic heterotrophic bacteria in a branching root system embedded in the bones of vertebrates, whereas a trunk and anterior palps extend into the surrounding water. The unique life style within a bone environment is challenged by the high bacterial activity on, and within, the bone matrix possibly causing O(2) depletion, and build-up of potentially toxic sulphide. We measured the O(2) distribution around embedded Osedax and showed that the bone microenvironment is anoxic. Morphological studies showed that ventilation mechanisms in Osedax are restricted to the anterior palps, which are optimized for high O(2) uptake by possessing a large surface area, large surface to volume ratio, and short diffusion distances. The blood vascular system comprises large vessels in the trunk, which facilitate an ample supply of oxygenated blood from the anterior crown to a highly vascularised root structure. Respirometry studies of O. mucofloris showed a high O(2) consumption that exceeded the average O(2) consumption of a broad line of resting annelids without endosymbionts. We regard this combination of features of the respiratory system of O. mucofloris as an adaptation to their unique nutrition strategy with roots embedded in anoxic bones and elevated O(2) demand due to aerobic heterotrophic endosymbionts.

Do Acartia tonsa (Dana) eggs regulate their volume and osmolality as salinity changes?

Subitaneous eggs from an euryhaline calanoid copepod Acartia tonsa were challenged by changes in salinity within the range from full strength salinity, down to zero and up to >70 psu. Egg volume changed immediately, increasing from 2.8 × 10(5) µm(3) at full strength salinity (35 psu) to 3.8 × 10(5) µm(3) at 0 psu and back to its initial volume when gradually being returned to full strength salinity. Egg osmolality followed the molality of the surrounding water when challenged within a salinity range from 2 to 50 psu. Egg respiration was not affected when eggs kept at 35 psu was exposed to low salinity (2 psu). These results suggest that eggs are unable to regulate their volume or osmolality when challenged with changes in salinity. Gradual changes in salinity from 35 to 2 psu and back did not harm the eggs (embryos), since the hatching success remained unaffected by such changes in salinity. In contrast, extreme hyper-saline conditions (76 psu) made the eggs implode and killed the embryo. We propose that the embryo is protected from salinity stress by its plasma membrane and that water exchange driven by osmosis is restricted to the perivitelline space of the egg, which acts as a perfect osmometer in the salinity range of 5-35 psu. We hypothesize further that the embryo is able to keep its volume and osmolality constant due to the impermeability of the inner plasma membrane of the egg or by a combination of osmoregulation and reduced permeability of the inner plasma membrane.