Comparative study of domoic acid accumulation, isomer content and associated digestive subcellular processes in five marine invertebrate species.

Despite the deleterious effects of the phycotoxin domoic acid (DA) on human health, and the permanent threat of blooms of the toxic Pseudo-nitzschia sp. over commercially important fishery-resources, knowledge regarding the physiological mechanisms behind the profound differences in accumulation and depuration of this toxin in contaminated invertebrates remain very scarce. In this work, a comparative analysis of accumulation, isomer content, and subcellular localization of DA in different invertebrate species was performed. Samples of scallops Pecten maximus and Aequipecten opercularis, clams Donax trunculus, slippersnails Crepidula fornicata, and seasquirts Asterocarpa sp. were collected after blooms of the same concentration of toxic Pseudo-nitzschia australis. Differences (P < 0.05) in DA accumulation were found, wherein P. maximus showed up to 20-fold more DA in the digestive gland than the other species. Similar profiles of DA isomers were found between P. maximus and A. opercularis, whereas C. fornicata was the species with the highest biotransformation rate (∼10 %) and D. trunculus the lowest (∼4 %). DA localization by immunohistochemical analysis revealed differences (P < 0.05) between species: in P. maximus, DA was detected mainly within autophagosome-like vesicles in the cytoplasm of digestive cells, while in A. opercularis and C. fornicata significant DA immunoreactivity was found in post-autophagy residual bodies. A slight DA staining was found free within the cytoplasm of the digestive cells of D. trunculus and Asterocarpa sp. The Principal Component Analysis revealed similarities between pectinids, and a clear distinction of the rest of the species based on their capabilities to accumulate, biotransform, and distribute the toxin within their tissues. These findings contribute to improve the understanding of the inter-specific differences concerning the contamination-decontamination kinetics and the fate of DA in invertebrate species.

Sediment archives reveal irreversible shifts in plankton communities after World War II and agricultural pollution.

To evaluate the stability and resilience1 of coastal ecosystem communities to perturbations that occurred during the Anthropocene,2 pre-industrial biodiversity baselines inferred from paleoarchives are needed.3,4 The study of ancient DNA (aDNA) from sediments (sedaDNA)5 has provided valuable information about past dynamics of microbial species6-8 and communities9-18 in relation to ecosystem variations. Shifts in planktonic protist communities might significantly affect marine ecosystems through cascading effects,19-21 and therefore the analysis of this compartment is essential for the assessment of ecosystem variations. Here, sediment cores collected from different sites of the Bay of Brest (northeast Atlantic, France) allowed ca. 1,400 years of retrospective analyses of the effects of human pollution on marine protists. Comparison of sedaDNA extractions and metabarcoding analyses with different barcode regions (V4 and V7 18S rDNA) revealed that protist assemblages in ancient sediments are mainly composed of species known to produce resting stages. Heavy-metal pollution traces in sediments were ascribed to the World War II period and coincided with community shifts within dinoflagellates and stramenopiles. After the war and especially from the 1980s to 1990s, protist genera shifts followed chronic contaminations of agricultural origin. Community composition reconstruction over time showed that there was no recovery to a Middle Ages baseline composition. This demonstrates the irreversibility of the observed shifts after the cumulative effect of war and agricultural pollutions. Developing a paleoecological approach, this study highlights how human contaminations irreversibly affect marine microbial compartments, which contributes to the debate on coastal ecosystem preservation and restoration.

Effects of marine harmful algal blooms on bivalve cellular immunity and infectious diseases: A review.

Bivalves were long thought to be "symptomless carriers" of marine microalgal toxins to human seafood consumers. In the past three decades, science has come to recognize that harmful algae and their toxins can be harmful to grazers, including bivalves. Indeed, studies have shown conclusively that some microalgal toxins function as active grazing deterrents. When responding to marine Harmful Algal Bloom (HAB) events, bivalves can reject toxic cells to minimize toxin and bioactive extracellular compound (BEC) exposure, or ingest and digest cells, incorporating nutritional components and toxins. Several studies have reported modulation of bivalve hemocyte variables in response to HAB exposure. Hemocytes are specialized cells involved in many functions in bivalves, particularly in immunological defense mechanisms. Hemocytes protect tissues by engulfing or encapsulating living pathogens and repair tissue damage caused by injury, poisoning, and infections through inflammatory processes. The effects of HAB exposure observed on bivalve cellular immune variables have raised the question of possible effects on susceptibility to infectious disease. As science has described a previously unrecognized diversity in microalgal bioactive substances, and also found a growing list of infectious diseases in bivalves, episodic reports of interactions between harmful algae and disease in bivalves have been published. Only recently, studies directed to understand the physiological and metabolic bases of these interactions have been undertaken. This review compiles evidence from studies of harmful algal effects upon bivalve shellfish that establishes a framework for recent efforts to understand how harmful algae can alter infectious disease, and particularly the fundamental role of cellular immunity, in modulating these interactions. Experimental studies reviewed here indicate that HABs can modulate bivalve-pathogen interactions in various ways, either by increasing bivalve susceptibility to disease or conversely by lessening infection proliferation or transmission. Alteration of immune defense and global physiological distress caused by HAB exposure have been the most frequent reasons identified for these effects on disease. Only few studies, however, have addressed these effects so far and a general pattern cannot be established. Other mechanisms are likely involved but are under-studied thus far and will need more attention in the future. In particular, the inhibition of bivalve filtration by HABs and direct interaction between HABs and infectious agents in the seawater likely interfere with pathogen transmission. The study of these interactions in the field and at the population level also are needed to establish the ecological and economical significance of the effects of HABs upon bivalve diseases. A more thorough understanding of these interactions will assist in development of more effective management of bivalve shellfisheries and aquaculture in oceans subjected to increasing HAB and disease pressures.

A new diatom species P. hallegraeffii sp. nov. belonging to the toxic genus Pseudo-nitzschia (Bacillariophyceae) from the East Australian Current.

A new species belonging to the toxin producing diatom genus Pseudo-nitzschia, P. hallegraeffii sp. nov., is delineated and described from the East Australian Current (EAC). Clonal cultures were established by single cell isolation from phytoplankton net hauls collected as part of a research expedition in the EAC region in 2016 on the RV Investigator. Cultures were assessed for their morphological and genetic characteristics, their sexual compatibility with other Pseudo-nitzschia species, and their ability to produce domoic acid. Light and transmission electron microscopy revealed cells which differed from their closest relatives by their cell width, rows of poroids, girdle band structure and density of band straie. Phylogenetic analyses based on sequencing of nuclear-encoded ribosomal deoxyribonucleic acid (rDNA) regions showed this novel genotype clustered within the P. delicatissima complex, but formed a discrete clade from its closest relatives P. dolorosa, P. simulans, P. micropora and P. delicatissima. Complementary base changes (CBCs) were observed in the secondary structure of the 3' nuclear ribosomal transcribed spacer sequence region (ITS2) between P. hallegraeffii sp. nov. and its closest related taxa, P. simulans and P. dolorosa. Under laboratory conditions, and in the absence of any zooplankton cues, strains of P. hallegraeffii sp. nov. did not produce domoic acid (DA) and were not sexually compatible with any other Pseudo-nitzschia clones tested. A total of 18 Pseudo-nitzschia species, including three confirmed toxigenic species (P. cuspidata, P. multistriata and P. australis) have now been unequivocally confirmed from eastern Australia.

Exposure to the toxic dinoflagellate Alexandrium catenella modulates juvenile oyster Crassostrea gigas hemocyte variables subjected to different biotic conditions.

The Pacific oyster Crassostrea gigas is an important commercial species cultured throughout the world. Oyster production practices often include transfers of animals into new environments that can be stressful, especially at young ages. This study was undertaken to determine if a toxic Alexandrium bloom, occurring repeatedly in French oyster beds, could modulate juvenile oyster cellular immune responses (i.e. hemocyte variables). We simulated planting on commercial beds by conducting a cohabitation exposure of juvenile, "specific pathogen-free" (SPF) oysters (naïve from the environment) with previously field-exposed oysters to induce interactions with new microorganisms. Indeed, toxic Alexandrium spp. exposures have been reported to modulate bivalve interaction with specific pathogens, as well as physiological and immunological variables in bivalves. In summary, SPF oysters were subjected to an artificial bloom of Alexandrium catenella, simultaneously with a cohabitation challenge. Exposure to A. catenella, and thus to the paralytic shellfish toxins (PSTs) and extracellular bioactive compounds produced by this alga, induced higher concentration, size, complexity and reactive oxygen species (ROS) production of circulating hemocytes. Challenge by cohabitation with field-exposed oysters also activated these hemocyte responses, suggesting a defense response to new microorganism exposure. These hemocyte responses to cohabitation challenge, however, were partially inhibited by A. catenella exposure, which enhanced hemocyte mortality, suggesting either detrimental effects of the interaction of both stressors on immune capacity, or the implementation of an alternative immune strategy through apoptosis. Indeed, no infection with specific pathogens (herpesvirus OsHV-1 or Vibrio aesturianus) was detected. Additionally, lower PST accumulation in challenged oysters suggests a physiological impairment through alteration of feeding-related processes. Overall, results of this study show that a short-term exposure to A. catenella combined with an exposure to a modified microbial community inhibited some hemocyte responses, and likely compromised physiological condition of the juvenile oysters.

Physiological and pathological changes in the eastern oyster Crassostrea virginica infested with the trematode Bucephalus sp. and exposed to the toxic dinoflagellate Alexandrium fundyense.

Effects of experimental exposure to Alexandrium fundyense, a Paralytic Shellfish Toxin (PST) producer known to affect bivalve physiological condition, upon eastern oysters, Crassostrea virginica with a variable natural infestation of the digenetic trematode Bucephalus sp. were determined. After a three-week exposure to cultured A. fundyense or to a control algal treatment with a non-toxic dinoflagellate, adult oysters were assessed for a suite of variables: histopathological condition, hematological variables (total and differential hemocyte counts, morphology), hemocyte functions (Reactive Oxygen Species (ROS) production and mitochondrial membrane potential), and expression in gills of genes involved in immune responses and cellular protection (MnSOD, CAT, GPX, MT-IV, galectin CvGal) or suspected to be (Dominin, Segon). By comparing individual oysters infested heavily with Bucephalus sp. and uninfested individuals, we found altered gonad and digestive gland tissue and an inflammatory response (increased hemocyte concentration in circulating hemolymph and hemocyte infiltrations in tissues) associated with trematode infestation. Exposure to A. fundyense led to a higher weighted prevalence of infection by the protozoan parasite Perkinsus marinus, responsible for Dermo disease. Additionally, exposure to A. fundyense in trematode-infested oysters was associated with the highest prevalence of P. marinus infection. These observations suggest that the development of P. marinus infection was advanced by A. fundyense exposure, and that, in trematode-infested oysters, P. marinus risk of infection was higher when exposed to A. fundyense. These effects were associated with suppression of the inflammatory response to trematode infestation by A. fundyense exposure. Additionally, the combination of trematode infestation and A. fundyense exposure caused degeneration of adductor muscle fibers, suggesting alteration of valve movements and catch state, which could increase susceptibility to predation. Altogether, these results suggest that exposure of trematode-infested oysters to A. fundyense can lead to overall physiological weakness that decrease oyster defense mechanisms.

Physiological responses of Manila clams Venerupis (=Ruditapes) philippinarum with varying parasite Perkinsus olseni burden to toxic algal Alexandrium ostenfeldii exposure.

Manila clam stock from Arcachon Bay, France, is declining, as is commercial harvest. To understand the role of environmental biotic interactions in this decrease, effects of a toxic dinoflagellate, Alexandrium ostenfeldii, which blooms regularly in Arcachon bay, and the interaction with perkinsosis on clam physiology were investigated. Manila clams from Arcachon Bay, with variable natural levels of perkinsosis, were exposed for seven days to a mix of the nutritious microalga T-Iso and the toxic dinoflagellate A. ostenfeldii, a producer of spirolides, followed by seven days of depuration fed only T-Iso. Following sacrifice and quantification of protozoan parasite Perkinsus olseni burden, clams were divided into two groups according to intensity of the infection ("Light-Moderate" and "Moderate-Heavy"). Hemocyte and plasma responses, digestive enzyme activities, antioxidant enzyme activities in gills, and histopathological responses were analyzed. Reactive oxygen species (ROS) production in hemocytes and catalase (CAT) activity in gills increased with P. olseni intensity of infection in control clams fed T-Iso, but did not vary among A. ostenfeldii-exposed clams. Exposure to A. ostenfeldii caused tissue alterations associated with an inflammatory response and modifications in hemocyte morphology. In the gills, superoxide dismutase (SOD) activity decreased, and an increase in brown cell occurrence was seen, suggesting oxidative stress. Observations of hemocytes and brown cells in tissues during exposure and depuration suggest involvement of both cell types in detoxication processes. Results suggest that exposure to A. ostenfeldii disrupted the pro-/anti-oxidant response of clams to heavy P. olseni intensity. In addition, depressed mitochondrial membrane potential (MMP) in hemocytes of clams exposed to A. ostenfeldii suggests that mitochondrial functions are regulated to maintain homeostasis of digestive enzyme activity and condition index.