ß-1,3-Glucan/chitin unmasking in the Saccharomyces cerevisiae mutant, Δmnn9, promotes immune response and resistance of the Pacific oyster (Crassostrea gigas) to Vibrio coralliilyticus infection.

Yeast cells can play a crucial role in immune activation in fish and shellfish predominantly due to the cell wall component ß-1,3-glucan, providing protection against bacterial or viral infections. However, the immunostimulatory capacity of dietary yeast cells remains poorly studied in bivalves. To understand the role of yeast cell wall components (mannan, ß-glucan and chitin) as immune activators, this study characterized the surface carbohydrate exposure of the wild-type baker's yeast Saccharomyces cerevisiae (WT) and its Δmnn9 mutant, which presents a defective mannan structure, and compared these profiles with that of ß-glucan particles, using fluorescein isothiocyanate (FITC)-labeled lectin binding analysis. Then, a first trial evaluated the immunological response in Crassostrea gigas juveniles after being fed for 24 h with an algae-based diet (100A) and its 50% substituted version (based on dry weight) with WT (50A50WT) and Δmnn9 (50A50Y), and the posterior resistance of the juveniles against Vibrio coralliilyticus infection (trial 1). The mRNA expression was measured for ß-glucan-binding protein (CgßGBP), Toll-like receptor 4 (CgTLR4), C-type lectin receptor 3 (CgCLec-3), myeloid differentiation factor 88 (CgMyD88), nuclear factor-kappa B (CgNFκB), lysozyme (CgLys), interleukin 17-5 (CgIL17-5), and superoxide dismutase (CgSOD), in oysters, before and 24 h after the bacterial inoculation. A second trial tested the effect of incorporating Δmnn9 into the 100A diet for 24 h at different substitution levels: 0, 5, 10, 25, and 50% (100A, 95A5Y, 90A10Y, 75A25Y, and 50A50Y), followed by the bacterial challenge with V. coralliilyticus (trial 2). Our findings showed that the outer cell wall surface of WT is largely composed of mannan, while Δmnn9 presents high exposure of ß-glucan and chitin, exhibiting similar FITC-lectin binding profiles (fluorescence intensity) to ß-glucan particles. A significantly higher survival after the bacterial challenge was observed in oysters fed on 50A50Y compared to those fed 50A50WT and 100A in trial 1. This better performance of 50A50Y was supported by significantly higher gene expressions of CgLys, CgSOD, CgMyD88, and CgßGBP compared to 100A, and CgSOD and CgNFκB in relation to those fed on 50A50WT, prior to the bacterial inoculation. Furthermore, improved survival was observed in oysters fed 50A50Y compared to those offered lower Δmnn9 levels and 100A in trial 2. The superior performance of Δmnn9-fed oysters is mostly associated with the elevated presence of unmasked ß-glucans on Δmnn9 cell wall surface, facilitating their interactions with oyster hemocytes. Further studies are needed to evaluate administration dose and frequency of Δmnn9 to develop strategies for long-term feeding.

Comparing life history traits and tolerance to changing environments of two oyster species (Ostrea edulis and Crassostrea gigas) through Dynamic Energy Budget theory.

To predict the response of the European flat oyster (Ostrea edulis) and Pacific cupped oyster (Crassostrea gigas/Magallana gigas) populations to environmental changes, it is key to understand their life history traits. The Dynamic Energy Budget (DEB) theory is a mechanistic framework that enables the quantification of the bioenergetics of development, growth and reproduction from fertilization to death across different life stages. This study estimates the DEB parameters for the European flat oyster, based on a comprehensive dataset, while DEB parameters for the Pacific cupped oyster were extracted from the literature. The DEB parameters for both species were validated using growth rates from laboratory experiments at several constant temperatures and food levels as well as with collected aquaculture data from the Limfjorden, Denmark, and the German Bight. DEB parameters and the Arrhenius temperature parameters were compared to get insight in the life history traits of both species. It is expected that increasing water temperatures due to climate change will be beneficial for both species. Lower assimilation rates and high energy allocation to soma explain O. edulis' slow growth and low reproductive output. Crassostrea gigas' high assimilation rate, low investment in soma and extremely low reserve mobility explains the species' fast growth, high tolerance to starvation and high reproductive output. Hence, the reproductive strategies of both species are considerably different. Flat oysters are especially susceptible to unfavourable environmental conditions during the brooding period, while Pacific oysters' large investment in reproduction make it well adapted to highly diverse environments. Based on the life history traits, aquaculture and restoration of O. edulis should be executed in environments with suitable and stable conditions.

Can only one physiological trait determinate the adverse effect of green fluorescent protein (GFP) incorporation on Vibrio virulence?

Green fluorescent protein (GFP) has been used extensively for in situ animal studies that follow up bacterial infection under epifluorescence microscopy. It is assumed that GFP is acting as a "neutral" protein with no influence on the bacterial physiology. To verify this hypothesis, the virulence of Vibrio splendidus ME9, Vibrio anguillarum NB10, and their respective GFP-tagged strains ME9-GFP and NB10-GFP (transconjugants) was compared in vitro and tested in vivo towards blue mussel (Mytilus edulis) larvae. Results showed that the incorporation of GFP negatively impacted the growth and swimming motility of NB10 in vitro. Correspondingly, the mRNA levels of genes involved in bacterial swimming motility (flaA, flaE, and cheR) were significantly down-regulated in NB10-GFP. As for the strain ME9 on the other hand, GFP incorporation only had a negative effect on swimming motility. However, both the strains NB10-GFP and ME9-GFP showed almost the same virulence as their respective parental strain towards mussel larvae in vivo. Overall, the data presented here demonstrated that incorporation of GFP may cause modifications in cell physiology and highlight the importance of preliminary physiological tests to minimize the negative influence of GFP tagging when it is used to monitor the target localization. The study also supports the idea that the virulence of Vibrio species is determined by complex regulatory networks. Notwithstanding the change of a single physiological trait, especially growth or swimming motility, the GFP-tagged Vibrio strain can thus still be considered usable in studies mainly focusing on the virulence of the strain. KEY POINTS: • The effect of GFP incorporation on physiological trait of Vibrio strains. • The virulence in vibrios could be multifactorial. • The stable virulence of Vibrio strains after GFP incorporation.

Dynamic Immune Response to Vibriosis in Pacific Oyster Crassostrea gigas Larvae during the Infection Process as Supported by Accurate Positioning of GFP-Tagged Vibrio Strains.

As the immune system is not fully developed during the larval stage, hatchery culture of bivalve larvae is characterized by frequent mass mortality caused by bacterial pathogens, especially Vibrio spp. However, the knowledge is limited to the pathogenesis of vibriosis in oyster larvae, while the immune response to pathogenic microorganisms in this early life stage is still far from being fully elucidated. In this study, we combined green fluorescent protein (GFP)-tagging, histological and transcriptomic analyses to clarify the pathogenesis of experimental vibriosis and the mechanisms used by the host Pacific oyster Crassostrea gigas larvae to resist infection. The Vibrio strains first colonized the digestive system and rapidly proliferated, while only the transcription level of IκB kinase (IKK) and nuclear factor κB (NF-κB) associated with signaling transduction were up-regulated in oyster at 18 h post challenge (hpc). The mRNA levels for integrin ß-1, peroxinectin, and heat shock protein 70 (HSP70), which are associated with phagocytosis, cell adhesion, and cytoprotection, were not upregulated until 30 hpc when the necrosis already happened in the larval digestive system. This suggested that the immunity in the early stages of C. gigas is not strong enough to prevent vibriosis and future research may focus on the strengthening of the gastrointestinal immune ability to defend vibriosis in bivalve larvae.

Effect of pluronic block polymers and N-acetylcysteine culture media additives on growth rate and fatty acid composition of six marine microalgae species.

The efficiency of microalgal biomass production is a determining factor for the economic competitiveness of microalgae-based industries. N-acetylcysteine (NAC) and pluronic block polymers are two compounds of interest as novel culture media constituents because of their respective protective properties against oxidative stress and shear-stress-induced cell damage. Here we quantify the effect of NAC and two pluronic (F127 and F68) culture media additives upon the culture productivity of six marine microalgal species of relevance to the aquaculture industry (four diatoms-Chaetoceros calcitrans, Chaetoceros muelleri, Skeletonema costatum, and Thalassiosira pseudonana; two haptophytes-Tisochrysis lutea and Pavlova salina). Algal culture performance in response to the addition of NAC and pluronic, singly or combined, is dosage- and species-dependent. Combined NAC and pluronic F127 algal culture media additives resulted in specific growth rate increases of 38%, 16%, and 24% for C. calcitrans, C. muelleri, and P. salina, respectively. Enhanced culture productivity for strains belonging to the genus Chaetoceros was paired with an ~27% increase in stationary-phase cell density. For some of the species examined, culture media enrichments with NAC and pluronic resulted in increased omega-3-fatty acid content of the algal biomass. Larval development (i.e., growth and survival) of the Pacific oyster (Crassostrea gigas) was not changed when fed a mixture of microalgae grown in NAC- and F127-supplemented culture medium. Based upon these results, we propose that culture media enrichment with NAC and pluronic F127 is an effective and easily adopted approach to increase algal productivity and enhance the nutritional quality of marine microalgal strains commonly cultured for live-feed applications in aquaculture. KEY POINTS: • Single and combined NAC and pluronic F127 culture media supplementation significantly enhanced the productivity of Chaetoceros calcitrans and Chaetoceros muelleri cultures. • Culture media enrichments with NAC and F127 can increase omega-3-fatty acid content of algal biomass. • Microalgae grown in NAC- and pluronic F127-supplemented culture media are suitable for live-feed applications.

Bivalves are NO different: nitric oxide as negative regulator of metamorphosis in the Pacific oyster, Crassostrea gigas.

BACKGROUND: Nitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and although NO pathways have been comparatively well-investigated in gastropods, annelids and crustaceans, there has been very limited research on the effects of NO on metamorphosis in bivalve shellfish. RESULTS: In this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas. The nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induced metamorphosis at 75, 76 and 83% respectively, and operating in a concentration-dependent manner. Additional induction of up to 54% resulted from exposures to 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, with which NO interacts to catalyse the synthesis of cyclic guanosine monophosphate (cGMP). Conversely, high concentrations of the NO donor sodium nitroprusside dihydrate in combination with metamorphosis inducers epinephrine, MK-801 or SMIS, significantly decreased metamorphosis, although a potential harmful effect of excessive NO unrelated to metamorphosis pathway cannot be excluded. Expression of CgNOS also decreased in larvae after metamorphosis regardless of the inducers used, but intensified again post-metamorphosis in spat. Fluorescent detection of NO in competent larvae with DAF-FM diacetate and localisation of the oyster nitric oxide synthase CgNOS expression by in-situ hybridisation showed that NO occurs primarily in two key larval structures, the velum and foot. cGMP was also detected in the foot using immunofluorescent assays, and is potentially involved in the foot's smooth muscle relaxation. CONCLUSION: Together, these results suggest that the NO pathway acts as a negative regulator of metamorphosis in Pacific oyster larvae, and that NO reduction induces metamorphosis by inhibiting swimming or crawling behaviour, in conjunction with a cascade of additional neuroendocrine downstream responses.

The blue mussel inside: 3D visualization and description of the vascular-related anatomy of Mytilus edulis to unravel hemolymph extraction.

The blue mussel Mytilus edulis is an intensely studied bivalve in biomonitoring programs worldwide. The lack of detailed descriptions of hemolymph-withdrawal protocols, particularly with regard to the place from where hemolymph could be perfused from, raises questions regarding the exact composition of aspirated hemolymph and does not exclude the possibility of contamination with other body-fluids. This study demonstrates the use of high resolution X-ray computed tomography and histology combined with 3D-reconstruction using AMIRA-software to visualize some important vascular-related anatomic structures of Mytilus edulis. Based on these images, different hemolymph extraction sites used in bivalve research were visualized and described, leading to new insights into hemolymph collection. Results show that hemolymph withdrawn from the posterior adductor muscle could be extracted from small spaces and fissures between the muscle fibers that are connected to at least one hemolymph supplying artery, more specifically the left posterior gastro-intestinal artery. Furthermore, 3D-reconstructions indicate that puncturing hemolymph from the pericard, anterior aorta, atria and ventricle in a non-invasive way should be possible. Hemolymph withdrawal from the heart is less straightforward and more prone to contamination from the pallial cavity. This study resulted simultaneously in a detailed description and visualization of the vascular-related anatomy of Mytilus edulis.

Does Ralstonia eutropha, rich in poly-ß hydroxybutyrate (PHB), protect blue mussel larvae against pathogenic vibrios?

The natural amorphous polymer poly-ß-hydroxybutyrate (PHB-A: lyophilized Ralstonia eutropha containing 75% PHB) was used as a biological agent to control bacterial pathogens of blue mussel (Mytilus edulis) larvae. The larvae were supplied with PHB-A at a concentration of 1 or 10 mg/L for 6 or 24 hr, followed by exposure to either the rifampicin-resistant pathogen Vibrio splendidus or Vibrio coralliilyticus at a concentration of 105 CFU/ml. Larvae pretreated 6 hr with PHB-A (1 mg/L) survived a Vibrio challenge better relative to 24 hr pretreatment. After 96 hr of pathogen exposure, the survival of PHB-A-treated mussel larvae was 1.41- and 1.76-fold higher than the non-treated larvae when challenged with V. splendidus and V. coralliilyticus, respectively. Growth inhibition of the two pathogens at four concentrations of the monomer ß-HB (1, 5, 25 and 125 mM) was tested in vitro in LB35 medium, buffered at two different pH values (pH 7 and pH 8). The highest concentration of 125 mM significantly inhibited the pathogen growth in comparison to the lower levels. The effect of ß-HB on the production of virulence factors in the tested pathogenic Vibrios revealed a variable pattern of responses.

Ralstonia eutropha, containing high poly-ß-hydroxybutyrate levels, regulates the immune response in mussel larvae challenged with Vibrio coralliilyticus.

Marine invertebrates rely mainly on innate immune mechanisms that include both humoral and cellular responses. Antimicrobial peptides (AMPs), lysozyme and phenoloxidase activity, are important components of the innate immune defense system in marine invertebrates. They provide an immediate and rapid response to invading microorganisms. The impact of amorphous poly-ß-hydroxybutyrate (PHB-A) (1 mg PHB-A L-1) on gene expression of the AMPs mytimycin, mytilinB, defensin and the hydrolytic enzyme lysozyme in infected blue mussel larvae was investigated during "in vivo" challenge tests with Vibrio coralliilyticus (105 CFU mL-1). RNAs were isolated from mussel larvae tissue, and AMPs were quantified by q-PCR using the 18srRNA gene as a housekeeping gene. Our data demonstrated that AMPs genes had a tendency to be upregulated in challenged mussel larvae, and the strongest expression was observed from 24 h post-exposure onwards. The presence of both PHB-A and the pathogen stimulated the APMs gene expression, however no significant differences were noticed between treatments or between exposure time to the pathogen V. coralliilyticus. Looking at the phenoloxidase activity in the infected mussels, it was observed that the addition of PHB-A significantly increased the activity.

Mixture toxicity in the marine environment: Model development and evidence for synergism at environmental concentrations.

Little is known about the effect of metal mixtures on marine organisms, especially after exposure to environmentally realistic concentrations. This information is, however, required to evaluate the need to include mixtures in future environmental risk assessment procedures. We assessed the effect of copper (Cu)-Nickel (Ni) binary mixtures on Mytilus edulis larval development using a full factorial design that included environmentally relevant metal concentrations and ratios. The reproducibility of the results was assessed by repeating this experiment 5 times. The observed mixture effects were compared with the effects predicted with the concentration addition model. Deviations from the concentration addition model were estimated using a Markov chain Monte-Carlo algorithm. This enabled the accurate estimation of the deviations and their uncertainty. The results demonstrated reproducibly that the type of interaction-synergism or antagonism-mainly depended on the Ni concentration. Antagonism was observed at high Ni concentrations, whereas synergism occurred at Ni concentrations as low as 4.9 µg Ni/L. This low (and realistic) Ni concentration was 1% of the median effective concentration (EC50) of Ni or 57% of the Ni predicted-no-effect concentration (PNEC) in the European Union environmental risk assessment. It is concluded that results from mixture studies should not be extrapolated to concentrations or ratios other than those investigated and that significant mixture interactions can occur at environmentally realistic concentrations. This should be accounted for in (marine) environmental risk assessment of metals. Environ Toxicol Chem 2017;36:3471-3479. © 2017 SETAC.

Isolation of Vibrionaceae from wild blue mussel (Mytilus edulis) adults and their impact on blue mussel larviculture.

The blue mussel (Mytilus edulis) is known as a robust bivalve species, although its larviculture appears to be highly susceptible to diseases. In this study, we isolated 17 strains from induced mortality events in healthy wild-caught blue mussel adults and demonstrated that they caused between 17% and 98% mortality in blue mussel larvae in a newly developed, highly controlled immersion challenge test model. Eight of the isolates belong to the Splendidus clade of vibrios, while the other isolates belong to the genus Photobacterium. The genomes of the most virulent Vibrio isolate and the most virulent Photobacterium isolate were sequenced and contained several genes encoding factors that have previously been linked to virulence towards bivalves. In vitro tests confirmed that all 17 isolates were positive for these virulence factors. The sequenced genomes also contained a remarkably high number of multidrug resistance genes. We therefore assessed the sensitivity of all isolates to a broad range of antibiotics and found that there were indeed many strong positive correlations between the sensitivities of the isolates to different antibiotics. Our data provide an ecological insight into mass mortality in blue mussels as they indicate that wild mussels contain a reservoir of pathogenic bacteria.

The impact of quorum sensing on the virulence of Aeromonas hydrophila and Aeromonas salmonicida towards burbot (Lota lota L.) larvae.

In this study, the link between quorum sensing in Aeromonas spp. and its virulence towards burbot (Lota lota) was investigated. High mortality occurred in burbot juveniles challenged with Aeromonas salmonicida HN-00, but not in juveniles challenged with Aeromonas hydrophila AH-1N. Meanwhile, both A. hydrophila AH-1N and A. salmonicida HN-00 were virulent towards larvae. The effect of quorum sensing on the virulence of A. hydrophila AH-1N towards burbot larvae was further investigated using quorum sensing mutants (N-(butyryl)-L-homoserine lactone production and receptor mutants). Challenge with these mutants resulted in higher survival of burbot larvae when compared to challenge with the wild type, and the addition of the signal molecule N-butyryl-L-homoserine lactone restored the virulence of the quorum sensing production mutant. Moreover, quorum sensing inhibitors protected the burbot larvae from both Aeromonas strains. Finally, the freshwater micro-algae Chlorella saccharophila and Chlamydomonas reinhardtii, which are able to interfere with quorum sensing, also protected burbot from the pathogens. However, QS interference was unlikely to be the only mechanism. This study revealed that the virulence of Aeromonas spp. towards burbot is regulated by quorum sensing and that quorum sensing inhibitors and micro-algae are promising biocontrol agents.

Energy vs. essential fatty acids: what do scallop larvae (Argopecten purpuratus) need most?

Larvae of the Chilean-Peruvian scallop Argopecten purpuratus were fed a Dunaliella tertiolecta diet (Dun-diet) supplemented with lipid emulsions, rich in 20:5n-3 (EmEPA), 22:6n-3 (EmDHA) or in saturated fatty acids (EmCOCO). A mixed algal diet of Isochrysis galbana (T-iso) and Chaetoceros neogracile served as a positive control (St-diet). Lipid supplementation to the Dun-diet improved the larval growth and increased the percentage of eyed larvae significantly, compared to the non-supplemented Dun-diet because of the extra energy supplied and not because of its fatty acid composition. No significant differences were observed between supplementation with EmEPA, EmDHA or EmCOCO. A mixture of 20% EmEPA+20% EmDHA (40% EmHUFA) was more efficient in raising the total lipid content in larvae than 40% EmCOCO. Both emulsions increased the triacylglycerol content in larvae compared to the non-supplemented Dun-diet. The best result, however, was obtained with the St-diet, probably because of a more suitable 18:2n-6/18:3n-3 ratio and a higher level of arachidonic acid. A positive relationship between the 18:2n-6/18:3n-3 ratio and the larval performance was found. No significant difference was observed in post-settlement larval size or percentage of metamorphosed larvae between the St-diet and the St-diet supplemented with 20% EmHUFA or 20% EmCOCO. The metamorphosed larvae had a constant DHA/EPA ratio of 3.7, independent from the diet, which suggested a metabolic control of the two fatty acids and a species-dependency of the ratio.