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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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 ± 1 °C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 ± 1 °C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed conditions was caused by a decrease in hyalinocytes and small agranular cells. In clams, the THC was significantly lower in comparison to those not acclimated, regardless of the conditions of the acclimatization. These findings demonstrate that hemocyte characteristics were differentially affected in both species by the tested conditions of acclimatization. The phagocytosis and ROS production in clams and phagocytosis in oysters were not different in those acclimated for 1 day under both conditions, i.e. emersed and submerged, and those sampled in the field (unacclimated). The THC was significantly affected by acclimatization conditions, so the differences between clams and oysters should be considered in studies where important concentrations of hemocytes are required. The difference in the immune response between both species could be related to their habitat (epifaunal vs. infaunal) and their ability of resilience to manipulation and adaptation to captivity. Our results suggest that functional characteristics of hemocytes should be analyzed in both oysters and clams during the first 1 or 2 days, preferably acclimated under emersed rather than submerged conditions., (Copyright © 2011. Published by Elsevier Ltd.)

Sydney rock oysters (SRO) Saccostrea glomerata suffer mass mortalities during summer and autumn as a result of infection by a protozoan parasite Marteilia sydneyi (QX disease). Mass selected disease resistant (QXR) lines have been used with some success in affected estuaries in recent years, with resistance attributed to oxidative defense systems. However, the role of hemocytes in resistance to QX by SRO has not been fully explored. In the present study, fifty QXR and fifty wild caught (WC) oysters were collected from a lease at Pimpama River during a QX outbreak in January 2011. Hemocytes characteristics (type, morphology) and functions (mortality, phagocytosis and oxidative activity) from both oyster lines were analyzed by flow cytometry in the context of infection intensity and parasite viability (determined histologically). Amongst the QXR oysters, 20% were diseased containing viable parasite, 74% had killed M. sydneyi and 6% were uninfected. In contrast, 86% of WC oysters were diseased, 2% had killed M. sydneyi and 12% were healthy. Significant differences in hemocyte number and physiology between the two oyster lines were found (ANOVA). Phagocytosis rate and the mean oxidative activity per cell were similar between both oyster lines. Higher numbers of infiltrating and circulating hemocytes, higher percentage of circulating granulocytes, their higher size and complexity in QXR oysters, and the production of reactive oxygen species were associated with the ability to kill the parasite. High abundance of M. sydneyi in the digestive tubule epithelium of both oyster lines implied inability to kill the parasite at the beginning of the infection. However, QXR oysters had the ability to kill M. sydneyi at the stage of sporangiosorae in the epithelium of digestive tubules. The similar phagocytic ability of hemocytes from both oyster lines, the size of the parasite at this infection stage, and its localization suggested that encapsulation is likely to be the main process involved in the eradication of M. sydneyi by QXR oysters., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Haliotis tuberculata mortality outbreaks have occurred in France since 1998 and were attributed to a pathogenic Vibrio harveyi. These mortalities were recorded in September, a month with abalone reproduction and characterised by high seawater temperatures. The importance of gonadal maturation and temperature increase on abalone immunity and susceptibility to V. harveyi infection needed to be clarified. Therefore, an immune survey analyzing a large panel of parameters was performed from June to September 2007 on abalone from the Bay of Brest. The data obtained were put in relation with abalone reproductive status and its susceptibility to V. harveyi. Most parameters showed clear patterns from early to late summer and during gametogenesis, phagocytosis and phenoloxidase activity were reduced, whereas basal reactive oxygen species production and agglutination titres were significantly increased. Total haemocyte counts went up after the partial spawning event at the end of June, and cell complexity diminished. Using a Principal Component Analysis, the "haemolymph profile" was shown to decrease in parallel with spawning and gonadal maturation processes, and reached a minimum just after total spawning. A significant correlation between this "haemolymph profile" and disease susceptibility allowed us to establish for the first time in abalone, a clear concordance between maturation and spawning processes, immune status and abalone susceptibility to V. harveyi.

This work presents the first detailed microscopic and functional analysis of the haemocytes of an abalone; the European Haliotis tuberculata. It is shown that in contrast to the situation in bivalves, only very few basophilic "granulocytes" could be found and exclusively with a histological stain. Neither flow cytometry, phase contrast observation nor transmission electron microscopy were able to detect any granular cells. The large majority of cells was constituted of "hyalinocytes", which could be sorted by flow cytometry, for the first time, into small (blast-like) and large cells. This permits a detailed analysis of haemocytes and especially of the lowly represented blast-like cells. The differences in haemolymph cell composition between bivalves and gastropods is reviewed in depth and discussed in view of the new data we present. Most of the abalone haemocytes analysed harbour many vacuoles, large glycogen deposits, lipid inclusions and acidic compartments. However, although the number of these "inclusions" was rather variable in between individual hyalinocytes, these experiments did not allow to discern subpopulations using these criteria, and the population appears more as a "differentiation continuum". Haemocytes adhere very rapidly and are immunologically active as they quickly phagocytose latex beads and zymozan particles. This study is the first step towards understanding the H. tuberculata immune system by adapting new tools to gastropods and in providing a first detailed morpho-functional study of their haemocytes.