Ceratothoa oestroides Infection in European Sea Bass: Revealing a Long Misunderstood Relationship.

Ceratothoa oestroides (Cymothoidea, Isopoda) is a generalist crustacean parasite that negatively affects the economic sustainability of European sea bass (Dicentrarchus labrax) aquaculture in the North-East Mediterranean. While mortalities are observed in fry and fingerlings, infection in juvenile and adult fish result in approximately 20% growth delay. A transcriptomic analysis (PCR array, RNA-Seq) was performed on organs (tongue, spleen, head kidney, and liver) from infected vs. Ceratothoa-free sea bass fingerlings. Activation of local and systemic immune responses was detected, particularly in the spleen, characterized by the upregulation of cytokines (also in the tongue), a general reshaping of the immunoglobulin (Ig) response and suppression of T-cell mediated responses. Interestingly, starvation and iron transport and metabolism genes were strongly downregulated, suggesting that the parasite feeding strategy is not likely hematophagous. The regulation of genes related to growth impairment and starvation supported the growth delay observed in infected animals. Most differentially expressed (DE) transcripts were exclusive of a specific organ; however, only in the tongue, the difference between infected and uninfected fish was significant. At the attachment/feeding site, the pathways involved in muscle contraction and intercellular junction were the most upregulated, whereas the pathways involved in fibrosis (extracellular matrix organization, collagen formation, and biosynthesis) were downregulated. These results suggest that parasite-inflicted damage is successfully mitigated by the host and characterized by regenerative processes that prevail over the reparative ones.

Immune priming depends on age, sex and Wolbachia in the interaction between Armadillidium vulgare and Salmonella.

The protection conferred by a first infection upon a second pathogenic exposure (i.e. immune priming) is an emergent research topic in the field of invertebrate immunity. Immune priming has been demonstrated in various species, but little is known about the intrinsic factors that may influence this immune process. In this study, we tested whether age, gender and the symbiotic bacterium Wolbachia affect the protection resulting from immune priming in A. vulgare against S. enterica. We firstly primed young and old, symbiotic and asymbiotic males and females, either with a non-lethal low dose of S. enterica, LB broth or without injection (control). Seven days post-injection, we performed a LD50 injection of S. enterica in all individuals and we monitored their survival rates. We demonstrated that survival capacities depend on these three factors: young and old asymbiotic individuals (males and females) expressed immune priming (S. enterica-primed individuals survived better than LB-primed and non-primed), with a general decline in the strength of protection in old females, but not in old males, compared to young. When Wolbachia is present, the immune priming protection was observed in old, but not in young symbiotic individuals, even if the Wolbachia load on entire individuals is equivalent regardless to age. Our overall results showed that the immune priming protection in A. vulgare depends on individuals' states, highlighting the need to consider these factors both in mechanistical and evolutionary studies focusing on invertebrate's immunity.

Modulations of immune parameters caused by bacterial and viral infections in the terrestrial crustacean Porcellio scaber: Implications for potential markers in environmental research.

The terrestrial crustacean Porcellio scaber (Crustacea: Isopoda) is an established invertebrate model in environmental research. Preceding research using isopods did not widely use immune markers. In order to advance their use in research, knowledge of the reference values in control animals as well as variations during infections is of importance. This study presents, for the first time, the morphology, and ultrastructure of the three main haemocyte types of Porcellio scaber as semigranulocytes (SGCs), granulocytes (GCs), and hyalinocytes (HCs), with the latter having two subtypes, using various light and electron microscopy approaches. The modulation of selected immune cellular and humoral parameters of P. scaber in symptomatic phases of Rhabdochlamydia porcellionis and Iridovirus IIV-31 infections is presented. A clear difference in the immune responses of bacterial and viral infections was shown. Remarkable changes in total haemocyte count (THC) values and the proportions of three different haemocyte types were found in animals with a viral infection, which were not as significant in bacterially infected animals. Modified NO levels and SOD activity were more pronounced in cases of bacterial infection. Knowledge of the morphological and ultrastructural features of distinct haemocyte types, understanding the baseline values of immune parameters in control animals without evident symptoms of infection, and the influence that infections can have on these parameters can serve as a basis for the further use of P. scaber immune markers in environmental research.

Pathogenesis, tissue distribution and host response to Rhabdochlamydia porcellionis infection in rough woodlouse Porcellio scaber.

Rhabdochlamydia porcellionis is a known intracellular pathogen in digestive glands of the terrestrial isopod crustacean Porcellio scaber. To describe the pathogenesis, tissue distribution and host response to R. porcellionis, we conducted microscopic observations and localization of infection in tissues by Fluorescent In Situ Hybridization (FISH). Digestive glands were confirmed as the primary site of infection. From there, R. porcellionis disseminates either through the apical membrane of infected cells into the lumen of digestive glands and further throughout the digestive tract or into the surrounding hemocoel by rupture of the basal membrane and lamina of infected digestive gland cells. Once in the hemocoel, R. porcellionis infects hindgut cells, hemocytes and hemopoetic tissues while the ventral nerve cord and gonads seem to be devoid of infection despite the presence of rhabdochlamydia on the surface of these organs. The host response to R. porcellionis includes aggregation of hemocytes around the infected cells and formation of multilayered melanized nodules exhibiting endogenous fluorescence. The structure of nodules is asymmetric when hemocytes are deposited on the basal side of infected gut and digestive glands cells, or symmetric, when nodules entrapping clusters of rhabdochlamydiae are deposited on other organs in the hemocoel. The study also revealed a high prevalence of infection in P. scaber populations (up to 27%) and confirmed its detrimental effect on the host. Although agility, behavior and molting cycle of infected animals appear unaffected, in the later stages R. porcellionis infection manifests as severe damage to the digestive system and decreased feeding, which eventually lead to the death of the host organism.

Modulation of host immunity and reproduction by horizontally acquired Wolbachia.

The Wolbachia are symbiotic bacteria vertically transmitted from one host generation to another. However, a growing amount of data shows that horizontal transfers of Wolbachia also frequently occur within and between host species. The consequences of the arrival of new symbionts on host physiology can be studied by their experimental introduction in asymbiotic hosts. After experimental transfers of the eight major isopod Wolbachia strains in the isopod Porcellio dilatatus only two of them (wCon and wDil) were found to (1) have no pathogenic effect on the host and (2) be able to pass vertically to the host offspring. In the present work, we studied the influence of these two strains, able to complete an horizontal transfer, on immunity and reproduction of P. dilatatus at two stages of the transfer: (1) in recipient hosts that encounter the symbionts: to test the influence of symbiont when acquired during host life and (2) in vertically infected offspring: to test the influence of a symbiotic interaction occurring all lifelong. The impact of Wolbachia varied depending on the stage: there were clearer effects in vertically infected individuals than in those that acquired the symbionts during their lives. Moreover, the two Wolbachia strains showed contrasted effects: the strain wCon tended to reduce the reproductive investment but to maintain or increase immune parameters whilst wDil had positive effects on reproductive investment but decreased the investment in some immune parameters. These results suggest that horizontally acquisition of Wolbachia can influence the balance between host immune and reproductive traits.

Feminizing Wolbachia: a transcriptomics approach with insights on the immune response genes in Armadillidium vulgare.

BACKGROUND: Wolbachia are vertically transmitted bacteria known to be the most widespread endosymbiont in arthropods. They induce various alterations of the reproduction of their host, including feminization of genetic males in isopod crustaceans. In the pill bug Armadillidium vulgare, the presence of Wolbachia is also associated with detrimental effects on host fertility and lifespan. Deleterious effects have been demonstrated on hemocyte density, phenoloxidase activity, and natural hemolymph septicemia, suggesting that infected individuals could have defective immune capacities. Since nothing is known about the molecular mechanisms involved in Wolbachia-A. vulgare interactions and its secondary immunocompetence modulation, we developed a transcriptomics strategy and compared A. vulgare gene expression between Wolbachia-infected animals (i.e., "symbiotic" animals) and uninfected ones (i.e., "asymbiotic" animals) as well as between animals challenged or not challenged by a pathogenic bacteria. RESULTS: Since very little genetic data is available on A. vulgare, we produced several EST libraries and generated a total of 28 606 ESTs. Analyses of these ESTs revealed that immune processes were over-represented in most experimental conditions (responses to a symbiont and to a pathogen). Considering canonical crustacean immune pathways, these genes encode antimicrobial peptides or are involved in pathogen recognition, detoxification, and autophagy. By RT-qPCR, we demonstrated a general trend towards gene under-expression in symbiotic whole animals and ovaries whereas the same gene set tends to be over-expressed in symbiotic immune tissues. CONCLUSION: This study allowed us to generate the first reference transcriptome ever obtained in the Isopoda group and to identify genes involved in the major known crustacean immune pathways encompassing cellular and humoral responses. Expression of immune-related genes revealed a modulation of host immunity when females are infected by Wolbachia, including in ovaries, the crucial tissue for the Wolbachia route of transmission.

The immune cellular effectors of terrestrial isopod Armadillidium vulgare: meeting with their invaders, Wolbachia.

BACKGROUND: Most of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes. METHODOLOGY/PRINCIPAL FINDINGS: In A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×10(3)Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearson's test = 12 822.98, df = 2, p<0.001). CONCLUSIONS/SIGNIFICANCE: The characteristics of the isopod immune system fell within the range of those known from aquatic crustaceans. The colonization of the haemocytes by Wolbachia seemed to stand from the haematopoietic organs, which may act as a reservoir to discharge Wolbachia in the haemolymph, a known route for horizontal transfer. Wolbachia infection did not affect the haemocyte density, but the quantity of granular haemocytes decreased by one third. This may account for the reduced prophenoloxidase activity observed previously in these animals.

Variations of immune parameters in terrestrial isopods: a matter of gender, aging and Wolbachia.

Ecological factors modulate animal immunocompetence and potentially shape the evolution of their immune systems. Not only environmental parameters impact on immunocompetence: Aging is one major cause of variability of immunocompetence between individuals, and sex-specific levels of immunocompetence have also been frequently described. Moreover, a growing core of data put in light that vertically transmitted symbionts can dramatically modulate the immunocompetence of their hosts. In this study, we addressed the influence of gender, age and the feminising endosymbiont Wolbachia (wVulC) on variations in haemocyte density, total PO activity and bacterial load in the haemolymph of the terrestrial isopod Armadillidium vulgare. This host-symbiont system is of particular interest to address this question since: (1) wVulC was previously shown as immunosuppressive in middle-aged females and (2) wVulC influences sex determination. We show that age, gender and Wolbachia modulate together immune parameters in A. vulgare. However, wVulC, which interacts with aging, appears to be the prominent factor interfering with both PO activity and haemocyte density. This interference with immune parameters is not the only aspect of wVulC virulence on its host, as reproduction and survival are also altered.

Is activated hemocyanin instead of phenoloxidase involved in immune response in woodlice?

In the Common woodlouse Porcellio scaber (Crustacea: Isopoda: Oniscidea), experimental immune challenge did not induce the expression of pro-phenoloxidase that, in most other invertebrates studied thus far, can be activated into phenoloxidase via an activation cascade upon immune challenge. Instead, Porcellio hemocyanin proved to exhibit catecholoxidase activity upon activation. However, none of the activating factors known from other invertebrates other than SDS-treatment resulted in activation of hemocyanin into a functional phenoloxidase in vitro. The distinct characteristics of isopod hemocyanin are reflected by the quaternary structure of the hemocyanin dodecamers that differs from that of other crustacean hemocyanins in that the two hexamers share a common 3-fold rotation axis and have an angular offset of 60 degrees against each other. Accordingly, the sequence of Porcellio hemocyanin can be distinguished clearly from other crustacean hemocyanins and in a phylogenetic analysis forms a cluster with other isopod and amphipod hemocyanins. We propose a peracarid-type hemocyanin that may have evolved in response to its required multiple functions in respiration and immune response, while phenoloxidase sensu strictu is lacking.