The Immune Cellular Effectors of Terrestrial Isopod Armadillidium vulgare: Meeting with Their Invaders, Wolbachia

PLOS ONE, Apr 2011

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±0.7×103 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.

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The Immune Cellular Effectors of Terrestrial Isopod Armadillidium vulgare: Meeting with Their Invaders, Wolbachia

Wolbachia. PLoS ONE 6(4): e18531. doi:10.1371/journal.pone.0018531 The Immune Cellular Effectors of Terrestrial Isopod Armadillidium vulgare : Meeting with Their Invaders, Wolbachia Fre de ric Chevalier 0 Juline Herbinie` re-Gaboreau 0 Joanne Bertaux 0 Maryline Raimond 0 Franck Morel 0 Didier Bouchon 0 Pierre Gre` ve 0 Christine Braquart-Varnier 0 David M. Ojcius, University of California Merced, United States of America 0 1 Universite de Poitiers, Laboratoire Ecologie , Evolution, Symbiose, UMR CNRS 6556, Poitiers , France , 2 Universite de Poitiers, Laboratoire Inflammation, Tissus Epitheliaux et Cytokines , EA 4331, Poitiers , France 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 (6.4560.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.660.76103 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. - Funding: This research was funded by the CNRS, the University of Poitiers (Action Incitative: Diversite bacterienne et immunocompetence des ho tes) and the Agence Nationale de la Recherche (EndoSymbArt ANR-06-BLAN-0316 - ADaWOL ANR-09-JCJC-0109). FC and JHG were supported by a fellowship from the French Ministe`re de lEducation Nationale et de la Recherche. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Lacking the memory of vertebrate immunity, invertebrates largely depend upon their innate defensive mechanisms to protect themselves against pathogens and invading organisms. Immune cellular responses include early non-self recognition [1], phagocytosis, cellular encapsulation and nodulation [13]. Immune humoral responses involve clotting and coagulation reactions [3,4], the production of antimicrobial peptides [5] and the prophenoloxydase cascade [6]. In crustaceans all these processes are conducted by, or originate from haemocytes which are considered as the cornerstone of their immune system [3,7,8]. Most of our knowledge on crustacean immune system stands from decapods, such as freshwater crayfishes, shrimps or crabs which live in aquatic ecosystems. Meanwhile, some isopods (Oniscidae) have evolved a terrestrial lifestyle, which could have impacted their immune system. The latter could thus stand closer to that of other terrestrial arthropods because of similar environmental constraints during their evolution. But for now, the immune system of such terrestrial crustaceans remains poorly described. Concerning the cellular effectors, to this date nothing has been published on the different haemocyte types and their origins. Regarding molecular effectors, only a few papers have been published since 2005 [912]. In Armadillidium vulgare, an antimicrobial peptide acting against Gram positive bacteria was characterized [9] as well as numerous proteins known to be involved in both aspects of the immune response in crustacean decapods and other arthropods [10]. In Porcellio scaber, hemocyanin is suggested to fulfil functions of phenoloxidase in addition to serving as a respiratory pigment [11]. In the same species phagocytosis is highly specific upon priming [1 (...truncated)


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Frédéric Chevalier, Juline Herbinière-Gaboreau, Joanne Bertaux, Maryline Raimond, Franck Morel, Didier Bouchon, Pierre Grève, Christine Braquart-Varnier. The Immune Cellular Effectors of Terrestrial Isopod Armadillidium vulgare: Meeting with Their Invaders, Wolbachia, PLOS ONE, 2011, 4, DOI: 10.1371/journal.pone.0018531