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

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.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).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

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

International audienceBackground 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

Phagocyted ink particles within lysosomes in haemocytes (TEM).

<p>Ink particles were observed in lysosomes (arrowhead) from hyaline (<b>A</b>) and semi-granular haemocyte (<b>B</b>). n: nucleus, arrowhead: ink particles in a lysosome, asterisk: primary endosome.</p

Layout of the haematopoietic organ (TEM).

<p>In the haematopoietic organ (<b>A</b>, transversal section and schematic layout), the compactness of the tissue and the morphology of the cells allowed discriminating between the central area (a, not visible here but see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018531#pone-0018531-g007" target="_blank">Fig. 7C</a>), and the internal (b), central (c) and external (d) cortex. The central area contained stem cells (dividing, <b>B</b>) isolated and steeped in matrix (<b>C</b>, <b>D</b>). The granules appeared in the haemocytes from the central cortex on, though the granular types could not be distinguished (<b>E</b>). The external cortex contained mostly cells with granules (<b>F</b>). A diapedesis figure (arrowhead) across the basal membrane (*) indicated a probable route for haemocyte release from the external cortex (**) into the haemolymph (<b>G</b>).</p

TEM characterization of three haemocyte types.

<p>The hyaline type (<b>A</b>) did not contain granules, contrary to the semi-granular (<b>B</b>) and granular (<b>C</b>) types. n: nucleus, cy: cytoplasm, g: granules, m: mitochondria, ga: Golgi apparatus, rer: rough endoplasmic reticulum.</p

FISH detection of <i>Wolbachia</i> in circulating haemocytes and haematopoietic organs.

<p>In infected animals, <i>Wolbachia</i> (in red) colonized many haemocytes (<b>A</b>, <b>B</b>) and the central area (*) as well as the cortex (**) of the haematopoietic organ (<b>C</b>), although groups of cells remained uncolonized (Arrowheads, <b>D</b>–<b>F</b>). The control uninfected animals presented only rare <i>Wolbachia</i>-like artefacts (haemocytes, <b>G–H</b>, haematopoietic organ, <b>I</b>). <b>A</b>–<b>C, G–H</b>: red: <i>Wolbachia</i>, green: Actin; blue: Nuclei. <b>A</b>, <b>B</b>, <b>G</b>, <b>H</b>: average intensity Z-projections. <b>B</b> and <b>H</b>: Close-ups. <b>D</b>–<b>F</b>: 3D analysis (ImageJ 3D viewer) of image <b>C</b>. <b>D</b>: tilted volume rendering of <i>Wolbachia</i> (red) and the nuclei (turquoise) in the central area extracted from the Z-stack. <b>E</b> and <b>F</b>: volume rendering of <i>Wolbachia</i> (grey-scale) from the whole haematopoietic organ. <b>E</b>: front view corresponding to image <b>C</b>, <b>F</b>: tilted view (180°).</p

Separation of circulating haemocyte populations.

<p>The flow cytometry FSC <i>vs.</i> SSC dotplot shows two populations: P1 and P2. After separation on a Percoll gradient, TEM confirmed that P1 contained few hyaline (*) and semi-granular haemocytes (**) and P2 only granular haemocytes. P1 and P2 ellipses drawn manually.</p

TEM detection of <i>Wolbachia</i> in haematopoietic organs and circulating haemocytes.

<p><i>Wolbachia</i> (*) in a non-differentiated cell in an haematopoietic organ (<b>A</b>) and in a semi-granular haemocyte (<b>B</b>).</p