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

Abstract

BackgroundMost 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 FindingsIn 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/SignificanceThe 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

“…In A. vulgare , it has recently been shown that Wolbachia impact immune cellular processes [10,11,65]. We show here that Wolbachia symbiosis leads to a down-regulation of some A. vulgare immune genes.…”

“…In A. vulgare , Wolbachia colonize not only the ovaries but also other tissues, particularly the immune cells [65,84]. Therefore, perturbation of the host immune gene expression could be a direct effect of the bacteria on immunity.…”

Cannibalism and Predation as Paths for Horizontal Passage of Wolbachia between Terrestrial Isopods

“…Fluorescence in situ Hybridization was performed according to Chevalier et al [46] on a total of 4 µL for each pool fixed with 1% paraformaldehyde-PBS solution (137 mM NaCl, 8 mM Na 2 HPO 4 , 12H 2 O, 1.5 mM KH 2 PO 4 , 3 mM KCl, pH 7.3), at 35% formamide with the probes W1-Cy3 (5′-AATCCGGCCGARCCGACCC-3′) and W2-Cy3 (5′-CTTCTGTGAGTACCGTCATTATC-3′) (30 ng.mL −1 each) targeting Wolbachia 16S rRNA [47]. As in Chevalier et al [46] the samples were further stained with FITC-phalloidin (20 ng, Sigma) mixed into the hybridization buffer and with DAPI (Sigma) mixed into the mounting medium (AF1 antifading, Citifluor, England, 2.5 mg.mL −1 ). Detection was performed with an epifluorescent microscope (Axio Observer-A1, Zeiss) with Apotome (structured illumination) equipped with a 63X/1.25 objective (oil immersion) and with the AxioVision 4.8.1 software (Zeiss).…”

Polyester synthesis genes associated with stress resistance are involved in an insect–bacterium symbiosis

“…Recent studies on diverse insect-microbe symbiotic associations have revealed that the host organisms regulate infection densities of their symbionts by means of phagocytosis, antimicrobial peptides, lysozymes, reactive oxygen species, etc. (39)(40)(41)(42). These innate immune mechanisms may also comprise host-related stress factors for the symbionts.…”