A permanent association between hosts and bacteria implies the existence of control mechanisms that regulate the symbiont population to avoid uncontrolled proliferation and subsequent host damage by the bacteria and, conversely, to support the maintenance of the bacteria at a certain level, presumably by providing nutrients and modulating immunity. Since the work of Buchner (5
), insect intracellular symbiosis has been extensively described, but it has generally been described without regard to the interplay between the insect innate immune system and bacterial virulence. Nevertheless, the restricted area of symbiont growth within the bacteriocytes raises the question of whether, and how, these specific cells perceive, control, and maintain the bacteria.
Recently, we have studied S. zeamais
symbiosis, and we have discovered an intriguing bacteriocyte immune response to the endosymbiont: the expression of a member of the PGRP
gene family (21
). This family includes inducible and constitutive proteins that are involved either in activation or down-regulation of the host immune response (3
). In the context of weevil symbiosis, endosymbiont control may rely upon a regulated system that senses bacterial cell fluctuation and physiological status during insect development. To characterize wPGRP
with respect to gene regulation, we have measured its expression in response to P. aeruginosa
and E. coli
bacterial challenge of aposymbiotic naive larvae. Northern blot analysis showed clearly that the wPGRP
transcript steady-state level is greatly increased after a bacterial challenge (Fig. ), thus demonstrating that nonsymbiont gram-negative bacteria can induce wPGRP
gene expression. Interestingly, this expression did not show identical profiles with E. coli
and with P. aeruginosa
(Fig. ), and wPGRP
transcript accumulation was shown to parallel bacterial growth in the aposymbiotic-infected larvae (Fig. ). These findings strongly support that wPGRP
gene expression depends on bacterial growth, which assigns to wPGRP
the status of an immune gene suitable for the host perception of endosymbiont cell density and growth.
Among the attributes of endosymbionts in the insect world is the absence of virulence and tissue invasion. However, Dale et al. (10
) have recently shown in S. zeamais
that the endosymbiont SZPE expresses, during the host nymphal phase, inv/spa
genes that are involved in the type three secretion system associated with cell invasion. The reason for this remains unclear, but it may be related to physiological and hormonal changes that occur during this phase of metamorphosis. One possibility is that endosymbiont inv/spa
gene up-regulation is the consequence of bacterial release from the bacteriocytes and the subsequent invasion of the surrounding cells. To explore this hypothesis, and to test whether the wPGRP
gene expression evolves according to the change in the endosymbiont physiological status, we have conducted FISH experiments to monitor SZPE location in parallel with a real-time RT-PCR wPGRP
transcript survey at different stages in the host life cycle.
Two striking observations were made: (i) a wPGRP transcript accumulation within the bacteriome, and (ii) concomitant wPGRP gene induction and SZPE release from the bacteriocytes in the nymphal phase.
(i) This is the first time that we have been able to demonstrate an up-regulation of an immune defense gene in insect bacterial intracellular symbiosis. wPGRP transcript accumulation in the bacteriome is interpreted as being a response of the insect immune system to the symbiont.
However, the function of the wPGRP
gene remains speculative and needs more investigation for us to understand whether this gene triggers antibacterial peptide synthesis via an Imd-like pathway or whether it possesses amidase activity and, at the opposite end, whether it downregulates an Imd-like pathway. It is likely that the wPGRP protein possesses amidase activity, since the five amino acid residues required for this enzymatic activity (30
) are well conserved in this weevil gene (21
). In this case, the overexpression of the wPGRP
gene in the bacteriocytes would prevent activation of an Imd-like pathway and therefore would help symbiont persistence in these symbiont-bearing cells. This hypothesis is strongly supported by the function of the wPGRP
ortholog in Drosophila
) that is transcribed mostly in the larval gut as PGRP-SC1
genes, probably to prevent a permanent activation of the Imd pathway in response to gut microbial flora (3
(ii) Insect primary endosymbionts have always been described as being intracellular during the insect life cycle. In Sitophilus
spp., several authors have described the bacteriome dissociation during nymphosis and the bacteriocyte “migration” along the intestine (19
). However, neither these authors nor those of other studies have firmly demonstrated whether SZPE exits from the bacteriocytes during this phase. The observation that SZPE increases up to 20-fold the expression of inv/spa
virulence genes during metamorphosis (10
) raises the question of whether the change in SZPE transcription parallels bacterial release and whether, and then how, the weevil immune system responds to this symbiont physiological change.
In this work, FISH experiments have shown that SZPE remains intracellular until the last larval stages (Fig. , image 8). However, as soon as metamorphosis begins in the pre-nymph stage the larval bacteriome dissociates, and numerous bacteria were shown outside the bacteriocytes (Fig. , image 9). Bacterial growth and invasion are limited at the intestine side exposed to the adipocyte tissue, probably due to the immune activity of this tissue, while many bacteria are found in the gut lumen (Fig. , images 10 and 11). This finding calls into question the formation of the adult midgut caeca bacteriomes that may result from cell reinfection along the intestine, in addition to (or instead of) the bacteriocyte migration described previously (19
In parallel to bacterial cell release in the nymphal phase, a high level of wPGRP
gene up-regulation was shown (Fig. ). This gene up-regulation, along with bacterial inv/spa
gene induction (10
), indicates that host-symbiont interaction involves the interplay between host immune systems and bacterial virulence genes. However, the function of both immune and virulence genes needs to be determined in the context of mutualistic associations. Whatever the role of the wPGRP
gene (activator or downregulator), its induction could be regarded as an adaptive immune response to bacterial release in order to prevent bacterial invasion into insect tissues and complete bacterial clearance. The relatively high prevalence of SZPE in the digestive tract, and its absence from the adipocyte tissue, are in line with this assumption and suggest, instead, bacterial elimination from and by this immunocompetent tissue.
In conclusion, the study described here shows for the first time that the PGRP
gene family is prone to interact with insect endosymbiosis. The wPGRP
gene is regulated by gram-negative bacteria, its expression changes according to bacterial growth, it is up-regulated in the bacteriocytes, and it responds actively to SZPE release from the bacteriocytes and/or to the change in SZPE gene transcription. The future investigation of the molecular mechanisms of wPGRP
regulation and activity (activator or down-regulator) and of wPGRP
-SZPE interaction will contribute to the understanding of how prokaryotes and eukaryotes interact to favor mutualism at the initial step of symbiosis. So far, no data have reported innate immune genes in those associations, except the recent up-regulation of an i-type lysozyme gene recently described in the aphid bacteriocytes (32