The maternally transmitted alfa-Proteobacterium Wolbachia pipientis (Rickettsiales) is a widespread endosymbiont of filarial nematodes and arthropods, including crustaceans, mites, spiders, scorpions, and especially insects, where it is estimated to infect up to 66% of the species (Werren et al., 2008).

While in worms Wolbachia are obligate symbionts, in arthropods they induce several reproductive manipulations in order to increase the number of infected females. Effects of the infection include cytoplasmic incompatibility, parthenogenesis, feminization of genetic males, and male-killing, as well as influences on host longevity and fecundity (Stouthamer et al., 1999; Werren et al., 2008). Such a host phenotypic variability is generally linked to the high genome plasticity of Wolbachia. However, experimental data also suggest a role for the symbiont in modulating the host sexual phenotypes by interaction with the hormonal signaling pathway. In particular, the various phenotypic effects may be due to differences in host physiology, and in particular to endocrine-related processes governing growth, development, and reproductive behavior which display a high variability in insects. In particular, a number of evidence links Wolbachia symbiosis to insulin and steroid (i.e., ecdysteroid) signaling. Many studies report antagonistic or cooperative relationships between steroid hormones and insulin. Indeed, an extensive crosstalk between the two hormonal signaling pathways has been demonstrated in regulating metabolism, development, and reproduction. For example, in the fly Drosophila melanogaster there is a mutual antagonistic relationship between these metabolic networks for nutrient homeostasis (Colombani et al., 2005). At a molecular level, for example, the expression of the ecdysone receptor (EcR) co-activator DOR (which is misregulated in diabetic mammals) is controlled by insulin signaling via the forkhead transcription factor FOXO (Francis et al., 2010). The two hormonal signaling pathways may also cooperate: in mosquitoes they act in combination for the yolk protein precursor gene expression required for vitellogenesis (Roy et al., 2007). In the larval prothoracic gland, the insulin-signaling pathway modulates ecdysone release and influences both the duration and rate of larval growth (Shingleton, 2005); in adults, insulin-like peptides may trigger steroid synthesis by the follicular cells in insect ovaries (Wu and Brown, 2006). A parallel regulation by ecdysone and insulin has also been demonstrated in the Drosophila’s ovary where the hormones modulate the proliferation and self-renewal of germ-line stem cells independently (Ables and Drummond-Barbosa, 2010).

In the following sections, data on the involvement of Wolbachia in modulating both host hormonal pathways are discussed. Due to the complexity of such metabolic pathways, at present, it is not clear whether Wolbachia operates by attacking one pathway, thus affecting other related metabolic networks, or if the symbiont targets both pathways, even interacting at several points in each of them.

The insulin/IGF signaling (IIS) pathway plays key roles in growth, metabolism, reproduction, and longevity in different organisms. Recently, a specific interaction between IIS and Wolbachia has been demonstrated in D. melanogaster, where insulin-like peptide mutants display an extended lifespan if they harbor the symbiont (Grönke et al., 2010). Another research involves Drosophila insulin receptor mutants, characterized by a reduction in IIS signaling with pleiotropic effects on many traits, including extreme dwarfism, sterility, increased fat levels, and shortened lifespan: interestingly, in presence of Wolbachia the IIS-related mutant phenotypes resulted in significant moderate effects (e.g., reduced fecundity and extended lifespan), suggesting that the symbiont acts to increase insulin signaling itself (Ikeya et al., 2009).

Wolbachia also seems to interact with chico, a gene encoding an insulin receptor substrate (Böhni et al., 1999). Drosophila carrying homozygous chico¹ alleles are sterile, but in presence of Wolbachia the females produce progeny, even if significantly smaller than their heterozygous siblings (Clark et al., 2005; Richard et al., 2005).

Additional data on the possible interaction between the symbiont and host IIS are provided by studies on crustaceans. Wolbachia is known to infect several species of crustaceans, and especially (but not exclusively) terrestrial isopods where the symbiont can induce the feminization of genetical males through an interaction with host hormonal signaling pathways (Bouchon et al., 2008). Crustacea are by default females, and male sex differentiation is triggered by an androgenic hormone (AH) secreted by the androgenic gland (AG; Legrand et al., 1987; Sagi and Khalaila, 2001). The current hypothesis about the feminizing action of Wolbachia is that the symbiont interacts either with the AG differentiation process or the AH receptors (Rigaud and Juchault, 1998; Bouchon et al., 2008). However, if Wolbachia bacteria are experimentally inoculated in adult males, the host soon develops female structures, despite the presence of the AG, which becomes even hypertrophic (Martin et al., 1973, 1999). This suggests that the AH receptors are no longer functional, favoring the hypothesis that Wolbachia induces feminization by targeting the receptor of the AH. Interestingly, the AH has been proven to be an insulin-like peptide (Manor et al., 2007): in vivo silencing of the gene induces an arrest of spermatogenesis, prevents the regeneration of male secondary sexual characteristics, and also induces a lag in molt and a growth reduction (Ventura et al., 2009). In sequential hermaphrodites, the silencing of the AG insulin-like factor induces the feminization of male-related phenotypes too (Rosen et al., 2010).

Among invertebrates Wolbachia is known to infect exclusively (!) Arthropoda and Nematoda, two Phyla belonging to the Ecdysozoa, a clade of animals which share the ability to replace the exoskeleton. This process is called ecdysis and is controlled hormonally by a class of steroids called ecdysteroids (Ewer, 2005).

In filarial worms, antibiotic curing of Wolbachia “infection” inhibits nematode fertility and development, suggesting a specific role for the symbiont in host oogenesis, embryogenesis, and molting (Casiraghi et al., 2002; Arumugam et al., 2008; Frank et al., 2010). In arthropods, several data suggest that the phenotypic effects induced by Wolbachia may be linked to steroid-related processes. As it is well known, insect steroids play key roles in the coordination of multiple developmental processes, and in adults they control important aspects of reproduction.

In particular, during development, insect molting is induced by the systemic hormone 20-hydroxyecdysone (20E). 20E acts on members of an evolutionarily conserved family of nuclear receptors: it binds to the heterodimeric EcR/Usp receptor composed of EcR and USP (ultraspiracle, homologous to the vertebrate retinoid-X receptor), which shares many commonalities with the human thyroid hormone receptor. Then the EcR/USP complex activates the transcriptional processes underlying the cellular and morphogenetic molting cascade events (Gilbert et al., 2002). A biological action of 20E binding of un-partnered EcR has also been demonstrated (Spindler et al., 2009).

Despite the fact that ecdysteroids are present throughout the entire life of insects, their role in adults is quite elusive. Ecdysteroids, for example, may have a role in lifespan (Tricoire et al., 2009; Schwedes et al., 2011) or in stress responses, such as nutritional shortage, high temperature, dry starvation, and oxidative stress (Hirashima et al., 2000; Terashima et al., 2005; Ishimoto and Kitamoto, 2011). In adult flies ecdysone-mediated signaling is also involved in stressful social interactions and in homeostatic sleep regulation (Ishimoto and Kitamoto, 2011). Another unconventional role of the “molting” hormone is the control of important aspects of reproduction, including ovarian development and oogenesis (Raikhel et al., 2005). In many insect species 20E is directly involved in the regulation of vitellogenin biosynthesis by the female fat body, a metabolic tissue functionally analogous to the vertebrate liver; and it can also induce vitellogenin synthesis in males (Huybrechts and De Loof, 1977; Bownes et al., 1983; Zhu et al., 2007). The 20E has also been shown to affect sexual behavior (Ganter et al., 2007).

De Loof (2006) proposes that ecdysteroids may also act as sex hormones. In particular, 20E secreted by the follicle cells of the insect ovary could be the physiological equivalent of vertebrate estrogens, while E – the precursor of the active molting hormone 20E – should act as a distinct hormone, being the physiological equivalent of vertebrate testosterone (De Loof and Huybrechts, 1998; De Loof, 2006). Indeed, E can regulate a set of genes that are distinct from those controlled by 20E, thus confirming that it may exert different biological functions from 20E (Beckstead et al., 2007). However, in insects the existence of sex hormones is under debate, as the differentiation of primary and secondary sexual characteristics is generally considered under the exclusive control of the genotype of each single cell (Steinmann-Zwicky et al., 1989; Schütt and Nöthiger, 2000). However recent data demonstrate that in insects, as well as in vertebrates, non-autonomous (=hormonal) sex determination controls sex dimorphism (DeFalco et al., 2008; Casper and Van Doren, 2009). Thus, if ecdysteroids function as molting and sex hormones, this could explain why Wolbachia interferes with insect development and reproduction, as discussed in the following section.

Last but not least, it is worth noting that Wolbachia establish themselves in many host steroidogenic tissues, including the fat body and the ovarian follicular epithelium, as demonstrated in many insect species and shown in Figure 1 (Sacchi et al., 2010; Gonella et al., 2011; Negri and Pellecchia, in press).

Many experimental data support the role of Wolbachia in modulating the insect sexual phenotypes by interaction with the host hormonal signaling pathway. Indeed, the various phenotypic effects observed may be due to differences in host physiology and in particular to endocrine-related processes governing growth, development, and reproductive behavior.

In particular, a number of evidences links Wolbachia symbiosis to insulin and ecdysteroid signaling.

Several studies report an extensive crosstalk between the two hormonal signaling pathways, which may work antagonistically, jointly or even independently. Like many other symbiotic bacteria, Wolbachia could operate by attacking one crucial pathway in their hosts, thus affecting other metabolic networks, or by targeting both pathways, even interacting at several points in each of them for its own benefit (that is the manipulation of host reproduction and development in order to increase the number of infected females). In view of the interplay between hormone signaling and epigenetic machinery, a direct influence of the “infection” on hormonal signaling involving ecdysteroids might be achievable through the manipulation of host epigenetic pathways.

Although further work is needed to fully clarify the genetic and molecular bases of such an interaction, new work hypotheses have been now offered for the study of the mechanisms used by symbionts to dialog with their hosts. Likewise, the Wolbachia–host interaction should become an emerging model system for the study of hormone signaling orchestration made by microbial symbionts playing with nuclear receptors, and for shedding light on the role of NRCs in integrating the transcriptional co-regulation with the epigenetic regulation.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.