Wolbachia and its host have a long history of co-evolution, and the interaction between both parters is very complex, which has not been clearly clarified so far [30]. Wolbachia is mainly located in the insect host reproductive system [26], including female ovary and male testis which makes them good materials for studying Wolbachia-host interaction. Previous results based on the transcriptomic data of the testis tissue of the third instar larvae of Drosophila have shown that Wolbachia infection can affect the expression of genes related to spermatogenesis and thus may induce CI [27]. In this study, we focused on the gene expression in the adult testes of the Wolbachia-infected and Wolbachia-free Drosophila melanogaster to investigate the possible effects of Wolbachia on the host reproductive system during the process of sperm maturity. The results showed that the expression of genes involved in innate immune system and multiple metabolic pathways, especially lipid and carbohydrate metabolism, were significantly different between Wolbachia-infected and Wolbachia-free Drosophila adult testis. We speculate that Wolbachia may competes with its host for carbohydrate and lipid metabolism resources, on the other hand, Wolbachia also provides vitamin for the host (Fig. 4).
Wolbachia is associated with the high expression of innate immune genes in native host
When insects are infected by pathogenic bacteria, the host innate immune responses such as Toll and IMD signaling pathways are activated and then produce a variety of antibacterial peptides (AMPs) [31]. Wolbachia can survive in host cell, and some data have shown that it does not induce immune responses in its native host [12, 32–34]. However, our results showed that Wolbachia was related in enhanced immune responses in its native host testis, including multiple genes in the Toll and IMD pathways, such as DptB, CG9673, spheroide, Takl1 and Drsl3. For example, the protein encoded by the DptB gene is an antimicrobial peptide induced by the IMD signal pathway, which is specifically produced in insect fat bodies and can resist gram-negative bacteria infection [35], indicating that Wolbachia, as a gram-negative bacteria, can still induce the immune response in its native host. In addition, spheroide and Takl1 are usually involved in activating Toll signaling pathways in resisting gram-positive bacteria and fungal infections [36]. It can be seen that in the naturally infected host, Wolbachia may still lead to an increase in its immune response. The enhanced host's immune system may be a "double-edged sword" for Wolbachia, which is harmful to itself and limits infection with other pathogenic bacteria, prevents the pathogenic bacteria for snatching intracellular resources. Of course, Wolbachia may also escape the host ’s immune system in various ways. The expression level of peptidoglycan recognition protein PGRP-SC2 is significantly higher in Wolbachia infected testis, PGRP-SC2 can negatively regulate the IMD signaling pathway by hydrolyzing peptidoglycan, preventing the activation of the constitutive IMD pathway, thereby maintaining the balance between immune tolerance and immune response for Wolbachia infection [37]. Our subsequent transcriptome data (unpublished) shows that the expression levels of Toll and IMD pathway related genes are not different between infected and uninfected Drosophila female ovaries, so it can be speculated that the relationship between Wolbachia and male or female hosts is different. Wolbachia may escape the host's immune system in other ways in females, for example, some studies have pointed out that Wolbachia itself can encode Peptidoglycan Amidase to avoid the host's immune system [38].
Wolbachia is associated with the more active carbohydrate metabolism process in the host
In this study, we noticed that the carbohydrate metabolism was more active in the Wolbachia-infected sample, which might be associated with the infection of Wolbachia. First, the expression levels of Mal-A1, Mal-A3, and Mal-A4 genes related to starch and sucrose hydrolase activity were significantly up-regulated in Wolbachia infected Drosophila testis. These genes can accelerate the formation of D-glucose, which is the initial substrate of glycolysis. It has been reported that insect associated Wolbachia exhibits a complete glycolysis metabolic pathway [23], so it is possible that Wolbachia compete with the host to consume the glycolysis substrate-glucose, resulting in a more active sugar metabolism in the host.
Second, the expression levels of UDP-glycosyltransferase genes including CG5724, CG5999, and Ugt86Dh were significantly up-regulated in Wolbachia-infected testes. Actually, there is no gene involved in UDP-glycosylation in the Wolbachia genome [23], even though Wolbachia need this function. Wolbachia must synthesize its own cell wall and Lipopolysaccharides (LPS) is an essential component of the cell wall [11]. The glycosylation reaction is a very important step in the biosynthesis of LPS, in which the host glycosyltransferase plays vital function for Wolbchia to survive [39]. Our results that the glycosyltransferase genes were up-regulated in the Wolbachia-infected host indicated that Wolbachia might heavily rely on the host in the process of LPS synthesis.
Wolbachia is associated with the more active lipid metabolism in the host
We detected significantly differentially expressed genes in lipid metabolism between Wolbachia-infected and Wolbachia-uninfected Drosophila testes. Two genes including CG5804 and CG8628 were significantly up-regulated in Wolbachia-infected Drosophila testis, both of which belonged to the acetyl-CoA binding protein (ACBP) family which were involved in regulating the expression of genes related to lipid metabolism. Interestingly, we detected that lipolysis-related genes such as mag and CG10116 were also up-regulated in Wolbachia-infected Drosophila testis, and this genes can regulate the storage of triacylglycerol (TAG) and maintain the balance of fat metabolism [40, 41]. Finally, the expression levels of genes Npc2f and Npc2d involved in intracellular cholesterol transport were also significantly up-regulated in Wolbachia-infected testis. All of these results indicated that the existence of Wolbachia was related to the high expression of genes related to lipid metabolism and transport in the host, suggesting that it may induce a more active fat metabolism in the host.
On the contrary, a gene (CG10097) in lipid derivatives synthesis was significantly down-regulated in Wolbachia-infected Drosophila testis tissue. Both GO molecular functional analysis and KEGG analysis showed that this gene encoded fatty acyl-CoA reductase (alcohol-forming), which was the key enzyme in the biosynthesis of insect epidermal wax esters or insect cutin and wax. This indicated that during the period of proliferation of Wolbachia in the host, the host might concentrate fatty acid resources for necessary lipid metabolism for survival, which thus restricted its conversion process to other lipid derivatives.