The interaction between Wolbachia and its host is dynamic and complex and has yet to be fully clarified . Wolbachia are mainly located in the reproductive system of their insect hosts , including the female ovaries and male testes, which makes them good tissues for studying Wolbachia-host interactions. In recent years, the interactions between Wolbachia and its hosts have been widely studied. Most studies have focused on the mechanisms of CI induced by Wolbachia. Zheng et al. showed that Wolbachia induced high expression of key gene in adult D. mel testis, but low expression levels in female ovaries infected with Wolbachia. Overexpression of key in the testes significantly reduced the embryo hatching rate, and Wolbachia infected females could rescue this defect. Overexpression of key also altered the expression of some immunity-related genes and increased ROS levels in male testes. These results suggest that Wolbachia may induce fertility defects through immune-related pathways . This is similar to the results of the study on Hira gene by Zheng et al. . Zheng et al. performed an analysis of small RNAs in adult D. mel testes infected with Wolbachia and uninfected testis and showed that Wolbachia may negatively regulate psq by upregulating nov-miR-12, resulting in male fertility disorders . Yuan et al. conducted a proteome analysis of female Drosophila spermathecae and seminal receptacles (SSR) and showed that Wolbachia infection significantly altered the expression of various proteins in males, including immune-, metabolic-, and reproductive-related proteins. Wolbachia infection leads to downregulated expression of male reproductive-related proteins . Compared with the findings of Zheng et al. and other transcriptome data from Drosophila testes, our transcriptome data from the testes of Wolbachia-infected and uninfected male Drosophila are different. Our transcriptome data did not show differential expression of reproductive-related genes in DmelW and DmelT. In contrast, we found that carbohydrate metabolism, proteolysis and immune-related genes were greatly upregulated in DmelW, which was consistent with the results of Zheng et al. . In particular, in our transcriptome data, the expression of genes key, Hira and Ance, which were reported to be related to the reproduction of male D. mel and may be the cause of Wolbachia-induced CI [27, 39] were not significantly different between Wolbachia-infected and uninfected male testes. Zheng et al. studied the effects of Wolbachia on spermatogenesis in the larval stages of Drosophila , and we believe that the effects of Wolbachia on spermatogenesis in the larval and adult stages are different, especially as not all sperm contain Wolbachia in the early stages of spermatogenesis, but almost 100% can induce CI . Wolbachia is thought to affect spermatogenesis by secreting its own substances. The dynamic changes in Wolbachia gene expression in the different life cycles of D. mel, identified by Darbyac et al. showed that the genes related to the Wolbachia secretory system were upregulated in the larval and pupal stages. During this period, Wolbachia may secrete more proteins that participate in Wolbachia-host interactions . Based on the results above, we believe that Wolbachia may have different interactions with the host during the larval and adult stages. Wolbachia may have a more significant effect on male testes in the early stage of spermatogenesis through protein secretion, and this effect may be weaker in the adult stage.
Wolbachia infection is associated with more active carbohydrate metabolism in the host
Pyruvate is the most essential metabolic molecule in prokaryotes, and it is also necessary for Wolbachia survival. Pyruvate is produced by glycolysis, and the Wolbachia genome lacks three key enzymes to produce pyruvate. However, Wolbachia retains the complete pyruvate pathway to generate energy through the tricarboxylic acid (TCA) cycle [44, 45]. Glycolysis is accelerated in Wolbachia-infected nematodes and that nematodes provide pyruvate for their symbiotic bacteria . In our study, we noticed that carbohydrate metabolism was more active in Wolbachia-infected samples. The expression levels of the Mal-A2, Mal-A1, Mal-A3, and Mal-A4 genes, which are involved in starch and sucrose metabolism, were significantly upregulated in DmelW testes. The hydrolytic enzymes encoded by these genes can accelerate D-glucose formation, which is the initial substrate of glycolysis . This is similar to the results reported by Zheng et al. . Therefore, Wolbachia may compete with the host to consume the glycolysis substrate glucose, resulting in accelerated production of glucose in the host.
Wolbachia may rely on the host lysosome pathway for amino acids
Due to its intracellular lifestyle, Wolbachia lacks many essential biosynthetic pathways, many of which are involved in amino acid production [47, 48]. Therefore, Wolbachia must obtain amino acids from their hosts [47–50]. Previous studies have shown that Wolbachia is highly dependent on host proteolysis via ubiquitination and the endoplasmic reticulum-associated protein degradation (ERAD) pathway . Yuan et al. also speculated that Wolbachia might alter the abundance of proteins in the SSR by affecting ubiquitin-proteasome-mediated proteolysis . Although there is no significant difference in ubiquitin-related genes between DmelM and DmelT, we found significant differences in lysosome-related genes between the two strains, indicating that lysosome activity was significantly enhanced in Wolbachia-infected testes and that lysosomes play an important role in the process of intracellular protein degradation . Thus, we speculate that amino acids may be acquired by Wolbachia via the lysosomal degradation pathway of the host. This is consistent with the results of previous studies in Tetranychus urticae .
Besides, several serine protease-encoding genes were upregulated in DmelW, including betaTry, alphaTry, zetaTry, iotaTry, and etaTry. KEGG enrichment analysis showed that the neuroactive ligand-receptor interaction pathway was significantly enriched in Wolbachia-infected testes. Considering these results, we speculate that Wolbachia can increase host catabolism. Similar results were reported by Zheng et al. .
Wolbachia infection is associated with more active lipid metabolism in the host
Lipid metabolism is likely to be critical to the Wolbachia-host relationship. Both Wolbachia and insects lack cholesterol biosynthesis genes, and the Wolbachia genome also lacks fatty acid synthesis genes, so there is likely to be resource competition between Wolbachia and the host . Our study showed that several genes involved in lipid synthesis were significantly upregulated in DmelW. The process of fatty acid synthesis was significantly upregulated in DmelW, which may supply fatty acids due to a resource shortage caused by competition with Wolbachia. Two studies also found that Wolbachia abundance was correlated with increased odd-chain fatty acids and increased mRNA expression of fatty acid synthase [4, 53]. At the same time, we found that several intracellular cholesterol transport genes were significantly upregulated in DmelW, including Npc1b, Npc2f, and Npc2d, which may lead to changes in intracellular cholesterol transport. Cholesterol is vital for membrane stability and cellular signaling in insects. Wolbachia replication is also cholesterol-dependent, as cholesterol-rich host membranes are required to form the vacuole surrounding each bacterium , so there is likely to be resource competition between Wolbachia and the host, resulting in accelerated cholesterol transport in the host. Cholesterol also plays an important role in pathogen blockage by Wolbachia. Previous studies have reported that Wolbachia can regulate intracellular cholesterol transport to resist DENV infection .
In contrast, some peroxisome-related genes, including CG17560, CG13091, CG10097, and CG1441, were significantly downregulated in DmelW. GO molecular functional analysis showed that these genes have fatty acyl-CoA reductase (alcohol-forming) activity and are involved in the biosynthesis of insect cutin and wax. This result further indicated that infection with Wolbachia may influence the lipid metabolism of the host in various ways.
Wolbachia infection is associated with high expression levels of innate immunity genes in native host
When Wolbachia is transferred into a novel host, such as a mosquito, it causes a strong immune response in the host . However, in their native hosts, such as Drosophila, Wolbachia does not induce an immune response due to the mutually beneficial relationship [57–59]. Wolbachia can also improve the native host’s resistance to pathogens in other ways [55, 60]. However, one study showed that Wolbachia could induce an innate immune response in D. mel adult testis .
Our results showed that the presence of Wolbachia was related to enhanced immune responses in the testes of its native host, including multiple genes in the Toll and IMD pathways, such as, the protein encoded by the DptB gene is an antimicrobial peptide induced by the IMD signaling pathway that promotes resistance to gram-negative bacterial infection . Drs and Drsl3 encode antimicrobial peptides under the control of the Toll signaling pathway induced by fungal infection . Although Wolbachia is surrounded by the host membrane, it has a nearly complete peptidoglycan precursor lipid II synthesis pathway . Wolbachia also encodes an amidase (AmiD) that cleaves its own peptidoglycan to evade the host immune response . We speculate that due to the higher density of Wolbachia in the testis compared to other tissues, during the rapid proliferation of Wolbachia, it is impossible for it to completely evade the host immune system, resulting in immune recognition. For example, the expression of the peptidoglycan recognition protein gene PGRP-SC2 is significantly higher in Wolbachia-infected testes, and PGRP-SC2 can negatively regulate the IMD signaling pathway by hydrolyzing peptidoglycan, preventing activation of the constitutive IMD pathway, thereby maintaining the balance between immune tolerance and immune response in Wolbachia infection .
Wolbachia infection is not associated with oxidative stress in the native host
In addition to the above results showing that Wolbachia may affect the host’s Toll and IMD pathways, Wolbachia may also affect the level of reactive oxygen species (ROS) in the host [59, 66]. ROS is a natural byproduct of oxidative phosphorylation, and it can cause severe cell damage. However, ROS also plays an important role in immune response, not only participating in the transmission of immune signals, but also directly killing pathogenic microorganisms . Zug et al. reported that there is a close relationship between Wolbachia and ROS in the host. In a novel host, Wolbachia induces ROS production, resulting in significant upregulation of host antioxidant genes. However, in their native hosts, Wolbachia not only induces ROS production and oxidative stress, but also the expression of antioxidant genes (from Wolbachia, the host, or both) to restore oxidative homeostasis . The study by Molloy et al. on ROS in naturally infected versus antibiotic-cured Aedes albopictus supports this hypothesis. They found that Wolbachia infection status had no significant effect on ROS and antioxidant enzyme gene expression levels in A. albopictus. The author speculated that Wolbachia might not enhance host resistance to pathogens through the ROS-induced immune pathway .
We hypothesize that Wolbachia does not induce oxidative stress in their native hosts. To verify this hypothesis, we detected the expression levels of antioxidant genes in Wolbachia-infected and uninfected D. mel adult testes. We found that the expression levels of several genes related to ROS degradation were not significantly different between DmelW and DmelT testes, which was consistent with the results of Molloy et al. . Our results indicate that Wolbachia infection does not induce oxidative stress in its native hosts at least in the adult stage.
In summary, our RNA-seq data collected from adult D. mel testes showed numerous DEGs between Wolbachia-infected and Wolbachia-free samples, and these genes are mostly involved in carbohydrate metabolism, lysosome, lipid metabolism, immune response, and peroxisome, and no differentially expressed genes putatively associated with spermatogenesis were discovered. These data provide useful molecular information for the study of Wolbachia-host intracellular relationships. Subsequent analysis of transcriptome data in Drosophila ovaries would be helpful to further understand the differences in Wolbachia-host molecular interactions between male and female hosts.