The Association of Wolbachia on the Gene Expression in Drosophila Adult Testis


 Background: Wolbachia is a type of intracellular symbiotic bacteria widely distributed in arthropods including most insects and nematodes. These maternally inherited bacteria can regulate the host's reproductive system in various ways for their own vertical transmission. Since the identification of Wolbachia in many insects, the relationship between Wolbachia and host has attracted great interest. Wolbachia must rely on the host cells to survive, and they can also improve the fitness of the host through a variety of ways. However, the molecular basis of interaction between Wolbachia and their host has not been well resolved so far. Results: We performed transcriptome sequencing on testis tissues of adults of Wolbachia-infected and Wolbachia-free Drosophila melanogaster. Comparison of gene expression profiles revealed 471 significantly differentially expressed genes that involved in cell metabolism, cell membrane component correlation and hydrolysis process.Conclusions: Our results show that lipid and carbohydrate metabolism are more active in Wolbachia-infected testis than in Wolbachia free testis. This work strengthens our general understanding of the Wolbachia-host intracellular relationship and may provide a new perspective for Wolbachia-mediated virus-blocking.


Background
Wolbachia is a class of intracellular bacteria that are widely spread in arthropods and nematodes, and this maternally transmitted bacteria can regulate the host's reproductive system in a variety of ways to increase its chances of transmission, such as cytoplasmic incompatibility (CI), parthenogenesis inducing (PI), male killing (MK), femalization and enhancement of female fertility [1][2][3][4],and these manipulations are considered as a kind of "sel sh strategies" to increase their infection rate in the host population and reduce host tness [1,5]. But in other ways Wolbachia can signi cantly improve host tness. For example, Wolbachia is necessary for nematode growth and development, as the adult worms were retarded, ovaries degenerated, and embryos could not form when Wolbachia was remove from nematodes by antibiotics [6]. In insect hosts, Wolbachia can increase the host's fertility [7][8][9][10][11], and enhance the insect host's resistance to many pathogens (such as RNA virus) [12][13][14]. Wolbachia can also provide vitamins to the host and give the host a clear growth advantage in the face of adversity [1,15]. In addition, the host longevity, olfactory response, immunity and stem cell proliferation were also affected by Wolbachia [16][17][18]. It is expected that Wolbachia have evolved mutualistic relationship with many of their hosts [19] .
Wolbachia infect a remarkable range of insect hosts suggests their interaction with various host metabolic pathways for their successful intracellular maintenance within a host [20]. This interactive relationship is complex and has received extensive attention. Recent transcriptomic, genomic, and proteomic data have revealed that Wolbachia affects many physiological processes in the host. Previous studies that combined genome-wide RNAi screening and new high-throughput uorescence in situ hybridization technology in Drosophila cell lines have found that a large number of host genes may be involved in the alteration of Wolbachia level. These genes are involved in lipid metabolism, transport, protein degradation, translation, and cell cycle [21,22], indicating that many host's metabolic pathways have an obvious impact on the survival of Wolbachia. A genome-wide analysis of various insect-related Wolbachia strains has indicated that Wolbachia has a complete and conserved glycolysis pathway and ribo avin (vitamin B2) synthesis pathway [23]. Ribo avin synthesized by Wolbachia has a signi cant contribution to the growth and development of bed bugs [15]. However, Wolbachia is highly dependent on the host carbon source (such as phosphoglyceride), amino acids (such as alanine) and phospholipids [23]; Wolbachia lacks lipid metabolism genes, especially lipid A synthesis genes [24], so Wolbachia must obtaining these lipids from the host to support cell growth. In addition, the proliferation of Wolbachia in the host is cholesterol-dependent, and Wolbachia lacks cholesterol synthesis genes, so their survival is likely related to the transport of host cholesterol. A recent study also showed that Wolbachia can be localized in the endoplasmic reticulum, which is the main site of lipid metabolism, affecting the distribution of the endoplasmic reticulum and obtaining vacuole membranes from it [25]. Therefore, the above studies have indicated that Wolbachia has a close nutritional symbiosis relationship with the host, so lipids and carbohydrate metabolism is likely to play an extremely important role in the symbiotic relationship between Wolbachia and the host. However, the interaction between Wolbachia and the host in lipid and carbohydrate metabolism has not been studied in details.
Wolbachia is mainly localized in the reproductive tissues of host [26], which is a good material for studying the interaction between Wolbachia and host. Previous studies have showed that Wolbachia affects the early spermatogenesis process [27]. The gene expression pattern between the Drosophila melanogaster larval testis in the Wolbachia-infected and the Wolbachia-free samples observed differential expression of genes related to metabolism, immunity, reproduction and other functions, some of which that may be related to spermatogenesis, including Ance, lola and Mst84Db, were down-regulated in Wolbachia-infected sample, inducing abnormal spermatogenesis that causes Wolbachia to "modify" sperm, which may be the cause of CI. However, in Drosophila, mature sperms are continuously produced in the testis after eclosion but not in the larval stage [28]. Therefore, the purpose of this study is to explore the putative effect of Wolbachia in the process of sperm maturation, based on the gene expression in the testis tissue of Drosophila adults.
We selected Wolbachia-infected and Wolbachia-free Drosophila adult testis tissues for RNA-seq, and analyzed the differentially expressed genes. A total of 472 genes are different expressed at least a 2 fold change (q-value < 0.1%) between Wolbachia-infected and Wolbachia-free testis, involving a variety of physiological processes such as innate immune immunity, carbohydrate metabolism, and lipid metabolism. Most of the differentially expressed genes involved in innate immunity including Toll and IMD pathway were up-regulated in the presence of Wolbachia. More interestingly, lipid and carbohydrate metabolism related gene were signi cant up-regulated in the presence of Wolbachia, while epidermal wax ester synthesis related genes were down-regulated by Wolbachia. Our research found for the rst time that the presence of Wolbachia is associated with more active lipid metabolism and carbohydrate metabolism in the host. These results can provide important evidence for elucidating the mechanism of Wolbachia-host intracellular relationship.

Result
Transcriptome sequencing data of Drosophila adult testis We performed transcriptome sequencing on testis tissues of adults (one day after eclosion) of Wolbachia-infected (WInM) and Wolbachia-free (WUnM) Drosophila melanogaster. Comparison of gene expression pro les revealed 471 signi cantly differentially expressed genes (at least 2fold, q-value < 0.1%), with 402 genes up-regulated and 69 genes down-regulated in the presence of Wolbachia infection.
The signi cantly differentially expressed genes between Wolbachia-infected (WInM) and Wolbachia-free (WUnM) samples are involved in many metabolic pathways We performed Gene Ontology classi cation analysis on the 472 signi cantly differentially expressed genes and found that they were involved in cell metabolism, cell membrane component correlation and hydrolysis process (Fig. 1a, b). Results of KEGG enrichment analysis showed that up-regulated genes in WInM were mostly involved in transport, signal transduction, immune response, glucose metabolism, lipid metabolism and digestion processes, while down-regulated genes in WInM was involved in the insect epidermal wax ester synthesis pathway (Fig. 2).
We detected altogether six signi cantly up-regulated genes in WInM were involved in the innate immune pathway of the Drosophila (Table 1 & Fig. 3a), all of which including DptB, CG9673, spheroide, Takl1, Drsl3, and the peptidoglycan recognition protein gene PGRP-SC2 were functional in Toll and IMD pathways. Table 1 Genes related to immune response that are differentially expressed (≥ 2 fold changes, q-value < 0.1%) in testes of Wolbachia-infected ies compared to Wolbachia-free ies.  (Fig. 3b). In addition, the expression of UDP-glycosyltransferase genes such as CG5724, CG5999, Ugt86Dh were also signi cantly up-regulated in Wolbachia-infected testis (Fig. 3d), and these genes are necessary for Wolbachia in the synthesis of LipidA and further synthesis of lipopolysaccharide (LPS) [29].
We also found that the several genes involved in lipid metabolism were signi cantly up-regulated in Wolbachia-infected Drosophila adults testis, among which the genes of mag and CG10116 were involved in lipid degradation, genes of CG5804 and CG8628 encoded acetyl-CoA binding proteins, gene of CG8834 was related in fatty acid synthesis, gene of Acox57D-d was functional in fatty acid degradation, and genes of Npc2f and Npc2d were involved in intracellular cholesterol transportation ( Table 2 & Fig. 3c). In contrast, the genes of CG10097, CG1441, CG13091, and CG17560, which were involved in the synthesis of other lipid derivatives such as fatty alcohols from fatty acid, were signi cantly down-regulated in Wolbachia-infected testis, which were annotated to have fatty acyl-CoA reductase (alcohol-forming) activity, or be involved in the biosynthesis of insect cutin and wax (Table 2).

Discussion
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 clari ed 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 signi cantly 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][33][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 speci cally 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 signi cantly 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 signi cantly 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 signi cantly up-regulated in Wolbachia-infected testes. Actually, there is no gene involved in UDPglycosylation 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 signi cantly differentially expressed genes in lipid metabolism between Wolbachia-infected and Wolbachia-uninfected Drosophila testes. Two genes including CG5804 and CG8628 were signi cantly 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 signi cantly 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 signi cantly 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.

Conclusions
Wolbachia and the insect host has had a long co-evolutionary history that formed a mutually bene cial symbiosis. Wolbachia provides the necessary nutrients for the host, and its survival is strictly dependent on the host. Our RNA-seq data based on Drosophila adult testis indicated that Wolbachia may affect various physiological pathways of the host, such as immunity, glucose metabolism and lipid metabolism. These data provide important molecular evidence for the Wolbachia-host intracellular relationship. Subsequent analysis of transcriptome data in Drosophila ovaries may help further understand the differences in Wolbachia-host molecular interactions between male and female hosts.

Fruit y rearing
Drosophila melanogaster in this laboratory was kindly donated by Prof. Hu Haoyuan in Anhui Normal University. The standard medium of Drosophila corn our was used for feeding. 60 g corn our, 30 g brown sugar, 5 g sucrose, 1 g sodium benzoate, 6 g agar powder, 800 ml water was added and boiled for 5 min. After cooling, 5 g yeast powder was added to make fruit y medium. The fruit ies were reared in an arti cial climate box (Ningbo Jiangnan Instrument Factory), light: dark = 14L: 10D, relative humidity 40%, light 7000Lux [42].

Wolbachia -free fruit y strains
We used the MLST method to detect the Wolbachia strain in fruit ies [43] and found that the Drosophila melanogaster naturally infected the Wolbachia wMel strain, and the Wolbachia-infected Drosophila melanogaster strain was named WIn. We prepared tetracycline stock solution 10 mg/mL, adding 2 mL tetracycline stock solution per 100 g corn our medium [44]. After continuous treatment for three generations, by PCR detection of Wolbachia wsp, ftsz, and 16 s genes, we obtained Wolbachia-free fruit y strain WUn and then transferred them to standard medium and continuously cultivated for more than 5 generations to remove the effects of antibiotics.

RNA-seq
The WIn and WUn male fruit ies that had been reared in corn our medium for one day after eclosion then dissected in RNase-free water, and the complete testis was placed in RNAhold and stored at -80 °C. Total RNA was extracted by using TansZol Up Plus RNA Kit, about 10 Drosophila testes were used for each sample. Using PE150bp pair-end transcriptome sequencing in BGISEQ-500 platform (BGI, Shenzhen, China), the amount of sequencing data requires 6G for each sample. The sequnces were submitted to NCBI with the accession number of PRJNA639180.

Quantitative real-time PCR (qPCR)
To further investigate the differentially expressed genes identi ed by RNA-seq, 17 genes were selected for qRT-PCR analysis. Speci c primers for the 17 genes and RP49 (Ribosome protein 49 reference gene) were designed by NCBI primer-BLAST (Additional le 1).
RNA extraction from WIn and WUn testis was carried out using TransZol Up Plus RNA Kit (TransGen, Beijing, China) according to the instructions, RNA reverse transcription was performed using Transscript One-Step gDNA Renover and cDNA Synthsis SuperMix (TransGen, Beijing, China), 1 µg RNA was used for reverse transcription, gDNA remover 1µL, Oligo(dT) 1µL, 2xTS Reaction Mix 10µL, TransScript RT/RI Enzyme Mix 1µL, added Nuclease-free water for total volume 20µL. Reaction conditions : 42℃ for 30 min, then 85℃ for 5sec. QPCR veri es differential gene expression. The primers were shown in Table   S1. We used the ΔΔCt method, by using the PerfectStart Green qPCR SuperMix Kit (TransGen, Beijing, China), the reaction system is cDNA 1µL, Forward/Reverse Primer each 0.4µL (10 µM/L), 2xPerfectStart Green qPCR SuperMix 10µL, and Nuclease-free water 8.2µL. Reaction conditions: Pre-reaction at 94 ° C for 30 s, then 40 cycles of 94 ° C for 5 s, 60 ° C for 30 s, and then the dissolution step was performed.

Data analysis
For data analysis, multiple t test in Graphpad prism8 was used to analyze the signi cance of differences between two groups. p-value of less than 0.05 was considered signi cant.

Declarations
The datasets generated and analyzed during the current study are available in the SRA database at NCBI, with the accession number of PRJNA639180.  Figure 1 Functional GO enrichment analysis of the down-regulated genes(a) or up-regulated genes(b) in Wolbachia-infected Drosophila melanogaster adult testis compared to Wolbachia-free sample.  Quantitative PCR validation on some of the signi cantly different expressed genes between Wolbachiainfected and Wolbachia-uninfected Drosophila testis in innate immune response(a); carbohydrate metabolism(b,d) and lipid metabolism(c). Statistical signi cance was determined with multiple t-test in Prism8, "***" and "****" indicate signi cant differences with p-value<0.001and p-value<0.0001, respectively, n=3.