Gut symbiont-derived sphingosine modulates vector competence in Aedes mosquitoes

Sun, Xiaomei; Wang, Yanhong; Yuan, Fei; Zhang, Yanan; Kang, Xun; Sun, Jian; Wang, Pengcheng; Lu, Tengfei; Sae Wang, Fanny; Gu, Jinbao; Wang, Jinglin; Xia, Qianfeng; Zheng, Aihua; Zou, Zhen

doi:10.1038/s41467-024-52566-1

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Published: 19 September 2024

Gut symbiont-derived sphingosine modulates vector competence in Aedes mosquitoes

Nature Communications volume 15, Article number: 8221 (2024) Cite this article

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Abstract

The main vectors of Zika virus (ZIKV) and dengue virus (DENV) are Aedes aegypti and Ae. albopictus, with Ae. aegypti being more competent. However, the underlying mechanisms remain unclear. Here, we find Ae. albopictus shows comparable vector competence to ZIKV/DENV with Ae. aegypti by blood-feeding after antibiotic treatment or intrathoracic injection. This suggests that midgut microbiota can influence vector competence. Enterobacter hormaechei_B17 (Eh_B17) is isolated from field-collected Ae. albopictus and conferred resistance to ZIKV/DENV infection in Ae. aegypti after gut-transplantation. Sphingosine, a metabolite secreted by Eh_B17, effectively suppresses ZIKV infection in both Ae. aegypti and cell cultures by blocking viral entry during the fusion step, with an IC₅₀ of approximately 10 μM. A field survey reveals that Eh_B17 preferentially colonizes Ae. albopictus compared to Ae. aegypti. And field Ae. albopictus positive for Eh_B17 are more resistant to ZIKV infection. These findings underscore the potential of gut symbiotic bacteria, such as Eh_B17, to modulate the arbovirus vector competence of Aedes mosquitoes. As a natural antiviral agent, Eh_B17 holds promise as a potential candidate for blocking ZIKV/DENV transmission.

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Introduction

Aedes mosquitoes are the main vectors of highly pathogenic arboviruses, such as Zika virus (ZIKV), dengue virus (DENV), and Chikungunya virus (CHIKV), which are public health threats. Ae. aegypti is widely distributed worldwide, especially in tropical and subtropical environments, and is closely associated with urban areas^1,2. Ae. albopictus is highly adaptable to new environments. It has expanded its distribution into temperate regions and become an increasingly important vector. This range expansion provides new opportunities for viruses to establish and cause epidemics in Ae. aegypti-free areas³. Studies have shown that while both Ae. aegypti and Ae. albopictus can transmit ZIKV and DENV, Ae. albopictus is a minor vector of ZIKV and DENV as compared to Ae. aegypti⁴. Nonetheless, the underlying mechanisms remain unexplored.

ZIKV, closely related to DENV, is a single-stranded, positive-sense RNA Flavivirus⁵. Many clinical symptoms are caused by ZIKV and these range from asymptomatic or mild symptoms to severe neurologic complications, including Guillain-Barré syndrome and microcephaly^6,7,8,9.

Flaviviruses are primarily transmitted to humans by mosquitoes. After mosquitoes acquire viruses from an infected host via blood feeding, the viruses replicate and diffuse through midgut epithelium and systemically spread from the hemocoel to other tissues, including the fat bodies, nervous system and salivary glands. Infected mosquitoes can then bite and spread the virus to other hosts¹⁰. Therefore, the mosquito midgut, hemocoel, and salivary glands are important organs for virus replication or transmission.

The midgut is the site of multi-taxon interactions in the host. These include symbiotic microbes and pathogens which will ultimately affect the pathogen infection¹¹. The intestinal symbiotic bacterium Chromobacterium reduces the susceptibility of mosquitoes to the malaria parasite Plasmodium falciparum and DENV by producing stable bioactive factors¹¹. Recolonization of Proteus sp. Prsp_P in the midgut through a sugar meal resulted in a significant reduction of DENV titers¹¹. A Talaromyces fungus (Tsp_PR), isolated from wild Ae. aegypti, facilitated DENV infection by inhibiting trypsin transcription and enzyme catalysis¹². These results suggest that gut microbes can play a critical role in the regulation of microbial and viral pathogen infections.

Commensal bacteria can affect host metabolism and physiology, such as lipid metabolism^13,14. Sphingolipids (SLs) can serve as receptors for toxins, viruses, and bacteria^15,16. SLs are common in eukaryotes, but only a few bacteria can produce them, such as species in the Bacteroidetes and Chlorobi phyla¹⁷. Previous studies have demonstrated that bioactive lipids produced by gut microbiota may cross through the epithelial barrier and interact with the host metabolism¹⁸.

Sphingosine (sph), the key molecule of SLs, is a major membrane component that influences many complex biological processes, including cell growth, differentiation, development, autophagy and apoptosis^15,19,20. Sph has strong antibacterial activity against many pathogens and this has been studied for Staphylococcus aureus and Pseudomonas aeruginosa^21,22,23. However, the direct effects of sph on viruses have not been studied until recently. Endogenous sph prevented herpes simplex virus 1 (HSV-1) infection by forming intraluminal vesicles to limit fusion of viral membrane endosomes²⁴. Moreover, sph blocked infection of human epithelial cells with SARS-CoV-2 pseudoviral particles by binding to ACE2, the cellular receptor of SARS-CoV–2²⁵. However, it is unknown if sph can inhibit flavivirus infection.

Here, we show that the midgut microbiota of Aedes mosquitoes regulates their vector competence. We isolate Enterobacter hormaechei_B17 (Eh_B17) from field-collected Ae. albopictus and discover its production of sph, a metabolite that inhibits ZIKV infection in vitro (C6/36 and Vero cells) by limiting viral fusion with the membrane. Field studies reveal that Eh_B17 preferentially colonizes Ae. albopictus compared to Ae. aegypti. Our results demonstrate the ability of symbiotic colonization by sph-producing bacteria to influence the vector competence of Aedes mosquitoes, highlighting its potential importance in mosquito-borne disease control strategies.

Results

Gut commensal bacteria are key determinants in virus infection of Ae. albopictus and Ae. aegypti

Both Ae. aegypti and Ae. albopictus can transmit ZIKV and DENV, however Ae. albopictus is a minor vector of ZIKV and DENV as compared to Ae. aegypti^4,26. To investigate the mechanism, we inoculated lab-adapted Ae. aegypti (UGAL/Rockefeller strain) and Ae. albopictus (Jiangsu strain) mosquitoes with ZIKV MR766 strain via blood-feeding or the intrathoracic route to identify the infection barrier of ZIKV in mosquitoes. Viral RNA levels in the whole mosquitoes and virus titers in saliva were measured at 7 d and 10 d post infection, respectively. ZIKV RNA levels in mosquitoes and virus titers in saliva were 1.9- and 6.6-fold higher in Ae. aegypti than in Ae. albopictus via the blood-feeding route (Fig. 1a,b). Surprisingly, via the intrathoracic route, the ZIKV RNA levels in Ae. albopictus was 1.1-fold higher than that in Ae. aegypti and no significant difference was found in virus titers of saliva (Fig. 1a,b). Thus, we speculated that the vector competent difference between Ae. aegypti and Ae. albopictus lies in the midgut and the commensal microbiota may be involved.

**Fig. 1: Intestinal symbiotic bacteria act as essential contributors to the differential vector competence of *Ae. albopictus* and *Ae. aegypti* for ZIKV transmission.**

To test this hypothesis, Ae. aegypti and Ae. albopictus were both treated with antibiotics (penicillin and streptomycin) for 5 d and then orally infected with the ZIKV MR766 strain (Supplementary Fig. 1a,b). The ZIKV RNA levels in whole mosquitoes were determined at 7 d post infection. Compared to the untreated control group, after antibiotic treatment, the ZIKV RNA levels increased 1.3- and 1.7-fold, and the prevalence increased 1.1- and 1.5-fold for lab-adapted Ae. aegypti (UGAL/Rockefeller strain) and Ae. albopictus (Jiangsu strain), respectively. And, no significant difference was observed between antibiotic-treated Ae. aegypti and Ae. albopictus (Fig. 1c). In keeping to the lab-adapted strains, by antibiotic treatment, field-derived Ae. aegypti (Haikou strain) and Ae. albopictus (Haikou strain) showed 1.1- and 1.5-fold higher levels of ZIKV RNA, and 0- and 1.3-fold higher levels of prevalence, respectively, compared to the control group. And the trend is consistent with the observation from lab-adapted mosquitoes with antibiotic treatment (Fig. 1d). These results suggested the involvement of gut commensal bacteria in limiting viral invasion of the midgut barrier.

Intestinal flora from Aedes mosquitoes inhibits ZIKV infection in Ae. aegypti

Field Ae. albopictus were collected from Hainan Province, China to identify gut commensal bacteria that affect ZIKV vector competence. Thirteen bacterial species were isolated from the midguts and identified by 16S ribosomal RNA (rRNA) sequencing (Supplementary Table 1). The efficacy of these commensal bacteria on the ZIKV vector competence of Aedes mosquitoes was individually assessed by transplantation into lab-adapted Ae. aegypti via blood feeding, followed by oral ZIKV infection. Among the 13 tested bacteria, Eh_B17 and Enterobacter sichuanensis_B36 (Es_B36) suppressed ZIKV RNA levels in Ae. aegypti by 1.5- and 1.2-fold, virus prevalence by 1.3- and 1.1-fold, respectively (Fig. 2a). Then, a similar bacterial species of Eh_B17 was also isolated from lab-adapted Ae. aegypti, named Enterobacter hormaechei_B56 (Eh_B56). However, Eh_B56 only suppressed ZIKV RNA levels in Ae. aegypti by 1.2-fold, virus prevalence by 1.1-fold through midgut transplantation, respectively (Fig. 2a). Moreover, Eh_B17 reduced the salivary virus titers in antibiotic-treated Ae. aegypti mosquitoes 5.8-fold (Fig. 2b).

**Fig. 2: Gut symbiotic bacteria are involved in ZIKV infection in lab-adapted mosquitoes.**

The antiviral effect was further confirmed in field-derived Ae. aegypti (Haikou strain) mosquitoes by feeding ZIKV-containing blood after colonization of Eh_B17. A 1.1- and 1.7-fold inhibition of viral RNA levels in midguts and carcasses were observed in the Eh_B17-treated group, respectively, as compared to the untreated control group (Fig. 2c). Apart from that, 5-fold reduction of virus titers in saliva was detected upon Eh_B17-treatment.

Pathogenicity assays showed that none of these three strains affected the lifespan, egg deposition and egg hatchability of Ae. aegypti. This suggested that Eh_B17, Es_B36, and Eh_B56 did not exert their antiviral activity by impacting host longevity and reproduction (Supplementary Fig. 2a–c).

The genomes of the Eh_B17, Es_B36, and Eh_B56 were sequenced and phylogenomic analysis revealed that Eh_B17 and Eh_B56 belong to the species E. hormaechei, while Es_B36 belong to E. sichuanensis (Fig. 2d). The colonization assay showed that Eh_B17, Es_B36, and Eh_B56 could stably remain in the female midgut by 5.5 × 10⁷ CFU, 1.9 × 10⁷ CFU, and 3.8 × 10⁷ CFU per midgut at 3 d post feeding (Fig. 2e). A green fluorescent protein (GFP) gene with an apramycin (Apr) resistance gene was integrated into the chromosome of Eh_B17 (Eh_B17-GFP) in order to further confirm the colonization of Eh_B17 (Supplementary Fig. 2d). Insertion of the GFP gene did not affect bacterial growth kinetics in LB medium (Fig. 2f). Eh_B17-GFP remained stably present in the female midgut 7 d after blood feeding, with an average of 4.2 × 10⁴ CFU per midgut (Fig. 2g), a characteristic that potentially created a favorable environment for them to exert their antiviral effects.

Metabolites secreted from bacteria inhibit ZIKV infection

Symbiotic bacteria use their effectors, such as secreted proteins, metabolites or cellular components, to interact with their hosts²⁷. The bacterial culture supernatant was separated into retention (secreted proteins) and flow-through fractions (secreted metabolites) using a 3-kDa centrifugal filter in order to identify the antiviral components. C6/36 cells were infected with ZIKV at MOI 0.01 in the presence of a 1% concentration of retention or flow-through fractions, which had no effect on the cell viability as shown in Supplementary Fig. 3a. The addition of flow-through, but not retention of Eh_B17, Es_B36, and Eh_B56 supernatants strongly inhibited ZIKV RNA levels in the C6/36 supernatant and cell lysates, indicating that the existence of substance shared by these bacteria might be capable to reduce ZIKV infection (Fig. 3a and Supplementary Fig. 3b). Consistently, similar antiviral effect of flow-through was observed in Vero cells (Supplementary Fig. 3c). The most pronounced antiviral capability was observed in Eh_B17 flow-through fraction (Fig. 3a). The antiviral effect was then confirmed in Ae. aegypti mosquitoes by co-feeding ZIKV in the presence of Eh_B17 fractions. A 2.0- and 1.8-fold inhibition of viral RNA levels and virus prevalence was detected in the flow-through fraction, respectively, while no significant reduction was observed in the retention and lysates fraction (Fig. 3b,c). The virus titers in the salivary were significantly inhibited by 8-fold with the addition of flow-through fraction, compared to the LB-treated group (Supplementary Fig. 3d).

**Fig. 3: Metabolites secreted by Eh_B17, Es_B36, and Eh_B56 blocks ZIKV infection.**

The flow-through fractions of Eh_B17, Es_B36, and Eh_B56 culture supernatants were further analyzed by LC-MS/MS. No protein was detected, suggesting that the effector may be a metabolite(s). The flow-through of Eh_B17 was then separated into six fractions by semi-preparative high performance liquid chromatography (SPHPLC) according to the peak time at 210-, 225-, 280-, and 320-nm detection wavelengths (Supplementary Fig. 3e) and verified the antiviral activity (Fig. 3d). Fraction 6 (F6) decreased the ZIKV RNA levels and prevalence in antibiotic-treated Ae. aegypti mosquitoes by 2.1- and 1.6-fold, respectively (Fig. 3e). The addition of F6 also reduced salivary virus titers by 14-fold as compared to LB treatment (Supplementary Fig. 3f).

To narrow down the range of target metabolites, F6 components of Eh_B17, Es_B36, and Eh_B56 were analyzed for metabolic profiling using an ultra-performance liquid chromatography (UPLC) system. Venn diagrams identified 530 and 136 common metabolites in positive and negative ion modes, respectively (Supplementary Fig. 3g, Supplementary Data 1 and 2). Only 38 metabolites, among these three bacteria, had a KEGG ID in both of the mode (Fig. 3f). A Sankey dot plot of these 38 common metabolites revealed that most were enriched in the sphingolipid metabolism and drug metabolism-cytochrome P450 pathway (Fig. 3g), among which 24 compounds were commercially available (Supplementary Table 2).

The sphingosine inhibits ZIKV infection in vitro and in vivo

C6/36 cells were infected with ZIKV (MOI 0.01) in the presence of an individual metabolite (10 μM), then viral RNAs in the cell lysates were quantified at 48 h post infection. Among the 24 commercial compounds, sph, psph, and mox significantly reduced ZIKV RNA levels in supernatant and cell lysates (Fig. 4a). Further evaluation of these three metabolites in Vero cells revealed that mox supplementation exerted no remarkable effect on viral infection (Fig. 4b). A MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that sph had no obvious impact on the viability of C6/36 cells at the experimental concentration, while psph exhibited strong cytotoxicity (Supplementary Fig. 4a). We then measured the levels of sph and psph secreted by these three bacteria. The results demonstrated that the levels of sph secreted by Eh_B17 was 1.7- and 2.6-fold higher than that of Es_B36, and Eh_B56, respectively, which was associated with the best antiviral activity of Eh_B17 among three strains (Fig. 4c). However, no significant difference in psph levels was observed among these three bacteria (Fig. 4c). More importantly, the levels of sph secreted by Eh_B17 was 17-fold higher than that of psph (Fig. 4c). Thus, we speculated that sph is a key antiviral metabolite secreted by these three bacteria. Next, we found that the MS/MS spectra of sph generated in Eh_B17, Eh_B36 and Eh_B56 exhibited remarkable consistency with that of the commercially synthesized product, indicating that the structure of the bacterial-derived sph was in concordance with the synthesized product (Supplementary Fig. 4b).

**Fig. 4: Sphingosine inhibits ZIKV infection in mosquitoes and cells.**

From a genomic perspective, the conservation of serine palmitoyl transferase (SPT), the initiating enzyme of the sphingolipid metabolism pathway, demonstrated the presence of sphingolipid metabolism in Eh_B17, Es_B36, and Eh_B56 (Supplementary Table 3 and Supplementary Data 3). However, despite performing standard BLASTP homolog searches across the genomes of these bacterial strains, we were unable to identify reliable homologs of key sph synthesis and degradation enzymes in eukaryotes, such as ceramidases (CDases), ceramide synthase (CerS), sphingosine kinases (SK), and sphingosine phosphate phosphatase (SPPase), indicating potential differences in sphingolipid metabolic mechanisms between bacteria and eukaryotes (Supplementary Fig. 5a). This observation is consistent with previous studies in Bacteroides²⁸. To further explore the role of sph in ZIKV infection, we disrupted the SPT gene in Eh_B17, generating a mutant strain (Eh_B17-mutant) deficient in the sphingolipid metabolism pathway (Supplementary Fig. 5b-5e). This knockout of SPT did not impact bacterial growth kinetics in LB medium (Supplementary Fig. 5f). Notably, the mutant strain exhibited a substantial reduction in sph levels in the flow-through fraction, specifically 1.8-fold lower compared to those of the Eh_B17 strain (Supplementary Fig. 5g). This finding indicates that the SPT gene plays an important role in regulating sphingolipid metabolic pathways in bacteria. It also suggests the existence of alternate pathways in bacteria, which are distinct from those found in eukaryotes. We also found significantly elevated sph level in the midgut of Ae. aegypti (UGAL/Rockefeller strain) mosquitoes treated with Eh_B17. Specifically, sph levels exceeded those observed in mosquitoes treated with LB medium and the Eh_B17-mutant strain by 1.6- and 1.2-fold, respectively, further confirming the importance of SPT in sphingolipid regulation (Supplementary Fig. 5h). More importantly, in Ae. aegypti mosquitoes, the antiviral efficacy of Eh_B17 surpassed that of the Eh_B17-mutant strain, with a remarkable 1.4-fold greater suppression of ZIKV RNA levels, demonstrating a strong link between the bacterial impact on ZIKV infectivity and sph levels (Fig. 4d).

In order to further examine the antiviral effect of sph in vivo, antibiotic-treated Ae. aegypti mosquitoes were blood-fed with ZIKV in addition to increasing amounts of sph. The virus prevalence decreased in a dose-dependent manner from 86% to 50% at 5 μM, 38% at 10 μM and 36% at both 20 μM and 40 μM of sph (Fig. 4e). The addition of 40 μM sph reduced salivary virus titers by 11.3-fold as compared to the DMSO-treated group (Fig. 4f). The inhibitory activity of sph against ZIKV was then determined in C6/36 cells by measuring the ZIKV RNAs in the cell lysates resulting in a 50% inhibitory concentration (IC₅₀) of 5.9 μM (Fig. 4g). Taken together, sph is a key effector conferring ZIKV resistance in vitro and in vivo.

Sphingosine blocks ZIKV and DENV infection during membrane fusion step

We added sph at different time points during ZIKV infection in C6/36 or Vero cells in order to determine which step of the virus life cycle is targeted by sph (Fig. 5a). In the virus-treatment (virus mixed with sph for 30 min, then infected cells), cell-treatment (cells were treated with sph, washed, then infected with virus), and co-treatment (cells were immediately infected with a mixture of sph and virus) groups, sph significantly decreased ZIKV virus levels in both C6/36 and Vero cells (Fig. 5b). However, the post-treatment (cells infected with virus and then treated with sph) showed no obvious effect on ZIKV infection (Fig. 5b). Correspondingly, the levels of E protein were significantly reduced in the virus-treatment, cell-treatment, and co-treatment groups, but not in the post-treatment group (Fig. 5c). Similar results were observed for DENV (Supplementary Fig. 6a). These results indicate that sph blocks ZIKV and DENV entry at an early stage of infection.

**Fig. 5: Sphingosine suppressed ZIKV infection by affecting membrane fusion.**

We performed an attachment, membrane insertion, and membrane fusion assay to further investigate the step of entry that was specifically affected by sph. Prechilled C6/36 cells were incubated with ZIKV mixed with 0 or 10 μM sph for 30 min and RNA was extracted from the cells. Sph supplementation exerted no obvious effect on virus attachment to the host cell surface (Fig. 5d). Liposomes, ZIKV, and 50 µM sph were then treated at pH 5.75 to induce virus-membrane insertion and separated on sucrose gradients by ultracentrifugation at the same pH. ZIKV floated with liposomes to the top after pH 5.75 treatment, while those at neutral pH (pH 8.0) remained in the bottom. This result suggested a low-pH dependent membrane insertion (Fig. 5e); however, addition of sph had no apparent effect on the floatation of ZIKV with liposomes at low pH, indicating that sph does not play a role in ZIKV membrane insertion (Fig. 5e). DENV or ZIKV were pre-attached to Vero cells on ice and fusion was triggered at 37 °C by low pH with or without sph to reveal the effect of sph on viral membrane fusion. The fused cells were measured using focus forming assay. The membrane fusion of ZIKV and DENV was inhibited by sph in a dose-dependent manner with IC₅₀ values of 6.73 μM and 10.19 μΜ, respectively, (Fig. 5f and Supplementary Fig. 6b). The physiological concentrations of sph in the midgut of lab-adapted Ae. albopictus (Jiangsu strain) mosquitoes was 7.9 μM, which is significantly higher than the 5.5 μM of Ae. aegypti (UGAL/Rockefeller strain), but decreased to 5.7 μM by antibiotic treatment (Fig. 5g). We obtained similar results in field-derived Ae. albopictus (Haikou strain), Ae. aegypti (Haikou strain) and antibiotic-treated Ae. albopictus, with sph concentrations in the midgut of 8.1 μM, 5.4 μM, and 5.5 μM, respectively. Therefore, sph inhibited ZIKV/DENV infection by restricting virus-membrane fusion during entry.

Eh_B17 levels are potentially associated with vector competence of field Aedes mosquitoes

The above results revealed that sph strongly inhibited both ZIKV and DENV infection at a physiological concentration. The gut concentration of sph in the competent vector Ae. aegypti is lower than that of the less competent Ae. albopictus. Thus, the preferential colonization of sph-producing bacteria may modulate the vector competence of Aedes mosquitoes. To test this hypothesis, 11 field Aedes strains including 4 Ae. aegypti and 7 Ae. albopictus were collected from China and Thailand. The ZIKV vector competence of field-derived Aedes mosquitoes were evaluated with Ae. aegypti (Haikou strain), Ae. albopictus (Haikou strain), Ae. albopictus (Lingshui strain), and Ae. albopictus (Shenzhen strain). After blood-feeding infection, ZIKV RNA levels in the midgut of Ae. aegypti increased significantly to 1.6-, 1.4-, and 1.5-fold higher than those observed in Ae. albopictus strains from Haikou, Lingshui, and Shenzhen, respectively. Similarly, ZIKV RNA levels in Ae. aegypti carcasses were significantly elevated, being 2.6-, 2.0-, and 2.2-fold higher than the corresponding Ae. albopictus strains. In addition, virus titers in saliva of Ae. aegypti exceeded those of the corresponding Ae. albopictus strains by 4.9-, 5.6-, and 5.9-fold, respectively (Fig. 6a). These results confirmed that field-derived Ae. aegypti (Haikou strain) was more competent in ZIKV transmission than Ae. albopictus strains from Haikou, Lingshui, and Shenzhen.

**Fig. 6: Intestinal colonization of Eh_B17 and Es_B36 modulates the vector competence of field-derived *Aedes* mosquitoes.**

The prevalence of sph-producing Eh_B17 and Es_B36 was measured using qPCR. Eh_B17 was detected in 100% of Ae. albopictus at four locations, with 94%, 52%, and 60% at the other three sites; however, it was only detected in 47%, 40%, 69%, and 29% of the Ae. aegypti mosquitoes at four locations tested (Fig. 6b). Similarly, Es_B36 was significantly more abundant in Ae. albopictus strains than in Ae. aegypti strains, and it was not detected in two Ae. aegypti strains (the Ae. aegypti Zhanjiang and Danzhou strains) (Fig. 6c). These data indicated that sph-producing bacteria preferentially colonized in Ae. albopictus as compared to Ae. aegypti.

Discussion

DENV, ZIKV, CHIKV, and yellow fever virus (YFV) are medically important arboviruses transmitted by Aedes spp. The internal factors that affect the vector competence for an arbovirus include vector genetics, immune responses and commensal microbiota²⁹. Previous studies showed that the DENV viral loads in the midguts of antibiotic-treated mosquitoes were twice higher than that in the untreated mosquitoes, suggesting that the mosquito midgut microbiota might inhibit viral infection³⁰. Furthermore, Wolbachia is an obligate endosymbiont that naturally infects various arthropods, including various mosquito species, such as Ae. albopictus, Culex pipiens, and Culex quinquefasciatusm, except Ae. aegypti³¹. Wolbachia mediates pathogen blockade (PB), thereby reducing the ability of arboviruses to infect, replicate, and transmit, and it becomes an essential factor influencing the vector competence between Ae. aegypti and Ae. albopictus³². In this study, we focus on the role of gut commensal bacteria in the vector competence of Aedes species.

The ZIKV/DENV infection and transmission rate of Ae. albopictus is similar to that of Ae. aegypti via intrathoracic injection to bypass the midgut barrier or by blood-feeding after antibiotic treatment (Fig. 1). This demonstrated that commensal microbiota mainly affects its vector competence. In this study, we identified several sph-producing bacteria preferentially colocalized in Ae. albopictus that confer viral resistance through secreted sph (Figs. 2a and 4a). This further confirmed that vector competence among Aedes species can be modulated by gut symbiotic bacteria. The vector competence and geographic distribution of Aedes mosquitoes heavily affect epidemics of ZIKV and DENV. Ae. aegypti is widely distributed worldwide, typically in tropical and subtropical environments. It is responsible for the global transmission, and most pandemics, of ZIKV and DENV^1,2. In contrast, Ae. albopictus is an invasive mosquito that has spread quickly from the tropics to the temperate zone. It has become a globally important vector due to its rapid adaptation to new environments. Thus, Ae. albopictus is often responsible for DENV and ZIKV outbreaks in Ae. aegypti -free areas, including the United States and China³. The primary vector of CHIKV is Ae. aegypti, but the 2005-2006 epidemic on Reunion Island in the Indian Ocean was spread by Ae. albopictus^33,34. An E1-A226V mutation occurred during this epidemic, contributing to increased fitness of CHIKV in Ae. albopictus^35,36. This adaptation has fueled global pandemics in Asia, the Indian subcontinent, and Europe over the past decade³⁷. Thus, if sph-resistant mutations emerge in DENV or ZIKV, they might also help Ae. albopictus adapt to be a more efficient vector and expand the range of the viruses.

Commensal gut bacteria can influence pathogen transmission and development through secreting small peptides, proteins and metabolites. The secretions most often studied are bacterial peptides called bacteriocins. The bacteriocins include thuricin CD, nisin, lacticin 3147, and subtilosin A and these peptides have a relatively narrow antimicrobial spectrum^{38,39,40,41,42}. Serratia marcescens can also facilitate DENV infection in Ae. aegypti through a secreted protein, SmEnhancin²⁷. Some bacterial metabolites can directly suppress pathogen growth. For example, Clostridium scindens can reduce Clostridium difficile infection in mice by producing secondary bile acids⁴³. An intestinal commensal bacterium, Chromobacterium (Csp_P), possesses anti-Plasmodium and anti-DENV activities and acts by generating stable bioactive factors⁴⁴. The present study demonstrated that gut symbiotic Eh_B17, Es_B36, and Eh_B56, can effectively confer ZIKV/DENV resistance in Ae. albopictus via the secreted metabolite, sph (Figs. 2a, 4a, c). Field-derived Ae. aegypti (Haikou strain) displayed higher ZIKV RNA levels and virus titers than Ae. albopictus strains from Haikou, Lingshui, and Shenzhen (Fig. 6a), and the Eh_B17 and Es_B36 preferentially colonized Ae. albopictus compared to Ae. aegypti (Fig.6b,c). It exhibits the variation in vector competence among Aedes species.

Our results suggest that sph inhibits ZIKV/DENV infection by limiting virus-membrane fusion with an IC₅₀ of 6.73 μM for ZIKV and 10.19 μM for DENV (Fig. 5f and Supplementary Fig. 6b). The mass spectrometry result showed that the average concentration of sph in the midgut of lab-adapted Ae. albopictus (Jiangsu strain) and field-derived Ae. albopictus (Haikou strain) were 7.9 and 8.1 μM, respectively, which can inhibit ZIKV/DENV infection (Fig. 5g). Sphingolipids are important cell membrane components involved in cellular processes such as differentiation, proliferation, apoptosis, signal transduction, and membrane trafficking⁴⁵. Endogenous sphingosine is also a component of the cell membrane and released from ceramide by ceramidases. Antiviral activity against SARS-CoV-2 can occur by inhibition of the binding with the receptor ACE2²⁵. Sph can also inhibit HSV-1 infection in macrophages by trapping the virus in endosomes and then shuttling it to lysosomal degradation sites²⁴. By adding sph at multiple infection/entry stages, we demonstrated that sph blocked ZIKV/DENV infection during membrane fusion (Fig. 5f). It is plausible that sph blocks fusion by docking into a pocket in the E protein or by altering the curvature of host or viral membranes. These possibilities need further investigation.

Most organisms require intestinal symbionts to provide essential nutrient supplementation such as essential amino acids, vitamins, and nitrogen fixation^46,47,48. The sphingolipids produced by gut bacteria can be hydrolyzed and absorbed by the host to maintain sphingolipid homeostasis. For example, sphingolipids extracted from Acinetobacter and administered orally to mice are readily absorbed and metabolized in the liver to complex sphingolipids⁴⁹. Bacterial-derived palmitoyl coenzyme A can alter the rate of host sphingolipid synthesis, resulting in modified ceramide metabolism in the liver and skeletal muscle⁵⁰. Our results revealed that the content of sph in the midgut of lab-adapted Ae. albopictus (Jiangsu strain) was significantly higher than that in the midgut of Ae. aegypti (Fig. 5g). However, the levels in antibiotic-treated lab-adapted Ae. albopictus (Jiangsu strain) and Ae. aegypti (UGAL/Rockefeller strain) were similar, indicating that more sph-producing bacteria colonize the midgut of Ae. albopictus (Fig. 5g). Therefore, it is possible that differences in the synthetic sphingolipid pathway between Ae. albopictus and Ae. aegypti may result in an excess of sph-producing bacteria in the midgut of Ae. albopictus that provide its sphingolipid requirements.

However, there are still some limitations in this study. We observed a strong correlation between the abundance of Eh_B17 as well as Eh_B36 and the vector competence of both lab-adapted and field-derived Aedes mosquitos. Nevertheless, there might be multiple factors influencing the vector competence of Aedes mosquitoes, such as genetic and immunological background⁵¹, different virus genotypes⁴, as well as other symbiotic bacteria like Wolbachia³², which shouldn’t be neglected.

Commensal bacteria are promising agents for controlling arbovirus transmission. The release of Wolbachia-carrying mosquitoes in field trials has been effective in controlling wild mosquito populations^32,52,53,54. Wolbachia-infected males successfully decreased wild Ae. albopictus populations on two islands in Guangzhou, China, from 2014 to 2017. Comparable results were obtained on wild Ae. aegypti populations in Australia from 2017 to 2018^54,55. Eh_B17 could be easily transplanted into Ae. aegypti via the oral route by mixing with 10% sucrose, and this could suppress ZIKV/DENV infection and prevalence. Thus, sph can be used as a transmission inhibitor of ZIKV and DENV in Ae. aegypti populations.

Methods

Mosquito rearing and antibiotic treatment

Lab-adapted Ae. aegypti (UGAL/Rockefeller strain) and Ae. albopictus (Jiangsu strain) were reared as previously described^56,57,58,59.Field-derived Ae. aegypti (Haikou strain), Ae. albopictus (Haikou strain), Ae. albopictus (Lingshui strain), and Ae. albopictus (Shenzhen strain) were also maintained under the same conditions. Briefly, adult mosquitoes were continuously fed with a solution of water and 10% (w/v) sucrose under 28 °C and 80% relative humidity conditions. Mosquito strains were maintained by feeding them on chicken blood from chickens raised by our laboratory to promote egg development. Female mosquitoes can feed on blood and transmit viruses.

Mosquitoes were reared on 10% (wt/vol) sucrose and ddH₂O with 100 U/mL of penicillin and 100 µg/mL of streptomycin (Cat. 15140122, Gibco, USA) for 5 d to eliminate intestinal bacteria. Mosquitoes were starved for 15–18 h before viral infection using an in vitro membrane-based feeding system. The colony forming unit assay was used to confirm successful elimination of enteric bacteria. All of the virus and mosquito assays were performed under Biosafety Level 2 (BSL2) and approved by the Bioethics Committee of the Institute of Zoology, Chinese Academy of Science (IOZ20190061).

Bacteria were reintroduced into antibiotic-treated Ae. aegypti via sucrose meals. Briefly, bacteria with an OD₆₀₀ of 0.001 were incubated overnight in LB medium at 37 °C. The final bacterial concentration was approximately 1.0 × 10⁹ CFU/mL. The bacteria were then mixed with sucrose solution (5% wt/vol) at a ratio of 1:9 (1.0 × 10⁸ CFU/mL) and fed to female mosquitoes for 72 h.

Cells, viruses, antibodies and reagents

C6/36 cells (ATCC, CRL-1660) were grown in RPMI medium containing 8% fetal bovine serum (FBS, Cat. 10091148, Invitrogen™, USA) at 28 °C and 5% CO₂. Vero cells (ATCC, CCL-81) were maintained in DMEM plus 8% FBS at 37 °C and 5% CO₂. The ZIKV (MR766 strain; GenBank sequence accession number, HQ234498) and DENV2 (New Guinea C strain; GenBank accession number, M29095) were passaged into C6/36 cells. The plaque formation assay was used to determine the titer of ZIKV. The 4G2 (Cat. E2691, GeneTex, USA) is a mouse monoclonal antibody (MAb) capable of recognizing all of the flavivirus E protein fusion peptides, including DENV and ZIKV^60,61. ( ± )-Methyl jasmonate (Cat. T4900, TargetMol, USA), caprylic acid (Cat. T3946, TargetMol, USA), hexadecanedioic acid (Cat. HY-W018161, MedChemExpress, USA), D-erythro-Sphingosine (Cat. HY-101047, MedChemExpress, USA), valproic acid (Cat. HY-10585A, MedChemExpress, USA), phytosphingosine (Cat. B8653, Apexbio, USA), 4-(2,6,6-Trimethyl-1-cyclohexen-1-yl)-2-butanone (Cat. Y-ZH-7475, Xinyan Bomei, China), Leukotriene E4 (Cat. 20410, Cayman, UK), Dibutyl phthalate (Cat. HY-Y0304, MedChemExpress, USA), 5-Hydroxypentanoic acid sodium salt (Cat. M094849, Mreda, China), Moxifloxacin (Cat. B1218, Apexbio, USA), Glycocholic acid (Cat. HY-N1423, MedChemExpress, USA), Astemizole (Cat. T1278, TargetMol, USA), Procainamide (Cat. B25856, Yuanye Bio, China), α-Ionone (Cat. I0076, TCI, Japan), β-Ionone (Cat. I830244, Macklin, China), Eplerenone (Cat. GC15831, GLPBIO, USA), Geranylacetone (Cat. BQS162724, Gersion Bio, China), Diisobutyl phthalate (Cat. CFN96846, ChemFaces, China), Palmitoylethanolamide (Cat. S4708, Selleck, USA), Oleamide (Cat. HY-N2327, MedChemExpress, USA), Sulfamethazine (Cat. IS0310, Solarbio, China), Nimodipine (Cat. T0343, TargetMol, USA), and β-Sinensal (Cat. S487310, TRC, Canda) were purchased. 4-but: 4-(2,6,6-Trimethyl-1-cyclohexen-1-yl)-2-butanone; le-e4: leukotriene E4; dibph: dibutyl phthalate; 5-ha: 5-hydroxypentanoic acid; ca: caprylic acid; va: valproic acid; mj: methyl jasmonate; mox: moxifloxacin; ha: hexadecanedioic acid; ga: glycocholic acid; ast: astemizole; pro: procainamide; α-ion: α-ionone; β-ion: β-ionone; epl: eplerenone; ger: geranylacetone; diiph: diisobutyl phthalate; pal: palmitoylethanolamide; psph: phytosphingosine; ole: oleamide; sul: sulfamethazine; nim: nimodipine; sph: sphingosine; β-sin: β-sinensal.

Isolation and characterization of gut bacteria from wild mosquitoes

The electric mosquito catchers were used to collect wild mosquitoes from different locations (Supplementary Table 1). To remove the midgut, mosquitoes were first surface disinfected with 75% ethanol and rinsed twice in sterile 1 × PBS buffer. Individual midguts were dissected and streak-cultured on LB agar plates under field conditions. Then, bacteria were inoculated onto new LB agar plates for bacterial isolation under laboratory conditions. Individual colonies were identified by 16S rRNA sequencing⁶². Primers used are listed in Supplementary Data 4.

Viral infection

Three- to four-day-old adult females were starved for 15–18 h before viral infection via an in vitro membrane blood feeding system⁶³. Under different experimental conditions, LB medium, DMEM, bacterial culture supernatant (retention and flow-through), bacterial cell lysates, different parts of flow-through, and antiviral effectors were mixed with mouse blood containing 1.0 × 10⁵ FFU/mL virus (1.0 × 10⁶ FFU/mL virus in Fig. 2a). The mixture was then preheated at 37 °C for 30 min and fed to mosquitoes at 37 °C for 1 h using a membrane feeder. The mosquitoes with a full blood meal were transferred to a new container for further experiments.

RNA extraction and virus detection

Total RNA was isolated from whole mosquito carcasses, pooled tissues, or cells using the TRIzol kit (Cat. 15596018, Invitrogen™, USA). A TIANamp Virus RNA Kit (Cat. DP315-R, TIANGEN, China) was used to extract RNA from the virus supernatant. A one-step SYBR PrimerScript reverse transcription PCR protocol (Cat. RR066A, Takara Bio Inc, Japan) on the Applied Biosystems Step-One Plus system (Applied Biosystems, USA) was applied to quantify viral and housekeeping gene RNA. The qPCR parameters were as follows: reverse transcription at 42 °C for 5 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s on a Step-One Plus system (Applied Biosystems, USA). Ribosomal protein gene S7 (rps7) gene and monkey-actin were used to normalize the viral RNA load. Primers used are listed in Supplementary Data 4.

Collection of saliva from mosquitoes

Fifteen females, aged at 10 d post infection, were anesthetized at 4 °C for 15 min, and saliva was then obtained by placing the mouth parts in a 10-μL pipette tip with 5 μL FBS for 30 min. Samples were dissolved in 300 μL DMEM plus 2% FBS and centrifuged at 13,000 × g at 4 °C for 30 min. Focus-forming assays on Vero cells were used to determine virus titers in individual saliva samples.

Focus-forming assay

The Vero cells were inoculated into a 96–well plate at 1.5 × 10⁴ cells per well using DMEM medium plus 8% FBS at 37 °C with 5% (vol/vol) CO₂ for 24 h. Ten-fold serial dilutions of viral supernatant (the saliva sample) were added and cells were infected for 2 h (100 μL/well). Each concentration was replicated in two wells. Cells were then cultured in 2% FBS-DMEM with 1% penicillin/streptomycin, and 20 mM NH₄Cl at 28°C for 72 h. After incubation in 4% paraformaldehyde (Cat. E672002-0500, BBI, China) for 30 min, the cells were permeabilized with 0.5% Triton X-100 for 10 min. Then, cells were washed with 1 × PBS, blocked with 3% BSA for 1 h at room temperature (RT). Then, they were incubated with mouse mAb 4G2 (Cat. E2691, GeneTex, USA) at 4 °C overnight. The cells were washed again and then incubated with fluorescence conjugated anti-mouse secondary antibody (Alexa Fluor 488, Cat. A11001, Invitrogen™, USA) (1:500) for 1 h. Virus foci were counted under a fluorescence microscope (Nikon Eclipse Ti, Japan).

Separation of proteins and metabolites secreted by bacteria

Bacteria were grown in LB medium at 37 °C until OD₆₀₀ reached 0.6, and then transferred to 16 °C and incubated for 48 h. The bacterial supernatant was collected and concentrated using vacuum freeze-drying (ALPHA LDplus, Germany). The dry powder was resuspended in ddH₂O in a 1/20 volume of the original supernatant. The resuspended supernatant was separated into retention (mainly protein) and flow-through (mainly metabolites) by a 3 kDa centrifugal filter. The fluids were stored at −80 °C for further experiments.

Toxicity detection of secreted effectors to C6/36 cells with MTT assay

A 96-well plate containing 1.0 × 10⁵ C6/36 cells per well was added with 100 µL of diluent and incubated at 28 °C for 96 h. Each concentration was replicated in three wells, and the negative control was RIPM 1640 medium plus 2% FBS. Cells were incubated for 4 h at 28 °C with 20 µL of 5 mg/mL MTT added to each well. MTT was then removed and incubated with 150 μL DMSO for 30 min at 28 °C to dissolve the formazan crystals. Liquid absorbance was measured at 570 nm.

Infection of cells with metabolites and viruses

Three to four × 10⁵ C6/36 cells were inoculated into a 24-well plate using RIPM 1640 medium plus 8% FBS at 28 °C with 5% (vol/vol) CO₂ for 24 h. Virus-treatment: Virus (ZIKV or DENV, MOI 0.01) was mixed with 10 µM sph or DMSO, and incubated for 30 min at RT. Cells were incubated with the mixture for 2 h at 28 °C and then cultured in 2% FBS-RIPM 1640 for 48 h. Cell treatment: Cells were pretreated with 10 μM sph or DMSO for 2 h at 28 °C. The cells were infected with virus (ZIKV or DENV, MOI 0.01) for 2 h and then incubated in 2% FBS-RIPM 1640 for 48 h. Co-treatment: Virus (ZIKV or DENV, MOI 0.01) mixed with 10 µM sph was added to each well and incubated for 2 h at 28 °C. The supernatant was then replaced with 2% FBS-RIPM 1640 for 48 h. Post-treatment: After virus infection (ZIKV or DENV, MOI 0.01) for 2 h, cells were treated with 10 µM sph or DMSO in 2% FBS-RIPM 1640 for 48 h at 28 °C. Viral loads in cell supernatants and lysates were measured by qPCR and western blot.

Colonization and quantification of bacteria in the mosquito midgut

Bacteria were grown in LB medium at 37 °C until OD₆₀₀ was 0.6. Bacteria were then harvested by centrifugation at 4000 × g for 15 min and resuspended in sterile LB medium until OD₆₀₀ reaching 1. These bacteria were then mixed with 5% (wt/vol) sucrose solution at a ratio of 1:9 and fed to antibiotic-treated mosquitoes for 72 h. Mosquitoes were surface disinfected with 75% ethanol and washed twice with sterile 1 × PBS. For each group, individual midguts were aseptically removed from mosquito abdomens aseptically and homogenized in 100 μL sterile LB medium. Ten-fold serial dilutions of homogenate were applied to LB agar plates and kept overnight at 37 °C, followed by bacterial colony count. At least three independent experiments were performed.

Eh_B17-GFP were counted by the same method as above. However, it was cultured in LB agar plates containing 100 μg/mL apramycin. Fluorescent colonies were counted by fluorescence microscopy.

Mosquitoes were surface disinfected with 75% ethanol and washed twice with sterile 1 × PBS. For each group, individual midguts were aseptically removed from mosquito abdomens aseptically and total DNA was isolated using the DNeasy Blood & Tissue Kit (Cat. 69506, QIAGEN, Germany). Bacterial and housekeeping gene DNA was quantified using SuperReal premix plus (Cat. FP205-03, TIANGEN, China). The qPCR parameters were as follows: 95 °C for 3 min for initial denaturation, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s on a Step-One Plus system (Applied Biosystems, USA). The rps7 gene was used to normalize the burden of the gut microbiota. Primers used are listed in Supplementary Data 4.

Generation of Eh_B17-GFP strain

To introduce the GFP gene into the chromosome of Eh_B17, the upstream fragment of the HU gene was first amplified with Eh_B17-5F and Eh_B17-5R primers, and the downstream fragment of the HU gene was amplified with Eh_B17-3F and Eh_B17-3R primers. The upstream fragment, GFP, Apr resistance gene, and downstream fragment were linked using overlapping extension PCR. Then, the long fragment was cloned into the pCVD442 suicide vector named pCVD442_HU-GFP-Apr. Then, on LB plates containing ampicillin (100 μg/mL), diaminopimelic acid (0.5 mM), E. coli β2155 cells transfected with pCVD442_HU-GFP-Apr was screened. pCVD442_HU-GFP-Apr was transferred to Eh_B17 using the E. coli β2155 conjugation system. Transconjugants were selected on LB plates containing apramycin (100 μg/mL). The pCVD442 suicide vector was then triggered by the addition of 10% (wt/vol) sucrose. Finally, the HU-GFP-Apr-Eh_B17 was tested by PCR using the Eh _B17-out F and Eh_B17-out R primers. HU-GFP-Apr-Eh_B17 were also visualized under a confocal microscope (Zeiss LSM 710, Germany). The primers are shown in Supplementary Data 4.

Construction of the SPT knockout Eh_B17 mutant strain

First, a standard BLASTP search revealed that gene2221 and gene3898 in Eh_B17 are homologs of the serine palmitoyl transferase (SPT) gene (Supplementary Table 3 and Supplementary Data 3). Subsequently, the Eh_B17 mutant strain lacking gene2221 and gene3898 was constructed. The upstream and downstream fragments of gene2221 were amplified from the Eh_B17 genome using primers gene2221-5F, gene2221-5R, gene2221-3F, and gene2221-3R. The chloramphenicol resistance gene (Cm) was also amplified from the pACYC184 plasmid using primers gene2221-CmF and gene2221-CmR. The upstream and downstream fragments of gene2221 were fused to the Cm resistance gene through fusion PCR, generating the complete targeting construct Δgene2221::Cm (upstream fragment-Cm resistance gene-downstream fragment). This targeting construct was cloned into the suicide plasmid pCVD442, yielding the targeting plasmid pCVD442-Δgene2221::Cm. Through electroporation, the targeting plasmid pCVD442-Δgene2221::Cm was transduced into the E.coli β2155 strain, resulting in the donor strain β2155/pCVD442-Δgene2221::Cm. A conjugation experiment was then performed between the donor strain β2155/pCVD442-Δgene2221::Cm and the Eh_B17 recipient strain. Chloramphenicol-resistant Eh_B17 clones were selected on a chloramphenicol-containing plate and designated Eh_B17/pCVD442-Δgene2221::Cm, indicating successful integration of the targeting plasmid into the genome. To facilitate the excision of the plasmid and selection of the gene knockout clones, Eh_B17/pCVD442-Δgene2221::Cm cultures were plated on LB agar containing 10% sucrose and 20 μg/ml of chloramphenicol, but without NaCl. After the formation of monoclonal colonies, PCR screening was performed using primers gene2221-outF, gene2221-outR, gene2221-inF, and gene2221-inR. The clones that showed successful replacement of gene2221 with the Cm resistance gene were identified and designated Eh_B17/Δgene2221::Cm.

We used the same experimental approach to mutate the gene3898. Specifically, we amplified the upstream and downstream fragments of the gene3898 sequence from the Eh_B17 genome using primers gene3898-5F, gene3898-5R, gene3898-3F, and gene3898-3R. In addition, we isolated the gentamicin resistance gene (Gm) from the pJQ200SK plasmid using primers gene3898-GmF and gene3898-GmR. These genetic components were subsequently fused using fusion PCR technology, generating a comprehensive targeting construct Δgene3898::Gm (upstream fragment-Gm resistance gene-downstream fragment). The targeting construct was then cloned into the suicide plasmid pCVD442, resulting in the generation of the targeting plasmid pCVD442-Δgene3898::Gm. We then used electroporation to introduce the targeting plasmid pCVD442-Δgene3898::Gm into the E.coli β2155 strain, yielding the generation of the donor strain β2155/pCVD442-Δgene3898::Gm. A conjugation experiment was performed between the donor strain β2155/pCVD442-Δgene3898::Gm and the previously mutated recipient strain Eh_B17/Δgene2221::Cm. Selection on gentamicin-containing plates resulted in the isolation of gentamicin-resistant Eh_B17 clones, designated Eh_B17/Δgene2221::Cm/pCVD442-Δgene3898::Gm. To facilitate plasmid excision and selection of double mutant clones, multiple Eh_B17/Δgene2221::Cm/pCVD442-Δgene3898::Gm clones were plated on LB agar supplemented with 10% sucrose. After the formation of monoclonal colonies, PCR-based screening was performed using the gene3898-outF, gene3898-outR, gene3898-inF, and gene3898-inR primers to identify clones in which the gene3898 locus had been successfully replaced by the gentamicin resistance gene, resulting in the final mutant strain designated Eh_B17/Δgene2221::Cm/Δgene3898::Gm. Primers used are listed in Supplementary Data 4.

Metabolite extraction

Bacteria were grown overnight in LB medium at 37 °C with an OD₆₀₀ of 0.6. Then, 200 μL bacterial solution was applied to LB plates and maintained for 3 d. The bacterial colonies were collected in 1 mL methanol and then put in a water bath at 80 °C until the methanol evaporated completely. Samples were reconstituted in 0.5 mL of methanol and centrifuged at 12,000 × g for 3 min to collect the supernatant. The liquid was evaporated using a rotary evaporator, and the dry powder was dissolved in ddH₂O to at a concentration of 50 mg/mL. Liquid was filtrated with a membrane filter (0.45 µm) and stored at −20 °C for further testing. At least three independent experiments were performed.

The Bligh and Dyer method was used to extract sphingolipids⁶⁴. Mosquito midguts (300 for each group) were collected in 3 mL of an ice-cold mixture of chloroform and methanol (2/1, v: v), homogenized with 1 mm silica beads using Tissuelvser-24, and extracted by sonication at 4 °C for 15 min. A 1 mL volume of ultrapure water (containing 20 mM ammonia) was added and centrifuged at 4000 × g for 15 min. After collecting the lower organic phase, 2 mL chloroform/methanol was added and the procedure was repeated twice. The organic phases were combined and evaporated (Cat. RVC 2-18, Christ, Germany). The dried material was extracted with 1 mL methanol solution containing 0.1 M NaOH, shaken for 15 s, followed by a water bath at 55 °C for 1 h. Then 0.2 mL methanol solution containing 1 M HCl and 1 mL hexane was added. The sample was centrifuged, at 4000 × g for 15 min, and the bottom layer was aspirated into a new 15 mL centrifuge tube and evaporated. The evaporated material was redissolved with 3 mL of chloroform/methanol solution and the previous extraction steps were repeated. The dried sample was redissolved with 200 μL methanol/ethyl acetate (95/5, V/V), and was filtrated with a 0.45-µm membrane filter. The final sample was stored at −20 °C. At least three independent experiments were conducted.

Collect 1 mL of culture medium and lyophilize in a 2 mL centrifuge tube. Reconstitute the dried specimen with 300 μL methanol-water solution (1/4, V/V) by vertexing for 30 s and sonication for 3 min. Allow the mixture to stand at −20 °C for 2 h. The mixture is then centrifuged at 13,000 × g for 10 min at 4 °C. Filter the supernatant through a 0.45 µm membrane filter and store at −20 °C until further assay.

Separation of metabolites by semi-preparative high performance liquid chromatography

The separation of the flow-through of Eh_B17 was performed at RT using an SPHPLC system (Agilent, USA) with an Agilent HC-C18 column (4.6 × 250 mm, 5 μm). The column temperature was 60 °C, injection volume was 2 µL and the flow rate was 1 mL/min. Mobile phase A was methanol (0-50 min, 80% A; 50-55 min 100% A), and mobile phase B was UPLC-grade water. The effluent was detected at 210-, 225-, 280-, and 320-nm, and was divided into six parts according to the peak time. The parts were named F1 (0–6.5 min), F2 (6.5-10 min), F3 (10–15.5 min), F4 (15.5–20 min), F5 (20-25.5 min), and F6 (25.5–55 min). The collected fractions were then evaporated to dryness under a rotary evaporator (Cat. RVC 2-18, Christ, Germany) at RT, redissolved with LB, and stored at −20 °C for further testing.

HPLC-Q-TOF conditions

Mass spectrometry experiments as shown in Fig. 4c: Bacterial metabolites were extracted using the metabolite extraction method described above (n = 3 biologically independent replicates for each experiment). The metabolites were then detected using high-performance liquid chromatography coupled with quadrupole/time-of-flight (HPLC-Q-TOF) mass spectrometry. In brief, metabolites were profiled using a 6520 quadrupole/time-of-flight (Q-TOF) instrument (Agilent, USA) coupled to an Agilent 1200 HPLC system with a ZORBAX Eclipse XDB-C18 column (4.6 × 50 mm, 1.8 μm, Agilent). The mass spectrometer was instrumented with an electrospray ionization source (Agilent, USA) and performed in the positive ionization mode with a scan range of m/z 50-1000. The ESI positive mode was used and the mobile phase A was acetonitrile and the mobile phase B was UPLC grade water containing 1.5‰ ammonium formate and 1.5‰ formic acid (0–3 min, 5% A; 3–23 min, 95% A; 23–28 min 95% A; 28–30 min 5% A; 30–33 min 5% A). The flow rate was 0.5 mL/min and injection volume were 5 µL. Other parameters of the mass spectrometry were as follows: capillary voltage of 4000 V, fragmenter voltage of 100 V, nebulizer pressure (40 psi), collision voltage of 10 V, drying gas temperature of 300 °C, and gas flow of 11 L/min. The relative abundance of sph and psph in mass spectrometry was quantified by peak area using Agilent Mass Hunter software (version B.07.00; m/z 300.2897 for sph; m/z 318.3003 for psph) and statistically analyzed with GraphPad Prism statistical software.

Mass spectrometry experiments as shown in Fig. 5g and supplementary 5h: Sphingolipid levels in the midgut of mosquitoes were extracted using the metabolite extraction method described above (Bligh and Dyer method; n = 3 biologically independent replicates for each experiment). The metabolites were then detected, quantified, and analyzed using the same assay as shown in Fig. 4c. However, the concentration of sph, as shown in Fig. 5g, was calculated using the peak area of the sph standard product.

Mass spectrometry experiments as shown in supplementary Fig. 5g: Bacterial metabolites in the culture medium were extracted using the metabolite extraction method described above (n = 3 biologically independent replicates for each experiment). The metabolites were then detected, quantified, and analyzed using the same assay as shown in Fig. 4c.

Non-targeted metabolomic analysis of Eh_B17-F6, Es_B36, and Eh_B56

Non-targeted metabolomic analysis was performed by Beijing Protein Innovation Co., Ltd. All samples were acquired by the LC-MS system according to the machine instructions (n = 3 biologically independent replicates for Eh_B17-F6; n = 1 for both Es_B36 and Eh_B56). All of the chromatographic separations were performed using an UPLC system (SCIEX, USA). An XBridge BEH C18 column (3.5 μm, 2.1 mm × 100 mm, Waters, USA) was used for the reversed-phase separation. The column oven was maintained at 50 °C. The flow rate was 0.3 mL/min and the mobile phase consisted of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The gradient conditions were set as follows: 0–0.5 min, 5% phase B; 0.5–5 min, 5–80% phase B; 5–7 min, 80–100% phase B; 7–8 min,100% phase B; 8–8.1 min, 100–5% phase B; 8.1–10 min, 5% phase B. The injection volume for each sample was 10 μL.

A high-resolution tandem mass spectrometer TripleTOF5600 (SCIEX, USA) was used to detect metabolites eluted from the column. The Q-TOF was operated in both positive ion and negative ion modes. For the positive ion mode, the capillary voltages were set to 5 kV. For the negative ion mode, the capillary voltages were set to −4.5 kV. Mass spectrometry data were acquired in IDA mode. The TOF mass range was 50 to 1200 Da. The top 8 precursors were fragmented for MS/MS detection. In addition, a quality control sample (pool of all of the samples) was acquired after every 10 samples to evaluate the stability of the LC-MS throughout the acquisition. The data underwent a series of processes, including peak alignment, peak extraction, normalization, and deconvolution using XCMS software⁶⁵. HMDB database (https://hmdb.ca/) was used to identify adduct masses. The KEGG web server (https://www.genome.jp/kegg/) was used for KEGG enrichment analysis.

Whole-genome sequencing of Eh_B17, Es_B36, and Eh_B56

Constructed PCR-free SMRT bell libraries are sequenced on the PacBio SMRT Technology platform by Berry Genomics (China)⁶⁶. After library construction, the library insert size was checked on a Bioanalyzer® 2100, and the sequencing primers and enzymes are scaled to the SMRT Bell template according to PacBio Calculator calculations. Finally, loading sequencing was performed by MagBead.

Phylogenetic analysis

The Clustal W program (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalW/) was used to align partial sequences of SPT homologs, and cladograms were constructed by neighbor-joining analysis and displayed using MEGA⁶⁷. SPT sequences were obtained from NCBI and Eh_B17, Es_B36, and Eh_B56. No. of Bootstraps replications values was set to 1000 (Supplementary Data 5). Accession numbers are listed in Supplementary Data 5. Phylogenetic trees were then visualized using the iTOL web server (https://itol.embl.de/).

Influence of bacteria on mosquito lifespan, fecundity and fertility

Cultured bacteria (OD₆₀₀ = 1) were mixed with 5% (wt/vol) sucrose solution at a ratio of 1:9 and fed to 30 mosquitoes after antibiotic-treatment. Mosquito survival was monitored at 24 h intervals. For the measurement of fecundity and fertility, 30 antibiotic-treated female mosquitoes were fed with bacteria for 72 h, and LB was used as the control. The mosquitoes were then fed with chicken blood. Individual females were transferred to a separate 10-mL centrifuge tube for egg collection after 3 d of blood feeding. Females that died during oviposition were excluded from the study. After 3 d, the eggs were counted and subsequently these eggs were classified as either unhatched or hatched. Three independent experiments were conducted for each condition.

Western Blot

RIPA buffer (Cat. CW2333S, CWBIO, China) containing 1 × protease inhibitor (Cat. 78425, Pierce, USA) was used for cell extraction. Samples were electrophoresed on a pre-cast gel (Cat. BE6929-11, EASYBIO, China), transferred to a polyvinylidene fluoride membrane (Cat. IPVH00010, Millipore, USA) and incubated with mouse mAb 4G2 (Cat. E2691, GeneTex, USA) and anti-mouse IgG-HRP-conjugated secondary antibody (1:10000) (Cat. BE0102-100, EASYBIO, China). Anti-mouse monoclonal GAPDH antibody (Cat. 137959, Absin, China) (1:5000) was used as a loading control. Images were visualized with SuperSignal West Pico Substrate (Cat. 34577, Pierce, USA) and X-ray film.

Attachment assays

The 1.2 × 10⁶ C6/36 cells were chilled for 15 min at 4 °C and incubated with ZIKV at an MOI of 0.01 containing DMSO and 10 µM sph for 30 min at the same temperature. Then, the cells were washed with pre-cooled 2% FBS medium. Total RNA was then extracted and ZIKV loads were detected using qPCR.

Insertion assay

Liposomes were obtained by freeze-thawing and pressing through a polycarbonate filter (200 nm) using a previously described method⁶⁸. Liposomes consisted of 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE), sphingomyelin (Sigma, Japan), and cholesterol (Sigma, Japan) in a molar ratio of 1:1:1:3.

Liposome co-floating was used to identify virus-membrane contact⁶¹. Eighty microliters ZIKV (5.0 × 10⁵ FFU/mL) was incubated with 50 μM sph for 10 min at RT. A final concentration of 1 mM liposomes was then added and the mixture was adjusted to pH 5.75 with 0.3 M MES buffer or maintained at pH 8.0, and incubated for 30 min at RT. The mixture was adjusted to 20% sucrose. Ultracentrifuge tubes were layered from bottom to top with 40%, 20%, 15%, and 5% sucrose in a ratio of 3:4:12:2. All of the sucrose solutions were dissolved in pH 8.0 TAN buffer (20 mM TEA, 130 mM NaCl). Dil (Cat. 30423, Cayman, USA) was added to determine the floatability of the liposomes. The gradients were centrifuged at 49,000 × g for 3 h at 4 °C and separated from top to bottom in a ratio of 7:4:1. The total RNA was extracted from these three fractions and ZIKV loads were detected by qPCR.

Fusion assay

The fusion assay was conducted as the method described⁶⁹. Vero cells were first seeded on 24-well plates. The cells were washed with binding medium (RPMI without bicarbonate, 0.2% BSA, 10 mM Hepes, and 20 mM NH₄Cl, pH 7.9), which was pre-chilled. ZIKV (1.0 × 10⁵ FFU/mL) was diluted with the binding medium and incubated with the cells on ice with gentle shaking for 3 h. Following two washes with binding medium, cells were pulsed with a medium buffer (RPMI without bicarbonate, 0.2% BSA, 10 mM Hepes, pH 6.0 or 7.9) for 2 min at 37 °C in the presence of the indicated concentration of sph. Infected cells were incubated in MEM plus 2% FBS and 50 mM NH₄Cl for 3 h at 37 °C, and then cultured in MEM plus 2%FBS at 37 °C for 72 h. The number of infected cells was quantitated by focus forming assay as above described.

In brief, virus-infected cells were incubated in 4% paraformaldehyde (Cat. E672002-0500, BBI, China) for 30 min, followed by permeabilization with 0.5% Triton X-100 for 10 min. The cells were then blocked with 3% BSA for 1 h at room temperature. Subsequently, they were incubated with mouse mAb 4G2 (Cat. E2691, GeneTex, USA) at 4 °C overnight. The cells were washed again and incubated with Alexa Fluor 488 goat anti-mouse secondary antibody (Cat. A11001, Invitrogen™, USA) (1:500) for 1 h at room temperature. Virus foci were counted under a fluorescence microscope (Nikon Eclipse Ti, Japan). Infections observed at pH 6.0 and pH 7.9 were used as positive and negative controls, respectively.

Statistical analysis

The 16S rDNA amplicon deep sequencing and metabolomics data used http://www.bioinformatics.com.cn for analysis and visualization (Fig. 3f, g, Supplementary Fig. 3g). The Venn diagram in Fig. 3f and Supplementary Fig. 3g and the Sankey dot plot in Fig. 3g were implemented by the matplotlib python package (3.7.0). Phylogenetic trees were generated using MEGA and iTOL web server (https://itol.embl.de/). The MetaboAnalyst 5.0 web server (https://www.metaboanalyst.ca/) or KEGG web server (https://www.genome.jp/kegg/) were used for KEGG enrichment analysis and the p-value was determined by a two-sided Fisher’s exact test with Benjamini–Hochberg adjustment. HMDB database (https://hmdb.ca/) was used to identify adduct masses. GraphPad Prism statistical software was used for statistical analysis of the remaining data. Statistical test methods included the two-sided Mann-Whitney test, Kruskal-Wallis test followed by Dunn’s post hoc tests, two-sided unpaired t-test, one-way ANOVA test followed by Dunnett’s post hoc tests, two-sided Fisher’s exact test, one-way ANOVA test followed by Tukey’s post hoc tests, two-way ANOVA test followed by Tukey’s post hoc tests, two-sided multiple unpaired t-test, log-rank test (Mantel-Cox test) as shown in each Figure legend. Data are presented as mean ± SEM unless otherwise noted. The number of experiments (n) and number of replicates for each experiment are indicated in each Figure legend. A P < 0.05 was regarded as statistically significant.

Data availability

The 16S rRNA sequence data generated in this study have been deposited in the NCBI database under accession code PRJNA1151268. Non-targeted metabolomic analysis data are available at the NIH Common Fund’s National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench⁷⁰, https://www.metabolomicsworkbench.org, where it has been assigned Study ID ST003440 (https://doi.org/10.21228/M86G0K). The assembled genome sequences of Eh_B17, Es_B36, and Eh_B56 are provided in the Supplementary Data 6, Supplementary Data 7, and Supplementary Data 8, respectively. Non-targeted metabolomic analysis data are also deposited in China NMDC with accession numbers NMDCM0000047, NMDCM0000048 and NMDCM0000076. These whole-genome data are also deposited in China NMDC with accession numbers NMDC60106058, NMDC60106092, and NMDC60106093. All data, supplementary information, and Source Data files are available in this article. Source data are provided with this paper.

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Acknowledgements

This work was supported by National Key Plan for Scientific Research and Development of China (Grant No.2022YFC2601603, 2023YFA1801002), National Science Foundation of China (Grant No. 32370522, 82372288), the specific research fund of The Innovation Platform for Academicians of Hainan Province (YSPTZX202148). Non-targeted raw metabolomic analysis has been uploaded to the Metabolomics Workbench/National Metabolomics Data Repository (NMDR) (Grant No. U2C-DK119886 and OT2-OD030544).

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These authors contributed equally: Xiaomei Sun, Yanhong Wang, Fei Yuan.

Authors and Affiliations

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Xiaomei Sun, Yanhong Wang, Fei Yuan, Yanan Zhang, Tengfei Lu, Aihua Zheng & Zhen Zou
CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
Xiaomei Sun, Yanhong Wang, Yanan Zhang, Tengfei Lu, Aihua Zheng & Zhen Zou
NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University, Haikou, Hainan, China
Xun Kang & Qianfeng Xia
Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Jian Sun & Pengcheng Wang
Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
Fanny Sae Wang
Guangdong Provincial Key Laboratory of Tropical Disease Research, Department of Pathogen Biology, School of Public Health, Southern Medical University, Guangzhou, China
Jinbao Gu
Yunnan Tropical and Subtropical Animal Viral Disease Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming, China
Jinglin Wang

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Xiaomei Sun
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X.S, Y.W, F.Y, Q.X, A.Z, and Z.Z designed the study; X.S and F.Y performed assay and analyzed data; X.S, Y.W, F.Y, Y.Z, J.S, P.W, X.K, T.L, F.W, J.G, J.W, Q.X, A.Z, and Z.Z contributed to reagents/materials/analysis tools; X.S, Y.W, F.Y, Q.X, A.Z, and Z.Z wrote the manuscript.

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Correspondence to Qianfeng Xia, Aihua Zheng or Zhen Zou.

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Sun, X., Wang, Y., Yuan, F. et al. Gut symbiont-derived sphingosine modulates vector competence in Aedes mosquitoes. Nat Commun 15, 8221 (2024). https://doi.org/10.1038/s41467-024-52566-1

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Received: 08 October 2023
Accepted: 11 September 2024
Published: 19 September 2024
DOI: https://doi.org/10.1038/s41467-024-52566-1

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