cGAS-like receptors drive a systemic STING-dependent
host response in Drosophila

Xianlong Ai, Huimin Deng, Xiaoyan Li, Ziming Wei, Yuqiang Chen, Ting Yin, Junhui Zhang, Jingxian Huang, Haoming Li, Xiaoqing Lin, et al.

► To cite this version:

Xianlong Ai, Huimin Deng, Xiaoyan Li, Ziming Wei, Yuqiang Chen, et al. cGAS-like receptors drive a systemic STING-dependent host response in Drosophila. Cell Reports, 2024, 43 (12), pp.115081. 10.1016/j.celrep.2024.115081. hal-04896527

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<figure><img src="image_1.png" /></figure>

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Cell Reports

# cGAS-like receptors drive a systemic STING-dependent host response in *Drosophila*

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Xianlong Ai, Huimin Deng, Xiaoyan Li, ..., Carine Meignin, Jean-Luc Imler, Hua Cai

## Correspondence

jl.imler@ibmc-cnrs.unistra.fr (J.-L.I.),
caihua@gzhmu.edu.cn (H.C.)

## In brief

Ai et al. show that cGAMP injection or viral infections induce STING activation in a restricted set of tissues, including the central nervous system. They provide evidence of a systemic contribution of cyclic dinucleotides to antiviral immunity and show that viral infection alters sleep in a cGLR- and STING-dependent manner.

## Highlights

* Viral infection activates STING signaling in non-infected cells and remote tissues

* Ectopic fat body-specific expression of cGLR2 restricts Nora infection in the gut

* A virus-induced, cGLR1/2-dependent activity can be transferred to female flies during mating

* Sleep is regulated by viral infection in a cGLR- and STING-dependent manner

<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /></figure>

## Article

# cGAS-like receptors drive a systemic STING-dependent host response in *Drosophila*

Xianlong Ai,<fnref n="1" />,<fnref n="4" /> Huimin Deng,<fnref n="1" />,<fnref n="4" /> Xiaoyan Li,<fnref n="1" />,<fnref n="4" /> Ziming Wei,<fnref n="1" /> Yuqiang Chen,<fnref n="1" /> Ting Yin,<fnref n="1" /> Junhui Zhang,<fnref n="1" /> Jingxian Huang,<fnref n="1" /> Haoming Li,<fnref n="1" /> Xiaoqing Lin,<fnref n="1" /> Long Tan,<fnref n="1" /> Di Chen,<fnref n="1" /> Xiaohan Zhang,<fnref n="2" /> Xiuqing Zhang,<fnref n="2" /> Carine Meignin,<fnref n="3" /> Jean-Luc Imler,<fnref n="1" />,<fnref n="3" />,* and Hua Cai<fnref n="1" />,<fnref n="5" />,*

## SUMMARY

cGAS-like receptor (cGLR)-stimulator of interferon genes (STING) recently emerged as an important pathway controlling viral infections in invertebrates. However, its exact contribution at the organismal level remains uncharacterized. Here, we use STING::GFP knockin reporter *Drosophila* flies to document activation of the pathway in vivo. Four tissues strongly respond to injection of the cyclic dinucleotide 3'2'- cyclic guanosine monophosphate-adenosine monophosphate (cGAMP): the central nervous system, midgut, Malpighian tubules, and genital ducts. The pattern of STING::GFP induction in flies injected with 3'2'-cGAMP or infected by two viruses with different tropism suggests that the reporter is induced by a systemic signal produced in virus-infected cells. Accordingly, ectopic expression of cGLR2 in the fat body induces STING signaling in remote tissues and a cGLR1/2-dependent activity is transferred to females during mating. Furthermore, viral infection can alter sleep in a cGLR1/2- and STING-dependent manner. Altogether, our results reveal a contribution of cyclic dinucleotide signaling to a systemic host response to viral infection in *Drosophila*.

## INTRODUCTION

Viral infections pose a ubiquitous threat to all living organisms. Investigating strategies for combating viruses in a broad range of organisms is crucial to understand host-virus interactions and to get a full grasp of the diversity of existing antiviral strategies.<fnref n="1" /> Innate immunity participates in the control of infections in both invertebrates and vertebrates. It is activated by pattern recognition receptors (PRRs) that sense microbial molecules shared between large classes of microbes and activate signaling to mount an efficient antimicrobial response.<fnref n="2" /> Whereas the response to bacterial and fungal infections involves signaling pathways that have been evolutionarily conserved between insects and mammals (e.g., Toll/interleukin-1 receptor or immune deficiency (IMD)/tumor necrosis factor receptor pathways), regulating transcriptional factors of the nuclear factor κB (NF-κB) family, antiviral innate immunity was, until recently, thought to rely on different mechanisms in vertebrate and invertebrate animals.<fnref n="3" />,<fnref n="4" /> While vertebrates rely on strong transcriptional responses orchestrated by cytokines of the interferon (IFN) family,<fnref n="5" /> the invertebrate model organisms *Caenorhabditis elegans* and *Drosophila melanogaster* exploit the potent mechanism of RNA interference (RNAi) to silence and neutralize viral nucleic acids.⁶ Antiviral RNAi in *Drosophila* involves the RNasIII

enzyme Dicer-2, which senses viral double-stranded RNA and cleaves them into 21-nt-long small interfering RNA (siRNA) duplexes. These are loaded onto the enzyme Argonaute (AGO) 2, where one of the strands of the duplex is cleaved and discarded, while the other serves as a guide and allows AGO2 to target viral RNAs with complementary sequences (reviewed in Guo et al.<fnref n="5" />). The paradigm of RNA-based vs. protein-based antiviral strategies in invertebrates and vertebrates, respectively, has recently been challenged by the identification of potent inducible responses to viral infection or virus-derived molecular patterns in invertebrates⁷⁻⁹ and recognition for a contribution of RNAi in some tissues in mammals.¹⁰,¹¹ In particular, the evolutionarily conserved cyclic guanosine monophosphate (GMP)- adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-stimulator of IFN genes (STING) signaling pathway has recently emerged as an important player in antiviral immunity in insects (reviewed in Cai et al.¹²).

In mammals, cGAS senses the presence of double-stranded DNA (dsDNA) in the cytosol and subsequently produces the second messenger 2'3'-cGAMP.¹³ This cyclic dinucleotide (CDN) binds to STING, resulting in activation of TRAF-associated NF-κB activator binding kinase 1 and the transcription factors IRF3 and NF-κB, which induce IFNs and other antiviral genes, as well as inflammation.¹⁴ In the fruit fly *D. melanogaster*, three

<figure><img src="image_5.png" /></figure>

<footnote n="1">¹Sino-French Hoffmann Institute, State Key Laboratory of Respiratory Disease, School of Basic Medical Science, Guangzhou Medical University, Guangzhou, China</footnote>

<footnote n="2">²Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China</footnote>

<footnote n="3">³Université de Strasbourg, CNRS UPR9022, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France</footnote>

<footnote n="4">⁴These authors contributed equally</footnote>

<footnote n="5">⁵Lead contact</footnote>

<footnote>*Correspondence: jl.imler@ibmc-cnrs.unistra.fr (J.-L.I.), caihua@gzhmu.edu.cn (H.C.)</footnote>

<footnote>https://doi.org/10.1016/j.celrep.2024.115081</footnote>
cGAS-like receptors (cGLRs) have been identified, two of which (cGLR1 and cGLR2) have been shown to participate in antiviral immunity.<sup>15-17</sup> These receptors can be activated by dsRNA rather than dsDNA and, intriguingly, produce up to four different CDNs: 2'3'-cGAMP, 3'2'-cGAMP, 2'3'-c-di-AMP, and 2'3'-c-di-GMP. These CDNs bind to STING and activate signaling leading to activation of Relish, a transcription factor of the NF-κB family, to induce antiviral immunity.<sup>15,18-22</sup> Other invertebrates have been shown recently to express multiple cGLRs producing different isomers of CDNs.<sup>23</sup>

The identification of a bona fide NF-κB-driven transcriptional response to viral infection in *Drosophila* raises the question of the respective contributions of the RNAi and STING pathways in the control of viruses. Indeed, both pathways can be activated by dsRNA, excluding the possibility that they serve complementary functions like MDA5 and cGAS in mammals, which sense dsRNA and dsDNA in the cytosol, respectively (reviewed in Dalskov et al.<sup>24</sup>). In addition, neither Dicer-2 nor cGLRs contain a signal peptide or a transmembrane domain that could allude to a role in surveillance of the endosomes, as seen for mammalian Toll-like receptor 3 (TLR3) and TLR9, which senses dsRNA and dsDNA, respectively, in this compartment in mammalian cells (reviewed in Barbalat et al.<sup>25</sup>). An additional possibility would be that the two pathways operate in different tissues in a manner reminiscent of the essential contribution of nucleic acid-sensing TLRs in plasmacytoid dendritic cells, whereas RLRs and cGAS play a central role in other mammalian cells.<sup>26</sup>

To identify the tissues in which the STING pathway is activated in the context of virus infection, we generated STING::GFP knockin flies, taking advantage of the fact that STING expression is upregulated upon activation of the pathway.<sup>19</sup> Using these flies, we report here that the STING pathway is strongly activated in the central nervous system, the digestive tract, the excretory system, and the reproductive tract of male and female flies upon CDN injection or viral infection, revealing a new function of the cGLR-STING pathway in inter-organ communication.

## RESULTS

### STING::GFP knockin flies reveal activation of STING signaling in the digestive tract, Malpighian tubules, central nervous system, and reproductive tract

We generated STING::GFP knockin flies by fusing the sequence coding GFP upstream of the stop codon of the STING gene using the CRISPR-Cas technology (Figure 1A). We verified that STING-dependent signaling was still functional in homozygous STING::GFP flies, as illustrated by the increase in mRNA levels for STING, STING::GFP, and the STING-regulated gene *Srg1* in flies injected with 2'3'-cGAMP or 3'2'-cGAMP (Figures 1B and S1A). These flies provide a tool to reveal the tissues in which gene expression is turned on in response to activation of the STING pathway. Confocal examination of sagittal sections of STING::GFP flies injected with Tris buffer or 3'2'-cGAMP revealed bright fluorescence in the brain, the ventral nerve cord (VNC), and the anterior midgut of CDN-injected flies (Figure 1C). No obviously specific fluorescence was observed in *w*<sup>1118</sup> control flies examined under the same conditions (Figure S1B). In male flies, accumulation of fluorescence was observed in the

ejaculatory duct (Figure 1C). We also noted fluorescence in the posterior part of the abdomen of female flies. Diffuse fluorescence could be seen in the abdomen of male and female flies, suggesting induction of the pathway in additional tissues (Figure 1C).

To document this more accurately and quantify the activation of the pathway, we dissected the flies and examined isolated tissues. In the digestive tract, fluorescence appeared as early as 6 h post injection of 3'2'-cGAMP in the proventriculus and decreased by 7 days. By contrast, fluorescence increased over time in enterocytes and possibly other cell types in the R0 and R1 regions of the midgut, accumulating in dot-like structures in R1 (Figure 2A). Dissection also revealed induction of fluorescence in the Malpighian tubules, which increased gradually and was highest on day 7 (Figures 2B and S1C). A similar pattern was observed in the ventral nerve cord and the brain (Figures 2B, S1D, and S1E). STING::GFP was primarily induced in glial cells but also in neurons (Figure S1F). Green fluorescence was also strong in the ejaculatory duct of male STING::GFP flies 6 h post 3'2'-cGAMP injection and started to decrease at 48 h (Figures 2B and S1G). In the female reproductive tract, fluorescence rapidly increased in the oviduct and remained high on day 7 (Figures 2B and S1H). In addition, STING::GFP fluorescence was visible in the fat body at 12 h (Figures 2C and S1I).

In summary, our data reveal a set of tissues, including the central nervous system, the midgut, and the reproductive tract, that respond strongly to a STING agonist and may play a role in STING biology.

### Similar pattern of STING::GFP induction in response to 3'2'-cGAMP and viral infection

We next challenged STING::GFP flies with *Drosophila* C virus (DCV), a natural *Drosophila* pathogen known to activate STING signaling. DCV was injected in the thorax of male and female flies, resulting in systemic infection. Intriguingly, the pattern of fluorescence induction was similar to injection of 3'2'-cGAMP in several tissues, including the Malpighian tubules, the ducts in the male and female reproductive tracts, the central nervous system, and the R0 and R1 regions of the midgut (Figures 2 and S1), although induction was overall not as strong, except in the R0 region of the midgut and in the oviduct (Figures 2A, 2B, and S1H). In addition, DCV infection did not trigger STING::GFP expression in the proventriculus, unlike 3'2'-cGAMP (Figure 2A). Strikingly, the induction of STING::GFP did not reflect the tropism of DCV, which is known to primarily target the fat body and smooth muscles.<sup>27-29</sup> To clarify the connection between DCV infection and induction of STING signaling, we used immunofluorescence. As expected, we observed co-localization of DCV capsid protein and STING::GFP in the fat body, although fluorescence was weak and was also observed in non-infected cells (Figure 3A). Foci of viral infection could be detected in the central nervous system 12 h post challenge and increased with time (Figures 3B, S2A, and S2B). STING::GFP fluorescence appeared at 12 h but exhibited a diffuse pattern in the brain and ventral nerve cord and was not restricted to the virus-infected cells. Remarkably, STING::GFP-expressing cells in the distal region of Malpighian tubules from DCV-challenged flies were free of viral antigens
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 1. STING::GFP knockin flies reveal tissue-specific induction of STING signaling**</figcaption></figure>

(A) The dSTING gene, located on the right arm of the second chromosome (2R-46B4), is shown together with its annotated transcripts. The sequence coding the fluorescent protein EGFP was inserted at the 3' end of the coding sequences, just upstream of the TAA stop codon.

(B) *w*<sup>1118</sup> and homozygous STING::GFP flies were injected with Tris, 2'3'-cGAMP, or 3'2'-cGAMP, and the mRNA levels of *Sting* and *Sting::gfp* were assessed by RT-qPCR 24 h later. Data are from three independent experiments, each performed in biological triplicates and shown with scatterplot and median ± quartile. Analysis using pairwise permutation test with false discovery rate (FDR) method for CDN compared to Tris injection is shown (\*p < 0.01 and \*\*p < 0.001).

(C) Confocal images of sagittal sections from adult male and female STING::GFP flies 48 h after injection with Tris buffer or 3'2'-cGAMP. Scale bar: 50 µm. See also Figure S1.

(Figures 3C and S2C). In the digestive tract, cells positive for the viral antigen were detected 12 h after DCV infection, although STING::GFP was already induced after 6 h (Figure S2D). Analysis by confocal microscopy confirmed the presence of DCV in the smooth muscle cells surrounding the midgut but not in enterocytes where the STING::GFP was induced (Figure 3D). The same observation was made in the oviduct of female flies, with STING::GFP detected earlier than the viral capsid protein and detection of viral proteins in the outer layer of cells, whereas STING::GFP was expressed in the epithelial layer (Figure S2E). In the ejaculatory duct of male flies, DCV and STING::GFP co-localized 24 h post infection, although STING::GFP was already activated 6 h after infection and was strongest at 12 h (Figures 3E and S2F).

We next performed natural per os infection with the enteric Nora virus<sup>30</sup> and monitored GFP expression 8 days later. Again, we observed induction of STING::GFP in the anterior midgut, central nervous system, Malpighian tubules, and reproductive ducts (Figures 4A, 4B, S3A, S3B, and S3D). However, we

did not observe STING::GFP expression above background in the fat body of most flies at this time point (Figure 4B). STING::GFP was not upregulated in the proventriculus of Nora-infected flies, as observed for DCV. Immunostaining with the monoclonal antibody J2, which detects dsRNA,<sup>31</sup> confirmed infection of midgut cells but failed to detect dsRNAs characteristic of viral replication in other tissues (Figures 4A and S3A). Our results therefore suggest that midgut infection by Nora virus triggers a systemic activation of STING signaling. To test this hypothesis, we compared the sensitivity of Nora-infected and control flies to DCV infection. We observed that Nora-infected flies resisted the challenge slightly better, with reduced accumulation of DCV RNA compared to naive flies. Importantly, pre-infection with Nora virus neither conferred protection against DCV nor affected viral load in STING mutant flies (Figures 4D and S3C).

To address the contribution of cGLR1 and cGLR2 in the induction of STING::GFP by virus in different tissues, we recombined the STING knockin locus with chromosomes mutant for cGLR1,
<figure><img src="image_10.png" /><figcaption>Figure 2. Tissue-specific dynamic regulation of STING::GFP in response to 3′-cGAMP or DCV injection</figcaption></figure>

(A) Representative confocal images of dissected anterior midguts from STING::GFP flies at the indicated time points after injection of Tris, 3′-cGAMP, or DCV. Delineation of the proventriculus (P) and R0 and R1 regions of the anterior midgut is shown. All flies were dead 7 days post DCV infection.

(B and C) Quantification of STING::GFP fluorescence in dissected tissues, shown with a boxplot with scatterplot. Images were collected from a total of 12 dissected flies collected in three independent experiments (n = 12) at 6, 12, 24, and 48h and 7 days post injection. Scale bar: 25 µm. a.u., arbitrary unit.
See also Figure S1.

cGLR2, or both. Loss of cGLR1 reduced STING::GFP expression in all tissues following DCV systemic infection, with cGLR2 playing a lesser role when cGLR1 is functional, as reported previously at the whole-organism level (Figure S4). Furthermore, cGLR1 appears to play a major role in induction of STING::GFP in response to Nora infection, with contribution of cGLR2 especially when cGLR1 is absent (Figure S3D). Thus, we did not find evidence of tissue-specific contributions of the two functionally characterized cGLRs in *D. melanogaster*.

In summary, the pattern of STING::GFP expression in DCV- and Nora-challenged flies is globally similar to that of 3'-cGAMP-injected flies and does not correlate with virus-infected cells. Altogether, our findings suggest that local sensing of virus infection triggers a systemic STING-dependent response.

## Fat body-specific expression of cGLRs can upregulate STING::GFP in remote tissues

To test the hypothesis that virus sensing in specific tissues triggers a non-cell autonomous response, we took advantage of

our prior observation that ectopic overexpression of cGLRs in transgenic flies is sufficient to initiate CDN production and STING signaling.¹⁶ We used the fat body-specific Ipp-Gal4 driver to direct expression of wild-type or catalytically dead (AFA mutant) versions of cGLR2. As expected, Ipp-Gal4 efficiently drove expression of a UAS-GFP transgene in the fat body but failed to induce fluorescence in the central nervous system, Malpighian tubules, ejaculatory duct, and midgut (Figure 5A). Driving expression of cGLR2, but not the inactive version, with this driver resulted in some STING::GFP expression in the fat body, consistent with local production of CDN and activation of STING, although the fluorescence was weaker than for the UAS-GFP transgene. Interestingly, a strong induction of fluorescence was observed in the ventral nerve cord, brain, Malpighian tubules, ejaculatory duct, and midgut (Figure 5A and 5B). We confirmed by RT-qPCR that *srg1* was upregulated in the gut and the brain when cGLR2 was overexpressed in the fat body (Figure 5C). Accordingly, driving cGLR2 expression specifically in the fat body was sufficient to impair the accumulation of viral RNA and trigger strong protection
<figure><img src="image_11.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>**Figure 3. Induction of STING::GFP in response to DCV infection**</figcaption></figure>

(A) Partial colocalization between viral antigen and STING::GFP expression in the fat body. GFP and Cy3 (DCV capsid) intensity across the white straight line is shown on the right. Areas of colocalization are shown by arrows. Autofluorescence in the rows of oenocytes is indicated by an asterisk.

(B and C) Partial or lack of colocalization between viral antigen and STING::GFP expression in the brain (B) or Malpighian tubules (C), respectively.

(D) Detection of STING::GFP expression in epithelial cells of the midgut upon DCV infection. Vertical sections on the right show the presence of DCV antigen in smooth muscle cells but not in enterocytes. y-z images were scanned at the position indicated by white straight lines.

(E) Colocalization of viral antigen and STING::GFP in the epithelial cells lining the ejaculatory duct. Note that the viral antigen clusters at the apical side of the cells, whereas STING::GFP is diffuse in the cytoplasm. y-z images were scanned at the position indicated by white straight lines. Scale bar: 25 µm.

See also Figures S2 and S4.

of flies against DCV infection, as reported previously with the broadly expressed Actin5C driver (Figures 5D and S5A).¹⁶ Furthermore, we observed that flies expressing cGLR2 in the fat body and orally challenged with Nora virus exhibited reduced viral RNAs compared to controls 8 days later (Figure 5E). Overall, our experiments suggest that CDN production by cGLRs in virus-infected cells can be exported and trigger activation of STING signaling and antiviral immunity in remote tissues.

## Male-to-female sexual transfer of CDN-mediated immunity

The systemic nature of the response to 3′2′-cGAMP and the induction of STING signaling in reproductive ducts led us to wonder whether the CDN could be transferred during mating. To test this hypothesis, we injected 3′2′-cGAMP into male control flies. 24 h later, naive STING::GFP female flies were introduced into the tube for 4–6 h. Mated female flies were recovered
<figure><img src="image_17.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>Figure 4. Infection with the enteric Nora virus triggers systemic STING induction</figcaption></figure>

(A) Representative confocal images from midguts dissected from flies 8 days post feeding on sucrose or sucrose contaminated with Nora virus and stained with anti-dsRNA antibodies (J2). Scale bar: 25 µm.

(B) Percentage of GFP expression categories in dissected tissues 8 days following oral challenge with Nora virus. Images were collected from three independent experiments, each with 4 dissected flies (n = 12).

(C) Percentage of GFP expression categories in dissected tissues 12 h following injection with DCV virus. Images were collected from three independent experiments, each with 4 dissected flies (n = 12).

(D) $w^{1118}$ and STING$^{-/}$ flies were fed either sucrose or Nora for 8 days. Subsequently, they were injected with Tris or DCV, and their daily survival was monitored. Data were collected from three independent experiments, each comprising three groups of around 10 flies. Log rank test with Benjamini-Hochberg (BH) correction was used to compare the survival curves. ***p < 0.001. ns, non-significant.
See also Figures S3 and S4.

and analyzed 24 h later (Figure 6A). Strikingly, we observed fluorescence in the oviduct but also central nervous system, midgut, and Malpighian tubules (Figure 6A). Accordingly, STING mRNA was significantly upregulated in whole $w^{1118}$ female flies 24 h after mating with 3'2'-cGAMP-injected males (Figure 6B). This upregulation of STING was also observed in cGLR1; cGLR2 double knockout female flies, ruling out a possible cGLR-dependent signal amplification step (Figure S5B). Furthermore, these $w^{1118}$ female flies restrict DCV replication and better resisted a DCV challenge, with decreased viral RNA loads, than females mated with Tris-injected males. Such protection was not observed in STING mutant females, suggesting that 3'2'-cGAMP can be transferred from male to female during mating (Figures 6C and S5C). Finally, we tested whether expression of STING::GFP can be induced in females after mating with virus-infected males. We observed induction of fluorescence in the oviduct of flies after mating with DCV- or Nora-infected males (Figure 6D). This response was reduced or abolished when the females were crossed with cGLR1;cGLR2 double-mutant male flies, suggesting that the CDNs produced in response to viral infection in the males, rather than the virus itself, were transferred to the females. Overall, these results support the conclusion that circula-

tion of CDNs is a key feature of cGAS-STING signaling in *Drosophila*.

## STING signaling affects sleep in *Drosophila*

We monitored a number of parameters to connect the pattern of STING::GFP expression to biological function. We observed increased food intake in control but not in STING mutant flies injected with 3'2'-cGAMP compared to Tris (Figures 7A and S6A). We also noted that Tris-injected mutant flies ingested more food than the controls. The weight of STING mutant flies was reduced compared to control flies, although the mutants did not show increased sensitivity to starvation (Figures 7A, S6B, and S6C). We monitored fecundity and did not observe an effect on the quantity of progeny when 3'2'-cGAMP was injected both in male and female flies (Figures 7A and S6D). We also performed classical climbing assays to assess motor functions and did not notice an effect of 3'2'-cGAMP injection (Figures 7A and S6E).

Finally, we evaluated sleep by monitoring locomotor activity of single flies. Strikingly, the pattern of sleep was altered upon 3'2'-cGAMP injection, resulting in a significant decrease of sleep during both day and night (Figure 7B). 3'2'-cGAMP injection reduced the length of sleep and the number of sleeps and
<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>**Figure 5.** Fat body-specific expression of cGLR2 triggers STING::GFP expression in distant tissues, resulting in antiviral immunity</figcaption></figure>

(A) Confocal images of dissected tissues from flies in which the *lpp-Gal4* driver directs expression of either GFP (top row) or wild-type or mutant (AFA) cGLR2 in STING::GFP flies (center and bottom rows). Autofluorescence in the rows of oenocytes is indicated by an asterisk. Scale bar: 25 µm.

(B) Quantification of green fluorescence in different tissues dissected from flies of the indicated genotypes and shown with violin plots; the lines inside the violin plots denote median. Images were collected from four dissected flies in three independent experiments (n = 12).

(C) The expression of *Srg1* was monitored in the dissected fat body, gut, and brain from flies expressing GFP, wild-type, or mutant *cGLR2* under the control of the *lpp-Gal4* driver. The data are derived from three independent experiments, each experiment dissected at least 30 flies for fat body and gut and 120 flies for brain, and results are shown as mean ± SD. The *p* values were calculated using two-way ANOVA (***p < 0.0001).

(D) Fat body-specific expression of wild-type or mutant *cGLR2* flies injected with DCV; daily survival was monitored. Data are from 3 independent experiments, each involving 30 flies (n = 90). Log rank test with BH correction was used to compare the survival curves. ***p < 0.001.

(E) Flies with fat body-specific overexpression of either wild-type or mutant *cGLR2* were fed sucrose or Nora virus, and the viral RNA load was monitored 8 days later. Data are from four independent experiments indicated by different shapescolors, each involving three groups of four flies. Results are shown as mean ± SD. The *p* value was calculated using two-tailed Mann-Whitney test.

In (C) and (E), expression was normalized to the housekeeping gene *RpL32*.

increased the latency between sleep bouts (Figure S7A). This effect was abolished in STING mutant flies (Figures 7C and S7B). Furthermore, the quality of sleep in STING mutant flies was altered compared to controls, even in the absence of CDN injection (Figure S7C). Ectopic expression of STING in neurons using the elav-Gal4 driver was sufficient to decrease sleep, while a more modest effect was observed when driving STING expression in glial cells using the Repo-Gal4 driver (Figures S7E and

S7F). We next monitored DCV-infected flies and noticed a reduction of sleep starting 36–48 h post infection, depending on the experiment. The reduction of sleep in DCV-infected flies was highly reproducible between 48 and 72 h post infection, before locomotion decreased as infection progressed (Figures 7D and S7D). Importantly, no such effect of DCV on sleep was observed when STING or cGLR1/2 were not functional (Figures 7D and 7E). Overall, these findings reveal that systemic STING signaling
<figure><img src="image_23.png" /><figcaption>Figure 6. Transfer of STING-inducing activity from males to females during mating</figcaption></figure>

(A) The scheme of the experiment is shown at the top. STING::GFP is induced in the dissected oviduct, VNC, midgut, and Malpighian tubule from STING::GFP female flies 24 h after mating with w^1118^ male flies injected with 3'2'-cGAMP but not Tris. Scale bar: 25 µm.

(legend continued on next page)
triggered upon cGLR-dependent sensing of viral infection results
in physiological changes in *Drosophila*.

DISCUSSION

The central nervous system, midgut, Malpighian
tubules, and reproductive ducts participate in STING
biology

We used a STING::GFP knockin line to shed light on the *in vivo* dynamics of STING-dependent gene expression in *Drosophila*. This strategy monitors expression of a single gene and may miss some tissue-specific responses. In spite of this caveat, our results reveal that STING expression is strongly induced upon injection of 3'2'-cGAMP in four tissues: the central nervous system, the anterior midgut, the Malpighian tubules, and the ducts of the male and female reproductive tracts. These observations point to a set of tissues likely to play a central role in STING biology. Indeed, prior studies pointed to a role of STING in the digestive tract. In pupae, mobilization of the retrotransposon Gypsy during metamorphosis in the proventriculus, midgut, and hindgut has been proposed to mediate Relish-dependent induction of antiviral immunity in adults.<sup>32</sup> In addition, STING can sense CDNs produced by the microbiota in the adult midgut and is required for resistance against an oral viral challenge.<sup>22</sup> STING-dependent activation of Relish in response to enteric viral infection has also been proposed recently to induce intestinal stem cell proliferation, resulting in gut dysplasia.<sup>33</sup> The strong upregulation of STING expression in the brain and ventral nerve cord is also worthy of attention, considering the association of cGAS-STING signaling with neuroinflammation and neurodegeneration in mammals.<sup>34-37</sup> Our observations raise interesting perspectives for future studies exploiting the assets of the *Drosophila* model to shed light on the mechanism connecting cGAS-STING to the function of the nervous system in normal and pathologic conditions. Indeed, a recent report connected STING signaling to the phenotype of flies mutant for the early onset Parkinson disease gene *parkin*.<sup>38</sup> The two other tissues in which we detected strong STING::GFP induction, the Malpighian tubules and reproductive ducts, had not been previously associated with STING.

Compared to these strongly responding tissues, upregulation
of STING was modest in the fat body in response to 3'2'-cGAMP
or DCV infection, although this tissue plays a major role in immu-
nity and is a primary target for DCV replication,<sup>27-29</sup> and STING
has been shown to participate in one of its other functions: lipid
metabolism.<sup>39</sup> Of note, a similar situation was observed in the
midgut, where we did not see induction of STING::GFP in Nora
virus-infected cells. Yet, our data indicate that infection by
DCV or Nora virus was detected by cGLRs in the fat body or in

the gut, respectively, and resulted in STING activation in other
tissues. This may be explained by viral suppression of host cell
gene expression. Alternatively, RNAi triggered in virus infected
cells may be incompatible with an antiviral transcriptional
response, as reported in mammals.<sup>10,40,41</sup>

Physiological responses to 3′2′-cGAMP beyond
regulation of antiviral effectors

Of note, the pattern of STING induction we observed suggests that, in addition to induction of immunity genes encoding antiviral factors, CDNs may also upregulate genes that modify the physiology or the behavior of the flies. Indeed, following up on the observation of STING::GFP induction in the central nervous system, we show here that activation of the STING pathway affects sleep in *Drosophila*. It is interesting to note that the sleep-promoting gene *nemuri* is induced by bacterial infection and has been associated with improved survival of infection in *Drosophila*.<sup>42</sup> It will therefore be interesting to investigate the impact of sleep on the control of viral infection and survival in flies.

Interestingly, the pattern of STING::GFP expression points to other organs where activation of the pathway may result in physiological adaptations that help the organism to cope better with the infection. For example, Malpighian tubules mediate a purge of hemolymphatic lipids to protect host tissues from excessive reactive oxygen species generated in response to infection.⁴³ They have also been shown recently to participate in the control of gut homeostasis during infection,⁴⁴ and activation of IMD-Relish signaling in this tissue promotes gut tumor-associated mortality as a consequence of impaired uric acid excretion.⁴⁵ Proventriculus, in which we observed a strong induction, produces the peritrophic matrix, a chitinous membrane isolating the ingested food and protecting the gut epithelium, in particular from viral infection⁴⁶,⁴⁷; interestingly, we note that one of the genes regulated by 2'3'-cGAMP is *crystallin*, which encodes a protein participating in the formation of the peritrophic matrix in adults.⁴⁶,⁴⁷ Along the same line, induction of STING signaling in the ejaculatory duct may modify the pattern of seminal fluid proteins secreted by this epithelium, which induce a set of physiological and behavioral changes in females, including enhanced immune responses.⁴⁸

Altogether, our data suggest that activation of the STING
pathway in *Drosophila* results in induction of genes required for
both control of virus replication in virus-infected cells and adap-
tation of host physiology to promote survival.

CDN-dependent systemic immunity in *Drosophila*

Strikingly, an overall similar pattern was observed when flies were
infected with DCV and Nora virus, irrespective of the tissue
tropism of the viruses. In particular, for both viruses, we observed

<footnote>(B) Expression of *Sting* was monitored by RT-qPCR in *w*<sup>1118</sup> female flies 24 h after mating with *w*<sup>1118</sup> male flies previously injected with Tris or 3′2′-cGAMP. Expression was normalized to the housekeeping gene *RpL32*. Data are from three independent experiments, each performed in biological triplicates (n = 9). Mean ± SD is shown. The *p* value was calculated using two-tailed Mann-Whitney test (***p < 0.0001).</footnote>

<footnote>(C) *w*<sup>1118</sup> or *STING*<sup>-/-</sup> mutant females mated with *w*<sup>1118</sup> male flies that had been injected 24 h before with Tris or 3′2′-cGAMP were injected with DCV and monitored daily for survival. Data are from 4 independent experiments, each with around 30 flies. Log rank test with BH correction was used to compare the survival curves. ***p < 0.001.</footnote>

<footnote>(D) Induction of STING::GFP in the oviduct from female flies 24 h after mating with w<sup>1118</sup> or cGLR1<sup>-/-</sup>, cGLR2<sup>-/-</sup> male flies that had been previously infected with DCV or Nora virus. The percentage of GFP expression categories in dissected oviducts from the respective fly lines is presented at the bottom. Images were collected from three independent experiments, each involving dissection of four flies (n = 12). Scale bar: 25 µm.</footnote>
A

<table><thead><tr><th></th><th>food intake</th><th>weight</th><th>starvation</th><th>offspring number</th><th>climbing ability</th></tr></thead><tbody><tr><td>Response to 3'2'-cGAMP</td><td>↑</td><td>—</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Phenotype of STING<sup>fl</sup></td><td>↑</td><td>↓</td><td>↑</td><td>—</td><td>ns</td></tr></tbody></table>

B

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

C

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

D

<figure><img src="image_28.png" /></figure>

E

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /><figcaption>**Figure 7. STING regulates the duration and quality of sleep in *Drosophila***</figcaption></figure>

(A) Summary of the biological processes assessed in STING-stimulated or mutant flies. —, not done. ns, no significant change. Arrows indicate an increase or decrease of 3'2'-cGAMP compared to Tris or mutant compared to control flies.

(B–D) Representative sleep profile of *w*<sup>1118</sup> and STING<sup>fl</sup> flies injected with Tris, 3'2'-cGAMP, or DCV. Mean and SEM at 30 min intervals are illustrated on the curves, and the statistical analysis of day and night sleep duration is shown on the right (n = 32).

(legend continued on next page)
induction of STING::GFP in non-infected cells, suggesting that a signal travels from infected to naive cells to alert of an infectious danger. This signal could be a protein, akin to IFNs in vertebrates, which activate a subset of genes common with those induced directly by PRRs upon sensing viral infection, thereby amplifying the response.⁴⁹ Alternatively, this signal might be the CDNs produced upon sensing viral RNAs in infected cells. Although we cannot fully rule out the first hypothesis, the following observations suggest that CDNs may mediate transfer of information from infected cells to non-infected tissues. (1) Injection of CDNs into the body cavity of flies, or feeding CDNs to flies, induces anti-viral immunity, indicating that they can be imported, at least in some cell types.¹⁵,¹⁷,¹⁸,²² (2) CDNs can be detected in the hemolymph of flies ectopically expressing cGLR1 or cGLR2, revealing that CDNs can be exported, at least by some cell types.¹⁵ (3) Male flies injected with CDNs or infected by DCV can trigger induction of STING::GFP in the oviduct and other tissues of female flies; this activation requires cGLRs in the virus-infected male flies and STING in the mated females, suggesting that CDNs are transferred from male to female during mating. (4) Many insect pox-ins—a family of nucleases degrading 2'3'-cGAMP that radiated from the order Lepidoptera, which includes moths and butterflies—contain signal peptides, suggesting extracellular roles in CDN regulation, which would support our observations in *Drosophila*.¹²,⁵⁰,⁵¹ Therefore, we propose that CDNs represent an efficient and rapid way to transfer information of a danger to non-infected cells. Because of their charge, CDNs cannot diffuse through the cell membrane. Transfer of CDNs between cells in *Drosophila* may be mediated by gap junctions between adjacent tissues, such as smooth muscles and epithelial cells in the midgut and the reproductive ducts, as already reported in mammals.⁵²,⁵³ Different transporters involved in CDN import⁵⁴⁻⁶¹ and export⁵⁴,⁶²,⁶³ have also been identified in mammals. In *Drosophila*, the concentrative nucleoside transporter CNT2 has been proposed to mediate uptake of CDNs in enterocytes and to participate in the control of enteric DCV and Sindbis virus infection.²² An ATP-dependent 2'3'-cGAMP export mechanism has also been observed in the cell line Sf9, derived from the fall army worm (*Spodoptera frugiperda*).⁶³ A full understanding of the systemic contribution of CDNs to STING biology will require the identification and characterization of the *Drosophila* exporters and importers involved. The challenge of identifying these transporters is likely to be worth the effort, as this will clarify the contribution of the different tissues to the control of viral infection. In addition, the characterization of the transporters might reveal specificity for some CDNs, which would indicate that some of them may be specifically dedicated to immunotransmitter functions, thus providing an explanation for the diversity of CDNs produced in flies in response to viral infection.¹⁵

## Concluding remarks

Whatever the precise mechanism, induction of a systemic immune response by circulating CDNs may represent a major asset

of the cGLR-STING pathway to control viral infections in insects and may explain its coexistence with the antiviral siRNA pathway. Indeed, RNAi is intrinsically not transitive or systemic in flies.⁶⁴ This is different from the nematode *C. elegans* and plants, which encode cellular RNA-dependent RNA polymerases that mediate production of secondary siRNAs that are instrumental for systemic RNA silencing.⁶⁵⁻⁶⁸ Yet, a systemic dimension has been proposed for RNAi in flies in the context of viral infections, involving the release of dsRNA from dying infected cells and possibly the production of secondary siRNAs.⁶⁹⁻⁷¹ Further studies are required to establish which of the siRNA or STING pathways is the most efficient to trigger a systemic response to viruses. Of note, induction of the two key components of the siRNA pathway, Dicer-2 and AGO2, is a conserved feature of the response to 2'3'-cGAMP in drosophilid flies,⁷² pointing to a possible mechanism by which CDN production and export by infected cells could prime the antiviral RNAi pathway in distant cells.

## Limitations of the study

Our study reveal a contribution of CDN signaling to a systemic host response to viral infection in *Drosophila*. A limitation of the study lies in the detection of circulating CDNs across cells and tissues and the elucidation of the mechanisms governing their transfer. The development and application of more sensitive and accurate methods for CDN detection, such as liquid chromatography-mass spectrometry, ELISA, or fluorescence labeling of CDNs, could significantly enhance our capacity to detect and track these molecules. Furthermore, employing screening assays with cellular or *Drosophila* models is crucial to effectively identify and characterize CDN transporters. In addition, 3'2'-cGAMP injection triggers STING::GFP expression in the proventriculus, unlike DCV or Nora infection, but our data do not elucidate this discrepancy. One possibility is that the quantity of CDN importers expressed in the proventriculus is limiting so that only high CDN levels, as provided by injection, results in STING activation. By contrast, DCV or Nora infection may not trigger the production of sufficient CDNs to activate STING in the proventriculus. Future investigations should also explore the specific neuronal circuits that engage STING signaling to modulate sleep.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hua Cai (caihua@gzhmu.edu.cn).

### Materials availability

Materials used in this study are available upon request.

### Data and code availability

* All data reported in this paper will be shared by the lead contact upon request.

<footnote>(E) Statistical analysis of day and night sleep duration in *w*¹¹¹⁸ and *cGLR1*⁻/⁻, *cGLR2*⁻/⁻ after injection with Tris or DCV(n = 32). In (B)–(E), the statistical results are presented as mean ± SD. The *p* values were calculated using two-way ANOVA (*p* < 0.05; ***p* < 0.01; **p* < 0.001; ****p < 0.0001). See also Figures S6 and S7.</footnote>
* The raw data corresponding to Figures 1B, 2B, 2C, 4D, 5D, 6C, and S1A and original code have been deposited in the Figshare repository (https://doi.org/10.6084/m9 figshare.27889668.v1) and are publicly available as of the date of publication. The DOI is also listed in the key resources table.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

We thank Joao Marques and Dominique Ferrandon for critical reading of the manuscript and useful suggestions and members of our laboratories in Strasbourg and Guangzhou for stimulating discussions. J.-L.I. and H.C. acknowledge support from the National Key R&D Program of China (2023YFE010770) and the Chinese National Overseas Expertise Introduction Center for Discipline Innovation (Project “111” [D18010]). J.-L.I. acknowledges financial support from CNRS, grants from ANR (ANR-10-IDEX-0002, ANR-11-EQPX-0022, ANR-17-EURE-0023, ANR-20-SFRI0012, and ANR-22-CE15-0019), and the Hoffmann-Infinitus Program. X.A. was supported by the Natural Science Foundation (32200121) and China Postdoctoral Science Foundation (2021M700941). H.C. was supported by the Natural Science Foundation (32370767 and 32000662), Guangdong Provincial Science Fund for Distinguished Young Scholars (2023B1515020098), Guangdong Provincial Young Scholars Academic Exchange Program (2022A0505030018), Youth Talent Support Program of the Guangdong Provincial Association for Science and Technology (SKXRC202229), and the Science and Technology Program of Guangzhou (202102020090). We thank Dr. Renjie Jiao, Dr. Dan Wang, and the Bloomington Stock Center for fly stocks.

## AUTHOR CONTRIBUTIONS

Experiments were conceived and designed by X.A., H.C., and J.-L.I. Experiments were conducted by X.A., H.D., and X.L. with help from Z.W., Y.C., T.Y., J.Z., J.H., H.L., Xiaohan Zhang, Xiuqing Zhang, C.M., X.L., L.T., and D.C. Data were analyzed by X.A., H.D., X.L., C.M., J.L.I., and H.C. The manuscript was written by X.A., H.C., and J.-L.I. with help from C.M. All authors contributed to editing the manuscript and supported the conclusions.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Fly lines and rearing

* METHOD DETAILS

* RNA extraction from flies and quantification of gene expression by RT-qPCR

* Fly imaging and immunohistochemistry

* Confocal imaging

* Quantification of fluorescence signal intensity

* Viral infections

* CDNs injection

* Male to female transmission assays

* Biological assays

* Sleep activity assays

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.115081.

Received: June 7, 2024

Revised: October 14, 2024

Accepted: November 26, 2024

Published: December 16, 2024

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>nc82</td><td>Developmental Studies Hybridoma Bank</td><td>Cat# nc82; RRID:AB_2314866</td></tr><tr><td>Rabbit anti-DCV</td><td>Dostert et al.<sup>7</sup></td><td>N/A</td></tr><tr><td>Alexa Fluor<sup>TM</sup> 594 phalloidin</td><td>Invitrogen</td><td>Cat# A12381; RRID:AB_2315633</td></tr><tr><td>Anti-dsRNA J2 monoclonal antibody (mAb)</td><td>English and Scientific Consulting Kft</td><td>Cat# 10010200; RRID: AB_2651015</td></tr><tr><td>Erioglaucine disodium salt</td><td>Sigma-Aldrich</td><td>Lot# SHBN8755</td></tr><tr><td>Alexa Fluor<sup>TM</sup> 568 goat anti-mouse IgG</td><td>Invitrogen</td><td>Cat# A-11004; RRID:AB_2534072</td></tr><tr><td>Alexa Fluor<sup>TM</sup> 488 goat anti-rabbit IgG</td><td>Invitrogen</td><td>Cat# A-11034; RRID:AB_2576217</td></tr><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>Drosophila C virus</td><td>Goto et al.<sup>19</sup></td><td>N/A</td></tr><tr><td>Nora virus</td><td>Cai et al.<sup>15</sup></td><td>N/A</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>3'2'-cGAMP</td><td>Biolog Life Science Institute</td><td>Cat# C 238</td></tr><tr><td>2'3'-cGAMP</td><td>Biolog Life Science Institute</td><td>Cat# C 161</td></tr><tr><td>4% Paraformaldehyde Fix Solution</td><td>Leagene Biotechnology</td><td>Cat# DF0135</td></tr><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>Evo M-MLV RT Kit with gDNA Clean for qPCR II</td><td>Accurate Biotechnology(Hunan)</td><td>Code. AG11711</td></tr><tr><td>SYBR Green Premix Pro Taq HS qPCR kit</td><td>Accurate Biotechnology(Hunan)</td><td>Code. AG11701</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>DrosDel w<sup>1118</sup></td><td>Cai et al.<sup>18</sup></td><td>N/A</td></tr><tr><td>STING::GFP</td><td>This study</td><td>N/A</td></tr><tr><td>STING<sup>-/-</sup></td><td>Cai et al.<sup>18</sup></td><td>N/A</td></tr><tr><td>cGLR1<sup>-/-</sup></td><td>Holleufer et al.<sup>16</sup></td><td>N/A</td></tr><tr><td>cGLR2<sup>-/-</sup></td><td>Holleufer et al.<sup>16</sup></td><td>N/A</td></tr><tr><td>UAS-cGLR2</td><td>Holleufer et al.<sup>16</sup></td><td>N/A</td></tr><tr><td>UAS-cGLR2AFA</td><td>Holleufer et al.<sup>16</sup></td><td>N/A</td></tr><tr><td>UAS-GFP</td><td>Bloomington Drosophila Stock Center</td><td>BDSC:4776</td></tr><tr><td>Ipp-Gal4</td><td>China Agricultural University, Laboratory of Dan Wang</td><td>N/A</td></tr><tr><td>UAS-mCherry</td><td>Sino-French Hoffmann Institute, Laboratory of Renjie Jiao</td><td>N/A</td></tr><tr><td>Repo-Gal4</td><td>Sino-French Hoffmann Institute, Laboratory of Dominique Ferrandon</td><td>N/A</td></tr><tr><td>elav-Gal4</td><td>Sino-French Hoffmann Institute, Laboratory of Renjie Jiao</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>Oligonucleotide primers for qPCR, See Table S1</td><td>This study</td><td>N/A</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>CFX Maestro Software (version 1.0)</td><td>Bio-Rad</td><td>https://www.bio-rad.com/zh-cn</td></tr><tr><td>GraphPad Prism8 (version 8.2.0)</td><td>GraphPad Software, LLC</td><td>www.graphpad.com</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>ImageJ (version 1.15s)</td><td>NIH</td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>Adobe Photoshop CS6</td><td>Adobe</td><td>N/A</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>http://matlab.p2hp.com/</td></tr><tr><td>Raw data and original codes</td><td>https://doi.org/10.6084/m9.fishshare.27889668.v1</td><td>This paper</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Fly lines and rearing

Flies were reared on standard sucrose-cornmeal-yeast agar medium at 25°C and a 12:12-h light:dark photoperiodic cycle. All utilized fly lines were free of *Wolbachia*. 3-5 days-old adult flies were employed for all experiments, unless specified otherwise in the figure legends. The STING::GFP line was engineered in the *w*¹¹¹⁸ background through CRISPR-Cas9 technology: the sequence coding the fluorescent protein GFP was integrated at the C terminus of STING. The STING::GFP line and mutant lines STING⁻¹⁻, cGLR1⁻¹⁻, cGLR2⁻¹⁻;cGLR1/2⁻¹⁻ were isogenized to the DrosDel *w*¹¹¹⁸ isogenic background as described previously.¹⁶,¹⁸ The UAS transgenic lines of wild-type and AFA mutant versions of cGLR2, UAS-cGLR2 and UAS-cGLR2*AFA*, have been described.¹⁶ *Ipp-Gal4* flies were obtained from Dan Wang (China Agricultural University, Beijing, China). *Repo-Gal4* from Dominique Ferrandon (IBMC, University of Strasbourg), *elav-Gal4* and *UAS-mCherry* from Renjie Jiao (SFHI, Guangzhou Medical University, China). *UAS-GFP* flies were obtained from the Bloomington *Drosophila* Stock Center (BL4776).

## METHOD DETAILS

### RNA extraction from flies and quantification of gene expression by RT-qPCR

RNA extraction, cDNA synthesis and RT-qPCR analysis were performed as described.¹⁸ Gene expression was analyzed with CFX Maestro Software (Bio-Rad). cDNA was reversed transcribed from RNA using the *Evo M-MLV* RT Kit with gDNA Clean for qPCR II (Code. AG11711). RT-qPCR was conducted using the SYBR Green Premix *Pro Taq* HS qPCR kit (Code. AG11701) on a CFX-384 (Bio-Rad) platform, with each reaction repeated in triplicate. The transcription level of the target gene was normalized to expression of the RNA encoding ribosomal protein L32 (RpL32) for each sample. Flies were collected in pools of 6 males and homogenized for RNA extraction and RT-qPCR, unless specified otherwise in the figure legends.

### Fly imaging and immunohistochemistry

To monitor STING::GFP fluorescence intensity, *Drosophila* tissues were dissected after fixing with 4% paraformaldehyde for 6 h. For immunofluorescence analysis, flies were placed in 4% paraformaldehyde and centrifuged for 10 min at 9,000g and then fixed for 6 h. Tissues (brain, ventral nerve cord, fat body, midgut, Malpighian tubule, oviduct and ejaculatory duct) were then dissected in PBS. Samples were washed 3 times for 5 min with 0.4% (brain, ventral nerve cord) or 0.1% (other tissues) Triton X-100 in PBS and then incubated with the same solution supplemented with 5% bovine serum albumin for 2 h. Samples were incubated with rabbit anti-DCV capsid antibody⁷ (1:1000), mouse Anti-nc82 monoclonal antibody (1:200; Developmental Studies Hybridoma Bank AB_2314866), mouse Anti-dsRNA J2 monoclonal antibody (mAb) (1:400; English and Scientific Consulting Kft Cat# 10010200), Alexa Fluor 594 phalloidin (1:500; Invitrogen A12381) antibodies for 48 (brain, ventral nerve cord) or 12 (other tissues) hours at 4°C. Subsequently, the samples were washed 5 times for 5 min in PBS with 0.4% or 0.1% Triton X-100. For the secondary antibodies, Alexa Fluor 568 goat anti-mouse IgG (1:500; Invitrogen A11004) or Alexa Fluor 488 goat anti-rabbit IgG (1:500; Invitrogen A11034) were used.

### Confocal imaging

Images were collected using Leica SP8 and Zeiss LSM 980 confocal microscopes. The figures were assembled in Adobe Photoshop CS6. To compare the STING::GFP fluorescence intensity, uniform microscopy conditions were maintained, and control and treatment images were edited in parallel by Adobe Photoshop CS6 with minor image adjustments for brightness and/or contrast.

### Quantification of fluorescence signal intensity

The fluorescence signal intensity was quantified using ImageJ software in the region of interest. Data were obtained from a minimum of 9 tissues per treatment across three independent experiments, and the fluorescence signal intensity for each tissue was presented as a data point.
## Viral infections

Nora and DCV viral stocks were prepared as described in 10 mM Tris-HCl, pH 7.5.⁷,³⁰ Infections were conducted on 3-5 days-old male flies. For systemic infections, flies were injected in the thorax (Nanoject III apparatus, Drummond Scientific) with 4.6 nL of DCV (5 (survival) or 500 (gene induction and viral load) PFU/fly). After the injection, flies were moved to vials with regular food, housing 10 flies per vial. For Nora oral infections, 1\*3 cm piece of filter paper was placed in the bottom of an empty vial and loaded with 250 µL of a mix of 30% virus extract and 70% 50 mM sucrose solution. Ten flies were placed per vial and left feeding for 3 days at 25°C. For mock oral infections, flies were exposed to a 50 mM sucrose solution. Following this infection phase, flies were transferred to fresh vials containing regular food for five days. After the infection, flies were maintained at 25°C for dissection, mating or survival daily assessments.

## CDNs injection

2′3′-cGAMP and 3′2′-cGAMP (Invivogen) were dissolved in 10 mM Tris pH 7.5 to reach a concentration of 0.9 mg\*mL⁻¹. 3–5 days old adult flies were injected with 69 nL of CDN solution or 10 mM Tris pH 7.5 (negative control) by intrathoracic injection using a Nanoject III apparatus. For experiments monitoring antiviral effects of 3′2′-cGAMP, the flies were injected with DCV (5pfu) 7 days after CDN injection, as described.¹⁵

## Male to female transmission assays

To assess the transmission of the injected CDNs, 3-5 days-old w¹¹¹⁸ males were injected with 3′2′-cGAMP. 24 h later, they were mated in single pairs to 3-5 days-old STING::GFP, w¹¹¹⁸ or STING⁻/⁻ knock-out virgin females. To monitor mating-dependent activation of STING in female flies, 3-day-old w¹¹¹⁸ or cGLR1⁻/⁻, cGLR2⁻/⁻ double mutant males were injected with DCV or fed with Nora virus and 24 h or 8 d later, males were mated in single pairs to 3-5 days-old STING::GFP or w¹¹¹⁸ virgin females. Approximately 120 pairs of each cross and control were set up. The pairs were observed for about 4 h and 20–50 pairs from each line were mated. Immediately after mating, females were introduced into new vials. After 24 h, the females were analyzed by confocal imaging, RT-qPCR and survival tests.

## Biological assays

For feeding assays, adult male flies were starved on 1% agar for 19 h. Subsequently, they were transferred to a vial containing 1% agar with 50 mM sucrose and 1mg\*mL⁻¹ Erioglaucine disodium salt dye (Sigma-Aldrich Lot# SHBN8755) for 10 min. Flies were collected in 1.5 mL tubes and frozen in liquid nitrogen for 10 min. After shaking, 22 carcasses without heads and legs were collected to homogenize in 500µL PBS, and then centrifuged (14,000g) for 20 min 100µL supernatants were used to measure the absorbance at 630 nm using Microplate Reader (BioTek Instruments, Inc.), with each sample measured in triplicate and averaged. For weighing experiments, 30 female and 15 male flies were reared in a tube (inner diameter, 4.5 cm) with standard sucrose-cornmeal-yeast medium at 25°C, and 30 male flies from the offspring were weighed before or after drying on a precision balance (Sartorius BSA124S-CW). Drying was performed overnight at 60°C on a layer of filter paper. For fecundity assays, three pairs of 5 days-old unmated flies were raised in a tube (inner diameter, 2 cm) with standard food at 25°C. The flies were transferred to a new tube daily, and the number of the pupa from the first three days was counted as the offspring number. For climbing assays, the climbing index was calculated as the average percentage of flies to reach a 6 cm line within 4 s after being tapped to the bottom of a vial. For each group, measurements were taken for 3 sets of 10 males, each set measured at least 3 times and averaged.

## Sleep activity assays

3-5 days-old male adult flies were maintained under a 12-h light/12-h dark cycle at 25°C. Flies were placed individually into a detecting tube (length, 65 mm; inner diameter, 5 mm) containing fly food at one end, and a cotton plug at the other. Experiments were conducted in an incubator maintained at 50% humidity. Flies activity was quantified using the *Drosophila Activity Monitor (DAM)* System 2 (Trikinetics, MA, U.S.), which records the infrared beam crossing of individual flies in each tube every minute. Sleep was defined as a period of 5 consecutive minutes of inactivity. Flies were kept in detecting tubes to adapt for 24–48 h, unless otherwise noted, and then, data were collected for at least 3 days with the DAM System. Sleep data were analyzed with MATLAB (MathWorks).

## QUANTIFICATION AND STATISTICAL ANALYSIS

The experimental data were analyzed using GraphPad Prism 8.2.0 (GraphPad Software). The Holm-Šídák method (two-way ANOVA) was performed making pairwise comparisons to a specific mock control (stated in the figure legends). The multiple comparison tests were all two-tailed and the *p*-values were corrected for multiple comparison. Unpaired two-tailed Mann-Whitney test was performed to compare the difference between only two groups. For gene expression and viral load, the data were analyzed by pairwise permutation tests in R using "coin" and "rcompanion" packages with Bonferroni's method. Normality was evaluated by QC plot analysis and data were log transformed for qPCR data to apply one or two-way ANOVA. Survival experiments were analyzed in R using the Survival package with Log rank test with Benjamini-Hochberg correction. Sample number for each experiment are stated in the figure legends. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.