The D. melanogaster fly lines used were the following: w¹¹¹⁸, Oregon-R, and yw. Fly stocks harbor the sensitive allele of Pastrel 3L:7350895 (Thr). Flies were reared on a standard cornmeal diet (Bloomington) at a constant temperature of 25°C and kept under a 12:12 photoperiod. All fly lines were cleaned of possible chronic infections (viruses and Wolbachia). In addition, fly stocks were analyzed by RT-PCR with pairs of primers specific for CrPV, DAV, DXV, DCV, FHV, and NoraV to confirm that they were not persistently infected by these viruses.

DCV stock was prepared in w¹¹¹⁸ flies. Flies were injected intrathoracically with 500 TCID50 per fly. When mortality started, flies were anesthetized and squashed in PBS (3 flies per 100 μl of PBS). The extract was frozen at −80°C, thawed and centrifuged for 15 min at 15,000 × g at 4°C. The supernatant was recovered and filtered to eliminate bacteria, aliquoted, and stored at −80°C.

FHV stock was prepared on low-passage S2 cells. When the cytopathic effect started, the supernatant was harvested and centrifuged.

Both stocks were titrated in S2 cells. Titers were measured by end-point dilution method and expressed as 50% Tissue culture Infective Dose (TCID₅₀). DCV stock: 1,18 × 10¹⁰ TCID₅₀/ml, FHV stock: 5 × 10⁹ TCID₅₀/ml).

Injections: flies were injected intrathoracically using a nanoject (Nanoject II apparatus; Drummond Scientific) with 50 nL of a viral suspension of 10 TCID₅₀ of Drosophila C virus or 100 TCID₅₀ of Flock house virus in 10 mM Tris, pH 7. An injection of the same volume of 10 mM Tris, pH 7 served as a mock-infected control. Infected flies were kept at 25°C and changed to fresh vials every 2 days.

Oral infections: flies were starved during 5 h in an empty tube. Then, flies were transferred to a tube containing a Whatman filter paper in the bottom embedded in a mix of viral stock in PBS (10% viral stock, 35% sucrose and 2% of blue dye). After 16 h, only the flies having a blue-belly (corresponding to blue dye in the gut due to ingestion) were placed in new media tubes, kept at 25°C and changed to fresh vials every 2 days.

For all the experiments, 4- to 7-day old adult female flies were used. For each experimental condition, flies were divided in two pools: one pool was used for viral titration and RNA extraction, and the other one for protein extraction. For every experiment presented, the analyses were based on three biological replicates. In Figure 2, 16 biological replicates were performed (except w¹¹¹⁸ in Figure 2B, n = 15). For western blots, only biological replicates were used. For RT-qPCR, three technical replicates per condition and per biological replicate were used.

Mortality of infected flies was measured daily by counting the number of dead flies in each test tube. Three biological replicates of 60 flies each were done per condition. Fly mortality at day 1 was attributed to damage invoked by injection and/or manipulation procedure, and excluded from further analyses.

For each time point and condition, total RNA was extracted from a pool of 4–12 flies depending on the biological replicate. Each pool was homogenized in 300 μl of PBS and 100 μl were used to perform RNA extractions using TRIzol reagent (Invitrogen).

The first-strand cDNAs were produced from 400 ng of RNA using Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase (Thermo Scientific) according to the manufacturer's instructions. For each sample, a negative control without the reverse transcriptase enzyme was performed, in order to check for potential genomic DNA contamination. Roche Universal Sybr Green Master Mix (Rox) was used for qPCR. The sequences of the primers used were:

Ago-2 F primer: 5′-GTGGTTTACACGCCTCCTCA-3′

Ago-2 R primer: 5′-GGGTAGTTGCGACTGTGGAA-3′

Dcr-2 F primer: 5′-GGGTGAACAGGGAGTGGATG-3′

Dcr-2 R primer: 5′-CAAAAAGACCTGGGCTGTGC-3′

Quantification was normalized to that of mRNA encoding the endogenous ribosomal protein Rp49 as previously reported (21). Data were calculated using the ΔΔCt method to compute relative gene expression. For each sample, 3 technical replicates plus 1 RT negative control were included in the qPCR plate. qPCR was performed in 384-well plates with a final volume of 10 μl with QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). The following program was used: Hold stage 50°C for 2 min, 95°C for 10 min. PCR stage: 40 cycles of 95°C for 15 s, 60°C for 1 min. A melt curve to confirm the specificity of the reaction was performed.

For each time point and condition, total proteins were extracted from pools of 4–8 flies, depending on the biological replicate, using 200 μl NP40 Buffer: 20 mM HEPES-KOH buffer pH 7.5; 100 mM KCl; 5% Glycerol; 0.05% NP40; 1 mM DTT (freshly added) and 1x complete, EDTA-Free Protease Inhibitor Cocktail (Roche) (freshly added). Each pool was homogenized with a pestle, centrifuged at 12,000 × g for 10 min and the supernatant was recovered and stored at −80°C. Ten microliters of each protein extract were ran in SDS-PAGE using Mini-PROTEAN TGX Stain-free gels 4–20% (BIO-RAD) according to the manufacturer's instructions. The gels were then activated for image acquisition using Molecular Imager Gel Doc XR+ (BIO-RAD) and the transfer was performed using Trans-Blot Turbo Transfer Pack nitrocellulose membranes and Trans-Blot Turbo Transfer System (BIO-RAD). The image acquisition of the total amount of proteins transferred was performed with Molecular Imager Gel Doc XR+ (BIO-RAD). For the immunoblots, the following primary antibodies were used: Anti-Dcr-2 Abcam AB4732 (1:1000); Anti-Ago-2 Siomi 9D6 (non-commercial antibody, 1:15) kindly provided by Haruhiko Siomi. The secondary antibodies were HRP-linked: anti-rabbit GE Healthcare NA9340V (1:10000); anti-mouse Abcam AB6728 (1:10000). The antibodies were diluted in a solution of PBS-BSA 3%, TWEEN 20 0.3%. Membranes were incubated overnight at 4°C with the primary antibodies, washed 3 times for 5 min with PBS-TWEEN 20 0.3%, incubated with secondary antibodies 1 h at room temperature and washed again 3 times for 5 min with PBS-TWEEN 20 0.3% before adding the ECL (SuperSignal West Pico Plus Chemiluminescent substrate—Thermo Scientific). The image acquisitions were performed with MyECL Imager (Thermo Scientific). The intensities of the bands corresponding to Dcr-2 and Ago-2 were normalized with the total amount of protein in their respective lanes using ImageStudioLite (LI-COR Biosciences) (Supplementary Figures 1, 2).

In order to compare results from different experiments, two types of normalization were employed. To study differences in the relative protein expression, all the data were normalized with the mock-infected time point 0 (Figures 5, 6). To analyze non-relative protein levels, all data were corrected for the experimental effect as previously shown (21). This procedure transforms the raw values into their deviation from the experimental mean, and the resulting adjusted values are centered on zero (Figures 2, 7).

All statistical analyses were performed with Prism 8—GraphPad. The following statistical tests were used: for grouped tables with two grouping variables, two-way ANOVA followed by Sidak's multiple comparisons test; for table with one grouping variable, the Mann-Whitney test; for survival curves, the log-rank (Mantel-Cox) test; to calculate the probability of deviations from a theoretically expected distribution, the binomial test.

To study the regulation of the siRNA pathway during viral infections we used different D. melanogaster strains, viruses and modes of infection in order to establish the commonality of the response. We challenged w¹¹¹⁸, Oregon-R, and yw D. melanogaster strains in two different infection conditions: (1) injection of Drosophila C Virus [DCV; (+)ssRNA Dicistroviridae], a natural Drosophila pathogen; and of Flock House Virus [FHV; bisegmented (+)ssRNA, Nodaviridae], a non-natural fly pathogen used as a control; and (2) oral infection by feeding with DCV. Survival curves showed that after DCV infection by injection, w¹¹¹⁸ flies had an intermediate survival rate compared to yw flies that were more susceptible, whereas Oregon-R were more resistant (Figure 1A). Injection of DCV resulted in the death of all flies at 8 days post-infection (dpi) for w¹¹¹⁸ and 4 dpi for yw flies, while 97% of Oregon-R flies died at 9 dpi (Figure 1A, left panel). In flies orally infected with DCV, the proportions of mortality 15 days after infection were of ~25% for w¹¹¹⁸, ~50% for yw, and ~6% for Oregon-R strains (Figure 1A, middle panel). Upon FHV injection, yw were still the most susceptible flies, with ~35% mortality at day 6 compared with ~9% for w¹¹¹⁸ and ~15% for Oregon-R (Figure 1A, right panel). We then measured viral loads by TCID₅₀ for all tested conditions at 0 dpi (15 min post-injection or 16 h post-feeding) and at 3 further times post-infection (Figure 1B and Supplementary Table 1). Following virus injection, both DCV and FHV reached higher titers in yw flies than w¹¹¹⁸ and Oregon-R flies at 2 dpi and 3 dpi, respectively, while DCV accumulated to higher levels at 3 days post-injection in w¹¹¹⁸ flies compared with Oregon-R (Figure 1B and Supplementary Table 1). However, no significant differences in DCV titers were found between the different fly strains during oral infections (Figure 1B and Supplementary Table 1). Taken together, these results show that these fly strains differ in their susceptibility to viral infections and that there is a correlation between the increase of viral loads and mortality (Figure 1B and Supplementary Table 1), indicating that flies are dying due to the physiological burden imposed by the infection.

We next asked if the differences in susceptibility and viral accumulation observed between w¹¹¹⁸, Oregon-R and yw flies might be due to differences in the basal levels of the siRNA pathway core proteins, Dcr-2 and Ago-2, before viral infection. To address this question, we measured the levels of these proteins in the three Drosophila strains by western blot. Dcr-2 basal levels were similar between strains (Figure 2A). A significant increase in Ago-2 basal levels between w¹¹¹⁸ and Oregon-R (Figure 2B) was observed, but it cannot explain the difference in susceptibility to virus infection observed for these strains. Taken together, these results show that the basal levels of Dcr-2 and Ago-2 do not play a critical role in fly survival and in the control of viral infections.

As we did not observe a correlation between Dcr-2 and Ago-2 proteins basal levels and infection outcomes in the D. melanogaster strains tested, we wanted to explore if a difference in gene expression would be noticeable as a result of the viral infection. We evaluated the mRNA expression levels of Dcr-2, Ago-2, and rp49 (housekeeping gene) by qPCR at four time points post-infection. The mRNA was extracted from the same pools of flies used in Figure 1B. We did not observe any significant difference in Dcr-2 (Figure 3) and Ago-2 (Figure 4) mRNA levels of infected flies compared with mock infected flies, independent of the fly strain, virus or mode of infection used.

To explore if mRNA expression levels correlate with proteins levels upon viral infection, we quantified the expression of Dcr-2 and Ago-2 proteins. Proteins were extracted from flies that were biological replicates to Figures 1B, 3, 4 and the presence and quantity of Dcr-2 and Ago-2 were analyzed by western blot. After normalization with the time point 0 of the mock-infected condition (see Materials and Methods for details on normalization protocol and Supplementary Figures 1, 2), Dcr-2 levels showed a relative increase in virus infected flies at the time point 0 (15 min post-injection or 16 h post-feeding) independently of the fly strain, virus and mode of infection used (Figure 5). These increments were statistically significant for w¹¹¹⁸ injected with DCV (Figure 5A), for yw injected with FHV (Figure 5B), and for Oregon-R and yw orally infected with DCV (Figure 5C). These significant increases in Dcr-2 protein expression were not observed throughout all the conditions; however, in the 27 western blots performed (3 biological replicates × 3 fly strains × 3 different modes of infection), 26 of them showed higher Dcr-2 levels in infected flies than in the mock infected control. The probability that this phenomenon is due to a random effect is only 0.00002% (binomial test assuming that, during a random phenomenon, the probability to be higher or lower with respect to the control is the same). On the contrary, at the last time point (3 days post-infection for injection and 6 days post-infection for feeding) we found that Dcr-2 protein relative levels in infected flies were lower compared to the mock-infected flies. This decrease was statistically significant for yw injected with FHV (Figure 5B), and for Oregon-R and yw orally infected with DCV (Figure 5C). In injected flies, we found this pattern in 14 of 15 experiments with a probability of 0.004% for this phenomenon to be due to a random effect. In orally infected flies, we found this pattern in 9 of 9 experiments, with a probability of 0.2%. Due to the limited amount of anti-Ago-2 antibody in our possession (non-commercial antibody), we chose to perform the analysis for Ago-2 protein levels only in the w¹¹¹⁸ strain. We observed the same trend as for Dcr-2, but without significant differences (Figure 6). From a total of 7 western blots performed, we found higher relative levels of Ago-2 7 times for the time point 0, with respect to mock infected flies, with a probability of 0.78% (binomial test) that this is due to a random effect.

Altogether, the results show that Dcr-2 and Ago-2 protein levels change promptly upon virus infection.

The data shown in Figure 5 correspond to Dcr-2 protein levels relative to the mock-infected time point 0. Therefore, we cannot differentiate if the change observed is due to an absolute Dcr-2 increase after the infection or to an absolute Dcr-2 decrease in the control flies due to the stress caused by the experimental procedure (injury in the case of injection, and starvation in the case of feeding—see modes of infection in Materials and Methods). To answer this question, we compared the absolute protein levels for each time point with the basal level of the non-injected control flies from Figure 2. Since we observed the same effect on DCV and FHV injected flies, we pooled the data of all infected flies. We then analyzed the absolute levels of Dcr-2 among strains for each time point. As expected, we did not observe any strain-dependent difference in the levels of Dcr-2 (Figure 7A), in agreement with the results reported in Figure 2. This allowed us to pool each treatment condition (mock infected, virus infected and non-injected) to analyze the absolute levels of Dcr-2 across time and independently of the fly strain. Figure 7B shows that, at time 0, Dcr-2 levels in mock-infected flies rapidly decrease compared to the non-injected control. In contrast, injection of virus increased the level of Dcr-2 protein. At 3 days post-infection, the tendency reversed with increased levels of Dcr-2 in mock-infected flies and decreased levels in virus-infected flies. The same analysis was performed for flies infected by feeding. Figure 7C shows the same trend with a prompt increase of Dcr-2 protein levels upon virus ingestion and a later decrease. We did not observe a significant decrease of Dcr-2 levels during mock infection at time point 0, but we did find a significant increase for the time point 3. This indicates that starvation, as well as injection, induces a change in the regulation of Dcr-2.

Altogether, these results put in evidence a change of Dcr-2 protein levels that is not only dependent on virus infection but also on infection procedure-related stress. While virus infection rapidly induces an increment of Dcr-2 protein concentration, the infection procedure-related stress immediately decreases Dcr-2 protein levels. The increase of Dcr-2 protein during viral infection is strong enough to mask the decrease produced by stress.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.