Micrornas from Snellenius Manilae Bracovirus Regulate Innate and Cellular Immune Responses of Its Host Spodoptera Litura

ARTICLE

https://doi.org/10.1038/s42003-020-01563-3 OPEN MicroRNAs from Snellenius manilae bracovirus regulate innate and cellular immune responses of its host Spodoptera litura

Cheng-Kang Tang1, Chih-Hsuan Tsai1, Carol-P. Wu1, Yu-Hsien Lin 1, Sung-Chan Wei1, Yun-Heng Lu1, ✉ 1234567890():,; Cheng-Hsun Li1 & Yueh-Lung Wu1

To avoid inducing immune and physiological responses in insect hosts, parasitoid wasps have developed several mechanisms to inhibit them during parasitism, including the production of venom, specialized wasp cells, and symbioses with polydnaviruses (PDVs). These mechanisms alter the host physiology to give the wasp offspring a greater chance of survival. However, the molecular mechanisms for most of these alterations remain unclear. In the present study, we applied next-generation sequencing analysis and identiﬁed several miRNAs that were encoded in the genome of Snellenius manilae bracovirus (SmBV), and expressed in the host larvae, Spodoptera litura, during parasitism. Among these miRNAs, SmBV-miR-199b- 5p and SmBV-miR-2989 were found to target domeless and toll-7 in the host, which are involved in the host innate immune responses. Microinjecting the inhibitors of these two miRNAs into parasitized S. litura larvae not only severely decreased the pupation rate of Snellenius manilae, but also restored the phagocytosis and encapsulation activity of the hemocytes. The results demonstrate that these two SmBV-encoded miRNAs play an important role in suppressing the immune responses of parasitized hosts. Overall, our study uncovers the functions of two SmBV-encoded miRNAs in regulating the host innate immune responses upon wasp parasitism.

✉ 1 Department of Entomology, National Taiwan University, Taipei 106, Taiwan. email: [email protected]

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arasitic wasps possess many effectors to regulate host phy- miR-2989 restored host immune responses and decreased the Psiological mechanisms during parasitization to ensure their pupation rate of wasps. These results demonstrate that miRNAs offspring successfully develop in the host. These include the produced by SmBV can inhibit the host immune responses to production of venoms, symbiosis with polydnaviruses (PDVs), and enable the successful growth of wasp eggs in the host. the release of teratocytes into the host body during egg hatching1–5. These parasitic factors help the wasp progeny to grow and to Results develop within the host by regulating the host immune responses, SmBV genome encodes abundant potential miRNAs that are inhibiting host growth and development, affecting endocrine hor- – associated with different physiological genes in the host. After mone levels, and regulating host nutrient metabolism6 9.PDVsare S. manilae had parasitized the host S. litura larva, SmBV was viruses belonging to the Polydnaviridae family and are divided into delivered into the host (Supplementary Fig. 1a). This resulted in two genera, bracoviruses (BVs) and ichnoviruses (IVs), according the inhibition of both cellular and humoral immunity, as reflected to its associated host. PDV genomes are circular double-stranded by the decrease in phagocytosis (Supplementary Fig. 1b) and the DNA and are around 190–500 kb in size. They are divided into decrease in expression of toll-7 and cecropin in the Toll pathway different segments and packaged into the capsid, forming viral – (Supplementary Fig. 1c). Since the expression of toll-7 was particles harboring different gene segments2,10 13. The association inhibited the most after 36 h of parasitism (Supplementary of PDVs with wasps enables the successful growth of the wasp in its Fig. 1c), the small RNA expression profile in S. litura larvae was host. During wasp oviposition, PDV particles enter the host along analyzed at 36 h post-parasitism using small RNA Hiseq next- with wasp eggs and express viral genes by adopting the host’s generation sequencing (Supplementary Fig. 2a–c). Clean miRNA transcription systems. These viral genes may alter the host phy- reads could be mapped to 855 known mature miRNAs in the siology and divert energy metabolism to providing energy to the – databases, of which 422 miRNAs were only expressed after wasp larvae, subsequently inhibiting host metamorphosis14 16, parasitism (accounting for 49.4% of the total) and 173 miRNAs and/or the host immune systems to provide a favorable environ- were exclusively expressed in uninfected hosts (accounting for ment for the wasp progeny6,17,18. 20.2% of the total) (Fig. 1a and Supplementary Data 1). There- Several studies have demonstrated that PDVs can regulate fore, it was hypothesized that certain miRNAs were produced the developmental functions of their hosts. The genome of when S. manilae parasitizes S. litura. The predicted targets of the TrIV, a PDV, contains a TrV family gene that has been miRNAs only expressed after parasitism were classified by Gene demonstrated to suppress the proliferation of host cells, thus Ontology analysis. The target genes of these miRNAs are involved inhibiting host development19. In a recent study, it was found in diverse biological processes (Fig. 1b), located in various cellular that CvBV expressed miRNAs that could inhibit ecdysone compartments from the extracellular matrix, extracellular region receptor (EcR) expression, resulting in delayed development of part, extracellular region, to the membrane part (Fig. 1c), and its host, Plutella xylostella20. PDVs have previously been shown have different molecular functions, including translation reg- to inhibit both the cellular and humoral immunity of the ulator activity, molecular transducer activity, receptor activity, host21. The former includes inhibition of encapsulation and a and nucleic acid binding transcription factor activity (Fig. 1d and decrease in cell adhesion that inhibits phagocytosis22,23,both Supplementary Fig. 2d). The target genes with functions related to of which result in the induction of apoptosis and, in severe the host immune system were selected for analyzing the differ- cases, the disruption of hemocytes. PDVs have been found to ential expression of their corresponding miRNAs. A total of 113 inhibit NF-κBs and to impair host innate immunity by miRNAs were found to target immune-related genes (Supple- blocking signaling pathways and inhibiting antimicrobial mentary Data 1) and those with a normalized log ratio (PSL-36/ protein syntheses or melanization in the host24,25. 2 HSL-CN) higher than 10 were selected. These selected miRNAs miRNAs are small RNA molecules (~22 nucleotides) that form were analyzed using miRbase to determine their known function stem-loop structures and function as silencers, i.e., they downregulate and structure; it was found that they mostly target genes related gene expression. miRNAs may target the promoter of messenger to the innate immune pathway or cellular immunity. For RNAs or degrade messenger RNAs26.ManymiRNAfunctionshave instance, miR-2989 targets the upstream genes, and miR-3871-5p, been identified in insects and are involved in metabolism, immune – miR-3552, and miR-8 affect the downstream AMP gene in the responses, social behavior, and virus-host interactions26 29.The Toll pathway of innate immunities. In the JAK/STAT pathway, parasitization of Diadegma semiclausum in Plutella xylostella larvae miR-151-5p, miR-34a-3p, miR-2808a-3p, miR-1621-5p, and was found to change the miRNA profile in the host30.Another miR-199b-5p target the upstream genes, miR-291a-5p simulta- parasitic wasp, Cotesia vestalis, and its associated bracovirus, CvBV, neously affects the upstream and downstream genes, and miR- produces miRNAs in P. xylostella larvae to alter the host physiology 219-3p affects the downstream AMP gene (Fig. 1e). In total, 11 and delay its growth20. So far, there is no evidence of PDV-produced miRNAs were selected for further study (Supplementary Table 1). miRNAs to regulate host immune responses. The endoparasitic wasp Snellenius manilae (Hymenoptera: Braconidae) is highly host-specific for Noctuidae larvae31. S. SmBV-derived miR-199b-5p and miR-2989 are expressed in manilae has a symbiotic relationship with a polydnavirus, S. parasitized hosts.Toconfirm the expression of the 11 miRNAs manilae bracovirus (SmBV). In this study, we investigated whe- obtained through NGS in S. litura after parasitism, a stem-loop ther the non-coding regions in the SmBV genome produce qPCR was used to compare the expression levels of the miRNAs in miRNAs along with their potential in regulating host immune parasitized larvae. Most miRNA candidates presented a lower or functions. To this end, next-generation sequencing (NGS) was comparable expression level to that of control let-7 miRNA; however, exploited to screen for miRNAs only expressed by SmBV in the miR-3552 and miR-2989 targeting the drosomycin and toll genes, host. Functional analysis of these miRNAs revealed their parti- respectively, in the Toll pathway and miR-199b-5p targeting dome- cipation in regulating different physiological mechanisms such as less in JAK/STAT pathway exhibited higher expression levels growth, metabolism, and immune responses. Specifically, we (Fig. 1f). To further confirm that these miRNA candidates were found that miR-199b-5p and miR-2989 inhibited the host derived from SmBV, their expression levels in SmBV-infected S. immune genes domeless and toll, respectively, and affected litura cell lines (SL1A cells) were analyzed. The results showed that encapsulation and phagocytosis in a cellular immunity assay. miR-199b-5p and miR-2989 were highly expressed at 48 h after Treatment using miRNA inhibitors targeting miR-199b-5p and SmBV infection, but the expression of miR-3552 was undetectable

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Fig. 1 Different miRNAs expression in parasitized and unparasitized S. litura.amiRNAs produced in unparasitized S. litura (HSL-CN) and S. manilae- parasitized S. litura (PSL-36; 36 h post-parasitism). Genes that may be affected by mature miRNAs expression in S. manilae-parasitized S. litura were identified by miRBase and classified according to their involved biological processes (b), cellular compartments (c), and molecular functions (d). e miRNAs produced in S. litura after S. manilae parasitism with gene targets predicted in the Toll and JAK/STAT pathways. f The expression levels of 11 predicted miRNAs were quantified by a stem-loop qPCR in S. litura third-instar larvae 48 h after S. manilae parasitism. The expression of let-7 miRNA serves as a positive control. g The expression levels of 11 predicted miRNAs were quantified using stem-loop qPCR in SL1A cells 48 h after SmBV infection, with let-7 miRNA as the positive control. A Ct value of 35 was set to be the cutoff for detection. h miR-199b-5p and miR-2989 are present on MdBV genome segments I and K. The red bars represent the locations of the two precursors. At least three repetitions were conducted for each group. p-value were calculated using Student’s t-test (*p < 0.05).

(Fig. 1g). To eliminate the possibility that these miRNAs were miR-199b-5p and miR-2989 are the main miRNAs suppressing derived from either S. litura or S. manilae, their expression levels the host immune system and target the JAK/STAT and Toll were analyzed in the following: S. litura injected with purified SmBV pathways, respectively. miR-199b-5p and miR-2989 were pre- (Supplementary Fig. 3a), female S. manilae alone (Supplementary dicted to target the genes domeless and toll-7, respectively. The Fig. 3b), and both parasitized and unparasitized S. litura (Supple- expression levels of domeless and toll-7 in SL1A cells infected with mentary Fig. 3c–d). Expression of miR-199-5p and miR-2989 was SmBV decreased at 48 h post infection compared to non-infected detected only in S. litura injected with purified SmBV or parasitized cells and cells in the initial stage of infection (Fig. 2a), suggesting that with the wasp. To further confirm the results from stem-loop PCR, a immune responses were inhibited upon SmBV infection. To con- northern blot analysis was performed to detect the expression of firm that miR-199b-5p and miR-2989 contributed to the decreased either the mature miRNA or the precursor miRNA of miR-199b-5p expression of domeless and toll-7, miR-199b-5p and miR-2989 and miR-2989. Our results showed that both mature and precursor mimics and a negative control miRNA were transfected into SL1A miRNAs were detected in SmBV infected or parasitized S. litura cells, respectively. Quantitative PCR results showed that miR-199b- (Supplementary Fig. 3e). However, they were not detected in non- 5p and miR-2989 mimics inhibited the gene expression of domeless parasitized S. litura (Supplementary Fig. 3e). These results indicated and toll-7, respectively (Fig. 2b), proving that miR-199b-5p and that the SmBV was indeed processed into miRNAs during the miR-2989 can downregulate the expression of these immune genes. infection of S. litura. A bioinformatics analysis was also performed, To assess whether the observed regulations resulted from direct and no corresponding precursor sequences were found on the miRNA base paring, the 3′UTR of both target genes (i.e., domeless genome of S. litura,confirming that miR-199b-5p and miR-2989 and toll-7) were fused to the 3′-end of an egfp gene to generate EGFP were derived from SmBV. reporter plasmids pKShE-199b-5p 3′UTR and pKShE-2989 3′UTR, We mapped these two miRNAs in the genome of MdBV, a respectively (Fig. 2c). Co-transfection of these EGFP reporter plas- close PDV to SmBV, and found these two miRNAs were mapped mids with corresponding miRNAs decreased EGFP levels as eval- to the proviral segments I and K, respectively, on the MdBV uated by Western blotting analysis (Fig. 2d) and fluorescence genome (Fig. 1h). These results show that miR-199b-5p and miR- intensity (Fig. 2e); this further confirmed that the expression of 2989 were derived from SmBV and were highly expressed only domeless and toll-7 were suppressed by miR-199b-5p and miR-2989, after SmBV entered the host. respectively.

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Fig. 2 miR-199b-5p and miR-2989 inhibit the humoral immunity pathway. a Expression levels of humoral immunity genes, domeless (Dome) and toll-7 (Toll-7), were quantified by qPCR in SL1A cells 48 h after infection with SmBV. b Expression levels of domeless (Dome) and toll-7 (Toll-7) were quantified by qPCR in SL1A cells 48 h after transfection with mimic of miR-199b-5p and miR-2989, and a control miRNA, respectively. c Reporter plasmids pKShE- 199b-5p 3′UTR and pKShE-2989 3′UTR in which the 3′-UTR of Domeless or Toll-7 were fused to the 3′-end of an EGFP coding region, respectively. These two reporter plasmids were transfected into SL1A cells with corresponding miRNA mimic or a control miRNA (Control). EGFP expression was detected at 48 h after transfection and quantified by d western blotting analysis and e fluorescent microscopy. Genes in JAK/STAT (f) and Toll (g) pathways decreased in expression after the transfection of miR-199b-5p- or miR-2989 mimic. The expression levels of genes in the signal cascade of the JAK/STAT pathway [domeless (DOME), STAT (STAT), and TEP (TEP)] and the Toll pathway [toll (TOLL), myd88 (MYD88), pelle (PELLE), dif (DIF), and drosomycin (DRO)] were determined by qPCR in SL1A cells transfected with miRNA mimics of miR-199b-5p and miR-2989, respectively. An actin signal was used as an internal control to normalize all readings. All experiments were performed with three biological replicates (n = 3). Data are expressed as the mean and standard deviation (SD). p-value were calculated using Student’s t-test (*p < 0.05).

The effects of miR-199b-5p and miR-2989 on the expression of inhibitor were synthesized. The specificity between miRNA other important genes downstream of the JAK/STAT and Toll mimic and its miRNA inhibitor was demonstrated by transfecting pathways, were also analyzed by transfecting miR-199b-5p and miRNA mimic and EGFP reporter plasmid into SL1A cells with miR-2989 mimics into SL1A cells. Gene expression analysis result or without the corresponding miRNA inhibitor. Transfection of showed that the expression of three immune genes in the JAK/ either miR-199b-5p- or miR-2989 mimic alone decreased the STAT pathway (domeless, STAT, and TEP) and five immune EGFP level, while co-transfection of miRNA mimic and its genes in the Toll pathway (toll, myd88, pelle, dif, and drosomycin) matching inhibitor did not have an effect on the EGFP level were decreased (Fig. 2f–g). These results demonstrate that two (Supplementary Fig. 4), thus demonstrating the specificity. miR- miRNAs derived from SmBV alter host humoral immunities by 199b-5p- or miR-2989 inhibitor were microinjected into the targeting upstream genes in the JAK/STAT and Toll pathways. hemolymph of SmBV-infected third-instar S. litura larvae. The The expression of the downstream genes was also reduced, and phagocytosis assay result showed that phagocytosis capacity was this may subsequently affect cellular immunities such as restored in insects injected with miR-199b-5p or miR-2989 encapsulation and phagocytosis that are induced by the down- inhibitor compared to insects injected with negative control stream gene expression. inhibitor or solely with virus infection (Fig. 3a, b). Similar results were obtained using flow cytometry to measure the phagocytic hemocytes (Supplementary Fig. 5a, b). An encapsulation assay miR-199b-5p and miR-2989 affect phagocytosis and encapsu- in vitro was performed to determine the binding of multiple lation in cellular immunity. Phagocytosis and encapsulation play hemocytes to foreign particles. It was found that hemocytes from important roles in cellular immunity in insects. They are induced insects injected with miR-199b-5p- or miR-2989 inhibitor by STAT in the JAK/STAT pathway and Pelle and Dif in the Toll increased the binding to Sephadex A-25 beads added into the cell pathway32. To examine the effects of miR-199b-5p and miR-2989 culture, compared to hemocytes added with NC inhibitor or with on cellular immunity, miR-199b-5p inhibitor and miR-2989 virus infection (Fig. 3c). In vivo encapsulation assays were also

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Fig. 3 Inhibition of SmBV miRNA expression increases cellular immune responses in S. litura. Third-instar S. litura larvae were injected with SmBV and miR-199b-5p or miR-2989 inhibitor; hemocytes were extracted 36 h after the microinjection. a The hemocytes were mixed with FITC-labeled E. coli and the phagocytosis activity of hemocytes was determined by green fluorescence emitted from the ingested E. coli. b The phagocytosis ratio (%) was derived from the proportion of FITC to DAPI; FITC can be seen as green fluorescence detected from FITC-labeled E. coli and DAPI can be seen as blue fluorescence detected from stained hemocytes. (The p-value of miR-199b-5p inhibitor: 0.00548445 and miR-2989 inhibitor: 0.04668200). c Encapsulation assay determining binding of multiple hemocytes to the Sephadex A-25 beads added in the cell culture. The encapsulation rate was calculated by KP assay. Bottom: representative images of one of the Sephadex A-25 beads added in each cell culture. (The p-value of miR-199b-5p inhibitor: 0.00078001 and miR- 2989 inhibitor: 0.00019559). d Encapsulation of C.elegans in the hemocoel of S. litura. 24 h after injection, encapsulated nematodes were recovered from S. litura. (The p-value of miR-199b-5p inhibitor: 0.00491668 and miR-2989 inhibitor: 0.00416911) NC inhibitor: negative control inhibitor. All experiments were performed with eight biological replicates (n = 8). Data are expressed as the mean and standard deviation (SD). p-values were calculated using Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.0005). performed to determine the binding ability of hemocytes to coli K-12 strain (1 × 105 colony-forming unit (CFU)) was injected nematodes. The nematodes were injected into S. litura larvae with into third-instar S. litura larvae. The survival rate of injected different treatments and were flushed out from S. litura hemocoel insects was monitored every hour for up to 144 h (Fig. 4c). A p- 24 h after the injection. We found that nematodes from the hosts value of less than 0.05 was considered statistically significant. injected with miR-199b-5p- or miR-2989 inhibitor were bound by Prior injection of either miR-199b-5p mimic (p < 0.001) or miR- the hemocytes of the host. (Fig. 3d). These results suggest that 2989 mimic (p < 0.01) significantly decreased the survival rate of miR-199b-5p and miR-2989 are responsible for the reduced cel- S. litura after infection with E. coli K-12 as compared to S. litura lular immunity in S. litura. not injected with miRNA mimic or injected with control mimic To further ensure that these two SmBV-encoded miRNAs (Fig. 4c). At 144 h, the survival rate of control mimic larvae was function to suppress host innate immunity, miR-199b-5p- or 50%, while that of larvae injected with either miRNA mimic was miR-2989 mimic was injected into third-instar larvae of S. litura less than 10%. These results demonstrate that miR-199b-5p and to assess their effects on immune gene expressions and immune miR-2989 can inhibit immune responses against foreign patho- responses, e.g., phagocytosis and encapsulation. Gene expression gens and increase mortality from E. coli infection. analysis result showed that the expression of immune genes in the JAK/STAT pathway and the Toll pathway were decreased after miRNA mimic injections (Supplementary Fig. 6). This, in turn, Inhibitors of miR-199b-5p and miR-2989 suppress the pupa- contributed to the suppressed phagocytic and encapsulation tion of S. manilae, resulting in normal growth of parasitized S. activity in insects injected with miR-199b-5p- or miR-2989 mimic litura larvae. Since the treatment of miR-199b-5p and miR-2989 (Figs. 4a, b). To further assess the effect of these two miRNA inhibitors restored the cellular immunity of S. litura, we further mimics on host immune responses to foreign pathogens, 24 h examined whether the immune suppression by miRNAs influ- after the microinjection of miR-199b-5p- or miR-2989 mimic, E. ences the growth status of parasitized S. litura larvae and the

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Fig. 4 SmBV-encoded miR-199b-5p and miR-2989 suppress cellular immune responses in S. litura. Third-instar S. litura larvae were microinjected with SmBV, miR-199b-5p or miR-2989 mimic. a Phagocytosis activity of injected S. litura larvae; green fluorescence is emitted from the ingested E. coli by hemocytes. The phagocytosis ratio (%) was derived from the ratio of FITC to DAPI. (The p-value of SmBV: 0.00210683, NC mimic: 0.30700539, 199b-5p mimic: 0.03159918, 2989 mimic: 0.04655791). b Encapsulation assay showing binding of multiple hemocytes to Sephadex A-25 beads added to the cell culture. The encapsulation rate was calculated by KP assay. Bottom: representative images of the Sephadex A-25 beads added to each cell culture. (The p- value of SmBV: 0.00168336, NC mimic: 0.25959650, 199b-5p mimic: 0.01188009, 2989 mimic: 0.00211812). c Survival rate of larvae in response to infection by E. coli K12. Kaplan–Meier survival curve with log-rank test (Wilcoxon–Breslow–Gehan Test) comparing survival of S. litura larva infected with E. coli K12. A p-value of less than 0.05 was considered statistically significant. Pairwise comparison: with NC mimic vs. with miR-199b-5p, p < 0.0001; with NC mimic vs. with miR-2989, p < 0.01. NC mimic: negative control mimic. All experiments were performed with five biological replicates (n = 5). Data are expressed as the mean and standard deviation (SD). p-value were calculated using Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.0005). successful pupation of S. manilae in S. litura. The determination showed that neither miRNAs could suppress development of body length and head capsule width of S. litura showed that (Fig. 5f–h). In conclusion, our results demonstrated that inhi- control larvae without any treatment presented normal develop- biting the actions of both SmBV-encoded miRNAs by miRNA ment, whereas parasitized larvae without treating with miRNA inhibitors restored the host immune responses, thus suppressing inhibitor exhibited arrested development (Fig. 5a–c). Notably, the S. manilae development in the host. parasitized larvae injected with each miRNA inhibitor presented normal development, in contrast to the parasitized larvae injected with inhibitor control (Fig. 5a–c). In addition, treatments of Discussion miRNA inhibitor in parasitized S. litura larvae suppressed the PDVs have been shown to assist parasitoid wasps in successfully development of S. manilae larvae. The pupation rates of S. parasitizing their hosts. PDVs produce protein products that can manilae larvae in miRNA inhibitor treatment groups were lower directly block or compete for receptors in the physiological and than 20% in comparison to the nearly 95% pupation rate of metabolic pathways of the host, causing delayed growth and control groups (Fig. 5d). The encapsulation activity of S. manilae arrested development18,33. PDVs also express viral proteins to egg and larvae was higher in both miRNA inhibitor treatment disrupt the adhesion and dissemination of immune cells, which groups than in the control groups (SmBV infected or SmBV disable phagocytosis and encapsulation in hemolymph18,22,23,34–36. infected with inhibitor control) (Fig. 5e). These results confirmed Furthermore, PDVs inhibit melanization and humoral immunity that miRNA inhibitor treatments recovered the host cellular pathways, resulting in the inhibition of AMP synthesis by the immune responses, which obstruct the development of S. manilae host3,21,25,37. In addition to viral genes and proteins affecting host larvae. To further ensure that both SmBV-encoded miRNAs functions, non-coding genes of viruses have also been shown to suppress host innate immunity rather than larval developmental play important regulatory roles. Recently, miRNAs produced by pathways, we injected each miRNA mimic into second-instar PDVs were shown to delay the growth of the host20. In this study, larvae of S. litura and assessed their development. The results we demonstrated that miRNAs encoded by the non-coding regions

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Fig. 5 Inhibition of SmBV miRNA expression decreases S. manilae parasitism rate. a Third-instar S. litura larvae were microinjected with miRNA inhibitors before parasitism by S. manilae wasps for 48 h. Arrow: S. manilae. The developments of S. litura larvae were measured by their b body sizes (lengths) and c head capsule width after parasitism (Control: unparasitized S. litura) (Green square: Control, orange circle: Parasitized, gray star: Parasitized + NC inhibitor, yellow triangle: Parasitized + miR-199b-5p inhibitor, blue diamond: Parasitized + miR-2989 inhibitor). (The p-value in b on 3–9 days post infection are 0.00000635, 0.00000006, 0.00001210, and 0.00000030) (The p-value in c on 5–9 days post infection are 4.79293E−28, 0.00020260, and 0.00038115). d The pupation rate of S. manilae wasps in S. litura (n = 3) over 10 continuous days. The number of wasps that successfully pupated in the group without miRNA inhibitor injection was set as 100%. (The p-value of Control: 0.00075194, miR-199b-5p inhibitor: 0.00075194, and miR-2989 inhibitor: 0.00561026). e Encapsulation assay determining binding of multiple hemocytes to S. manilae eggs (upper panel) and 4-day-old S. manilae larvae (lower panel) added in the cell culture. Arrow: encapsulated and melanized S. manilae egg or larvae. f Second-instar S. litura larvae were microinjected with miRNA mimics and monitored over a 9 days period post-injection. (Control: S. litura without miRNA mimic injection.) The developments of S. litura larvae were measured by their g body sizes (lengths) and h head capsule width after miRNA mimic injection (Green square: Control, yellow triangle: miR-199b-5p mimic, blue diamond: miR-2989 mimic). All experiments were performed with three biological replicates. Data are expressed as the mean and standard deviation (SD). p-value were calculated using Student’s t-test (***p < 0.005). of SmBV play key roles in the suppression of the host’s immune viruses may suppress host immunity and enable wasps to evade systems, which eventually benefits the pupation of S. manilae. the host immune responses. In SL1A cells parasitized by wasps or At present, approximately 300 types of viruses are known directly infected by SmBV, the miRNAs miR-199b-5p and miR- to produce miRNAs, including herpes simplex virus, baculovirus, 2989 were both found to downregulate immune response path- adenovirus, and nudivirus38,39. These viral miRNAs play ways and subsequently suppress the immune systems of S. litura. key roles in virus-host interactions40,41 as they can directly These two miRNAs targeted the Domeless in the JAK/STAT change host physiology and interfere with host defense pathway and Toll receptor in the Toll pathway, indicating that mechanisms26,29,42. For instance, viral miRNAs KUN-miR-1 and PDVs can transcribe miRNAs to regulate the different routes of OvHV-2-miR-5 can affect host gene expression and up-regulate host immune responses. It has previously been reported that IEP1 virus infectivity and replication in the host38. Previous studies and IEP2 produced by CkBV and CpBV-lectin molecule pro- have noted the changes in the expression levels of some miRNAs duced by CpBV interfered with the recognition and adhesion of before and after the parasitism of Lepidoptera larvae by parasitic wasp eggs by immune cells (granulocytes, plasmatocytes, and wasps; however, the functions of these miRNAs remained oenocytoid cells) in the host hemolymph44,45. Additionally, unclear30,43. In this study, miRNAs that are specifically expressed MdBV has been found to produce GLC, PTP, and ANK proteins in S. litura after parasitism by S. manilae and SmBV were iden- to decrease immune cell dissemination and adhesion13,23,24,46–49. tified by small RNA Hiseq NGS. Those that were involved in In the present study, impaired phagocytosis and encapsulation immune processes were further analyzed. It was found that the from hosts infected with SmBV was observed (Fig. 3). Injection of expression levels of miRNAs inhibiting immune responses were miR-199b-5p and miR-2989 inhibitors into host hemolymph higher than those involved in other processes, suggesting that restored phagocytosis and encapsulation (Fig. 3b, c), thus proving

COMMUNICATIONS BIOLOGY | (2021) 4:52 | https://doi.org/10.1038/s42003-020-01563-3 | www.nature.com/commsbio 7 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-01563-3 that miR-199b-5p and miR-2989 are indeed the suppressors of concentration of stock plasmid DNA was measured using a NanoDrop these host immune responses. 2000 spectrophotometer (Thermo Fisher Scientific) and the amount of DNA sample was determined to be equivalent to 1010 plasmid copy number using the During induction of encapsulation, Dif in the Toll pathway fi κ DNA Copy Number and Dilution Calculator (Thermo Fisher Scienti c). Ten-fold induces NF- B to activate encapsulation, whereas STAT in the serial dilution of the DNA sample ranging from 1010 to 101 plasmid copy number JAK/STAT pathway increases the encapsulation signals. Thoet- was performed, and the serially diluted samples were used as templates in qPCR. A kiattikul et al. reported that a PDV expressed two inhibitor kappa standard curve was obtained by plotting diluted template DNA to the corre- 53 6 B(IκB)-like proteins, H4 and H5, from the ANK gene family to sponding Ct value . To infect S. litura larvae, SmBV solution containing 10 virus copy number was injected into second-instar larvae. The expression level of SmBV- inhibit the expression of Dif in the Toll pathway and Relish in the encoded miRNAs was analyzed 36 h post-injection by stem-loop qPCR. Imd pathway25, eventually suppressing the encapsulation activity κ 17,50 and humoral immunities related to NF- B . This is similar to Small RNA Hiseq next-generation sequencing. We collected a total of 20 the findings presented in the present study, namely that the second-instar S. litura: 10 were parasitized by S. manilae and 10 were unpar- products expressed by PDVs effectively suppress the insect asitized. Total RNA was extracted using Trizol reagent (Geneaid), and small RNAs immune system and suggests that PDVs possess mechanisms to (~18–30 nt) were isolated by a denaturing polyacrylamide gel electrophoresis (PAGE) as per the method of Lagos-Quintana et al.20,54,55. Small RNA sequencing alter the host immune system to provide a survival advantage. libraries were constructed using a TruSeq Small RNA Library Preparation Kit The present study identified miRNAs (miR-199b-5p and miR- (Illumina) and sequenced using an Illumina HiSeq 2000/2500. The obtained 2989) transcribed by an SmBV which can directly alter humoral sequences were aligned with various known bioinformatics databases to classify immunity by decreasing the expression of immune genes. miR- and annotate the small RNAs. Non-annotated small RNAs were further analyzed by MIREAP and miRDeep to predict potential new miRNAs. Target genes were 199b-5p and miR-2989 produced by SmBV not only suppressed predicted for miRNAs using RNAhybrid, miRanda, TargetScan, and PITA. We humoral immunity but also indirectly blocked STAT and Dif analyzed their differential expression and predicted the loci of these target genes. signal transduction, decreasing phagocytosis and encapsulation Additionally, we analyzed the gene functions and pathways involved using Gene (Fig. 6). To our knowledge, this is the first report showing that Ontology functional annotation and KEGG pathway annotation. miRNAs derived from a PDV can target the host immune system, and that this immune suppression benefitted the parasitism of S. Stem-loop qPCR. miRNA cDNAs were synthesized from total RNA using fi manilae larvae within the S. litura host. miRNA-speci c stem-loop primers (Supplementary Table 2) according to previously described criteria56. Expression of the mature miRNA was analyzed by qPCR as described previously57. Briefly, cDNA was diluted 10 times and added into fi Methods a qPCR reaction mixture with a nal volume of 20 µL, containing 2× SYBR Green qPCR Master Mix (Fermentas), 0.5 µM each of forward and reverse primers, 1 μL Insects . Spodoptera litura were reared in cages and kept in growth chambers at 29 of cDNA, and 7 μL of ddH O. The qPCR was performed with the following pro- ± 1 °C with a photoperiod of 12:12 h (light: dark). The main components of the 2 fi gram: 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Expression of let-7a, a arti cial diet consisted of 90 g kidney bean powder, 36 g yeast extract, and 33 g miRNA normally expressed in most insect cells, was used as a positive control. wheat germ blended first with 480 ml of distilled water, and then with an additional 60 ml of nutrition mix (0.36g L-cysteine, 3.6 g L-ascorbic acid, and 0.225 g strep- tomycin sulfate). We then mixed 9.9 g agar powder and 300 ml distilled water in a Total RNA extraction, cDNA synthesis, and qPCR. The total RNA of miRNA 1000 ml beaker to form an agar mixture that was dissolved and boiled in a mimic-injected S. litura or miRNA mimic transfected SL1A cells was extracted microwave. The agar mixture was allowed to cool down to 70 °C before it was using Trizol reagent (Geneaid)58. Samples were homogenized with 1000 μLof added to the artificial diet mixture. Finally, 3 ml propionic acid was added to the Trizol reagent, then kept at room temperature for 5 min. 200 μL of chloroform was artificial diet mixture to prevent mold growth. The mixture was then poured into a added into homogenized samples and vortex for 15 s. After incubated for 3 min at plastic tank to cool, and thereafter stored in a 4 °C refrigerator.S. manilae were room temperature, the samples were centrifuged at 12,000 × g for 15 min at 4 °C. reared in growth chambers at 29 ± 1 °C with a photoperiod of 12:12 h (light: dark). The aqueous phase was transferred into a new eppendorf, then added 500 μLof Glucose solution (15%) was used for feeding51. 100% isopropanol and incubated for 10 min at room temperature. The RNA pellet was spin down at 12,000 × g for 15 min at 4 °C and washed by 1 mL of 75% ethanol two times at 7500 × g for 5 min at 4 °C. Finally, RNA pellet was dried at SpinVac at Cells, SmBV particles extraction, and viral copy number determination. Spo- 45 °C for 3 min and dissolved in 50 μL of RNAse-free water. The concentration of doptera litura SL1A cells were cultured in TC-100 medium (USBio) containing the extracted RNA was determined using the Nanodrop 2000. cDNAs were syn- 10% FBS (Gibco BRL) and cultured in a 26 °C incubator. SmBV virions were thesized using a PrimeScript™ RT Reagent Kit (Takara) with the following reaction collected from the ovaries of female S. manilae wasps, as previously described3. The components: 500 ng of RNA, 2 μL of 5× PrimerScript™ buffer, 0.5 μL of Primer- ovaries were dissected in pre-chilled phosphate-buffered saline (PBS), and the calyx Script RT Enzyme Mix I, 0.5 μL of Oligo dT primer, and 0.5 μL of random hex- was punctured to release the content into the PBS. The solution was filtered amers. Quantification of the expression of miRNA target genes was performed through a 0.45-μm filter, and the filtrate was centrifuged at 20,000 × g for 1 h52. using SYBR green (Bioline)-based qPCR on an ABI Plus One real-time system After centrifugation, the resulting pellet containing the virus was re-suspended in (StepOnePlus, Applied Biosystems). Each qPCR reaction contained 10 μL of SYBR PBS. A plasmid containing the C fragment of SmBV was constructed to serve as the green reagent, 0.4 μM each of forward and reverse primers, 2 μL of cDNA, and μ standard in qPCR to quantify the genome copy number of SmBV. The 6.4 L of ddH2O. The qPCR cycling conditions were as follows: stage 1, 95 °C for

Fig. 6 SmBV-encoded microRNAs as regulators in host immune responses. Upon the parasitism of wasp S. manilae to its host S. litura, the symbiotic SmBV integrates viral DNA into the host genome in hemocytes, and subsequently expresses miR-199b-5p and miR-2989 that inhibit signal transductions in JAK/STAT and Toll pathways, respectively. These suppressions indirectly block the function of STAT and Dif, alongside AMP synthesis inhibition, resulting in decreased immunity in plasmatocytes and increased pupation rate of the wasp larvae in the host.

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10 min; stage 2, 40 cycles of 95 °C for 15 s and 60 °C for 1 min; stage 3, 95 °C for 15 observe S. manilae larvae encapsulation, we allowed S. manilae to parasitize s. A list of primer sequences used in this study is given in Supplementary Table 3. second-instar S. litura and dissected them after 4 days to retrieve the S. manilae larvae. Following two to three washes in PBS, 50 μL of hemocytes was mixed with S. manilae larvae and added to individual wells of a 96-well plate. The 96-well plate Plasmid construction. EGFP reporter plasmids pKShE-199b-5p 3′UTR and was then sealed with Parafilm (M® All-Purpose Laboratory Film Bemis) and cul- pKShE-2989 3′UTR were constructed using the plasmid pKShE in which an EGFP tured at 26 °C for 30 min. Encapsulation was observed by a microscope (Nikon gene is driven by the hsp 70 promoter in the backbone of pBluescript II KS(−) Inverted Microscope-Eclipse TS100) after incubation. Using Nikon NIS auto- (Stratagene)59,60. The 3′UTRs of domeless and toll-7 were amplified by PCR from measurement software, encapsulated objects were counted at 400x magnification. the cDNA of S. litura and inserted downstream to egfp in the plasmid pKShE to Each experimental sample was counted in three different fields under the micro- generate the plasmids pKShE-199b-5p 3′UTR and pKShE-2989 3′UTR, respec- scope, and the data was presented as the average number of encapsulated objects tively. The primer sets and 5′-GGTTAGTACCTATATGTGAATA-3′ and 5′-AGC from these 3 fields. ACGTCTATTGAATGTTGCTGTC-3′ were used for the 3′UTR of domeless and 5′-AGAGGCGTGGGCCAAACCGAC-3′ and 5′-CACAAATTCCCCCCTCGGC AA-3′ were used to amplify the 3′ UTR of toll-7. Encapsulation of nematodes in vivo. An aliquot of nematode (strain: N2 Bristol) suspension was autoclaved (121 °C, 1.2 atm, 20 min) to eliminate possible interference from symbiosed E. coli. Approximately 500 dead C. elegans L1 larvae in fl fi 5 DNA transfection and uorescence quanti cation. SL1A cells (2 × 10 cells per sterile M9 buffer were injected into a third-instar S. litura larva using a 1 mL well) were seeded per well in 24-well culture plates (Corning). 0.25 μg of reporter ″ 61 ′ ′ tuberculin syringe with a 26G × ½ needle . S. litura larva were dissected along the plasmids pKShE-199b-5p 3 UTR or pKShE-2989 3 UTR were transfected alone or centerline 24 h after injection and nematodes in the hemocoel were flushed out with 10 pmol of the corresponding miRNA mimic or control miRNA (Control) ’ using phosphate-buffered saline. The obtained nematodes were observed under a using Cellfectin (Invitrogen) according to the manufacturer s protocol (Gibco microscope and were counted as either unencapsulated or encapsulated. BRL). EGFP expression was detected at 48 h after transfection by a fluorometer, and the images of cells were captured by a fluorescent microscope. Bacterial injection. Third-instar larvae were cold-anesthetized prior to injection. A fine glass needle was inserted into the dorsal vessel and 1 μL (containing 1 × 105 Western blotting analysis. After reporter plasmid and miRNA mimic (10 pmol) colony-forming unit (CFU)) of cultured bacteria was injected into the hemocoel62. 5 transfection, SL1A cells (2 × 10 ) were placed in the wells of a 24-well plate, washed For survival curve, 150 larvae were divided into five groups: NC mimic-injected with μ twice with PBS buffer, and lyzed in RIPA lysis buffer (100 L). Sodium dodecyl E. coli K12, miR-199b-5p mimic-injected with E. coli K12, miR-2989 mimic-injected sulfate (SDS) sample buffer was mixed with the cell samples in a 1:4 ratio. The with E. coli K12, E. coli K12-injected only group, and an PBS medium-injected group mixture was then heated to 100 °C for 10 min and then separated on a 10% SDS- (Control). The survival rate of experimental larvae in each group was recorded every polyacrylamide gel. Proteins on the gel were transferred onto a PVDF membrane 12 h until 144 h post infection. Statistical tests were performed using the online tool (Millipore) by electroblotting for 1 h in transfer buffer. The PVDF membrane was OASIS 2, and weighted log-rank test (Wilcoxon–Breslow–Gehan test) for deter- blocked with PBS containing 5% skim milk, 0.05% Tween 20 for 1 h and incubated mining significance63. with primary mouse anti-GFP (Millipore) or anti-Actin (Millipore) antibodies for another 1 h. The membrane was washed three times with PBS containing 0.05% Tween 20 (PBST) and then incubated with secondary antibody conjugated with Statistics and reproducibility. The relative expression levels of RNAs or miRNAs −ΔΔCt horseradish peroxidase (HRP) for 1 h. After three washes with PBST, the mem- were calculated using the 2 formula in which Ct is the cycle threshold value of ’ brane was rinsed with enhanced chemiluminescence (ECL) substrate and the qPCR, and the expression of 18S was used as a reference gene. Student s t-test was luminescence signal was detected using X-ray film exposure. used to compare each treatment group with the control group. Groups with significant differences versus the control group are marked with an asterisk (*)(*p < 0.05; **p < 0.01; ***p < 0.005) in Figs. 1, 5, including gene expression, phagocytosis Injection of miRNA inhibitor or miRNA mimic in S. litura larvae. Two micro- assay, encapsulation assay, and pupation test. liters of miRNA inhibitor (10 pmol) was administered to third-instar S. litura larvae by microinjection (80 larvae per treatment). Then, each larva was infected Reporting summary with 10 S. manilae for 48 h to establish parasitism, after which time the S. manilae . Further information on research design is available in the Nature were removed. The number of S. manilae that successfully pupated was calculated; Research Reporting Summary linked to this article. this was considered to be the viability of S. manilae under phagocytosis and encapsulation. The body length and head capsule width of hatched larva were also Data availability measured. miRNA mimic (10 pmol) was microinjected into second-instar S. litura Source data for the NGS and those underlying plots shown in figures are provided in larvae (20 larvae per treatment) for assay development, phagocytosis and encap- Supplementary Data 1 and 2. Full blots are shown in Supplementary Information. All sulation. Body lengths and head capsule widths of hatched larva were measured other data generated during and/or analyzed during the current study are available from over a 9 days period post-injection. All miRNAs mimic or miRNA inhibitor were the corresponding author on reasonable request. synthesized by MDbio Inc. The miRNAs or mutant miRNAs used in this study are as follows: SmBV-miR-199b-5p (5′-CCCAGUGUUUAGACUAUCUGUUCtt-3′); SmBV- Received: 3 January 2020; Accepted: 18 October 2020; miR-2989 (5′-GCACGUGAUGAGAACUCUGUtt-3′); miR-Control (5′-UUCUCC GAACGUGUCACGU-3′); SmBV-miR-199b-5p inhibitor (5′-GAACAGAUAGU CUAAACACUGGG-3′); SmBV-miR-2989 inhibitor (5′-ACAGAGUUCUCAUC ACGUGC-3′); miR-Control inhibitor (5′-ACGUGACACGUUCGGAGAA-3′). References Phagocytosis assay. Third-instar larvae were injected with SmBV (1 × 106 virus 1. Strand, M. R. & Burke, G. R. Polydnavirus-wasp associations: evolution, copy number). Hemocytes were extracted 36 h post-injection and seeded onto a 96- genome organization, and function. Curr. Opin. Virol. 3, 587–594 (2013). well plate (4 × 104 cells/well). FITC-labeled E. coli (4 × 104 cells/well) (BioParticles® 2. Strand, M. R. & Burke, G. R. 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61. Ono, M., Arimatsu, C., Kakinoki, A., Matsunaga, K. & Yoshiga, T. Comparison of Additional information cellular encapsulation with nematodes in two lepidopteran insects. Appl Entomol. Supplementary information is available for this paper at https://doi.org/10.1038/s42003- Zool. https://doi.org/10.1007/s13355-020-00687-6 (2020). 020-01563-3. 62. Lee, J., Hwang, S. & Cho, S. Immune tolerance to an intestine-adapted bacteria, Chryseobacterium sp., injected into the hemocoel of Protaetia Correspondence and requests for materials should be addressed to Y.-L.W. brevitarsis seulensis. Sci Rep-Uk 6, 3172210 (2016). 63. Han, S. K. et al. OASIS 2: online application for survival analysis 2 with Reprints and permission information is available at http://www.nature.com/reprints features for the analysis of maximal lifespan and healthspan in aging research. Oncotarget 7, 56147–56152 (2016). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements We thank Mr. Alexander Barton for kindly revising the manuscript. This research was Open Access This article is licensed under a Creative Commons funded by the grant MOST107-2311-B-002-024-MY3 to Y.L.W. from the Ministry of Attribution 4.0 International License, which permits use, sharing, Science and Technology, Taiwan. adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Author contributions Commons license, and indicate if changes were made. The images or other third party Guarantors of the integrity of the entire study, study concepts, and manuscript pre- material in this article are included in the article’s Creative Commons license, unless paration: C.K.T., C.H.T., Y.H.L., and Y.L.W. Study design, data acquisition/analysis, indicated otherwise in a credit line to the material. If material is not included in the literature research, and manuscript preparation: C.K.T., C.H.T., C.P.W., S.C.W., C.H.Lu, article’s Creative Commons license and your intended use is not permitted by statutory and Y.L.W. Data acquisition/analysis, manuscript editing, and revision: C.K.T., C.H.T., regulation or exceeds the permitted use, you will need to obtain permission directly from C.P.W., Y.H.Lin, and Y.L.W. All authors reviewed the manuscript. the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Competing interests The authors declare no competing interests. © The Author(s) 2021

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