1. IntroductionAlthough the practice of mass-produced insects has been long-standing (e.g., silkworm farming, apiculture, biocontrol agents) [1,2], its application has recently expanded to include uses as livestock and pet feed ingredients [3,4], protein for human consumption [5], chitin for numerous industrial applications [6], and remediation of wastes [7]. Due to the increasing popularity of and demand for insect-based products, there are considerable efforts to maximize insect mass-production [8]. For example, within rearing facilities, microbial pathogens (e.g., viruses, bacteria, fungi) can cause significant mortality, in addition to reducing fecundity and body size. Thus, increasing disease prevention and resistance of insects is essential to the success of this burgeoning industry [9,10,11].Despite the threat that entomopathogenic infections pose to insect mass-production, we know little about disease prevalence in these settings as systematic screening efforts are currently absent. Crickets (family: Gryllidae) are an especially popular farmed insect; however, they are known to be susceptible to multiple microbial pathogens that can cause disease outbreaks [12,13,14]. For example, the Acheta domesticus densovirus (AdDNV), a parvovirus, was implicated to have caused large disease outbreaks in farmed house cricket (Acheta domesticus) colonies globally, resulting in wholesale product losses. As a direct response to these outbreaks, many producers switched to farming alternative species, including Gryllodes sigillatus in North America, due to reports that they are less susceptible to AdDNV [15]. Furthermore, the number of reports of covert (or, silent) viral infections has increased in reared populations as molecular screening of viruses has expanded to asymptomatic populations [13]. Covert infections by a broad range of microorganisms and other infectious agents are widespread and can manifest as latent infections (e.g., remain within the host cell or integrate into the host genome) or persistent infections with low levels of replication [16]. Importantly, covert infections may become activated resulting in detectable pathology, including mortality.Beyond agricultural and industrial applications, several species of crickets have long been a model organism within several fields of research (e.g., evolutionary ecology, ecological immunology, insect physiology), where immune effectors have been evaluated across numerous contexts [17,18,19,20,21,22,23,24,25,26,27,28,29]. From these studies, we know that crickets mount an immune response to some microbial pathogens by, for example, exhibiting increased lysozyme-like activity of their hemolymph [30,31], producing a melanization and/or encapsulation response [32,33], and/or increasing circulating hemocytes [34] after controlled exposure to live, inactivated, or simulated (e.g., nylon filaments) pathogens. Although these studies provide insight on the functional downstream outcomes of infection, few have identified and quantified immune gene expression in response to pathogens in these insects [35,36], which is foundational to understanding the molecular basis of their defensive repertoire. Additionally, we do not yet know how crickets respond to naturally acquired pathogenic infections, as almost all assessments have been conducted following controlled inoculation.Insects possess a suite of cellular and humoral immune defenses in response to viral infection [37] and most of what we know about gene expression as the basis of these defenses comes from work in Drosophila melanogaster [38,39], lepidopterans [40], and several mosquito species [41]. Once a pathogen is detected by the insect host, a series of immune signaling pathways are activated intracellularly to respond to infection with a certain degree of specificity, which is in part attributed to the binding specificity of pattern recognition receptors (PRRs) to pathogen associated molecular patterns (PAMPs) (e.g., lipopolysaccharides and peptidoglycans) [42]. The main signaling pathways that mediate immunity in insects are the Toll, Immune deficiency (Imd), and Jak/STAT pathways. Within the Toll pathway, microbes are detected by PRRs that activate the ligand Spätzle, which then binds to Toll receptors and transduces the signals to Cactus-Dif (Dorsal-related immune factor) through a signaling complex containing the adapter MyD88 [43]. Cactus is then cleaved from Dorsal and/or Dif which then translocate into the nucleus and regulate the transcription of effector genes [44]. Within Imd, PRRs recognize invading pathogens and activate the adapter molecule Imd, which activates Relish [45]. Relish is then cleaved and its DNA binding domain translocates into the nucleus where it regulates the transcription of effector molecules [46]. The Jak/STAT pathway is activated as a response to cell stress and/or viral and fungal infection. In this pathway, Domeless is activated and then associated kinases recruit and phosphorylate STAT, which translocates into the nucleus to regulate the expression of downstream effector genes [47]. Additionally, STAT is negatively modulated by PIAS [48]. Activation of these signaling pathways leads to the production of downstream effector molecules, such as antimicrobial peptides (AMPs), that suppress a range of microbes, including viruses [49]. Further, lysozyme is a particularly potent antimicrobial effector in many insects, including crickets [50]; however, the potential antiviral activity of lysozyme has not been widely investigated [51]. Beyond these canonical immune signaling pathways, the RNAi (RNA interference) pathway plays a significant role in the antiviral response in Drosophila [52] and has been linked to Jak/STAT, suggesting coordination between these responses [53]. In this pathway, viral dsRNA is recognized by Dicer-2 proteins, which dice it into small siRNAs (small interfering RNAs) which are then loaded into an RNA induced silencing complex (RISC) by Dicer-2 and co-factor R2D2. RISC finds the target transcripts (by complementary sequence with the guide strand of the siRNA) and the Argonaute-2 protein (effector protein of RISC) degrades the target transcript [54].Recently, we characterized viral abundance in reared G. sigillatus crickets from two populations infected with cricket iridovirus (CrIV; family: Iridoviridae): one in which crickets were host to high amounts of viral copies and suffered from increased mortality and reduced fecundity while the other showed no apparent signs of disease and had very few viral copies present [13]. Thus, we consider the diseased population as one exhibiting an overt infection while the healthy population exhibited a covert infection of CrIV. Covert infections of invertebrate iridoviruses are reportedly more prevalent than overt lethal infections in some insect population [55,56,57], which could be due to several factors, including reduced virulence of the virus or increased tolerance of the host. Here, we quantified the expression of immune signaling pathway genes that have been shown to be important in anti-viral immune responses of insects across these two populations of crickets. We selected targets across Toll (MyD88, Cactus, Dorsal, and Dif), Imd (PGRP-LC, Imd, and Relish), and Jak/STAT (Domeless, PIAS, and STAT5B) signaling pathways in addition to a gene encoding for lysozyme. We also measured expression of targets within the RNAi pathway (Dicer-2, R2D2, and Argonaute-2). Finally, because the microbiome has been shown to influence viral dynamics in other insects [58], we quantified the amount of bacteria and fungi present via amplification of the 16S rRNA gene for bacteria and 18S rRNA for fungi to determine if total microbial load plays a role in viral dynamics. We also present TEM images of CrIV viral capsids to confirm active infection within crickets with an overt infection. 4. Discussion

Despite its importance in host response to pathogens, we still know little about the molecular basis of cricket immunity, and even less about immune responses within the context of covert infections. To improve our understanding of host–virus interactions in crickets, we evaluated canonical immune signaling pathways that have been shown in other arthropod systems to be involved in immunity to microbial organisms, including viruses. By quantifying gene expression across several facets of the invertebrate immune system, we have begun to characterize the immune response to overt cricket iridovirus (CrIV) infections in the popularly reared G. sigillatus cricket. Specifically, we found that crickets infected with high levels of cricket iridovirus had higher gene expression across the Toll, Imd, and Jak/STAT immune signaling pathways as well as within the RNAi pathway.

Activation of the Toll and Imd pathways are typically associated with defenses against pathogenic fungi, bacteria, and protozoa. Furthermore, these two pathways have been linked with the antiviral response in Diptera [73,74], but less is known about their role in orthopterans. Both Jak/STAT and RNAi pathways are known to play important roles in antiviral immunity and so it is not surprising that targets across these two pathways were upregulated in crickets with large CrIV viral loads in our study. Our study also evaluated an important antimicrobial effector, lysozyme, which has been found to have antiviral activity against dengue virus in mosquitoes [75] as well as against other viruses infecting eukaryotic hosts [51]. Our transcript level analyses show that the population of diseased crickets had significantly higher expression of lysozyme compared with the healthy population. This suggests that lysozyme might play a significant role in the cricket’s efforts to control the systemic replication of CrIV. Taken together, we can conclude that crickets with overt CrIV infection have an immune profile exhibiting strong induction of critical immune pathway components across Toll, Imd, Jak/STAT, and RNAi. At the same time, it begs the question of whether crickets tolerate viruses when they occur at a lower concentration and fully engage the immune system only when viral loads surpass a certain threshold. Unfortunately, our study is unable to fully answer this question given that our control (healthy population) also carried CrIV, albeit at significantly lower levels.A few previous studies have identified immune related genes in Orthoptera, including crickets; however, none to our knowledge have investigated an antiviral response in this order. An enzyme similar to the AMP prolixicin was discovered in A. domesticus suspected of being infected with a gregarine parasite and found to occur at higher concentrations in juveniles compared with adults [76]. In the black field cricket (Teleogryllus emma), researchers identified 58 differentially expressed unigenes and several AMPs following inoculation with E. coli [77]. In Gryllus bimaculatus, 4 inducible lysozymes and 6 AMPs were identified with similarities to defensin and diptericin, as well as pyrrhocoricin, prolixicin, and hemiptericin [78]. A comparative transcriptomic analysis of the immune response of migratory locusts challenged with Metarhizium fungi identified immune related unigenes including those involved with Toll, Imd, and Jak/STAT pathways, with 58 and 3 differentially expressed in the fat body and hemocytes, respectively [79]. It also found higher expression of lysozyme transcripts post-infection. While it is difficult to draw direct comparisons across these few studies, some patterns emerge, including the roles that the canonical immune signaling pathways and their effectors play in the Orthopteran immune system.Our findings add to the current understanding of the insect host immune response to iridovirus in crickets. Previous work has shown that lab reared Gryllus texensis crickets infected with an iridovirus have significantly lower phenoloxidase activity than uninfected crickets [80]. This contrasts with our findings of increased immune gene expression, but we did not assess any genes involved directly in the phenoloxidase cascade and phenoloxidase activity has been shown to be negatively associated with other components of immunity or reduced following immune activation in other insects [81]. Although, to our knowledge, there are no reports of studies that have investigated molecular markers of immune activation following an infection with CrIV, several studies have characterized host response to the closely related Invertebrate Iridescent virus 6 (IIV-6) in Drosophila [13,82]. From these, we know that the IIV-6 genome encodes for proteins that can inhibit insect host immune responses, including RNA silencing by the RNAi pathway (e.g., 340 L) [83], which is the primary defense against IIV-6 [84,85]. In our study, both Dicer-2 and Argonaute-2 were upregulated in crickets with overt CrIV infections, suggesting that the RNAi pathway is also important in antiviral defense for G. sigillatus. IIV-6 was also found to be able to inhibit both Imd and Toll pathways [86]. Interestingly, while there is no evidence that the Jak/STAT pathway confers immunity against IIV-6 infection in Drosophila [85], our study indicates significant induction of Jak/STAT pathway components in response to CrIV. Importantly, we found no evidence of viral inhibition of these responses at the transcriptional level in the present study.Why individuals from one population suffer from overt CrIV infections while the other maintains covert, asymptomatic infections remains an open question. One possibility is that the diseased population was exposed to an undetected microbe (e.g., bacteria or fungi) that made it more susceptible to an overt viral infection. Indeed, previous studies have demonstrated that co-infection with IIV-6 and a Gram-negative bacterium result in more rapid mortality in Drosophila [86]. Although we did not find differences in total microbial loads (Figure 4), we did not characterize microbiomes and therefore cannot rule out the possibility that bacterial or fungal composition are different between populations. Future studies will characterize the microbiome between populations with overt and covert infections to determine if the microbiome may play a role in promoting (or inhibiting) overt infections. While we previously found evidence of low viral loads of AdDNV in both populations of crickets [13], AdDNV has not previously been associated with disease in G. sigillatus [15]. Still, we did find that crickets with overt infections of CrIV had significantly higher (albeit relatively low) AdDNV viral loads. Additional studies will evaluate the impact of viral dynamics on infection outcomes. Another possibility is that intrinsic (e.g., inbreeding) or extrinsic (e.g., rearing environment) factors may impair immune function leading to the opportunistic reactivation of covert infections [87]. However, crickets with high levels of CrIV were able to mount an immune response across multiple pathways and so it is unlikely that crickets from the diseased population have a dysfunctional immune response, at least at the transcription level. Further studies probing post-transcriptional and post-translational outcomes will be essential to understanding the role immunity plays in regulating viral loads.In the present study, we assessed genes that are known to be important in immunity in other model insects (e.g., mosquitos, flies, moths). Future studies (e.g., RNAi knockdown experiments) will determine the importance of specific pathways on clearing or decreasing viral loads in these crickets. Furthermore, a comparison of complete transcriptomes between populations is required to obtain the global gene expression repertoire of infected crickets. These studies will be essential to fully characterize the defensive strategy of crickets at the transcript level and will improve our understanding of how crickets can tolerate low levels of CrIV and maintain covert infections. Hampering these efforts is the fact that few genetic resources for crickets currently exist [88], including the absence of a complete and annotated genome of G. sigillatus. Advancements in this field will greatly aid research efforts, including those seeking to improve production of reared beneficial insects.