Insect production is a rapidly growing industry globally. While the practice of farming insects has been around for millennia (e.g., silkworm farming and apiculture) (Defoliart, 1995), applications for mass-produced insects continue to expand beyond traditional uses (Castro-López et al., 2020; van Huis, 2020b), to include chitin production (Hahn et al., 2020), waste management and valorization (Surendra et al., 2016, 2020; Gasco et al., 2020), and use as feed for both pets, including cats and dogs (Bosch et al., 2014), and agricultural animals (Makkar et al., 2014; Henry et al., 2015; Tomberlin et al., 2015). Moreover, edible insects reared for direct human consumption (e.g., mealworms and crickets) are becoming an increasingly attractive solution to address the world’s critical need for novel and environmentally sustainable protein sources (van Huis et al., 2013; Hawkey et al., 2021). Worldwide, farmed insects have the potential to become a critically important commodity, buffering against food insecurities, providing additional revenue streams for rural and urban farmers, and offering sustainably produced resources in a growing number of applications (van Huis et al., 2013). Crickets (family: Gryllidae) are an especially popular group of insects due to their ease of rearing and nutritional profile (Wang et al., 2004; Zielińska et al., 2015; Stull et al., 2018) and are already used in many foods, including nutritional and functional additives (Hall et al., 2017; Osimani et al., 2018; Udomsil et al., 2019), and feed applications (van Huis, 2020a; Magara et al., 2021).

The demand for mass-produced insects is steadily increasing due to their utility with relatively low associated costs (Wilkie, 2018); however, there are several obstacles that hinder farmed insects from becoming an extensively utilized resource, including a dearth of rigorous empirical data (van Huis, 2017; Stull and Patz, 2020). Critical among these gaps is a lack of research on the entomopathogenic microbes that negatively impact reared insect colony health and production despite infectious disease outbreaks plaguing modern insect farms for decades (Eilenberg et al., 2018; Maciel-Vergara et al., 2021). As with traditional animal livestock, farmed insects are susceptible to parasites and pathogens, including viruses, bacteria, fungi, microsporidia, and nematodes (Kaya and Vega, 2012; Eilenberg et al., 2015). Pathogenic viruses can devastate reared insect populations (Maciel-Vergara and Ros, 2017) and especially so within cricket colonies, which are often reared in environments ideal for virus transmission (i.e., crowded, humid, and warm). For example, the Acheta domesticus densovirus (AdDNV), a small parvovirus, is responsible for causing severe epizootics throughout European, North American, and Asian cricket farms, resulting in massive mortality and product losses of the commonly utilized house cricket, A. domesticus (Styer and Hamm, 1991; Liu et al., 2011; Szelei et al., 2011; Weissman et al., 2012; Pham et al., 2013b). 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 were less susceptible to AdDNV (Weissman et al., 2012).

Beyond a single study (Weissman et al., 2012), we know little about viral disease and susceptibility in G. sigillatus, yet the list of entomopathogenic viruses infecting other farmed cricket species continues to grow to include iridoviruses, nudiviruses, and other dicistroviruses. Moreover, because there have not been wide-scale systematic surveillance efforts (de Miranda et al., 2021b), it is very likely that there are other pathogenic viruses among reared crickets still to be discovered. Here, we provide one of the first empirical reports of the identification and characterization of a viral pathogen in a diseased colony of lab-reared G. sigillatus crickets. We screened diseased colonies for known cricket viruses by measuring viral prevalence across sexes using both real-time PCR (qPCR) as well as Sanger sequencing and contrasted these results with a related population with no apparent signs of infection. Furthermore, we screened for novel pathogens by performing a non-targeted shotgun metagenomic analysis on guts and hemolymph from adult crickets. Metagenomic data corroborate our qPCR results and provide a complete genome of the suspected disease-causing agent, cricket iridovirus (CrIV) with significant similarities to the previously reported lizard–cricket iridovirus (Liz–CrIV) (Papp and Marschang, 2019). Our results add to the critical, but presently scarce, research on insect pathology of farmed crickets and highlight the importance of understanding viral infection and transmission dynamics in reared insect colonies.

Entomopathogenic viruses are known to cause significant losses to the reared insect industry (Maciel-Vergara and Ros, 2017); however, little is known about their diversity, biology, and host association. Using a range of molecular approaches in the present study, we were able to identify an cricket iridovirus (CrIV) as the likely causal agent of a disease affecting a colony of reared G. sigillatus, a species of growing importance for food, feed, and industrial purposes (Weissman et al., 2012). Metagenomic analyses revealed that our CrIV isolate, CrIV_USDA, was most closely related to lizard-cricket iridovirus (Liz-CrIV) (Papp et al., 2014; Papp and Marschang, 2019). Gross pathology included milky hemolymph, decreased fecundity, sluggish behavior, and melanotic lesions, all indicative of an active microbial infection. Our molecular identification and quantification assays indicated that Diseased crickets were supporting large numbers of CrIV. Diseased crickets were estimated to have about 1.5 trillion more copies of CrIV than Healthy crickets, based on the volume of DNA template extracted and used in our qPCR assays (Supplementary Table 1). Although our studies did not include a complete Koch’s postulate to confirm the causal agent infecting this colony of G. sigillatus, our molecular characterization based on shotgun metagenomics of hemolymph and gut samples and qPCR-based absolute quantification strongly suggests that CrIV is the main viral entomopathogen driving the pathology in the Diseased population. Future studies will focus on dissecting the intricacies of host-pathogen interactions, routes of transmission, and identifying the biological factors that might be associated with an active and covert infection. The latter consideration will be essential as we do not yet fully understand why CrIV presents as an active infection in one population but not the other.

Invertebrate iridescent viruses (family Iridoviridae) have non-occluded icosahedral particles, approximately 130 nm diameter, that contain a double-stranded DNA genome of about 140–210 kpb (Williams et al., 2000; İnce et al., 2018). They can infect a broad range of invertebrates, including terrestrial isopods, and have been isolated from several insect taxa (Kleespies et al., 1999; Jakob et al., 2002). Furthermore, certain invertebrate iridoviruses are known to cause disease in reptiles (Just et al., 2001; Weinmann et al., 2007; Marschang, 2011; Papp et al., 2014) and amphibians (Stöhr et al., 2016) via ingestion of infected insects, posing a particular health concern for the pet trade industry. In crickets, CrIV is known to cause disease in several species, including reared Gryllus texensis (Adamo et al., 2014), G. bimaculatus (Just and Essbauer, 2001), G. campestris, A. domesticus (Kleespies et al., 1999), and now G. sigillatus.

Our metagenomic analyses confirm the draft genome of Liz-CrIV from a previous study (Papp and Marschang, 2019) as a distinct virus from Invertebrate Iridescent virus 6 (IIV-6), although formal analyses are needed to determine if Liz-CrIV represents a strain of IIV-6 or a new species of invertebrate iridovirus (Papp and Marschang, 2019). Analyses from the current study also provide the most complete genome of Liz-CrIV. Importantly, our PCR primers were not able to discern between IIV-6 and Liz-CrIV and so future screening efforts should target areas of the genome that distinguish the two (see Supplementary Figure 1). Liz-CrIV was first discovered in Europe from commercially produced crickets in the mid to late 1990’s (Kleespies et al., 1999; Just and Essbauer, 2001). The populations used in the present study, descents of field-caught crickets in New Mexico, have been reared in a research lab setting since 2001, and have had no history of contact with commercially produced crickets. While we currently do not know the origins or the spread of this virus, our study confirms that Liz-CrIV is active across multiple continents.

In addition to active CrIV infections, our study found evidence for covert, or asymptomatic, infections of both CrIV and Acheta domesticus densovirus (AdDNV), the latter of which has been documented in G. sigillatus previously (Weissman et al., 2012). Covert infections of invertebrate iridovirus are reportedly more prevalent than active lethal infections in some insect populations (Williams, 1993, 2008; Tonka and Weiser, 2000). Although our study design did not allow us to examine in detail the effects that covert infections might have on cricket health, covert invertebrate iridovirus infections may have significant fitness consequences for hosts (e.g., increased development times and reduced fecundity) (Marina et al., 1999, 2003), which could have important ramifications for product yield in production facilities. Our study design prevents us from distinguishing between a persistent infection (with low levels of virus replication) or a latent infection and additional experiments (e.g., measuring transcription) could elucidate the nature of these covert infections (Williams et al., 2017).

Interestingly, we found a significant effect of the interaction between sex and population on the abundance of viral copies such that Healthy males had a lower abundance of AdDNV copies compared to Diseased males and females from either population. Although females are the larger sex (Sakaluk et al., 2019), our analysis normalized abundance values such that body size would not account for these differences. Thus, our results could be indicative of a higher tolerance of AdDNV by females, but controlled exposures would be needed to confirm this. We did not see the same sex-effect for CrIV. Few studies, if any, quantify viral loads across sex in reared and farmed insects, which makes generalizing this finding difficult. However, previous studies have demonstrated higher immune activity in female G. sigillatus compared with males (Gershman et al., 2010), which could contribute to differences in viral loads.

A key aspect of this system yet to be determined is the route of virus transmission, although per os is suspected to be the main route for CrIV (Williams et al., 2005). Adamo et al. (2014) found that CrIV can be transmitted horizontally via topical exposure in G. texensis and found no evidence of virus within the testes. Similar results were found by Just and Essbauer (2001) who found no signs of infection in the ovaries or testes in G. bimaculatus. However, both studies assessed transmission among populations experiencing active infections and so it is plausible that CrIV may be transmitted vertically when populations are exhibiting covert infections. Indeed, several classes of pathogenic viruses, including iridoviruses, are known to adopt mixed-mode transmission (both vertical and horizontal transmission) based on the relative fitness gains that are obtained via each transmission strategy (Ebert, 2013). Because horizontal transmission is riskier when hosts are rare but vertical transmission is constrained by host fitness, this mixed-mode transmission balances the likelihood of transmission with the constraints of host fitness. Thus, vertical transmission is favored when host densities are low while horizontal transmission is favored when host density is high. Further studies exploring the potential for mixed-mode transmission of CrIV across populations with active and covert infections will be critical and may explain the discrepancy between populations.

In conclusion, foundational research on the pathology of mass-produced insects, including crickets, will be essential to maintain the health and yield necessary for this growing industry. Here, we report the viral loads across sexes of active and covert infections of two pathogenic viruses (CrIV and AdDNV) in two populations of reared G. sigillatus. Importantly, these findings will inform future work addressing diagnostic, mitigation, and therapeutic interventions in reared insect colonies that could ultimately improve product yield and support this burgeoning sustainable industry.

Full genome of the CrIV isolate (Cricket iridovirus isolate Liz-CrIV_USDA_2019) can be found at GenBank (submission number: OK181107). Metagenomic sequences for hemolymph and guts are available at the NCBI Sequence Read Archive database (study number: PRJNA764167, SRA accession numbers: SAMN21469590, SAMN21469591, SAMN21469592, SAMN21469593, SAMN21469594, and SAMN21469595). The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

KD and JR contributed to conception, design of the study, and collected the data. KR and BO analyzed metagenomic data. KD performed statistical analyses and wrote the first draft of the manuscript. KR and JR wrote sections of the manuscript. BS, SS, JH, and RB provided necessary samples, materials, and additional expertise. All authors contributed to manuscript revision, read, and approved the submitted version.

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.

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