Journal of Invertebrate Pathology

Deformed wing virus (DWV) is an emerging insect pathogen efficiently transmitted through communicable and vector-borne routes with Apis mellifera. Continual transmission of DWV between hosts and vectors is required for maintenance of the pathogen within the population, and this vector-host-pathogen system offers unique disease transmission dynamics for pathogen maintenance between vector and a social host. In a series of experiments, we study how vector-vector, host-host and host-vector transmission routes maintain DWV in a honey bee population. We found co-infestations on shared hosts allowed for movement of DWV from mite to mite. Additionally, two social behaviors of the honeybee, trophallaxis and cannibalization of pupae, provide routes for communicable transmission from bee to bee. Communicable circulation of the virus solely amongst hosts was then shown to act as a reservoir of DWV for naive Varroa to acquire and subsequently vector the pathogen. Our findings illustrate communicable transmission between hosts can maintain and influence the future acquisition and vectoring of a pathogen by a vector. There are a handful of other infectious diseases, both zoonotic and which impact human health, that have potentially similar transmission dynamics.

The honey bee microbiome includes a wide variety of viruses. While most of them usually remain commensal, some can become pathogenic in specific contexts. Of these, one is that of deformed wing virus (DWV) and another, sacbrood virus (SBV). Although co-infection is the norm rather than the exception, most of the time these viruses have been studied independently. When investigated as co-infections, past studies have focused on their effects on the honey bee brood. In this study, we co-inoculated adult honey bees at emergence with DWV by injection and SBV orally (acting as the viral transmission by Varroa destructor and by trophallaxis or food, respectively), either simultaneously or sequentially. Using optical counters, we were able to track the survival and behaviour of these honey bees within colonies. Through regular in-hive sampling, we monitored the evolution of their viral loads as well as the expression of eight immune genes involved in honey bee anti-viral immunity. Here, we show that co-inoculations of DWV and SBV synergistically increase the virulence of DWV and conditionally promote the replication of both viruses. Finally, our results show that immune responses in adult honey bees depend on DWV genotypes and whether replication originates from a superinfecting virus or a virus already present in bees. Author SummaryHoney bees are highly social pollinators that live in crowded colonies. Their population density and the high frequency of interactions between individuals favours disease transmission and makes colonies susceptible to pathogen outbreaks. Many viruses commonly infect honey bees, however, they are often studied as single infections. As an effort to better understand interactions between honey bees and multiple viral populations, we co-inoculated young bees with two common honey bee viruses (deformed wing virus and sacbrood virus), released them in colonies and monitored their health and behaviour. Our findings show evidence of synergies between both viruses, as we show that a virus seemingly harmless for adult bees (sacbrood virus) may actually increase the virulence of another virus (deformed wing virus). These results highlight the importance of monitoring and studying multiple pathogens at once for a better understanding of the threat they represent to colony health and survival.

Viruses are a large class of honey bee pathogens that negatively affect colony health, yet their prevalence and transmission dynamics in commercial queen production operations remain poorly understood. To address this gap, we conducted a series of controlled queen monitoring experiments and surveys to understand the prevalence and viral loads of seven viruses across developmental stages of queens, drones, royal jelly, and workers from associated colonies. All viruses except SBV were detected, with BQCV, DWV-B, and LSVs showing consistently high prevalence. Eggs were frequently infected with LSVs, DWV-B, and CBPV, suggesting vertical virus transmission, and highlighting the importance of selecting healthy breeder queens. BQCV, on the other hand, dominated in queen larvae, pupae, and adult stages. Mated queens, particularly those maintained in bank colonies, exhibited higher prevalence and viral loads than virgin queens, with DWV-B and BQCV being most abundant. Worker bees from bank colonies also showed slightly higher viral loads compared to other colonies, indicating potential risks associated with queen banking. Drone samples revealed high BQCV and DWV-B prevalence, indicative of their potential role in venereal transmission. The results from hierarchical clustering and correlation analyses provided evidence that viral profiles of queens did not necessarily match those of their resident colonies, highlighting complex viral transmission dynamics. Collectively, these findings provide novel insights into virus transmission dynamics during queen production and emphasize the need to improve queen health. Author summaryHoney bee queen failure is a frequent challenge in beekeeping operations, and viral infections are increasingly recognized as an important contributor. Since no chemical therapeutics are available for managing viral diseases in honey bees, understanding routes of viral transmission is critical for developing strategies to minimize infections. However, the diversity of viruses infecting queens, their prevalence, and their main transmission routes have not been systematically investigated within the queen production industry. Therefore, in this study we investigated viral prevalence, loads, and transmission dynamics in commercial queen production operations, using a diverse array of samples, including queens at different developmental stages, worker bees from associated colonies, drones, and Varroa mites. Our analysis consistently indicates that vertical transmission from infected breeder queens, natural mating, and bank colonies with high Varroa infestations are key points where queens are most vulnerable and become infected with viruses. These findings offer novel insights into virus transmission dynamics in queens, emphasizing the importance of selecting healthy breeder queens and controlling Varroa infestations in drone source and bank colonies.

RNA viruses are common in tephritid fruit flies including the Queensland fruit fly, Australias most significant horticultural pest. For many their transmission, tissue tropism and load across host development remain unexplored. Yet these factors are important for host biology, ecology and pest management. We investigated Bactrocera tryoni orbivirus (OV), Bactrocera tryoni xinmovirus (XV), Bactrocera tryoni toti-like virus (TLV) and Bactrocera tryoni iflavirus species 2 (IVsp.2) that commonly coinfect B. tryoni laboratory populations. OV and XV transmission was vertical within and on eggs, while TLV transmission was vertical within eggs. IVsp.2 was not detected in eggs but was present in adults; however, IVsp.2 was horizontally transmitted, with viral load increasing with cohabitation time with infected flies. Horizontal transmission was not observed for the other viruses. OV had a similar load across all tissues, while XV was consistently more abundant in ovaries. TLV had a high viral load in the brain whereas IVsp.2 was abundant in the thorax, foregut and midgut. Besides differences in eggs, the viruses were detected in all other developmental stages, but viral load patterns differed: viral load remained constant for TLV, fluctuated for OV and XV, and was low in pre-adult stages and high in adults for IVsp.2. Our findings demonstrate distinct transmission strategies and tissue tropism among the viruses, providing new insights into their epidemiology and role in host biology. Furthermore, contrary to prevailing views that viruses are generally horizontally transmitted, most known RNA viruses of B. tryoni are vertically transmitted affecting the evolution of host-virus interactions.

Seasonal variation strongly influences honey bee colony dynamics, leading to time-dependent changes in behavioral and physiological phenotypes. However, the extent to which seasonal fluctuations affect the susceptibility and tolerance of honey bees to viral infection remains largely unexplored. To address this, we conducted a longitudinal study in which adult honey bee workers were collected monthly from research colonies and experimentally infected with Israeli acute paralysis virus (IAPV) over the course of a year. Our results showed significant seasonal variation in the mortality and IAPV load of inoculated bees, with bees challenged during the pre-overwintering period (i.e., fall) exhibiting the highest susceptibility and lowest tolerance to IAPV infection compared to bees challenged in spring, summer, or winter. To investigate factors underlying these seasonal differences, we conducted nutrition-based studies that determined: 1) the variation in lipid content of colonies throughout the year and its potential link to our observed trends in IAPV tolerance, and 2) the impact of seasonally collected pollen on the survivorship of IAPV-challenged bees. Our findings support that seasonal changes in honey bee physiology and nutritional status play key roles in influencing honey bee viral tolerance. We conclude that honey bee colonies are particularly vulnerable to viral infection during the pre-overwintering period, most likely as a result of reduced tolerance to pathogen stress when transitioning from a summer to winter worker population. We further hypothesize that this period of increased vulnerability to viral infection, in correlation with other disease factors such as Varroa mite abundance and available forage, likely contributes to the relatively high overwintering losses experienced by beekeepers. Given the recent reports of severe colony losses attributed to honey bee viruses, understanding the relationship between seasonality and viral tolerance in honey bees is crucial for better informing management strategies and improving overwintering success. AUTHOR SUMMARYWe explored how seasonal changes affect the ability of honey bees to withstand viral infections. Previous research has shown that there are physical and behavioral differences between summer and winter bees, but its unclear how these seasonal differences affect a honey bees ability to withstand viral infection. To investigate this, we collected honey bee workers monthly and infected them with Israeli acute paralysis virus (IAPV) over the course of a year. Our results showed that honey bees were most vulnerable to IAPV in the fall (i.e., prior to overwintering), as they showed the highest mortality rates and lowest viral tolerance, based on their IAPV loads, during this time. By following up with nutrition-based studies, we found that seasonal changes in bee nutrition in part explained the seasonal differences we observed in honey bee virus tolerance. Overall, our findings suggest that bees are less tolerant to viral infection during the pre-overwintering period when colonies transition from a summer to a winter worker population. This vulnerable period may help explain the high rates of colony losses experienced by beekeepers nationwide and demonstrates the importance of developing seasonally-dependent disease management strategies.

Understanding the interaction between rose rosette emaravirus (RRV) and its vectors is pivotal in addressing the epidemic outbreak of rose rosette disease. This study employed quantitative real-time RT-PCR to assess RRV genome copy numbers in Phyllocoptes fructiphilus and P. adalius, providing insights into the viral dynamics and vector competency. Our findings suggest active virus replication within P. fructiphilus, a confirmed vector species, unlike P. adalius, highlighting its non-vector status. Furthermore, the study highlights the variability in virus concentration in mites over time, underlining possible developmental stage-specific response and influence of mite lifestyle on RRV retention and replication. This research is the first step in understanding the virus-mite interactome, which is essential for developing effective management strategies against rose rosette disease.

Diaphorina citri, the Asian citrus psyllid, is the insect vector of the phloem-limited bacterium Candidatus Liberibacter asiaticus, which causes the most devastating citrus disease worldwide: Huanglongbing (HLB). An efficient cure for HLB is still not available and the management of the disease is restricted to the use of pesticides, antibiotics and eradication of infected plants. Plant- and insect-infecting viruses have attracted increasing attention for their potential to manipulate traits in insects, especially insect vectors of plant pathogens. However, so far there are no insect virus-based vectors available for use in D. citri. Cricket paralysis virus (CrPV) is a well-studied insect-infecting dicistrovirus with a wide host range and has been used as a model in previous translational studies. In this work, we demonstrate for the first time that CrPV is infectious and pathogenic to D. citri. We show that specific amino acid mutations in the CrPV primary cleavage DvExNPGP motif resulted in a viral mutant that was attenuated compared to wild-type CrPV during infection of either Drosophila cells line or adult D. citri insects. This attenuated CrPV mutant was then used as the backbone for engineering a recombinant CrPV-based vector to specifically alter D. citri gene expression via the RNA interference (RNAi) pathway, a technology called Virus Induced Gene Silencing (VIGS). As proof-of-concept, we engineered recombinant CrPV-based vectors carrying nucleotide sequences derived from a previously reported D. citri target gene: the inhibitor of apoptosis gene (IA). RT-qPCR analysis of insects either microinjected or fed with the recombinant CrPV mutants showed decreased IA gene expression as soon as viral replication was detected, indicating that the engineered CrPV-based VIGS system enables functional gene silencing in D. citri. This novel insect virus-based tool is easily amenable to genomic modification and represents a technical advance for understanding interactions between insect virus-based VIGS systems and D. citri.

Acute Bee Paralysis Virus (ABPV) is a key driver of honey bee colony losses that has been increasingly reported in non-bee hosts in the past decades. Ants have long been hypothesised to act as viral reservoirs, but most evidence comes from field surveys, and experimental tests are still scarce. Here, we combined survival and transmission assays, viral load measurements, viral replication assays, and host immune gene expression analyses to test whether the clonal raider ant Ooceraea biroi can harbour and transmit ABPV. ABPV-injected ants showed delayed development and elevated mortality. Although we found no evidence of viral replication, the virus was able to spread among colony members and viral particles persisted in colonies for several days. These results show that ants can acquire ABPV, incur fitness costs, and pass the virus within their colonies, suggesting that they may act as incidental viral reservoirs. By maintaining and disseminating honey bee viruses, even without supporting replication, ants could contribute to their environmental persistence and spillover across species.

Honeybees face increasing threats from biotic stress of viral pathogens that can severely impact colony health and contribute to global colony decline. However, comprehensive studies of biotic stress and composition of bee viruses across different environmental contexts and seasons remain scarce. This study aims to characterize and compare the diversity, abundance and composition of the Apis mellifera L. virome across three different landscapes (conventional, organic, and roadside) and seasonal gradients (early vs. late season) to better understand how environmental and temporal factors affect viral communities in honeybees. A. mellifera were collected from three different habitats (conventional farm, organic farm, and roadside habitat) during the spring and summer of 2024. Total RNA was extracted individually from whole honeybees and mRNA libraries were prepared, which were subsequently used for sequencing on an Illumina NovaSeq X Plus platform using paired-end 150 bp reads. Several bacteriophages, putative novel viruses, plant-, insect- and bee-associated viruses were detected in the honeybee viromes including Sacbrood virus, Black queen cell virus, Deformed wing virus-B (previously known as Varroa destructor virus-1) and Deformed wing virus. Furthermore, both habitat types and seasons influence viral abundance as majority of detected viruses showed higher abundance in the conventional farm and late season samples. The present findings provide novel insights into the ecological and seasonal dynamics of honeybee-virus interactions and contribute to strategies for improving honeybee health and resilience.

Commercial beekeepers in the US reported severe colony losses early in 2025, as colonies were being staged for their critical role in the almond pollination season in California. Average reported losses since the preceding spring exceeded 60%, with substantial variation among operations. Many colonies were still actively collapsing in January, 2025, when pooled and individual samples were collected then screened for levels of known honey bee pathogens and parasites. Deformed wing virus strains A and B, along with Acute bee paralysis virus, were found at unusually high levels, either in pooled colony samples or in individual bees exhibiting shaking behaviors and morbidity. Differences between these two analyses suggest that direct collections of morbid bees provide a superior diagnostic for causal viruses, a suggestion borne out by confirmation of symptoms and morbidity following isolation and new inoculations. Since these viruses are known to be vectored by parasitic Varroa mites, mites from collapsed colonies were in turn screened for resistance to amitraz, a critical miticide used widely by beekeepers. Miticide resistance was found in all collected Varroa, underscoring the urgent need for new control strategies for this parasite. While viruses are a likely end-stage cause of colony death, other stressors such as nutritional stress and agrochemicals may have also played significant roles.

Summary/abstractO_ST_ABSBackgroundC_ST_ABSWith the advent of high-throughput sequencing, large sets of insect-infecting RNA viruses producing apparent asymptomatic infections are being discovered. In the Mediterranean fruit fly (medfly) Ceratitis capitata, an agricultural key pest of a wide range of fruits, up to 13 different RNA viruses have been described. Recent analysis demonstrated a wide distribution of these viruses in different medfly strains collected worldwide, but little is known about the interactions between those viruses and the medfly host. Previous studies suggested that a higher abundance of Ceratitis capitata nora virus (CcaNV) decreased medfly developmental time. Here, we investigated the effect of CcaNV on a broad range of parameters related to host fitness and its interaction with other trophic levels. ResultsCcaNV purified from a naturally infected medfly strain was used to infect CcaNV-free flies orally and subsequently monitor pupal weight, adult emergence, flying ability and longevity. Our results revealed detrimental effects associated with a CcaNV infection in the medfly, in terms of reduced pupal weight and reduced adult longevity. Moreover, we tested the influence of a CcaNV infection in medflies on the parasitism performance of Aganaspis daci, a medfly endoparasitoid used in biological control programs against medflies. Our results showed that A. daci progeny increased when parasitizing on CcaNV-infected larvae. ConclusionsOur results proved that covert RNA viruses can impact on the insect ecology, directly affecting its insect host biology and indirectly influencing multitrophic interactions.

Exportin 1 (XPO1) is the major karyopherin-{beta} nuclear receptor mediating the nuclear export of hundreds of proteins and some classes of RNA and regulates several critical processes in the cell, including but not limited to, cell-cycle progression, transcription, translation, oncogenesis and longevity. Viruses have co-opted XPO1 to promote nucleocytoplasmic transport of viral proteins and RNA. Maize mosaic virus (MMV) is an Alphanucleorhabdovirus transmitted in a circulative propagative manner by the corn planthopper, Peregrinus maidis. MMV replicates in the nucleus of plant and insect hosts, and it remains unknown whether MMV co-opts P. maidis XPO1 (PmXPO1) to complete its life cycle. Because XPO1 plays multiple regulatory roles in cell functions and virus infection, we hypothesized that RNAi-mediated silencing of XPO1 would simultaneously and negatively affect MMV accumulation and insect physiology. Although PmXPO1 expression was not modulated during MMV infection, PmXPO1 knockdown negatively affected MMV accumulation in P. maidis at 12 and 15 days after microinjection. Likewise, PmXPO1 knockdown negatively affected P. maidis survival and reproduction. PmXPO1 exhibited tissue specific expression patterns with higher expression in the ovaries compared to the guts of adult females. Survival rate was significantly lower for PmXPO1 knockdown females, compared to controls, but no effect was observed for males. Adult females with PmXPO1 knockdown were heavier and had a larger abdomen compared to controls at 4, 8 and 12 days after dsRNA microinjection. Consistent with an increase in weight, glyceride content specifically and significantly increased in PmXPO1 knockdown female planthoppers. Ovary development was significantly inhibited, and mature eggs were not observed in adult females with PmXPO1 knockdown. Consistent with a major role of PmXPO1 in ovary function and egg production, oviposition and egg hatch in plants was dramatically reduced in dsRNA PmXPO1 treated insects compared with control. Altogether, these results suggest that PmXPO1 is a positive regulator of P. maidis reproduction and that it plays a proviral role in the insect vector supporting MMV infection.

The suppressed in-ovo virus infection trait (SOV) was the first trait applied in honey bee breeding programs aimed to increase resilience to virus infections, a major threat for colony survival. By screening drone eggs for viruses, the SOV trait scores the antiviral resistance of queens and its implications for vertical transmission. In this study, queens from both naturally surviving and traditionally managed colonies from across Europe were screened using a two-fold improved SOV phenotyping protocol. First, a gel-based RT-PCR was replaced by a RT-qPCR. This not only allowed quantification of the infection load but also increased the test sensitivity. Second, a genotype specific primer set was replaced by a primer set that covered all known deformed wing virus (DWV) genotypes, which resulted in higher virus loads and fewer false negative results. It was demonstrated that incidences of vertical transmission of DWV were more frequent in naturally surviving populations than in traditionally managed colonies, although the virus load in the eggs remained the same. Dynamics in vertical transmission were further emphasized when comparing virus infections with queen age. Interestingly, older queens showed significantly lower infection loads of DWV in both traditionally managed and naturally surviving colonies, as well as reduced DWV infection frequencies in traditionally managed colonies when compared with younger queens. Seasonal variation in vertical transmission was found with lower infection frequencies in spring compared to summer for DWV and black queen cell virus. Together, these patterns in vertical transmission suggest an adaptive antiviral response of queens aimed at reducing vertical transmission over time.

A significant number of insect-specific viruses (ISVs) have been discovered in agriculturally important insect pests, facilitated by high-throughput sequencing (HTS). Despite its global impact on tomato crops, the RNA virome of the South American tomato pinworm, Tuta absoluta, remains uncharacterized. In this study, we utilized meta-transcriptomics and bioinformatic approaches to discover the RNA virome of T. absoluta across worldwide populations. We identified ten novel ISVs, classified into six groups: Nidovirales, Bunyavirales, Mononegavirales, Virgaviridae, Iflaviridae, Nodaviridae, Solemoviridae, and Phasmaviridae. Notably, no core virus was consistently present across the studied populations, and field-collected samples revealed a greater diversity of ISVs compared to those from laboratory samples. In addition, we detected plant-infecting viruses and mycoviruses associated with the pest. This study represents the first description of the RNA virome associated with T. absoluta, providing valuable insights into its biological and ecological interactions. It also lay the foundation for future studies aimed to clarify the biological roles of ISVs.

Parasites can manipulate host behavior to increase their fitness while decreasing host fitness, a phenomenon known as an extended phenotype. Nucleopolyhedroviruses (NPVs), baculoviruses that infect Lepidopteran larvae, have been found to induce vertical climbing behavior and hyperactivity in exposed larvae. We quantified variation in the horizontal wandering behavior induced by different naturally-occurring pathogen isolates in the NPV that infects Dione (Agraulis) vanillae Linnaeus (Lepidoptera: Nymphalidae). Lab-raised larvae were infected with a constant dose of one of five different field-collected NPV isolates or a water control (n=98 larvae total), and placed in mazes to measure the horizontal distance wandered away from a food source. Virus-exposed larvae exhibited increased maximum distance of horizontal movement compared to the control, but did not significantly differ in the probability of wandering versus no movement. We also found variation in the distance wandered among the five virus isolates. However, grouping the five isolates into two previously-described viral strains or genogroups did not improve predicted differences in movement, perhaps due to the presence of within-strain genetic variation among isolates in the viral genes involved in controlling host behavior. Further work is needed to determine whether the observed between-isolate variation is the result of adaptive evolution. These results suggest that the NPV infecting D. vanillae manipulates larval behavior to increase horizontal wandering, which could lead to higher pathogen fitness by increasing long-distance dispersal of the virus across the landscape.

Jamestown Canyon Virus (JCV), a negative-sense arbovirus, is increasingly common in the upper Midwest of the USA. Transmitted by a range of mosquito genera, JCV has at its primary amplifying host, white-tailed deer. Aedes aegypti is the major transmitter globally of the positive-sense viruses dengue (DENV), Zika, chikungunya, and Yellow Fever. Ae. aegyptis distribution, once confined to the tropics, is expanding, in part due to climate change. Wolbachia, an insect endosymbiont, limits the replication of co-infecting viruses inside insects. The release and spread of the symbiont into Ae. aegypti populations has been effective in reducing transmission of dengue and other viruses to humans. The mechanism of Wolbachia-mediated viral blocking in vectors is still poorly understood, however. Here we explored JCV infection potential in Ae. aegypti, the nature of the vectors immune response, and interactions with Wolbachia infection. We show that Ae. aegypti is highly competent for JCV, growing to substantial loads and rapidly reaching the saliva after an infectious blood meal. The mosquito immune system responds with strong induction of RNAi and JAK/STAT. Neither the direct effect of viral infection nor the energetic investment in immunity appears to affect mosquito longevity. Wolbachia infection blocked JCV only in the early stages of infection. Wolbachia-induced immunity was small compared to that of JCV, suggesting innate immune priming does not likely explain blocking. We propose two models to explain why Wolbachias blocking of negative-sense viruses like JCV may be less than that of positive-sense viruses, relating to the slowdown of host protein synthesis and the triggering of interferon-like factors like Vago. In conclusion, we highlight the risk for increased human disease with the predicted future overlap of Ae. aegypti and JCV ranges. We suggest that with moderate Wolbachia-mediated blocking and distinct biology, negative-sense viruses represent a fruitful comparator model to other viruses for understanding blocking mechanisms in mosquitoes. Author SummaryJamestown Canyon Virus (JCV), a newly emerging virus in North America, causes disease when it spills out of its wild mammal hosts into human populations via the bite of infected mosquitoes. We show that the mosquito Aedes aegypti, known for transmitting many viral pathogens to humans globally, and whose distribution is creeping northward in the USA toward regions where JCV is present, is likely able to transmit the virus. Wolbachia is an endosymbiotic bacterium being released in wild mosquito populations of mosquitoes because it limits the replication of human viruses inside the mosquito, limiting their transmission to humans. We show that Wolbachia has a limited ability to control the replication of JCV, which is likely because Wolbachia-induced antiviral response is quite weak, and unique aspects of negative-sense virus biology make them less susceptible to blocking. Our findings suggest that JCV may serve as a comparative model to positive-sense viruses like dengue in dissecting the mechanism of Wolbachia-mediated virus blocking. It also warns that shifting mosquito distributions, as expected under a changing climate, could bring JCV and Aedes mosquitoes into greater contact, potentially increasing the incidence of JCV in humans.

High annual honey bee colony losses are associated with environmental and biological stressors, including virus infections. In insects, the octopamine pathway orchestrates the "fight-or-flight" response, regulating energy mobilization, temperature, and flight. We determined that sacbrood virus (SBV) infections induce expression of an octopamine receptor and enhance honey flight performance, whereas deformed wing virus (DWV) infections reduce flight performance, but how viruses interface with this pathway remained unknown. To elucidate the relationships between the octopamine response, virus infection, and flight, honey bees were infected with SBV or DWV and exposed to octopamine (OA), epinastine (EP)-an OA receptor antagonist, or both OA and EP; flight and gene expression were assessed. Pharmacologic manipulation revealed that octopamine supplementation rescued flight deficits in DWV-infected bees, but diminished performance in SBV-infected bees, while blocking octopamine receptors altered these effects. Transcriptome analyses indicated that SBV infections, and DWV infection with OA treatment, activated honey bee metabolic pathways, and that SBV infected bees had greater expression of genes involved in OA synthesis, unless treated with OA. These results provide a mechanistic explanation for virus-specific impacts on honey bee flight, which may have consequences on foraging efficiency, colony health and virus transmission.

Organisms are constantly at risk of being infected by pathogens such as viruses. Adaptations against viral infection include immune defenses encoded within the host genome and associations with defensive symbionts such as Wolbachia. Wolbachia is widely spread among insects, and its success in nature may be due to its antiviral effects, which can benefit hosts if viruses significantly reduce host fitness. Here, we conducted a meta-analysis to assess the degree to which viral infection affects the fitness of insect hosts, and which factors may influence the impact of viral infection on hosts such as if the insect host is a vector or not of viruses that cause diseases in humans and plants, and if the insect is a new or a natural host of a specific virus. We gathered 1,040 effect sizes from 150 studies. Our results show that viruses significantly reduce host fitness, especially their survival. The decrease in host fitness is higher in non-vector than in vector insects, and we found no difference in fitness decrease caused by viral infection between new and natural hosts. Moreover, we found that fitness effects caused by viruses vary between host order and fitness components. In conclusion, our results show that viruses exert severe harmful effects on hosts by decreasing their fitness. In this context, harboring symbionts that confer antiviral protection, such as Wolbachia, can be highly advantageous for hosts, enhancing their fitness. Conversely, Wolbachia can benefit from the presence of viruses, facilitating its spread in insect populations by offering antiviral protection.

The tropical house cricket Gryllodes sigillatus is a major species used in the edible insect farming industry. Despite the rapid expansion of this sector, diagnostic tools for detecting infections in these species remain limited. The lack of validated reference genes compromises the reliability of RT-qPCR-based gene expression analyses, which are essential for the development of molecular tools for disease diagnosis and health monitoring in insect production systems. To address this gap, we evaluated the expression stability of six candidate reference genes (ACTB, EF1, GAPDH, HisH3, RPL5, and 18SrRNA) across four body parts (abdomen, head, legs, and whole body) using a combination of complementary statistical approaches, including geNorm, NormFinder, BestKeeper, the {Delta}Ct method, the R statistical environment, and the integrated RefFinder tool. Candidate genes were identified and annotated using the recently published G. sigillatus genome, through sequence comparisons with closely related insect species using BLAST and reciprocal BLAST analyses, multiple sequence alignments. All procedures complied with MIQE 2.0 guidelines to ensure methodological rigor and transparency. The results showed that ACTB, EF1, RPL5, and 18SrRNA exhibited stable and consistent expression across all analyzed tissues, whereas GAPDH and HisH3 displayed high variability and were generally unsuitable for normalization, except in head tissue where GAPDH remained stable. This study provides the first validated set of reference genes for G. sigillatus, establishing a robust foundation for accurate, reproducible, and comparable gene expression analyses. Furthermore, these findings support the development of RT-qPCR-based diagnostic tools, contributing to improved health monitoring and biosafety in insect production systems.

RNA viruses establish persistent infections in insects through mechanisms that are not fully understood. We focused on three positive-sense RNA viruses that naturally establish persistent infections: Drosophila A virus (DAV), Drosophila C virus (DCV), and Nora virus. We examined how these viruses interact with their hosts during development and we found that pupal metamorphosis is a critical window where virus-host immune interactions influence persistence differently across viruses. Peak viral loads and replication occur during pupation, coinciding with increased endogenous reverse transcriptase activity. Notably, reverse transcription of viral RNA genomes produces viral DNA (vDNA) forms of DAV and DCV that are first detectable during pupation and are involved in persistence. In contrast, Nora virus achieves persistence without detectable vDNA. Immune responses during pupation are virus-specific, involving suppression of RNA interference components and varied regulation of JAK-STAT signaling. After metamorphosis, DAV continues producing vDNA into adulthood while DCV shows transient vDNA production, and Nora virus bypasses vDNA production altogether. These findings point to pupation as a key developmental stage for the establishment of persistent infections through distinct viral persistence strategies.