Vera I. D. Ros

Iflaviruses are arthropod‐infecting viruses classified in the family Iflaviridae. The most well‐studied iflaviruses are responsible for deleterious overt (symptomatic) infections in economically important insect species like honeybees and silkworms. The fact that iflaviruses are also found in mass reared insects such as crickets and flies, makes their study relevant for the food and feed industries and for biological control applications (e.g., mass reared insects for sterile insect techniques). In recent years it was found that iflaviruses are not restricted to insects, but are widely spread in many arthropod species, often causing covert (asymptomatic) infections. Fitness costs may be associated with these covert infections, as seen in a variety of host species. In this work we review the latest research on iflaviruses in respect to host range, viral genome organization, cycle of infection, tissue tropism, virus transmission strategies, fitness costs and pathogenic effects of both covert and overt infections, host immune responses to iflavirus infections, interactions between iflaviruses and other microorganisms, and the effect of iflavirus infections on host behavior.

Sterile Insect Technique (SIT) applications against major insect pests and disease vectors rely on the cost‐effective production of high‐quality sterile males. This largely depends on the optimal management of target pest colonies by maximizing the benefits provided by a genetically rich and pathogen‐free mother colony, the presence of symbiotic microorganisms, and efficient domestication, mass‐rearing, irradiation, and release processes. At the same time microbial (bacteria, fungi, microsporidia, and viruses) pathogen outbreaks should be minimized or eliminated, and the use of hazardous chemicals restricted. The optimization of the colony management strategies for different SIT target insects will ensure a standardized high‐quality mass‐rearing process and the cost‐effective production of sterile males with enhanced field performance and male mating competitiveness. The aims of the Coordinated Research Project (CRP) were to develop best practices for insect colony management for the cost‐effective production of high‐quality sterile males for SIT applications against major insect pests and disease vectors through a multidisciplinary approach involving entomologists, geneticists, ecologists, microbiologists, pathologists, virologists, and mass‐rearing experts.

Stress may be viewed as the disturbance of homeostasis of an organism. Stress may arise from the external or internal environment of living organisms and plays a significant role in the fight or flight responses of animals. An organism's potential to resist stress is determined by its ability to mount up an effective response against the stress factors. Therefore, stress‐induced biomolecules are useful indicators of a well‐functioning immune system. While the interactions between stress and immunity are well‐studied in vertebrate and plant systems, they are insufficiently documented among invertebrates, including dipteran insects that are mass‐reared for sterile insect technique (SIT) applications. Generally, mass‐reared insects may experience a variety of stress factors, which may affect various biological traits, including fecundity, weight of the progeny, adult emergence rates, flight propensity, mating ability, and their competitiveness with wild conspecifics. Many of these biological traits determine the costs and success of SIT programs. It is imperative to understand how stress impacts the quality of the reared insects and their biological traits, as well as the insect's defense responses to stress factors, to maintain robust and healthy colonies for successful release programs in SIT. Here, we review and discuss the sources and responses to biotic and abiotic stress in general in insects, while prioritizing literature on dipteran insects for SIT programs. We also coalesce genes and pathways that are modulated during stress and may be used as indicators to diagnose stress with the final aim to improve insect health in mass‐rearing colonies.

The sterile insect technique (SIT) for tsetse involves releasing sterilized males to outcompete wild males in mating, resulting in nonviable progeny. Balancing optimum sterility and male quality is crucial. While irradiation in hypoxia or anoxia is routine for tephritid flies, its effect on tsetse, especially postrelease, is not well understood. We conducted experiments to understand the impact of irradiation in hypoxia on the sterility of the F1 generation of Glossina morsitans morsitans ( Gmm ). Initially, we tested the impact of 1‐h preconditioning in hypoxia before irradiation and continued hypoxia up to 24, 48, and 72 h postirradiation on the emergence and flight propensity of treated males. We then assessed mating ability, survival, pupae per initial female (PPIF) and residual fertility of flies following irradiation at various doses after 1‐h hypoxia conditioning. Finally, we determined the PPIF and residual fertility of the F1generation and the emergence of treated flies to the F2 generation. Results show that short‐term conditioning (1 h) in hypoxia maintains or improves the qualities of the irradiated Gmm pupae. Regardless of irradiation in normoxia or hypoxia, higher residual fertility was observed in females than males in the F0 generation, and higher residual fertility in the F1 than the F0 generation for both sexes. However, the emergence rates of males decreased in the F2 generation compared with the F1 generation, a noteworthy finding for SIT programs for tsetse, implying diminishing populations of sterile flies.

Insects are associated with a wide variety of diverse RNA viruses, including iflaviruses, a group of positive stranded RNA viruses that mainly infect arthropods. Whereas some iflaviruses cause severe diseases in insects, numerous iflaviruses that were detected in healthy populations of butterflies and moths (order: Lepidoptera) do not show apparent symptoms. Compared to other hosts, only few iflavirus genomes for lepidopteran hosts could be found in publicly available databases and we know little about the occurrence of iflaviruses in natural and laboratory lepidopteran populations. To expand the known diversity of iflaviruses in Lepidoptera, we developed a pipeline to automatically reconstruct virus genomes from public transcriptome data. We reconstructed 1,548 virus genomes from 55 different lepidopteran species that we were identified as coding-complete based on their length. To include incompletely assembled genomes, we developed a reference-based patching approach, resulting in 240 patched genomes. By including publicly available genomes, we inferred a phylogeny consisting of 139 non-redundant iflavirus genomes. Of these, 65 represent novel complete genomes, of which 39 might even belong to novel virus species. Our analysis expanded virus host range, where highly similar viruses were found in the transcriptomes of different lepidopteran species, genera or even families. Additionally, we find two groups of lepidopteran species depending on the diversity of viruses that infect them: some species were only infected by closely related viruses, whereas other species are infected by highly diverse viruses from different regions of the phylogeny. Finally, we show that the evolution of one virus species, Iflavirus betaspexiguae , is impacted by recombination within the species, which is also supported by the co-occurrence of multiple strains within data sets. Our analysis demonstrates how data mining of publicly available sequencing data can be used at a large scale to reconstruct intra-family viral diversity which serves as a basis to study virus host range and evolution. Our results contain numerous novel viruses and novel virus-host associations, including viruses for relevant insect pests, highlighting the impact of iflaviruses in insect ecology and as potential biological control agents in the future.

Human African trypanosomiasis (HAT) and African animal trypanosomosis (AAT) are devastating diseases spread by tsetse flies (Glossina spp.), affecting humans and livestock, respectively. Current efforts to manage these diseases by eliminating the vector through the sterile insect technique (SIT) require transportation of irradiated late-stage tsetse pupae under chilling, which has been reported to reduce the biological quality of emerged flies. We therefore evaluated the impact of irradiation and transportation (including vibration and shock) on pupae at early-stage development (22 days of age) under ambient temperature and compared it to that on pupae at the late-stage development (29 days of age) under chilling, the current practice for tsetse in SIT programs. The quality of flies emerging from these transported pupae was assessed by their emergence rates, flight propensity, mating ability, insemination rates and survival rates (over ca. 100 days, and after specified shorter periods). Generally, flies emerging from the 22-day-old pupae had significantly (p < 0.05) higher values for the tested quality parameters, as compared to those emerging from 29-day-old pupae. Irradiation, transportation and the combination thereof significantly (p < 0.05) reduced all the tested quality parameters as compared with the untreated control within the 22-day-old pupae group. Further, vibration had a significant negative effect on the quality of flies, notwithstanding the age of the pupae. Irradiation and transportation of pupae at 22 days of age resulted in a higher proportion of flies of good biological quality as compared to those of 29 days of age, and hence may be considered for future SIT programs.

Numerous studies have demonstrated the presence of covert viral infections in insects. These infections can be transmitted in insect populations via two main routes: vertical from parents to offspring, or horizontal between nonrelated individuals. Thirteen covert RNA viruses have been described in the Mediterranean fruit fly (medfly). Some of these viruses are established in different laboratory-reared and wild medfly populations, although variations in the viral repertoire and viral levels have been observed at different time points. To better understand these viral dynamics, we characterized the prevalence and levels of covert RNA viruses in two medfly strains, assessed the route of transmission of these viruses, and explored their distribution in medfly adult tissues. Altogether, our results indicated that the different RNA viruses found in medflies vary in their preferred route of transmission. Two iflaviruses and a narnavirus are predominantly transmitted through vertical transmission via the female, while a nodavirus and a nora virus exhibited a preference for horizontal transmission. Overall, our results give valuable insights into the viral tropism and transmission of RNA viruses in the medfly, contributing to the understanding of viral dynamics in insect populations. IMPORTANCE: The presence of RNA viruses in insects has been extensively covered. However, the study of host-virus interaction has focused on viruses that cause detrimental effects to the host. In this manuscript, we uncovered which tissues are infected with covert RNA viruses in the agricultural pest Ceratitis capitata , and which is the preferred transmission route of these viruses. Our results showed that vertical and horizontal transmission can occur simultaneously, although each virus is transmitted more efficiently following one of these routes. Additionally, our results indicated an association between the tropism of the RNA virus and the preferred route of transmission. Overall, these results set the basis for understanding how viruses are established and maintained in medfly populations.