The Mediterranean fruit fly (medfly), Ceratitis capitata, is an agricultural pest of a wide range of fruits. The advent of high-throughput sequencing has boosted the discovery of RNA viruses infecting insects. In this article, we aim to characterize the RNA virome and viral sRNA profile of medfly. By means of transcriptome mining, we expanded the medfly RNA virome to 13 viruses, including two novel positive ssRNA viruses and the first two novel dsRNA viruses reported for medfly. Our analysis across multiple laboratory-reared and field-collected medfly samples showed the presence of a core RNA virome comprised of Ceratitis capitata iflavirus 2 and Ceratitis capitata negev-like virus 1. Furthermore, field-collected flies showed a higher viral diversity in comparison to the laboratory-reared flies. Based on the small RNA sequencing, we detected small interfering RNAs mapping to all the viruses present in each sample, except for Ceratitis capitata nora virus. Although the identified RNA viruses do not cause obvious symptoms in medflies, the outcome of their interaction may still influence the medfly’s fitness and ecology, becoming either a risk or an opportunity for mass-rearing and SIT applications.

The yellow mealworm, Tenebrio molitor, is currently one of the most important insect species produced for livestock feed and human consumption. High-density rearing conditions make the risk of disease and infections by parasitic symbionts a challenge in the mass production of these insects. However, certain symbionts are beneficial and should be favoured in order to promote healthy insect populations. Knowledge of parasitic symbionts and their management is essential for the insect rearing industry and its associated research. Here we review the documented microbial infectious agents, invertebrate parasites, and beneficial symbionts occurring in T. molitor. Furthermore, we discuss detection, prevention, and treatment methods for disease management in T. molitor production systems to inform future management and decision making in T. molitor rearing.

Current knowledge of the piRNA pathway is based mainly on studies on Drosophila melanogaster where three proteins of the Piwi subclade of the Argonaute family interact with PIWI-interacting RNAs to silence transposable elements in gonadal tissues. In mosquito species that transmit epidemic arboviruses such as dengue and chikungunya viruses, Piwi clade genes underwent expansion, are also expressed in the soma and cross-talk with proteins of recognized antiviral function cannot be excluded for some Piwi proteins. These observations underscore the importance of expanding our knowledge of the piRNA pathway beyond the model organism D. melanogaster. Here we focus on the emerging arboviral vector Aedes albopictus and we couple traditional approaches of expression and adaptive evolution analyses with most current computational predictions of protein structure to study evolutionary divergence among Piwi clade proteins. Superposition of protein homology models indicate possible high structure similarity among all Piwi proteins, with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the contrary, solvent-exposed surfaces showed low conservation, with several sites under positive selection. Analysis of the expression profiles of Piwi transcripts during mosquito development and following infection with dengue serotype 1 or chikungunya viruses showed a concerted elicitation of all Piwi transcripts during viral dissemination of dengue viruses while maintenance of infection relied on expression of primarily Piwi5. Opposite, establishment of persistent infection by chikungunya virus is accompanied by increased expression of all Piwi genes, particularly Piwi4 and, again, Piwi5. Overall these results are consistent with functional specialization and a general antiviral role for Piwi5. Experimental evidences of sites under positive selection in Piwi1/3, Piwi4 and Piwi6, that have complex expression profiles, provide useful knowledge to design tailored functional experiments. Argonautes are ancient proteins involved in many cellular processes, including innate immunity. Early in eukaryote evolution, Argonautes separated into Ago and Piwi clades, which maintain a dynamic evolutionary history with frequent duplications and losses. The use of Drosophila melanogaster as a model organism proved fundamental to understand the function of Argonautes. However, recent studies showed that the patterns and observations made in D. melanogaster, including the number of Argonautes, their expression profile and their function, are a rarity among Dipterans. In vectors of epidemic arboviruses such as dengue and chikungunya viruses, Piwi genes underwent expansion, are expressed in the soma, and some of them appear to have antiviral functions. Besides being an important basic question, the identification of which (and how) Piwi genes have antiviral functions may be used for the development of novel genetic-based strategies of vector control. Here we coupled population genetics models with computational predictions of protein structure and expression analyses to investigate the evolution and function of Piwi genes of the emerging vector Aedes albopictus. Our data support a general antiviral role for Piwi5. Instead, the detection of complex expression profiles with the presence of sites under positive selection in Piwi1/3, Piwi4 and Piwi6 requires tailored functional experiments to clarify their antiviral role.

Farmed insects can provide an alternative protein source for humans, livestock, and fish, while supporting adaptation to climate change, generating income for smallholder farmers, and reducing the negative impacts of conventional food production, especially in the tropics. However, the quantity, nutritional quality and safety of insects greatly relies on their feed intake. Tropical forages (grasses and legumes) can provide a valuable and yet untapped source of feed for several farmed insect species. In this perspective paper, we provide a viewpoint of how tropical forages can support edible insect production. We also highlight the potential of tropical forage-based diets over those using organic agricultural or urban by-product substrates, due to their versatility, low cost, and lower risk of microbial and chemical hazards. The main bottlenecks relate to dependence on the small number of farmed insect species, and in public policy and market frameworks regarding the use of edible insects as food, feed and in industrial processes. This perspective will serve interested stakeholders in identifying urgent issues at the research, ethical, marketing and policy levels that can prevent the emergence of new, insect-based value chains and business models, and the nutritional, economic and environmental benefits they promise.

A wide variety of insect-specific non-retroviral RNA viruses specifically infect insects. During viral infection, fragments of viral sequences can integrate into the host genomes creating non-retroviral endogenous viral elements (nrEVEs). Although the exact function of nrEVEs is so far unknown, some studies suggest that nrEVEs may interfere with virus replication by producing PIWI-interacting RNAs (piRNAs) that recognize and degrade viral RNAs through sequence complementarity. In this article, we identified the nrEVEs repertoire of ten species within the dipteran family Tephritidae (true fruit flies), which are considered a major threat to agriculture worldwide. Our results suggest that each of these species contains nrEVEs, although in limited numbers, and that nrEVE integration may have occurred both before and after speciation. Furthermore, the majority of nrEVEs originated from viruses with negative single-stranded RNA genomes and represent structural viral functions. Notably, these nrEVEs exhibit low similarity to currently known circulating viruses. To explore the potential role of nrEVEs, we investigated their transcription pattern and the production of piRNAs in different tissues of Ceratitis capitata. We successfully identified piRNAs that are complementary to the sequence of one nrEVE in C. capitata, thereby highlighting a potential link between nrEVEs and the piRNA pathway. Overall, our results provide valuable insights into the comparative landscape of nrEVEs in true fruit flies, contributing to the understanding of the intimate relation between fruit flies and their past and present viral pathogens.

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.