Non-retroviral Endogenous Viral Elements in Tephritid Fruit Flies Reveal Former Viral Infections Not Related to Known Circulating Viruses.

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.

Compatibility of mycorrhiza-induced resistance with viral and bacterial entomopathogens in the control of Spodoptera exigua in tomato.

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) are soil-borne microorganisms that establish mutualistic associations with roots of most terrestrial plants. This symbiosis results in nutritional and defensive benefits to the host plant, usually conferring protection against biotic stresses, but its indirect impact on third trophic levels is still unknown. In the present work, we explore whether the symbiosis of tomato plants with Funneliformis mosseae (and/or exposition to herbivory) influences the interaction of the generalist pest Spodoptera exigua (Lepidoptera: Noctuidae) with bacterial (Bacillus thuringiensis) and viral (baculovirus, SeMNPV) natural entomopathogens. RESULTS: Symbiosis with AMF and previous herbivory reduces the relative growth of S. exigua, increases its susceptibility to a sublethal dose of B. thuringiensis and has positive or neutral impact on the lethality of SeMNPV. Reduction of the phenoloxidase activity, a marker of the insect immune response, was associated with the larval feeding on plant material previously exposed to herbivory but not to the AMF. In addition, no changes in the insect gut microbiota could be associated with the observed changes in larval growth and susceptibility to the entomopathogens. CONCLUSION: Our findings provide the first evidence of compatibility of AMF symbiosis in tomato with the use of bacterial and viral entomopathogens, contributing to the development of novel approaches to combine the beneficial effect of AMF and entomopathogens in biological pest control. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Expanding the Medfly Virome: Viral Diversity, Prevalence, and sRNA Profiling in Mass-Reared and Field-Derived Medflies.

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.

Baculovirus infection affects caterpillar chemoperception.

Baculoviruses are double-stranded DNA entomopathogenic viruses that infect predominantly insects of the order Lepidoptera. Research in the last decade has started to disentangle the mechanisms underlying the insect-virus interaction, particularly focusing on the effects of the baculovirus infection in the host's physiology. Among crucial physiological functions, olfaction has a key role in reproductive tasks, food source detection and enemy avoidance. In this work, we describe that Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces expression changes in some odorant receptors (ORs) - the centrepiece of insect's olfaction - when infecting larvae from its natural host Spodoptera exigua (Lepidoptera: Noctuidae). Different ORs are up-regulated in larvae after SeMNPV infection, and two of them, SexiOR35 and SexiOR23, were selected for further functional characterization by heterologous expression in empty neurons of Drosophila melanogaster coupled to single-sensillum recordings. SexiOR35 appears to be a broadly tuned receptor able to recognise multiple and different chemical compounds. SexiOR23, although correctly expressed in Drosophila neurons, did not display any significant response to a panel of 58 stimuli. Behavioural experiments revealed that larvae infected by SeMNPV exhibit altered olfactory-driven behaviour to diet when it is supplemented with the plant volatiles linalool or estragole, two of the main SexiOR35 ligands, supporting the hypothesis that viral infection triggers changes in host perception through changes in the expression level of specific ORs.

Drosophila suzukii (Diptera: Drosophilidae): A Decade of Research Towards a Sustainable Integrated Pest Management Program.

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) also known as spotted-wing drosophila (SWD), is a pest native to Southeast Asia. In the last few decades, the pest has expanded its range to affect all major European and American fruit production regions. SWD is a highly adaptive insect that is able to disperse, survive, and flourish under a range of environmental conditions. Infestation by SWD generates both direct and indirect economic impacts through yield losses, shorter shelf life of infested fruit, and increased production costs. Fresh markets, frozen berries, and fruit export programs have been impacted by the pest due to zero tolerance for fruit infestation. As SWD control programs rely heavily on insecticides, exceedance of maximum residue levels (MRLs) has also resulted in crop rejections. The economic impact of SWD has been particularly severe for organic operations, mainly due to the limited availability of effective insecticides. Integrated pest management (IPM) of SWD could significantly reduce chemical inputs but would require substantial changes to horticultural management practices. This review evaluates the most promising methods studied as part of an IPM strategy against SWD across the world. For each of the considered techniques, the effectiveness, impact, sustainability, and stage of development are discussed.

ViR: a tool to solve intrasample variability in the prediction of viral integration sites using whole genome sequencing data.

BACKGROUND: Several bioinformatics pipelines have been developed to detect sequences from viruses that integrate into the human genome because of the health relevance of these integrations, such as in the persistence of viral infection and/or in generating genotoxic effects, often progressing into cancer. Recent genomics and metagenomics analyses have shown that viruses also integrate into the genome of non-model organisms (i.e., arthropods, fish, plants, vertebrates). However, rarely studies of endogenous viral elements (EVEs) in non-model organisms have gone beyond their characterization from reference genome assemblies. In non-model organisms, we lack a thorough understanding of the widespread occurrence of EVEs and their biological relevance, apart from sporadic cases which nevertheless point to significant roles of EVEs in immunity and regulation of expression. The concomitance of repetitive DNA, duplications and/or assembly fragmentations in a genome sequence and intrasample variability in whole-genome sequencing (WGS) data could determine misalignments when mapping data to a genome assembly. This phenomenon hinders our ability to properly identify integration sites. RESULTS: To fill this gap, we developed ViR, a pipeline which solves the dispersion of reads due to intrasample variability in sequencing data from both single and pooled DNA samples thus ameliorating the detection of integration sites. We tested ViR to work with both in silico and real sequencing data from a non-model organism, the arboviral vector Aedes albopictus. Potential viral integrations predicted by ViR were molecularly validated supporting the accuracy of ViR results. CONCLUSION: ViR will open new venues to explore the biology of EVEs, especially in non-model organisms. Importantly, while we generated ViR with the identification of EVEs in mind, its application can be extended to detect any lateral transfer event providing an ad-hoc sequence to interrogate.

Population genomics in the arboviral vector Aedes aegypti reveals the genomic architecture and evolution of endogenous viral elements.

Horizontal gene transfer from viruses to eukaryotic cells is a pervasive phenomenon. Somatic viral integrations are linked to persistent viral infection whereas integrations into germline cells are maintained in host genomes by vertical transmission and may be co-opted for host functions. In the arboviral vector Aedes aegypti, an endogenous viral element from a nonretroviral RNA virus (nrEVE) was shown to produce PIWI-interacting RNAs (piRNAs) to limit infection with a cognate virus. Thus, nrEVEs may constitute a heritable, sequence-specific mechanism for antiviral immunity, analogous to piRNA-mediated silencing of transposable elements. Here, we combine population genomics and evolutionary approaches to analyse the genomic architecture of nrEVEs in A. aegypti. We conducted a genome-wide screen for adaptive nrEVEs and searched for novel population-specific nrEVEs in the genomes of 80 individual wild-caught mosquitoes from five geographical populations. We show a dynamic landscape of nrEVEs in mosquito genomes and identified five novel nrEVEs derived from two currently circulating viruses, providing evidence of the environmental-dependent modification of a piRNA cluster. Overall, our results show that virus endogenization events are complex with only a few nrEVEs contributing to adaptive evolution in A. aegypti.

Coupling Transcriptomics and Behaviour to Unveil the Olfactory System of Spodoptera exigua Larvae.

Insect chemosensation is crucial for many aspects related to food seeking, enemy avoidance, and reproduction. Different families of receptors and binding proteins interact with chemical stimuli, including odorant receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), odorant binding proteins (OBPs) and chemosensory proteins (CSPs). In this work, we describe the chemosensory-related gene repertoire of the worldwide pest Spodoptera exigua (Lepidoptera: Noctuidae), focusing on the transcripts expressed in larvae, which feed on many horticultural crops producing yield losses. A comprehensive de novo assembly that includes reads from chemosensory organs of larvae and adults, and other larval tissues, enabled us to annotate 200 candidate chemosensory-related genes encoding 63 ORs, 28 IRs, 38 GRs, 48 OBPs and 23 CSPs. Of them, 51 transcripts are new annotations. Fifty ORs are expressed in larval heads based on RNA-seq and reverse transcription PCR analyses. Fourteen OBPs are expressed in larval, but not in adult heads. We also observe that expression profiles of ORs are strongly and non-specifically up-regulated upon pre-exposure of larvae to single volatile organic compounds (VOCs). Finally, we develop a behavioural assay to study the attraction/repellence to VOCs in S. exigua larvae and thus identify candidate ecologically relevant odours. A single-dose assay demonstrated that 1-hexanol triggers attraction and indole repels larvae at any timepoint. This work establishes the foundation for the study of chemosensation in S. exigua larvae, allowing further studies aimed to characterize chemosensory-related genes that underlie the ecologically relevant behaviours of larvae.

Structural and transcriptional evidence of mechanotransduction in the Drosophila suzukii ovipositor.

Drosophila suzukii is an invasive pest that prefers to lay eggs in ripening fruits, whereas most closely related Drosophila species exclusively use rotten fruit as oviposition site. This behaviour is allowed by an enlarged and serrated ovipositor that can pierce intact fruit skin, and by multiple contact sensory systems (mechanosensation and taste) that detect the optimal egg-laying substrates. Here, we tested the hypothesis that bristles present in the D. suzukii ovipositor tip contribute to these sensory modalities. Analysis of the bristle ultrastructure revealed that four different types of cuticular elements (conical pegs type 1 and 2, chaetic and trichoid sensilla) are present on the tip of each ovipositor plate. All of them have a poreless shaft and are innervated at their base by a single neuron that ends in a distal tubular body, thus resembling mechanosensitive structures. Fluorescent labelling in D. suzukii and D. melanogaster revealed that pegs located on the ventral side of the ovipositor tip are innervated by a single neuron in both species. RNA-sequencing profiled gene expression, notably sensory receptor genes of the terminalia of D. suzukii and of three other Drosophila species with changes in their ovipositor structure (from serrated to blunt ovipositor: Drosophila subpulchrella, Drosophila biarmipes and D. melanogaster). Our results revealed few species-specific transcripts and an overlapping expression of candidate mechanosensitive genes as well as the presence of some chemoreceptor transcripts. These experimental evidences suggest a mechanosensitive function for the D. suzukii ovipositor, which might be crucial across Drosophila species independently from ovipositor shape.

Functional transcriptome analyses of Drosophila suzukii antennae reveal mating-dependent olfaction plasticity in females.

Insect olfaction modulates basal behaviors and it is often influenced by the physiological condition of each individual such as the reproductive state. Olfactory plasticity can be achieved by modifications at both peripheral and central nervous system levels. Here we performed a genome-wide transcriptomic analysis of the main olfactory organ, the antenna, to investigate how gene expression varies with female mating status in Drosophila suzukii, a destructive and invasive soft fruit pest. We observed a wide mating-induced up-regulation of chemosensory-related genes in females, especially odorant receptor (Or) genes. We then used a candidate gene approach to define the comprehensive dataset of antenna-expressed chemosensory receptors and binding proteins, which showed many similarities with Drosophila melanogaster. Candidate gene approach was also used to finely quantify differential expression at Or isoform level, suggesting post-mating transcriptional modulation of genes involved in the peripheral olfactory system. We identified 27 up-regulated Or transcripts encoded by 25 genes, seven of them were duplications specific to D. suzukii lineage. Post-mating olfactory modulation was further supported by electroantennogram recordings that showed a differential response according to mating status to one out of eight odors tested (isoamyl-acetate). Our study characterizes the transcriptional mechanisms driven by mating in D. suzukii female antennae. Understanding the role of genes differentially expressed in virgin or mated females will be crucial to better understand host finding and the crop-damaging oviposition behavior of this species.

Cytokinin transfer by a free-living mirid to Nicotiana attenuata recapitulates a strategy of endophytic insects.

Endophytic insects provide the textbook examples of herbivores that manipulate their host plant's physiology, putatively altering source/sink relationships by transferring cytokinins (CK) to create 'green islands' that increase the nutritional value of infested tissues. However, unambiguous demonstrations of CK transfer are lacking. Here we show that feeding by the free-living herbivore Tupiocoris notatus on Nicotiana attenuata is characterized by stable nutrient levels, increased CK levels and alterations in CK-related transcript levels in attacked leaves, in striking similarity to endophytic insects. Using 15N-isotope labeling, we demonstrate that the CK N6-isopentenyladenine (IP) is transferred from insects to plants via their oral secretions. In the field, T. notatus preferentially attacks leaves with transgenically increased CK levels; plants with abrogated CK-perception are less tolerant of T. notatus feeding damage. We infer that this free-living insect uses CKs to manipulate source/sink relationships to increase food quality and minimize the fitness consequences of its feeding.

Transcriptome profiling reveals differential gene expression of detoxification enzymes in a hemimetabolous tobacco pest after feeding on jasmonate-silenced Nicotiana attenuata plants.

BACKGROUND: The evolutionary arms race between plants and insects has driven the co-evolution of sophisticated defense mechanisms used by plants to deter herbivores and equally sophisticated strategies that enable phytophagous insects to rapidly detoxify the plant's defense metabolites. In this study, we identify the genetic determinants that enable the mirid, Tupiocoris notatus, to feed on its well-defended host plant, Nicotiana attenuata, an outstanding model for plant-insect interaction studies. RESULTS: We used an RNAseq approach to evaluate the global gene expression of T. notatus after feeding on a transgenic N. attenuata line which does not accumulate jasmonic acid (JA) after herbivory, and consequently accumulates very low levels of defense metabolites. Using Illumina sequencing, we generated a de novo assembled transcriptome which resulted in 63,062 contigs (putative transcript isoforms) contained in 42,610 isotigs (putative identified genes). Differential expression analysis based on RSEM-estimated transcript abundances identified 82 differentially expressed (DE) transcripts between T. notatus fed on wild-type and the defenseless plants. The same analysis conducted with Corset-estimated transcript abundances identified 59 DE clusters containing 85 transcripts. In both analyses, a larger number of DE transcripts were found down-regulated in mirids feeding on JA-silenced plants (around 70%). Among these down-regulated transcripts we identified seven transcripts possibly involved in the detoxification of N. attenuata defense metabolite, specifically, one glutathione-S-transferase (GST), one UDP-glucosyltransferase (UGT), five cytochrome P450 (P450s), and six serine proteases. Real-time quantitative PCR confirmed the down-regulation for six transcripts (encoding GST, UGT and four P450s) and revealed that their expression was only slightly decreased in mirids feeding on another N. attenuata transgenic line specifically silenced in the accumulation of diterpene glycosides, one of the many classes of JA-mediated defenses in N. attenuata. CONCLUSIONS: The results provide a transcriptional overview of the changes in a specialist hemimetabolous insect associated with feeding on host plants depleted in chemical defenses. Overall, the analysis reveals that T. notatus responses to host plant defenses are narrow and engages P450 detoxification pathways. It further identifies candidate genes which can be tested in future experiments to understand their role in shaping the T. notatus-N. attenuata interaction.

Evolutionary Insights into Taste Perception of the Invasive Pest Drosophila suzukii.

Chemosensory perception allows insects to interact with the environment by perceiving odorant or tastant molecules; genes encoding chemoreceptors are the molecular interface between the environment and the insect, and play a central role in mediating its chemosensory behavior. Here, we explore how the evolution of these genes in the emerging pest Drosophila suzukii correlates with the peculiar ecology of this species. We annotated approximately 130 genes coding for gustatory receptors (GRs) and divergent ionotropic receptors (dIRs) in D. suzukii and in its close relative D. biarmipes We then analyzed the evolution, in terms of size, of each gene family as well of the molecular evolution of the genes in a 14 Drosophila species phylogenetic framework. We show that the overall evolution of GRs parallels that of dIRs not only in D. suzukii, but also in all other analyzed Drosophila Our results reveal an unprecedented burst of gene family size in the lineage leading to the suzukii subgroup, as well as genomic changes that characterize D. suzukii, particularly duplications and strong signs of positive selection in the putative bitter-taste receptor GR59d. Expression studies of duplicate genes in D. suzukii support a spatio-temporal subfunctionalization of the duplicate isoforms. Our results suggest that D. suzukii is not characterized by gene loss, as observed in other specialist Drosophila species, but rather by a dramatic acceleration of gene gains, compatible with a highly generalist feeding behavior. Overall, our analyses provide candidate taste receptors specific for D. suzukii that may correlate with its specific behavior, and which may be tested in functional studies to ultimately enhance its control in the field.

The Evolution of Olfactory Gene Families in Drosophila and the Genomic Basis of chemical-Ecological Adaptation in Drosophila suzukii.

How the evolution of olfactory genes correlates with adaption to new ecological niches is still a debated topic. We explored this issue in Drosophila suzukii, an emerging model that reproduces on fresh fruit rather than in fermenting substrates like most other Drosophila We first annotated the repertoire of odorant receptors (ORs), odorant binding proteins (OBPs), and antennal ionotropic receptors (aIRs) in the genomes of two strains of D. suzukii and of its close relative Drosophila biarmipes We then analyzed these genes on the phylogeny of 14 Drosophila species: whereas ORs and OBPs are characterized by higher turnover rates in some lineages including D. suzukii, aIRs are conserved throughout the genus. Drosophila suzukii is further characterized by a non-random distribution of OR turnover on the gene phylogeny, consistent with a change in selective pressures. In D. suzukii, we found duplications and signs of positive selection in ORs with affinity for short-chain esters, and loss of function of ORs with affinity for volatiles produced during fermentation. These receptors-Or85a and Or22a-are characterized by divergent alleles in the European and American genomes, and we hypothesize that they may have been replaced by some of the duplicated ORs in corresponding neurons, a hypothesis reciprocally confirmed by electrophysiological recordings. Our study quantifies the evolution of olfactory genes in Drosophila and reveals an array of genomic events that can be associated with the ecological adaptations of D. suzukii.

Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus.

Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity.

Midgut aminopeptidase N isoforms from Ostrinia nubilalis: activity characterization and differential binding to Cry1Ab and Cry1Fa proteins from Bacillus thuringiensis.

Aminopeptidase N (APN) isoforms from Lepidoptera are known for their involvement in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis. These enzymes belong to a protein family with at least eight different members that are expressed simultaneously in the midgut of lepidopteran larvae. Here, we focus on the characterization of the APNs from Ostrinia nubilalis (OnAPNs) to identify potential Cry receptors. We expressed OnAPNs in insect cells using a baculovirus system and analyzed their enzymatic activity by probing substrate specificity and inhibitor susceptibility. The interaction with Cry1Ab and Cry1Fa proteins (both found in transgenic insect-resistant maize) was evaluated by ligand blot assays and immunocytochemistry. Ligand blots of brush border membrane proteins showed that both Cry proteins bound mainly to a 150 kDa-band, in which OnAPNs were greatly represented. Binding analysis of Cry proteins to the cell-expressed OnAPNs showed that OnAPN1 interacted with both Cry1Ab and Cry1Fa, whereas OnAPN3a and OnAPN8 only bound to Cry1Fa. Two isoforms, OnAPN2 and OnAPN3b, did not interact with any of these two proteins. This work provides the first evidence of a differential role of OnAPN isoforms in the mode of action of Cry proteins in O. nubilalis.

Quantitative genetic analysis of Cry1Ab tolerance in Ostrinia nubilalis Spanish populations.

Tolerance to Bacillus thuringiensis Cry1Ab toxin in Spanish Ostrinia nubilalis populations was analyzed by quantitative genetic techniques, using isolines established from field-derived insects. F1 offspring was tested for susceptibility to trypsin activated Cry1Ab using a concentration that caused a mean larval mortality of 87% (±17% SD). The progeny of the most tolerant isolines (that had shown mortalities lower than 60%) was crossed to obtain the F2 generation that was exposed to the same Cry1Ab concentration. A clear reduction in mortality (62±17% SD) was observed. The upper limit for heritability was estimated to range between 0.82 and 0.90, suggesting that a high part of phenotypic variation in tolerance to Cry1Ab was attributable to genetic differences. An estimate of the minimum number of segregating factors indicated that the loci involved in tolerance to Cry1Ab were at least two. The role of the cadherin gene, which is a B. thuringiensis resistance gene in Lepidoptera, was assessed in the most tolerant isolines by using an EPIC-PCR marker specifically developed for this study. Association between cadherin and tolerance was obtained in one tolerant isoline; however it could be not confirmed by segregation analysis in the F2 progeny because F2 offspring was not viable. Our results indicate that the tolerance trait is common in Spanish field populations. Quantitative genetic techniques may be helpful for estimating the influence of genetic factors to Cry1Ab tolerance in O. nubilalis.

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design.

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.

Study of the aminopeptidase N gene family in the lepidopterans Ostrinia nubilalis (Hübner) and Bombyx mori (L.): sequences, mapping and expression.

Aminopeptidases N (APNs) are a class of ectoenzymes present in lepidopteran larvae midguts, involved in the Bacillus thuringiensis (Bt) toxins mode of action. In the present work, seven aminopeptidases have been cloned from the midgut of Ostrinia nubilalis, the major Lepidopteran corn pest in the temperate climates. Six sequences were identified as APNs because of the presence of the HEXXH(X)18E and GAMEN motifs, as well as the signal peptide and the GPI-anchor sequences. The remaining sequence did not contain the two cellular targeting signals, indicating it belonged to the puromycin-sensitive aminopeptidase (PSA) family. An in silico analysis allowed us to find orthologous sequences in Bombyx mori. A phylogenetic study of lepidopteran aminopeptidase sequences resulted in their clustering into nine classes. Linkage analysis revealed that the onapn genes as well as all bmapn genes clustered in a single linkage group. O. nubilalis aminopeptidases were expressed in all larval instars. In 5th instar larvae tissues, apns transcripts were found mainly in midguts while apn8 was also highly expressed in Malpighian tubules, and psa showed an ubiquitous expression pattern in O. nubilalis and B. mori. The sequence homology and gene organization of apns suggest a single origin from an ancestral lepidopteran apn gene.