Upon infection, the phenoloxidase system in arthropods is rapidly mobilized and constitutes a major defense system against invaders. The activation of the key enzymes prophenoloxidase (PPO) and their action in immunity through melanization and encapsulation of foreign bodies in hemolymph has been described in many insects. On the other hand, little is known about PPOs involvement in other essential functions related to insect development. In this paper, we investigated the function of the two PPOs of the crop pest, Spodoptera frugiperda (PPO1 and PPO2). We show that PPOs are mainly expressed in hemocytes with the PPO2 expressed at higher levels than the PPO1. In addition, these two genes are expressed in the same tissue and at the same stages of insect development. Through the generation of loss-of-function mutants by CRISPR/Cas9 method, we show that the presence of PPOs is essential for the normal development of the pupa and the survival of the insect.

Advances in viral metagenomics have paved the way of virus discovery by making the exploration of viruses in any ecosystem possible. Applied to agroecosystems, such an approach opens new possibilities to explore how viruses circulate between insects and plants, which may help to optimise their management. It could also lead to identifying novel entomopathogenic viral resources potentially suitable for biocontrol strategies. We sampled the larvae of a natural population of alfalfa weevils (Hypera postica), a major herbivorous pest feeding on legumes, and its host plant alfalfa (Medicago sativa). Insect and plant samples were collected from a crop field and an adjacent meadow. We characterised the diversity and abundance of viruses associated with weevils and alfalfa, and described nine putative new virus species, including four associated with alfalfa and five with weevils. In addition, we found that trophic accumulation may result in a higher diversity of plant viruses in phytophagous pests compared to host plants.

Meteorus pulchricornis (Ichneumonoidea, Braconidae) is an endoparasitoid wasp of lepidopteran caterpillars. Its parasitic success relies on vesicles (named M. pulchricornis Virus-Like Particles or MpVLPs) that are synthesized in the venom gland and injected into the parasitoid host along with the venom during oviposition. In order to define the content and understand the biogenesis of these atypical vesicles, we performed a transcriptome analysis of the venom gland and a proteomic analysis of the venom and purified MpVLPs. About half of the MpVLPs and soluble venom proteins identified were unknown and no similarity with any known viral sequence was found. However, MpVLPs contained a large number of proteins labelled as metalloproteinases while the most abundant protein family in the soluble venom was that of proteins containing the Domain of Unknown Function DUF-4803. The high number of these proteins identified suggests that a large expansion of these two protein families occurred in M. pulchricornis. Therefore, although the exact mechanism of MpVLPs formation remains to be elucidated, these vesicles appear to be "metalloproteinase bombs" that may have several physiological roles in the host including modifying the functions of its immune cells. The role of DUF4803 proteins, also present in the venom of other braconids, remains to be clarified.

Spodoptera frugiperda, the fall armyworm (FAW), is an important agricultural pest in the Americas and an emerging pest in sub-Saharan Africa, India, East-Asia and Australia, causing damage to major crops such as corn, sorghum and soybean. While FAW larvae are considered polyphagous, differences in diet preference have been described between two genetic variants: the corn strain (sf-C) and the rice strain (sf-R). These two strains are sometimes considered as distinct species, raising the hypothesis that host plant specialization might have driven their divergence. To test this hypothesis, we first performed controlled reciprocal transplant (RT) experiments to address the impact of plant diet on several traits linked to the fitness of the sf-C and sf-R strains. The phenotypical data suggest that sf-C is specialized to corn. We then used RNA-Se to identify constitutive transcriptional differences between strains, regardless of diet, in laboratory as well as in natural populations. We found that variations in mitochondrial transcription levels are among the most substantial and consistent differences between the two strains. Since mitochondrial genotypes also vary between the strains, we believe the mitochondria may have a significant role in driving strain divergence.

ABSTRACT The host microbiota may have an impact on pathogens. This is often studied in laboratory-reared hosts but rarely in individuals whose microbiota looks like that of wild animals. In this study, we modified the gut microbiota of the insect Tenebrio molitor by rearing larvae in soil sampled from the field. We showed by high throughput sequencing methods that this treatment modifies the gut microbiota so that it is more diversified than that of laboratory-reared insects, and closely resembled the one of soil-dwelling insects. To describe what the entomopathogenic bacterial symbiont Xenorhabdus (Enterobacteriaceae), vectored by the soil-dwelling nematode Steinernema, might experience in natural conditions, we studied the infestation of the soil-reared T. molitor larvae with three Steinernema–Xenorhabdus pairs. We performed the infestation at 18°C, which delays the emergence of new infective juveniles (IJs), the soil-dwelling nematode forms, but which is a temperature compatible with natural infestation. We analyzed by high throughput sequencing methods the composition of the bacterial community within the insect cadavers before the first emergences of IJs. These bacterial communities were generally characterized by one or two non-symbiont taxa. Even for highly lethal Steinernema–Xenorhabdus pairs, the symbiont does not dominate the bacterial community within the insect cadaver.

A successful biological invasion involves survival in a newly occupied environment. If a population bottleneck occurs during an invasion, the resulting depletion of genetic variants could cause increased inbreeding depression and decreased adaptive potential, which may result in a fitness reduction. How invasive populations survive in the newly occupied environment despite reduced heterozygosity and how, in many cases, they maintain moderate levels of heterozygosity are still contentious issues 1 . The Fall armyworm (FAW; Lepidoptera: Spodoptera frugiperda ), a polyphagous pest, is native to the Western hemisphere. Its invasion in the Old World was first reported from West Africa in early 2016, and in less than four years, it swept sub-Saharan Africa and Asia, finally reaching Australia. We used population genomics approaches to investigate the factors that may explain the invasive success of the FAW. Here we show that genomic balancing selection played a key role in invasive success by restoring heterozygosity before the global invasion. We observe a drastic loss of mitochondrial polymorphism in invasive populations, whereas nuclear heterozygosity exhibits a mild reduction. The population from Benin in West Africa has the lowest length of linkage disequilibrium amongst all invasive and native populations despite its reduced population size. This result indicates that balancing selection increased heterozygosity by facilitating the admixture of invasive populations from distinct origins and that, once heterozygosity was sufficiently high, FAW started spreading globally in the Old World. As comparable heterozygosity levels between invasive and native populations are commonly observed 1 , we postulate that the restoration of heterozygosity through balancing selection could be widespread among successful cases of biological invasions.

Background The holistic view of bacterial symbiosis, incorporating both host and microbial environment, constitutes a major conceptual shift in studies deciphering host-microbe interactions. Interactions between Steinernema entomopathogenic nematodes and their bacterial symbionts, Xenorhabdus , have long been considered monoxenic two partner associations responsible for the killing of the insects and therefore widely used in insect pest biocontrol. We investigated this “monoxenic paradigm” by profiling the microbiota of infective juveniles (IJs), the soil-dwelling form responsible for transmitting Steinernema - Xenorhabdus between insect hosts in the parasitic lifecycle. Results Multigenic metabarcoding (16S and rpoB markers) showed that the bacterial community associated with laboratory-reared IJs from Steinernema carpocapsae , S . feltiae , S . glaseri and S . weiseri species consisted of several Proteobacteria. The association with Xenorhabdus was never monoxenic. We showed that the laboratory-reared IJs of S . carpocapsae bore a bacterial community composed of the core symbiont ( Xenorhabdus nematophila ) together with a frequently associated microbiota (FAM) consisting of about a dozen of Proteobacteria ( Pseudomonas , Stenotrophomonas , Alcaligenes , Achromobacter , Pseudochrobactrum , Ochrobactrum , Brevundimonas , Deftia , etc.). We validated this set of bacteria by metabarcoding analysis on freshly sampled IJs from natural conditions. We isolated diverse bacterial taxa, validating the profile of the Steinernema FAM. We explored the functions of the FAM members potentially involved in the parasitic lifecycle of Steinernema . Two species, Pseudomonas protegens and P . chlororaphis , displayed entomopathogenic properties suggestive of a role in Steinernema virulence and membership of the Steinernema pathobiome. Conclusions Our study validates a shift from monoxenic paradigm to pathobiome view in the case of the Steinernema ecology. The microbial communities of low complexity associated with EPNs will permit future microbiota manipulation experiments to decipher overall microbiota functioning in the infectious process triggered by EPN in insects and, more generally, in EPN ecology.

Bathyplectes spp. are ichneumonid solitary larval parasitoids of the alfalfa weevil which have been classified in the subfamily Campopleginae and which harbor atypical virus particles. Despite the morphological differences between Bathyplectes spp. particles and the polydnaviruses carried by a number of related campoplegine species, called ichnoviruses, the process by which they are produced is very similar to that of ichnoviruses. To address the question of the nature and origin of these atypical particles, the Bathyplectes anurus ovary transcriptome has been analyzed. We found a number of highly expressed transcripts displaying similarities with genes belonging to the machinery involved in the production of ichnovirus particles. In addition, transcripts with similarities with repeat-element genes, which are characteristic of the packaged campoplegine ichnovirus genome were identified. Altogether, our results provide evidence that Bathyplectes particles are related to ichnoviruses.

Viral metagenomics and high throughput sequence mining have revealed unexpected diversity, and the potential presence, of parvoviruses in animals from all phyla. Among arthropods, this diversity highlights the poor knowledge that we have regarding the evolutionary history of densoviruses. The aim of this study was to explore densovirus diversity in a small arthropod pest belonging to Acari, the two-spotted spider mite Tetranychus urticae, while using viral metagenomics based on virus-enrichment. Here, we present the viromes obtained from T. urticae laboratory populations made of contigs that are attributed to nine new potential viral species, including the complete sequence of a novel densovirus. The genome of this densovirus has an ambisens genomic organization and an unusually compact size with particularly small non-structural proteins and a predicted major capsid protein that lacks the typical PLA2 motif that is common to all ambidensoviruses described so far. In addition, we showed that this new densovirus had a wide prevalence across populations of mite species tested and a genomic diversity that likely correlates with the host phylogeny. In particular, we observed a low densovirus genomic diversity between the laboratory and natural populations, which suggests that virus within-species evolution is probably slower than initially thought. Lastly, we showed that this novel densovirus can be inoculated to the host plant following feeding by infected mites, and circulate through the plant vascular system. These findings offer new insights into densovirus prevalence, evolution, and ecology.

Spodoptera frugiperda est un insecte ravageur de culture polyphage qui cree d’importants degâts sur le continent americain. Depuis quelques annees, elle est devenue invasive en Afrique et en Asie et pourrait bientot atteindre l’Europe. Pour mieux controler ce ravageur, de nouveaux moyens de lutte doivent etre proposes aux agriculteurs. L’UMR DGIMI etudie les mecanismes d’interactions entre cette noctuelle, son cortege de pathogenes/parasites et sa plante hote. Nous essayons notamment d’identifier des facteurs clefs impliques dans les defenses immunitaires des insectes ou dans leur adaptation a la plante. Le RNAi n’etant pas fonctionnel chez les lepidopteres, nous ne disposions pas jusqu’alors d’outils de validation fonctionnelle de genes in vivo chez Spodoptera frugiperda. Dans ce poster, nous vous presentons la mise en place de l’outil d’edition de genome CRISPR/Cas9 chez Spodoptera frugiperda. Nous avons genere deux lignees d’insectes portant chacune une mutation perte-de-fonction dans l’un des deux genes pro-phenoloxidase (Sf-PPO1 et Sf-PPO2). L’activite de l’enzyme active issue du clivage de PPO, la phenoloxidase, est fortement diminuee dans l’hemolymphe des deux lignees mutantes comparee a celle observee dans l’hemolymphe de larves sauvages. L’outil « CRISPR/Cas9 » est donc fonctionnel chez Spodoptera frugiperda et les deux lignees obtenues vont maintenant nous permettre de mieux caracteriser le role de chacune des PPO dans l’interaction de cet insecte avec les differents pathogenes etudies au sein de l’unite DGIMI.

Viral metagenomics is a powerful tool to decipher virus diversity and prevalence in agroecosystems, which may help to improve their functioning and their management. Towards this goal, we need to bring ecological concepts into virology starting by the inventory of “who is there, how many are they” to move then to question “what are they doing together”. To this aim, we established an inventory of viruses circulating between plants and insects in prototypic agroecosystems in Montpellier area. We chose two adjacent ecosystems, alfalfa fields and grassland. We sampled there plants and three invasive pest species, ie the cotton bollworm (Lepidoptera, Helicoverpa armigera), the alfalfa weevil (Curculionidae, Hypera postica) larvae and pea aphids (Hemiptera, Acyrthosiphon pisum). The viromes of about 4000 individuals, grouped in 169 samples, were obtained by viral metagenomics. We discovered 47 new arthropod and plant viral species associated with arthropod pests. We found that the composition in arthropod viruses differs completely between pest species, and plants viruses were shared by pest and plants. Finally, we analyzed the distribution of thirteen abundant viral species found in crop pests' viromes in arthropod communities using PCR approach. Arthropods were sampled, they represent twelve orders grouping about 3500 individuals. Five viruses out of the thirteen were found associated with other arthropod species, and were mainly present in predators. Our results showed that coupling viral metagenomics with PCR give insights into the viral diversity and distribution in arthropods.

Hyposoter didymator (Hymenoptera, Ichneumonidae) is a generalist solitary endoparasitoid of noctuid larvae. In the present work, we tested whether populations of H. didymator were divided in several genetically distinct taxa as described for many other generalist parasitoid species, and whether differences in H. didymator parasitism rates were explained by the insect host species and/or by the plant on which these hosts were feeding on. The genetic analysis of natural populations collected in different regions in France and Spain on seven different insect hosts and seven different host plants (775 individuals) showed that H. didymator populations belong to a unique single taxon. However, H. didymator seems to be somewhat specialized. Indeed, in the fields it more often parasitized Helicoverpa armigera compared to the other host species collected in the present work. Also, H. didymator parasitism rates in field conditions and semi-field experimental studies were dependent on the host plants on which H. armigera larvae are feeding. Still, H. didymator can occur occasionally on non-preferred noctuid species. One hypothesis explaining the ability of H. didymator to switch hosts in natura could be related to fluctuating densities of the preferred host over the year; this strategy would allow the parasitoid to avoid seasonal population collapses.

Alfalfa was cultivated in two potted soil series obtained from two sandy soils contaminated by Cu (SM) and metal(loids)/PAH (CD). Shoot production was monitored for 8 weeks. Then, larvae of Spodoptera exigua were reared on alfalfa of both soil series for eight days. A biotest (using Phaseolus vulgaris) was used to assess the soil phytotoxicity. Increasing soil contamination reduced P. vulgaris growth, but alfalfa growth was only reduced on the SM soil series. Exposure to the SM soil was mirrored by shoot Cu and Cr concentrations of alfalfa (respectively, in mg kg -1 DW, Cu and Cr ranged from 11.9 and 0.4 in the CTRL soil to 98.5 and 1.2 in the SM one). Exposure to the CD soil series was mirrored by shoot Zn concentrations (i.e., 48-91.6 mg kg-1 DW). Internal metal(loid) concentrations of S. exigua remained generally steady across both soil series (respectively Cd 0.05-0.16, Cr 0.5-3.3, Cu 5.8-98.5, Ni 0.6-1.6, Pb 0.4-1.3, and Zn 57-337 mg kg-1 DW), and most of the associated transfer factors were lower than 1. Here, due to the excluder phenotype of alfalfa across our TE contamination gradients, S. exigua could cope with high total metal(loid) concentration in both contaminated soils.

The Old World bollworm Helicoverpa armigera is now established in Brazil but efforts to identify incursion origin(s) and pathway(s) have met with limited success due to the patchiness of available data. Using international agricultural/horticultural commodity trade data and mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) and cytochrome b (Cyt b) gene markers, we inferred the origins and incursion pathways into Brazil. We detected 20 mtDNA haplotypes from six Brazilian states, eight of which were new to our 97 global COI-Cyt b haplotype database. Direct sequence matches indicated five Brazilian haplotypes had Asian, African, and European origins. We identified 45 parsimoniously informative sites and multiple substitutions per site within the concatenated (945 bp) nucleotide dataset, implying that probabilistic phylogenetic analysis methods are needed. High diversity and signatures of uniquely shared haplotypes with diverse localities combined with the trade data suggested multiple incursions and introduction origins in Brazil. Increasing agricultural/horticultural trade activities between the Old and New Worlds represents a significant biosecurity risk factor. Identifying pest origins will enable resistance profiling that reflects countries of origin to be included when developing a resistance management strategy, while identifying incursion pathways will improve biosecurity protocols and risk analysis at biosecurity hotspots including national ports.

Modifying the gut microbiota of insect larvea with a bacterial community from the soil. MicrobiOccitanie

During an insect sampling program in alfalfa crops near Montpellier, France in 2011, Lacanobia oleracea larvae were collected that died due to nucleopolyhedrovirus infection (LaolNPV). This virus was subjected to molecular and biological characterization. The virus was a multiple nucleocapsid NPV that showed similar restriction profiles to Mamestra configurata NPV-A (MacoNPV-A) but with significant differences. Polypeptide analysis demonstrated similar proteins in occlusion bodies and occlusion derived virions, to those observed in NPVs from Mamestra spp. Terminal sequencing revealed that the genome organization shared similarity with that of MacoNPV-A. The most homologous virus was MacoNPV-A 90/2 isolate (95.63% identity and 96.47% similarity), followed by MacoNPV-A 90/4 strain (95.37% and 96.26%), MacoNPV-B (89.21% and 93.53%) and M. brassicae MNPV (89.42% and 93.74%). Phylogenetic analysis performed with lef-8, lef-9, polh and a concatenated set of genes showed that LaolNPV and the Mamestra spp. NPVs clustered together with HaMNPV, but with a closer genetic distance to MacoNPV-A strains. The Kimura 2-parameter (K-2-P) distances of the complete genes were greater than 0.05 between LaolNPV and the MbMNPV/MacoNPV-B/HaMNPV complex, which indicates that LaolNPV is a distinct species. K-2-P distances were in the range 0.015-0.050 for comparisons of LaolNPV with MacoNPV-A strains, such that additional biological characteristics should be evaluated to determine species status. While MacoNPV-A was pathogenic to seven lepidopteran species tested, LaolNPV was only pathogenic to Chrysodeixis chalcites. Given these findings, Lacanobia oleracea nucleopolyhedrovirus should be considered as a new species in the Alphabaculovirus genus.

Ichnoviruses (IVs), unique symbiotic viruses carried by ichneumonid campoplegine wasps, derive from integration of a paleo-ichnovirus into an ancestral wasp genome. The modern 'genome' is composed of both regions that are amplified, circularized and encapsidated into viral particles and non-encapsidated viral genomic regions involved in particle morphogenesis. Packaged genomes include multiple circular dsDNAs encoding many genes mostly organized in gene families. Virus particles are assembled in specialized ovarian cells from which they exit into the oviduct lumen; mature virions are injected during oviposition into the insect host. Expression of viral proteins in infected cells correlates with physiological alterations of the host enabling success of parasitism.

The wasp Hyposoter didymator (Hymenoptera, Ichneumonidae) parasitizes several agricultural pest moths and could therefore be used in biological control. The 454 FLX Titanium pyrosequencing technology was used to define two distinct sets of multiplex combining 14 polymorphic microsatellite loci: 10 (referred to as HD) located within the genome of H. didymator and 4 (referred to as HdIV) located within the Ichnovirus genome which is integrated into the wasp genome. Genotyping of two populations collected in France on Helicoverpa armigera revealed that most of the loci are independent and at Hardy–Weinberg equilibrium.

Approches in silico et metagenomique pour caracteriser le role du microbiome associe aux nematodes entomopathogenes. Colloque de Genomique Environnementale Rennes 2013

Les nematodes des genres Heterorhabditis et Steinernema sont des pathogenes d’insectes vivants dans les sols et sont commercialises comme bio-insecticides pour lutter contre certains ravageurs de culture. Les stades infestants (IJs) vivent a l’etat libre dans le sol et transportent en symbiose dans leur tube digestif des enterobacteries des genres Xenorhabdus et Photorhabdus qui seront ensuite liberees dans les insectes parasites. Il s’agit d’associations tres specifiques, puisque chaque espece de nematode est generalement associee a une espece unique de bacteries. Les bacteries symbiotiques sont capables de secreter un tres grand nombre de toxines insecticides, de molecules permettant de neutraliser les defenses de l’insecte, de molecules antibiotiques et d’enzymes de degradation. Les nematodes utilisent ainsi leurs symbiontes bacteriens comme armes pour tuer l’insecte, pour eliminer les autres micro-organismes sensibles a leurs antimicrobiens et convertir la depouille en sources nutritives qui leur permettront de se developper et de se multiplier. Lorsque les ressources sont epuisees, les nematodes se reassocient a leurs bacteries et quittent la depouille de l’insecte a la recherche de nouvelles proies. Si le cycle biologique des nematodes et le role joue par les symbiontes lors du processus infectieux sont bien documentes, peu de choses sont connues sur l’ecosysteme microbien transporte par les larves infestantes du nematode. Il a ete montre que des bacteries appartenant a des genres tres varies (Acinetobacter, Ochrobactrum, Paenibacillus, Pseudomonas, Alcaligenes, etc…) etaient regulierement associees a des nematodes entomopathogenes (1, 2). Nous nous interessons au role de ce microbiome dans le processus infectieux du couple bacterio-helminthique, en combinant des approches de comparaison genomique in silico et des approches de metagenomiques descriptives et fonctionnelles. L’analyse de genomique comparative met en evidence que les bacteries Xenorhabdus et Photorhabdus peuvent partager de nombreux genes de leur genome accessoire avec des bacteries de l’environnement (sol et plantes), ce qui suggere des transferts genetiques horizontaux frequents dans une niche ecologique d’espace restreint (nematode ou cadavre de l’insecte). Les premiers resultats obtenus en PCR-TTGE ciblant l’ADNr 16S revele la presence d’un cortege bacterien inter-cuticulaire stable chez certains nematodes. Ces donnees preliminaires nous permettront d’engager un programme de sequencage a haut debit afin d’identifier en profondeur ce microbiome. A moyen terme, ce projet devrait nous permettre de caracteriser le pathobiome des nematodes entomopathogenes et mieux caracteriser le role de l’holobionte (macroorganisme et son cortege de microorganismes) dans le processus infectieux des nematodes entomopathogenes. (1) Gouge DH & Snyder JL (2006). Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria. J. Invertebrate Pathology 91: 147–157. (2) Babic I, Fischer-Le Saux M, Giraud E, Boemare N (2000). Occurrence of natural dixenic associations between the symbiont Photorhabdus luminescens and bacteria related to Ochrobactrum spp. in tropical entomopathogenic Heterorhabditis spp. (Nematoda, Rhabditida). Microbiology 146:709–718.