We report the complete CDS of five viruses: XiangYun picorna-like virus 4 (Iflaviridae), Protoambidensovirus dipteran1 (Parvoviridae), and three microviruses (Microviridae), detected by viromics surveillance of Aedes albopictus and Culex quinquefasciatus from Réunion Island. The protoambidensovirus detected in A. albopictus belongs to a clade previously reported only in Culex pipiens.

AbstractMosquitoes are major vectors of arboviruses, and host a wide diversity of insect viruses. Recent studies highlighted the impacts of mosquito-associated insect-specific viruses on the transmission of arboviruses. However, we still lack knowledge on the biotic and abiotic factors impacting the distribution dynamics of mosquito specific viruses, although such information has the potential to inform arbovirus surveillance efforts. To gain knowledge on the distribution of mosquito viruses in islands, we collected 13Aedes albopictusand 9Culex quinquefasciatuspooled larvae samples in the Réunion Island, described their whole viral communities at the family level by a viromic approach, and tested the impacts of mosquito species and spatial distance on the structure of their viral communities.Our study show that the composition of viral communities is more strongly linked to mosquito species than to the geographic origin of samples. Spatial disparities were only observed inAedes albopictusviromes. Finally, we described the genomes of five virus taxa (an iflavirus, an ambidensovirus and three potentially novel microvirus species). Notably, we detected the presence of a mosquito-infecting ambidensovirus, named CpDV, inAedes albopictus. It was previously only reported inCulex pipiens, implying that this densovirus may have a broader host range than currently estimated.Overall these results bring insights into the diversity and the distribution of mosquito viruses; their unexplored interactions with two major vectors of arboviral diseases warrant further studies.

Mosquitoes are major vectors of arboviruses, and host a wide diversity of insect viruses. Recent studies highlighted the impacts of mosquito-associated insect-specific viruses on the transmission of arboviruses. However, we still lack knowledge on the biotic and abiotic factors impacting the distribution dynamics of mosquito specific viruses, although such information has the potential to inform arbovirus surveillance efforts. To gain knowledge on the distribution of mosquito viruses in islands, we collected 13 Aedes albopictus and 9 Culex quinquefasciatus pooled larvae samples in the Rėunion Island, described their whole viral communities at the family level by a viromic approach, and tested the impacts of mosquito species and spatial distance on the structure of their viral communities. Our study show that the composition of viral communities is more strongly linked to mosquito species than to the geographic origin of samples. Spatial disparities were only observed in Aedes albopictus viromes. Finally, we described the genomes of five virus taxa (an iflavirus, an ambidensovirus and three potentially novel microvirus species). Notably, we detected the presence of a mosquito-infecting ambidensovirus, named CpDV, in Aedes albopictus . It was previously only reported in Culex pipiens , implying that this densovirus may have a broader host range than currently estimated. Overall these results bring insights into the diversity and the distribution of mosquito viruses; their unexplored interactions with two major vectors of arboviral diseases warrant further studies. ### Competing Interest Statement The authors have declared no competing interest.

Despite tight genetic compression, viral genomes are often organized into functional gene clusters, a modular structure that might favor their evolvability. This has greatly facilitated biotechnological developments such as the recombinant adenoassociated virus (AAV) systems for gene therapy. Following this lead, we endeavored to engineer the related insect parvovirus Junonia coenia densovirus (JcDV) to create addressable vectors for insect pest biocontrol. To enable safer manipulation of capsid mutants, we translocated the nonstructural (ns) gene cluster outside the viral genome. To our dismay, this yielded a virtually nonreplicable clone. We linked the replication defect to an unexpected modularity breach, as ns translocation truncated the overlapping 3' untranslated region (UTR) of the capsid transcript (vp). We found that the native vp 3' UTR is necessary for high-level VP production but that decreased expression does not adversely impact the expression of NS proteins, which are known replication effectors. As nonsense vp mutations recapitulate the replication defect, VP proteins appear to be directly implicated in the replication process. Our findings suggest intricate replication-encapsidation couplings that favor the maintenance of genetic integrity. We discuss possible connections with an intriguing cis-packaging phenomenon previously observed in parvoviruses whereby capsids preferentially package the genome from which they were expressed. IMPORTANCE Densoviruses could be used as biological control agents to manage insect pests. Such applications require an in-depth biological understanding and associated molecular tools. However, the genomes of these viruses remain difficult to manipulate due to poorly tractable secondary structures at their extremities. We devised a construction strategy that enables precise and efficient molecular modifications. Using this approach, we endeavored to create a split clone of Junonia coenia densovirus (JcDV) that can be used to safely study the impact of capsid mutations on host specificity. Our original construct proved to be nonfunctional. Fixing this defect led us to uncover that capsid proteins and their correct expression are essential for continued rolling-hairpin replication. This points to an intriguing link between replication and packaging, which might be shared with related viruses. This serendipitous discovery illustrates the power of synthetic biology approaches to advance our knowledge of biological systems.

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.

Densoviruses (DVs) are parvoviruses of arthropods and causative agents of natural epizootics in insects and crustaceans populations. Structurally simple, these small DNA viruses, display a large diversity of genomic sequences, structures and organizations. Such diversity, together with the diversity of their invertebrate hosts, from shrimps to mosquitoes and recently including sea stars, suggests that DVs are largely unknown and ubiquitous in the environment. Densoviruses are considered as a model of choice to study virus-host interactions and their evolution at different scales, from individuals to populations. This review summarizes the knowledge on densovirus biology obtained through mechanistic and global approaches. Finally, the potential use of these viruses as biological control agents against insect pests and disease-vectors are exposed.

The phosphatidylinositol-3-kinase (PI3K)/Akt/target of rapamycin (TOR) signalling pathway controls cell growth and survival, and is targeted by a number of viruses at different phases of their infection cycle to control translation. Whether and how insect viruses interact with this pathway remain poorly addressed. Here, we investigated the role of PI3K/Akt/TOR signalling during lethal infection of insect cells with an insect parvovirus. Using Junonia coenia densovirus (JcDV; lepidopteran ambidensovirus 1) and susceptible insect cells as experimental models, we first described JcDV cytopathology, and showed that viral infection affects cell size, cell proliferation and survival. We deciphered the role of PI3K/Akt/TOR signalling in the course of infection and found that non-structural (NS) protein expression correlates with the inhibition of TOR and the shutdown of cellular synthesis, concomitant with the burst of viral protein expression. Together, these results suggest that NS proteins control the cellular translational machinery to favour the translation of viral mRNAs at the expense of cellular mRNAs. As a consequence of TOR inhibition, cell autophagy is activated. These results highlight new functions for NS proteins in the course of multiplication of an insect parvovirus.

Background: Spodoptera frugiperda (Noctuidae) is a major agricultural pest throughout the American continent. The highly polyphagous larvae are frequently devastating crops of importance such as corn, sorghum, cotton and grass. In addition, the Sf9 cell line, widely used in biochemistry for in vitro protein production, is derived from S. frugiperda tissues. Many research groups are using S. frugiperda as a model organism to investigate questions such as plant adaptation, pest behavior or resistance to pesticides.Results: In this study, we constructed a reference transcriptome assembly (Sf_TR2012b) of RNA sequences obtained from more than 35 S. frugiperda developmental time-points and tissue samples. We assessed the quality of this reference transcriptome by annotating a ubiquitous gene family -ribosomal proteins - as well as gene families that have a more constrained spatio-temporal expression and are involved in development, immunity and olfaction. We also provide a time-course of expression that we used to characterize the transcriptional regulation of the gene families studied.Conclusion: We conclude that the Sf_TR2012b transcriptome is a valid reference transcriptome. While its reliability decreases for the detection and annotation of genes under strong transcriptional constraint we still recover a fair percentage of tissue-specific transcripts. That allowed us to explore the spatial and temporal expression of genes and to observe that some olfactory receptors are expressed in antennae and palps but also in other non related tissues such as fat bodies. Similarly, we observed an interesting interplay of gene families involved in immunity between fat bodies and antennae.

To evaluate densovirus potential against lepidopteran pests and their capacity to invade new hosts, we have characterised in vivo the infection and pathogenesis of the Junonia coenia densovirus (JcDNV) in the noctuid pest Spodoptera frugiperda. Here we show that infection starts with the ingestion of viral particles that cross the midgut epithelium without replicating. By quantitative PCR we established the kinetic and the route of infection, from virus ingestion to replication in visceral tracheae and hemocytes. JcDNV has a high particle-to-infection ratio mostly due to the barrier function of the midgut. Pathology and cytopathology suggested that infection of tracheal cells impairs oxygen delivery to demanding tissues leading to cytopathic effects in all the tissues. Finally, larval death results from several physiological shocks, including molting arrest and anoxia.

ABSTRACT Junonia coenia densovirus (JcDNV) is an ambisense insect parvovirus highly pathogenic for lepidopteran pests at larval stages. The potential use of DNVs as biological control agents prompted us to reinvestigate the host range and cellular mechanisms of infection. In order to understand the early events of infection, we set up a functional infection assay in a cell line of the pest Lymantria dispar to determine the intracellular pathway undertaken by JcDNV to infect a permissive lepidopteran cell line. Our results show that JcDNV particles are rapidly internalized into clathrin-coated vesicles and slowly traffic within early and late endocytic compartments. Blocking late-endocytic trafficking or neutralizing the pH with drugs inhibited infection. During internalization, disruption of the cytoskeleton, and inhibition of phosphatidylinositol 3-kinase blocked the movement of vesicles containing the virus to the nucleus and impaired infection. In summary, our results define for the first time the early endocytic steps required for a productive DNV infection.

Xanthogaleruca luteola Müller (Coleoptera, Chrysomelidae) is widely spread in Europe and North America and it is a dangerous pest of elm-trees. In Italy the elm-trees are often located in urban areas so the potential of 3 entomoparasitic nematodes (EPNs; 2 Italian strains of Steinernema carpocapsae and S. feltiae and 1 commercial strain of Heterorhabditis bacteriophora), was evaluated through field experiments for control of X. luteola. During the first 2 weeks of June 2006, S. carpocapsae (ItS-MR7), S. feltiae (ItS-CL2) and H. bacteriophora (Nematop®) in gel suspensions (with Idrosorb SR 200 and Xanthan gum) were sprayed on leaves and small branches heavily infested by X. luteola larvae in a periurban park of Bari city (Apulia Region, South Italy). The efficiency of these EPNs was then evaluated after 3 and 5 days. S. carpocapsae in Idrosorb + Xanthan gum controlled 67% of the larvae after 5 days, producing significantly greater larval mortality than S. feltiae and H. bacteriophora. Cultivation conditions of biocomplexes applicable to control Melolontha melolontha C. Sisak, Z. Kaskötő, T. Tóth, T. Lakatos ................................................................... 297 Abstract: Recently several nematode/bacterium complexes have been isolated in Hungary with potential to grubs of the European cockchafer (Melolontha melolontha). The cultivation of one of the most promising biocomplexes, Heterorhabditis downesi ’267’ + Photorhabdus temperata in liquid medium can be considered to be effective if the concentration of the dauer juveniles achieves 10,000/ml. A prerequisite for the elaboration of suitable conditions for cultivation is the development of optimal process conditions (aeration at low shear stress, medium composition, etc.) and to avoid the occurrence of phase variants of the bacterium symbiont. Cultivation experiments with P. temperata were performed first in shake flasks, in LB and TSY medium at 20-22°C. The aeration rate required by the cells during the logarithmic growth period was about 0.6-0.7 vvm. Applying a 5 litre mechanically stirred fermenter equipped with drafttube for bacterium growth studies, a special fedbatch technique has been elaborated. Cell numbers of the primary phase variant were sustained at high numbers over a period of 19 days. Medium optimization studies exchanged several protein components of LCM for lipid components and obtained considerably higher numbers of dauer juveniles and hermaphrodites. Rape seed oil was the most suitable lipid component. In the best medium, the initial nematode dauer juvenile number (1,000/ml) increased to 120,000/ml within 12 days. At present, scale-up studies of the biocomplex are in progress in column bioreactors of different type. Recovery of Steinernema carpocapsae and Steinernema feltiae in liquid culture A. Hirao, R.-U. Ehlers ................................................................................................. 301 Abstract: The development from infective Dauer juvenile (DJ) to third stage juvenile, so called “recovery”, occurs at almost 100% when DJs infect a living insect. In contrast, recovery is much lower, when DJs are inoculated in liquid culture media, in which the symbiotic bacteria were preincubated. For the purpose of obtaining an increase in recovery in liquid culture, recovery inducing factor for S. carpocapsae and S. feltiae was investigated. The influence of penetration behavior on recovery was checked. DJs injected into G. mellonella or inoculated into sand containing the insect recovered at almost 100%. The penetration activity is excluded to influence recovery of S. carpocapsae and S. feltiae. The high recovery in insects might to be due to a food signal present in the serum of G. mellonella. Inoculation of DJs in purified and antibiotics-treated serum was investigated. Whereas in vivo recovery of S. carpocapsae reached 100%, only 40% of S. feltiae recovered in sterilize serum. Whereas a food signal triggering recovery of S. carpocapsae is found in the serum, the composition of the G. mellonella serum seems suboptimal for recovery of S. feltiae. The influence of the density of the symbiotic bacteria, X. nematophila and X. bovienii (isolated from S. carpocapsae and S. feltiae, respectively) was investigated. The bacteria were proliferated in liquid medium for 36 and 48 hours. Then DJs at a density of 1,000 DJs/ml were inoculated at bacterial densities of 108, 109 and 1010 cells/ml. Recovery of S. carpocapsae was checked every two hours until 12 h post inoculation (hpi) and S. feltiae every two hours between 4 and 10 hpi. Comparing recovery at different bacterial densities, recovery of both nematodes increased with increasing bacterial density. However, the recovery of S. feltiae was delayed and lower compared to results obtained with S. carpocapsae. Culture supernatant of both bacterial symbionts contained food signal, however, the recovery was significantly higher when bacterial cells were present. Differential gene expression of recovery in Heterhorabditis bacteriophora A. Moshayov, H. Koltai, I. Glazer (Abstract only) ......................................................... 307 Expression of different desiccation tolerance related genes in various species of entomopathogenic nematodes V. S. Somvanshi, H. Koltai, I. Glazer (Abstract only) ................................................... 308 Genetic improvement of beneficial traits mixed population of Steinernema feltiae for enhancement of persistence and efficacy M. Chubinishvili, L. Salame, C. Chkhubianishvili, I. Glazer (Abstract only) ................. 309 Soil insect pests and miscellaneous When should we use biocontrol agents against leatherjackets (Tipula paludosa Meig.) R.P.Blackshaw ........................................................................................................... 313 Abstract: Recent work has demonstrated that biological control agents directed against the larvae of Tipula paludosa (leatherjackets) are more effective if applied against early instars in October rather than later instars in the late winter/early spring. In this paper simulation modelling is used to identify potential strategies for the deployment of biocontrol agents against leatherjackets in continuous grass, and in grass/arable rotations. It is concluded that the optimal prophylactic strategy in continuous grass lies between annual and biannual application frequencies, and that application in the autumn preceding cultivation for a spring crop suppresses populations better than applications earlier in the rotation. A decision tool based on economic thresholds is presented for grass/arable rotations. The results are discussed and it is concluded that development of a robust monitoring system is necessary if recommendations other than prophylactic applications are to be made. Biocontrol of Capnodis tenebrionis (L.) (Col., Buprestidae) with entomopathogenic fungi P. Marannino, C. Santiago Álvarez, E. de Lillo, E. Tarasco, O. Triggiani, E. Quesada Moraga ........................................................................................................ 319 Abstract: A study was carried out at ETSIAM to investigate the potential of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorok. for the control of the peach root-borer, Capnodis tenebrionis (L.), a major threat to stone-fruit orchards in several Mediterranean countries. The pathogenicity of 4 B. bassiana and 4 M. anisopliae isolates (from ETSIAM collection) against C. tenebrionis neonate larvae was assessed under endophytic conditions. Mortality rates varied from 23.5 to 100%, 10 days after inoculation by dipping in a suspension with 108 conidia/ml. The most virulent isolate, M. anisopliae EAMa 01/58-Su, was used as spore suspension (106, 107, 108 conidia/ml) for soil application on Prunus myrobalana Lois. potted seedlings 2 days before infesting them with C. tenebrionis neonates. The treatment caused a significant mortality (83.3 to 91.6%) on the larvae recovered from the roots 4 weeks later. No significant difference was detected among the doses. The susceptibility of C. tenebrionis adults to EAMa 01/58-Su was also assessed and a significant mortality (86.7%) was ascertained after immersion in a spore suspension (108 conidia/ml). No significant difference was pointed out between male and female mortalities. The effectiveness of the selected M. anisopliae isolate was proved also using non-woven fiber bands impregnated with conidia. Mortality of beetles that crossed the band varied from 85.7 to 100%. No significant correlation was found between time needed to cross the band and survival times. Our results demonstrate that EAMa 01/58Su is suitable to target successfully both peach-borer stages. Infectivity of Steinernema feltiae and Heterorhabditis bacteriophora towards first and second stage larvae of the forest cockchafer, Melolontha hippocastani K. Jung, A. Peters, J. Pelz .......................................................................................... 327 Abstract: At high population densities, the forest cockchafer, Melolontha hippocastani is a serious pest insect causing death to trees and seedlings. In the southern Rhine valley of Germany, a new gradation of M. hippocastani has built up since the 1980s. Despite considerable results on the control of grubs with entomopathogenic nematodes (EPN), so far no nematode was identified, which efficiently infect Melolontha spp. We herein report on laboratory assays with H. bacteriophora and S. feltiae, both alone, in combination with one another and with B. brongniartii against the 1st and 2nd instar larvae of M. hippocastani. First instar larvae did not seem to be more susceptible than second instar larvae. None of the nematode, alone or in combination caused high mortality, with H. bacteriophora being a little better than S. feltiae (28 compared to 4 % at 500.000 IJ’s/m2). In conclusion, the nematodes currently availa

Interleukin-17 (IL-17) is a proinflammatory cytokine produced by activated memory T cells, and it appears to play an upstream role in T cell-triggered inflammation by stimulating stromal cells to secrete other cytokines. We hypothesize that IL-17 plays a role in the recruitment of neutrophils in the bovine mammary gland during infection or immune-mediated inflammation. The rapid amplification of cDNA ends (RACE) method was used to obtain a cDNA of bovine IL-17 (BoIL-17) containing a 462-bp open reading frame (ORF) encoding a protein of 153 amino acids (aa) with a molecular mass of 17.2 kDa, a 23-residue NH2-terminal signal peptide, a single potential N-linked glycosylation site, and 6 cysteine residues. BoIL-17 protein shared 73.5% identity with the human protein and 67% with the mouse and rat proteins. Sf9 insect cells were transfected with BoIL-17 cDNA, and supernatant was tested for biologic activity on a primary culture of bovine mammary epithelial cells (MECs). mRNA synthesis of IL-6, IL-8, and growth-related oncogene alpha (Gro alpha) was induced, suggesting a functional role for IL-17 in mammary immunity.