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Thousands of Hymenopteran endoparasitoids have developed a unique symbiotic relationship with viruses named polydnavirus (PDVs). These viruses immunocompromise the lepidopteran host allowing the survival of the wasp eggs. In a previous work, we have shown the horizontal transfer of some polydnaviral genes into the genome of the Lepidoptera, Spodoptera exigua. One of these genes, BV2-5 (named gasmin) interferes with actin polymerization, negatively affecting the multiplication of baculovirus in cell culture. In this work, we have focused in the study of the effect of Gasmin expression on different aspects of the baculovirus production. In addition, and since actin polymerization is crucial for phagocytosis, we have studied the effect of Gasmin expression on the larval interaction with bacterial pathogens. Over-expression of Gasmin on hemocytes significantly reduces their capacity to phagocytize the pathogenic bacteria Bacillus thuringiensis. According to these results, gasmin domestication negatively affects baculovirus replication, but increases larvae susceptibility to bacterial infections as pay off. Although the effect of Gasmin on the insect interaction with other pathogens or parasitoids remain unknown, the opposite effects described here could shape the biological history of this species based on the abundance of certain type of pathogens as suggested by the presence of truncated forms of this protein in several regions of the world.

Insect innate immunity relies on numerous soluble and membrane-bound receptors, named pattern recognition proteins (PRPs), which enable the insect to recognize pathogen-associated molecular patterns. C-type lectins are among the best-studied PRPs and constitute the most diverse family of animal lectins. Here we have characterized two groups of Spodoptera exigua C-type lectins that differ in their phylogeny, domain architecture, and expression pattern. One group includes C-type lectins with similar characteristics to other lepidopteran lectins, and a second group includes bracoviral-related lectins (bracovirus-like lectins, Se-BLLs) recently acquired by horizontal gene transfer. Subsequently, we have investigated the potential role of some selected lectins in the susceptibility to Junonia coenia densovirus (JcDV). For this purpose, three of the bracoviral-related lectins were expressed, purified, and their effect on the densovirus infection to two different Spodoptera species was assessed. The results showed that Se-BLL3 specifically reduce the mortality of Spodoptera frugiperda larvae caused by JcDV. In contrast, no such effect was observed with S. exigua larvae. In a previous work, we have also shown that Se-BLL2 increased the tolerance of S. exigua larvae to baculovirus infection. Taken together, these results confirm the implication of two different C-type lectins in antiviral response and reflect the biological relevance of the acquisition of bracoviral genes in Spodoptera spp.

Biological insecticides based on Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) can efficiently control S. exigua larvae on field and greenhouse crops in many parts of the world. Spanish wild populations and laboratory colonies of S. exigua are infected by two iflaviruses (SeIV-1 and SeIV-2). Here we evaluated the effect of iflavirus co-infection on the insecticidal characteristics of SeMNPV occlusion bodies (OBs). Overall, iflavirus co-inoculation consistently reduced median lethal concentrations (LC50) for SeMNPV OBs compared to larvae infected with SeMNPV alone. However, the speed of kill of SeMNPV was similar in the presence or absence of the iflaviruses. A reduction of the weight gain (27%) associated with iflavirus infection resulted in a 30% reduction in total OB production per larva. Adult survivors of SeMNPV OB inoculation were examined for covert infection. SeMNPV DNA was found to be present at a high prevalence in all SeIV-1 and SeIV-2 co-infection treatments. Interestingly, co-inoculation of SeMNPV with SeIV-2 alone or in mixtures with SeIV-1 resulted in a significant increase in the SeMNPV load of sublethally infected adults, suggesting a role for SeIV-2 in vertical transmission or reactivation of sublethal SeMNPV infections. In conclusion, iflaviruses are not desirable in insect colonies used for large scale baculovirus production, as they may result in diminished larval growth, reduced OB production and, depending on their host-range, potential risks to non-target Lepidoptera., This study was funded by the Spanish Ministerio de Ciencia e Innovacio´n, project number AGL2011-30352-C02-01. AC received a student scholarship from the Ministerio de Economı´a y Competitividad.

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.

The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (pph) promoter. Additionally, the orf46 promoter was also tested in combination with the pph promoter, revealing an additive effect over the pph promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.

Analysis of the transcriptome of Spodoptera exigua larvae revealed the presence of several ESTs with homology to virus of the order Picornavirales and with the highest similarity to Infectious flacherie virus (Iflaviridae) that infects Bombyx mori larvae. Iflaviridae is a recently defined family of insect-infecting viruses that consist of positive single strand RNA genomes translated into a single polyprotein of around 3000 amino acids long. Using the sequence information derived from the obtained ESTs, we have completed the genomic sequence of this virus. The novel S. exigua iflavirus (SeIV-1) has a genome of 10.3 kb and codes for a 3222 aa polyprotein. Expression analysis has revealed the presence of the virus in all tissues tested and insect stages, being more abundant in the midgut of the larvae. High infectivity of this virus against S. exigua has been demonstrated after observing the presence of this virus in different colonies that were reared in the same chamber with the virus-infected colony, despite no evidence of pathological effects. Further study of viral covert infections of SeIV-1 could lead to a better understanding of its pathological effect as well as any possible interaction with other microbial pathogens used for the control of this pest.

Several expressed sequence tags (ESTs) with homology to chitin deacetylase-like protein (CDA) were selected from a group of Helicoverpa armigera genes whose expression changed after infection with H. armigera single nucleopolyhedrovirus (HearNPV). Some of these ESTs coded for a midgut protein containing a chitin deacetylase domain (CDAD). The expressed protein, HaCDA5a, did not show chitin deacetylase activity, but it showed a strong affinity for binding to chitin. Sequence analysis showed the lack of any chitin binding domain, described for all currently known peritrophic membrane (PM) proteins. HaCDA5a has previously been detected in the H. armigera PM. Such localization, together with its downregulation after pathogen infection, led us to hypothesize that this protein might be responsible for the homeostasis of the PM structure and that, by reduction of its expression, the insect may reduce PM permeability, decreasing the entrance of baculovirus. To test this hypothesis, we constructed a recombinant nucleopolyhedrovirus to express HaCDA5a in insect cells and tested its influence on PM permeability as well as the influence of HaCDA5a expression on the performance of the baculovirus. The experiments showed that HaCDA5a increased PM permeability, in a concentration-dependent manner. Bioassays on Spodoptera frugiperda and Spodoptera exigua larvae revealed that NPV expressing HaCDA5a was more infective than its parental virus. However, no difference in virulence was observed when the viruses were injected intrahemocoelically. These findings support the downregulation of a midgut-specific CDA-like protein as a possible mechanism used by H. armigera to reduce susceptibility to baculovirus by decreasing PM permeability.

Nematology , 2010, Vol. 12(1), 157-160 Short communication Species-specific polymerase chain reaction primers for simple detection of Bursaphelenchus fraudulentus (Nematoda: Parasitaphelenchidae) Anna F I L I P I A K 1 , ∗ , Agata J AKUBOW SKA 1 , 2 and Marek T OMALAK 1 Bursaphelenchus fraudulentus Ruhm, 1956 (Nema- toda: Parasitaphelenchidae) is a tree-inhabiting species frequently found in Europe (Ruhm, 1956; Schauer-Blume & Sturhan, 1989; Tomalak, 2004; Carletti et al ., 2005) and Asia (Braasch et al ., 2001). It is primarily found in dy- ing or dead deciduous trees (Ruhm, 1956; Schauer-Blume & Sturhan, 1989), although occasionally reported from conifers (Braasch et al ., 2001). Within the genus the spe- cific morphological characters (i.e. , vulval flap, shape of spicules, position of caudal papillae and presence of four incisures in the lateral fields) place B. fraudulentus into a distinct xylophilus -group which comprises eight other, morphologically similar, species, including the quarantine pest B. xylophilus (Gu et al ., 2008). Reliable methods of taxonomic identification of these species are therefore of particular interest to plant quarantine services. Several molecular techniques have been developed and used for identification of Bursaphelenchus spp. (Wang et al. , 1999; Matsunaga & Togashi, 2004; Burgermeister et al ., 2005; Castagnone et al. , 2005; Leal et al ., 2005, 2007) with ITS-RLFP analysis the most widely used in research and quarantine practice. An alternative method, based on a simple PCR amplification with primers specific for B. xylophilus and B. mucronatus (Matsunaga & Togashi, 2004), enables rapid and precise species identification, even from a single nematode (Filipiak et al ., 2007). Both native European species from the xylophilus - group, i.e. , B. mucronatus and B. fraudulentus , are rela- tively common and harmless to trees. They can be mor- phologically difficult to distinguish from each other and from the quarantine pest B. xylophilus. The provision of PCR primers specific for the DNA of B. fraudulentus , in addition to those already existing for B. xylophilus and B. 1 Department of Biological Pest Control and Quarantine, Institute of Plant Protection, Wladyslawa Wegorka 20, Poznan, 60-318, Poland 2 Department of Genetics, University of Valencia, Dr. Moliner 50, 46-100, Burjassot, Spain ∗ Corresponding author, e-mail: A.Filipiak@ior.poznan.pl Received: 22 October 2008; revised: 26 June 2009 Accepted for publication: 29 June 2009 Keywords: Bursaphelenchus xylophilus -group, diagnostics, molecular. mucronatus (Matsunaga & Togashi, 2004), enhances the utility of this method. The main objectives of our research were to sequence the ITS regions of the B. fraudulentus genome and to design specific primers for PCR amplifica- tion. Eight isolates of B. fraudulentus , two of B. mucrona- tus and one of B. xylophilus were examined. The Aus- trian (Osterreich), Russian (DE10w), German (Helmstedt and H26), and Hungarian (Ungarn) isolates of B. fraudu- lentus and the isolate of B. xylophilus (China) were ob- tained from the nematode collection of the Federal Re- search Centre for Cultivated Plants (formerly Federal Bi- ological Research Centre for Agriculture and Forestry – BBA), Braunschweig, Germany (Dr T. Schroder). All the remaining nematode populations were isolated from in- fested trees in Poland. The populations of B. fraudulentus originated from Poznan (PL-01 and PL-05) and Kornik (PL-04) and those of B. mucronatus from Nowy Tomysl (NTo-01) and Slawa Slaska (SlS-01). Their taxonomic identification was originally based on morphological and morphometric characteristics and subsequently confirmed by ITS-RLFP analysis according to the protocol described by Burgermeister et al. (2005). Prior to examination, all isolates were reared on Botrytis cinerea /malt agar (4.5%) at 25 ◦ C for ca 2 weeks. Propagated nematodes were col- lected by the Baermann funnel method and stored in 10 μ l H 2 O at − 20 ◦ C until use. Extraction of DNA was performed according to the method described by Iwahori et al. (1998), with mi- nor modifications to the composition of the lysis buffer: 100 mM Tris, pH 8.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, 1% β -mercaptoethanol, 100 μ g ml − 1 proteinase K per 100 μ l buffer. Before incubation, the mixture was © Koninklijke Brill NV, Leiden, 2010 DOI:10.1163/138855409X12469543767319 Also available online - www.brill.nl/nemy 157

Insect nucleopolyhedroviruses (NPVs) are studied widely as agents for biological control, as expression vectors for the production of heterologous proteins, and as transduction vectors for gene therapy applications. Most of these applications rely on the existence of cell lines that allow in vitro multiplication of the virus. The influence of pH in the medium culture on the multiplication of SeMNPV, HearSNPV and AcMNPV in different cell culture lines was investigated. The study showed a strong influence of the medium pH on the virus multiplication with the best results at pH 6.5, about half pH unit above the pH of insect culture media used most commonly. Additional experiments using a recombinant AcMNPV, expressing the green fluorescent protein, suggested that the enhanced virus multiplication at pH 6.5 is due mainly to a facilitated entry of the budded virions into the cells.

Virus transmission and the prevalence of infection depend on multiple factors, including the interaction with other viral pathogens infecting the same host. In this study, active replication of an iflavirus, Spodoptera exigua iflavirus 1 (order Picornavirales) was observed in the offspring of insects that survived following inoculation with a pathogenic baculovirus, Spodoptera exigua multiple nucleopolyhedrovirus. Tracking the origin of the iflavirus suggested the association of this virus with the occlusion bodies of the baculovirus. Here we investigated the effect of this association on the stability and infectivity of both viruses. A reduction in baculovirus pathogenicity, without affecting its infectivity and productivity, was observed when associated with the iflavirus. In contrast, viral association increased the infectivity of the iflavirus and its resistance to ultraviolet radiation and high temperature, two of the main factors affecting virus stability in the field. In addition, electron microscopy analysis revealed the presence of particles resembling iflavirus virions inside the occlusion bodies of the baculovirus, suggesting the possible co-occlusion of both viruses. Results reported here are indicative of facultative phoresis of a virus and suggest that virus–virus interactions may be more common than currently recognized, and may be influential in the ecology of baculovirus and host populations and in consequence in the use of baculoviruses as biological insecticides., This study received financial support from the Spanish Ministry for Science and Technology (AGL2011-30352-C02 and AGL2014-57752-C2). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The Douglas-fir tussock moth Orgyia pseudotsugata (Lepidoptera: Lymantriidae) is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus (SNPV) isolated from O. pseudotsugata larvae in Canada (OpSNPV) was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group II NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for

The satin moth Leucoma salicis L. (Lepidoptera, Lymantriidae) is a frequent defoliator of poplar trees (Populus spp.) in Europe and Asia (China, Japan). Around 1920 the insect was introduced into the USA and Canada. In this paper, a multicapsid nucleopolyhedrovirus isolated from L. salicis larvae in Poland (LesaNPV) was characterized and appeared to be a variant of Orgyia pseudotsugata (Op) MNPV. O. pseudotsugata, the Douglas fir tussock moth (Lepidoptera, Lymantriidae), occurs exclusively in North America. Sequences of three conserved baculovirus genes, polyhedrin, lef-8, and pif-2, were amplified in polymerase chain reactions using degenerate primer sets, and revealed a high degree of homology to OpMNPV. Restriction enzyme analysis confirmed the close relationship between LesaNPV and OpMNPV, although a number of restriction fragment length polymorphisms were observed. The lef-7 gene, encoding late expression factor 7, and the ctl-2 gene, encoding a conotoxin-like protein, were chosen as putative molecular determinants of the respective viruses. The ctl-2 region appeared suitable for unequivocal identification of either virus as LesaNPV lacked a dUTPase gene in this region. Our observations may suggest that LesaNPV, along with L. salicis, was introduced into O. pseudotsugata after introduction of the former insect into North America in the 1920s.

The turnip moth, Agrotis segetum (Lepidoptera, Noctuidae), is an important pest insect in Europe, Asia, and Africa. We have genetically characterized and classified a nucleopolyhedrovirus isolated from A. segetum larvae in Poland (AgseNPV-P). The restriction pattern of AgseNPV-P was distinct from an isolate from England/France (AgseNPV-UK and AgseNPV-F). Sequence analysis of three conserved baculovirus genes, polyhedrin, lef-8 and pif-2, revealed that AgseNPV-P differs substantially from the already described NPVs isolated from A. segetum and possibly represents a new NPV species. Phylogenetic analysis placed AgseNPV-P among group II NPVs and showed the closest relationship to Agrotis ipsilon (Agip) NPV and Spodoptera exigua (Se) MNPV.

Thaumetopoea pityocampa (pine processionary moth) is one of the most important pine pests in the forests of Mediterranean countries, Central Europe, the Middle East and North Africa. Apart from causing significant damage to pinewoods, T. pityocampa occurrence is also an issue for public and animal health, as it is responsible for dermatological reactions in humans and animals by contact with its irritating hairs. High throughput sequencing technologies have allowed the fast and cost-effective generation of genetic information of interest to understand different biological aspects of non-model organisms as well as the identification of potential pathogens. Using these technologies, we have obtained and characterized the transcriptome of T. pityocampa larvae collected in 12 different geographical locations in Turkey. cDNA libraries for Illumina sequencing were prepared from four larval tissues, head, gut, fat body and integument. By pooling the sequences from Illumina platform with those previously published using the Roche 454-FLX and Sanger methods we generated the largest reference transcriptome of T. pityocampa. In addition, this study has also allowed identification of possible viral pathogens with potential application in future biocontrol strategies., This research was supported by Karadeniz Technical University, Trabzon, Turkey (KTU-BAP 9102 and 9585) and the Spanish Ministry of Science and Innovation (AGL2011-30352-C02-02). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

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.

Viral covert infections in invertebrates have been traditionally attributed to sublethal infections that were not able to establish an acute infection. Recent studies are revealing that, although true for some viruses, other viruses may follow the strategy of establishing covert or persistent infections without producing the death of the host. Recently, and due to the revolution in the sequencing technologies, a large number of viruses causing covert infections in all type of hosts have been identified.The beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) is a worldwide pest that causes significant losses to agricultural and ornamental plant industries. In a previous project we used NGS to obtain a comprehensive transcriptome of the larval stage, revealing the presence of an important number of unigenes belonging to novel RNA viruses, most of them from the order Picornavirales. In order to characterize S. exigua viral complex, in this work we have completed the genomic sequences of two picorna-like viruses, and compared them to a SeIV1, a member of Iflaviridae previously described by our group. We performed additional studies to determine virus morphology, horizontal transmission, tissue and life stage distribution and abundance in the hosts. We discuss the role of virus persistent infections on insect populations. © 2014 Elsevier Inc., This research was supported by the Spanish Ministry of Science and Innovation (AGL2010-16809 and AGL2011-30352-C02-02)

Paenibacillus larvae, the etiological agent of the globally occurring epizootic American Foulbrood (AFB) of honey bees, causes intestinal infections in honey bee larvae which develop into systemic infections inevitably leading to larval death. Massive brood mortality might eventually lead to collapse of the entire colony. Molecular mechanisms of host-microbe interactions in this system and of differences in virulence between P. larvae genotypes are poorly understood. Recently, it was demonstrated that the degradation of the peritrophic matrix lining the midgut epithelium is a key step in pathogenesis of P. larvae infections. Here, we present the isolation and identification of PlCBP49, a modular, chitin-degrading protein of P. larvae and demonstrate that this enzyme is crucial for the degradation of the larval peritrophic matrix during infection. PlCBP49 contains a module belonging to the auxiliary activity 10 (AA10, formerly CBM33) family of lytic polysaccharide monooxygenases (LPMOs) which are able to degrade recalcitrant polysaccharides. Using chitin-affinity purified PlCBP49, we provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for members of the AA10 family. Using P. larvae mutants lacking PlCBP49 expression, we analyzed in vivo biological functions of PlCBP49. In the absence of PlCBP49 expression, peritrophic matrix degradation was markedly reduced and P. larvae virulence was nearly abolished. This indicated that PlCBP49 is a key virulence factor for the species P. larvae. The identification of the functional role of PlCBP49 in AFB pathogenesis broadens our understanding of this important family of chitin-binding and -degrading proteins, especially in those bacteria that can also act as entomopathogens., Author Summary American Foulbrood and its etiological agent, Paenibacillus larvae, pose a serious threat to global honey bee health. So far, molecular mechanisms of host-microbe interactions are poorly understood in this system and no key virulence factor for the entire species has been identified. Here, we demonstrate that P. larvae expresses a chitin-binding and -degrading protein PlCBP49 harboring one module that belongs to the auxiliary activity 10 (AA10) family of lytic polysaccharide monooxygenases (LPMOs). We provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for other members of the AA10 enzyme family. Using P. larvae mutants lacking PlCBP49 expression, we demonstrate that PlCBP49 is crucial for the degradation of the chitin-rich peritrophic matrix, a key step in pathogenesis of P. larvae infections. In the absence of PlCBP49 expression the peritrophic matrix remained nearly intact and about 95% of the infected larvae survived infection. This clearly indicated that PlCBP49 is a key virulence factor of P. larvae. These results constitute important progress in our understanding of both P. larvae pathogenesis and the biological role of LPMOs in entomopathogens. Furthermore, knowing PlCBP49 and its role in pathogenesis opens new possibilities to develop curative measures for this disease.

Made available in DSpace on 2014-12-03T13:11:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-12-02Bitstream added on 2014-12-03T13:22:24Z : No. of bitstreams: 1 WOS000327944500105.pdf: 2100332 bytes, checksum: 71ea7616e9a7949eed19cff4ce56111f (MD5) Spanish Ministerio de Ciencia e Innovacion European FEDER funds Generalitat Valenciana Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Host-pathogen interactions result in complex relationship, many aspects of which are not completely understood. Vip proteins, which are Bacillus thuringensis (Bt) insecticidal toxins produced during the vegetative stage, are selectively effective against specific insect pests. This new group of Bt proteins represents an interesting alternative to the classical Bt Cry toxins because current data suggests that they do not share the same mode of action. We have designed and developed a genome-wide microarray for the beet armyworm Spodoptera exigua, a serious lepidopteran pest of many agricultural crops, and used it to better understand how lepidopteran larvae respond to the treatment with the insecticidal protein Vip3Aa. With this approach, the goal of our study was to evaluate the changes in gene expression levels caused by treatment with sublethal doses of Vip3Aa (causing 99% growth inhibition) at 8 and 24 h after feeding. Results indicated that the toxin provoked a wide transcriptional response, with 19% of the microarray unigenes responding significantly to treatment. The number of up-and down-regulated unigenes was very similar. The number of genes whose expression was regulated at 8 h was similar to the number of genes whose expression was regulated after 24 h of treatment. The up-regulated sequences were enriched for genes involved in innate immune response and in pathogen response such as antimicrobial peptides (AMPs) and repat genes. The down-regulated sequences were mainly unigenes with homology to genes involved in metabolism. Genes related to the mode of action of Bt Cry proteins were found, in general, to be slightly overexpressed. The present study is the first genome-wide analysis of the response of lepidopteran insects to Vip3Aa intoxication. An insight into the molecular mechanisms and components related to Vip intoxication will allow designing of more effective management strategies for pest control. Univ Valencia, Dept Genet, Valencia, Spain UNESP, Dept Appl Biol, Jaboticabal, SP, Brazil UNESP, Dept Appl Biol, Jaboticabal, SP, Brazil Spanish Ministerio de Ciencia e InnovacionPBH2008-0113-PC Spanish Ministerio de Ciencia e InnovacionAGL2009-13340-C02-01 Spanish Ministerio de Ciencia e InnovacionAGL2012-39946-C02-01 Generalitat ValencianaGVPrometeo2011-044 CAPES: 194/09

Spodoptera exigua microarray was used to determine genes differentially expressed in S. exigua cells challenged with the species-specific baculovirus SeMNPV as well as with a generalist baculovirus, AcMNPV. Microarray results revealed that, in contrast to the host transcriptional shut-off that is expected during baculovirus infection, S. exigua cells showed a balanced number of up- and down-regulated genes during the first 36 hours following the infection. Many immune-related genes, including pattern recognition proteins, genes involved in signalling and immune pathways as well as immune effectors and genes coding for proteins involved in the melanization cascade were found to be down-regulated after baculovirus infection. The down-regulation of immune-related genes was confirmed in the larval gut. The expression of immune-related genes in the gut is known to affect the status of gut microorganisms, many of which are responsible for growth and development functions. We therefore asked whether the down-regulation that occurs after baculovirus infection affects the amount of gut microbiota. An increase in the gut bacterial load was observed and we hypothesize this to be as a consequence of viral infection. Subsequent experiments on virus performance in the presence and absence of gut microbiota revealed that gut bacteria enhanced baculovirus virulence, pathogenicity and dispersion. We discuss the host immune response processes and pathways affected by baculoviruses, as well as the role of gut microbiota in viral infection., Author Summary Baculoviruses are large DNA viruses that infect invertebrates, mainly insects from the order Lepidoptera. They were first discovered to cause insects' epizootics and are now used worldwide as biocontrol agents. Extensive studies on baculovirus biology led to the discovery that they can serve as expression vectors in insect cells; recently they have also been considered as vectors for gene therapy. Baculovirus infection, like many other oral infections, starts with the invasion of the gut by viruses; the gut is a compartment colonized by a community of resident microbiota. In this study, we observed that baculovirus infection leads to the decreased expression of immune-related genes in a Spodoptera exigua cell culture as well as in the larval gut. Gut microbial loads were found to increase after baculovirus infection. A series of bioassays showed that the baculovirus performs better in the presence of microbiota in the gut. Our study shows that baculovirus infection leads to increase of microbiota loads in the gut and that the gut microbiota play a significant role in insect immunity and susceptibility to viral infections. These findings suggest that gut microbiota can be manipulated to improve biocontrol strategies that employ baculoviruses.

We have obtained and characterized the transcriptome of Spodoptera exigua larvae with special emphasis on pathogen-induced genes. In order to obtain a highly representative transcriptome, we have pooled RNA from diverse insect colonies, conditions and tissues. Sequenced cDNA included samples from 3 geographically different colonies. Enrichment of RNA from pathogen-related genes was accomplished by exposing larvae to different pathogenic and non-pathogenic microbial agents such as the bacteria Bacillus thuringiensis, Micrococcus luteus, and Escherichia coli, the yeast Saccharomyces cerevisiae, and the S. exigua nucleopolyhedrovirus (SeMNPV). In addition, to avoid the loss of tissue-specific genes we included cDNA from the midgut, fat body, hemocytes and integument derived from pathogen exposed insects. RNA obtained from the different types of samples was pooled, normalized and sequenced. Analysis of the sequences obtained using the Roche 454 FLX and Sanger methods has allowed the generation of the largest public set of ESTs from S. exigua, including a large group of immune genes, and the identification of an important number of SSR (simple sequence repeats) and SNVs (single nucleotide variants: SNPs and INDELs) with potential use as genetic markers. Moreover, data mining has allowed the discovery of novel RNA viruses with potential influence in the insect population dynamics and the larval interactions with the microbial pesticides that are currently in use for the biological control of this pest.

Given the high similarity in genome content and organization between Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and Agrotis segetum nucleopolyhedrovirus (AgseNPV), as well as the high percentages of similarity found between their 30 core genes, the specificity of these NPVs was analysed for the respective insect hosts, S. exigua and A. segetum. The LD(50) for AgseNPV in second-instar A. segetum larvae was 83 occlusion bodies per larva and the LT(50) was 8.1 days. AgseNPV was orally infectious for S. exigua, but the LD(50) was 10 000-fold higher than for SeMNPV. SeMNPV was not infectious for A. segetum larvae when administered orally, but an infection was established by injection into the haemocoel. Bypassing midgut entry by intrahaemocoelic inoculation suggested that the midgut is the major barrier in A. segetum larvae for infection by SeMNPV. Delayed-early genes of SeMNPV are expressed in the midgut of A. segetum larvae after oral infections, indicating that the virus is able to enter midgut epithelial cells and that it proceeds through the first phases of the infection process. The possible mechanisms of A. segetum resistance to SeMNPV in per os infections are discussed.