Transcriptomic Survey of the Midgut of Anthonomus grandis (Coleoptera: Curculionidae)

Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase, and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information.Instituto de Microbiología y Zoología AgrícolaFil: Salvador, Ricardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Perez Principi, Ricardo Dario. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Berretta, Marcelo Facundo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Fernandez, Paula Del Carmen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Sciocco, Alicia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Light microscopy of microsporidian spores and baculovirus occlusion bodies in Epinotia aporema and Helicoverpa gelotopoeon larval tissues, respectively

Entomopathogens help regulate natural insect populations, and their use in pest management is the subject of active research. Classical and molecular techniques are currently available to elucidate disease etiology and pathogenesis. Here, we performed an Azan trichrome stain for light microscopic visualization of pathogens in the larval tissues of two Neotropical lepidopterans. Epinotia aporema larvae exhibiting symptoms of microsporidiosis were fortuitously detected in a rearing facility (IMyZA-INTA, Argentina), while Helicoverpa gelotopoeon specimens were experimentally infected with an Argentinean isolate of Helicoverpa armigera nucleopolyhedrovirus (HearNPV). Caterpillars were fixed in Duboscq-Brasil and paraffin embedded. Microtome sections (5 μm-thick) were deparaffinized and stained2 for observation under a Nikon Eclipse 80i microscope equipped with a Nikon DS-U3 digital camera.Inst. de Microbiología y Zoología Agrícola IMyZAFil: Arneodo Larochette, Joel Demian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Taibo, Catalina Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Centro de Investigación en Ciencias Veterinarias y Agronómicas. Servicio de Microscopía; ArgentinaFil: Sciocco, Alicia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; Argentin

Biological and molecular characterization of Rachiplusia nu single nucleopolyhedrovirus, a promising biocontrol agent against the South American soybean pest Rachiplusia nu

Rachiplusia nu (Lepidoptera: Noctuidae) is a key soybean pest in Argentina. Current management of this moth relies mainly on the use of synthetic insecticides and transgenic plants. In search of biological control-based alternatives, a baculovirus from R. nu (hereafter RanuNPV) was characterized and its insecticidal properties tested under laboratory conditions. RanuNPV occlusion bodies (OBs) were nearly tetrahedral, averaging 1.0 ± 0.2 μm in their longest edge and containing singly enveloped nucleocapsids. Histopathology of infected late-instar larvae revealed broad tissue tropism, where fat bodies and epidermis were the most affected organs. Phylogenetic analysis of concatenated polh, lef-8 and lef-9 partial sequences classified RanuNPV as a new species that clusters with other group II alphabaculoviruses infecting larvae of Plusiinae. Bioassays performed with R. nu neonates determined the median lethal dosage to be approximately 2.5 OBs/larva; most insects died within 4–5 days post inoculation showing typical baculovirus-induced liquefaction. No effects were observed in other lepidopteran species assayed, including Spodoptera frugiperda, Cydia pomonella and Diatraea saccharalis. High pathogenicity and host specificity make RanuNPV a good candidate for controlling R. nu.Instituto de Microbiología y Zoología Agrícola (IMYZA)Fil: Jakubowicz, Violeta. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de investigaciones Científicas y Tecnológicas. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Taibo, Catalina Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Centro de Investigación en Ciencias Veterinarias y Agronómicas. Servicio de Microscopía; ArgentinaFil: Sciocco, Alicia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Arneodo Larochette, Joel Demian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de investigaciones Científicas y Tecnológicas. Instituto de Agrobiotecnología y Biología Molecular; Argentin

Effect of the interaction between Anticarsia gemmatalis multiple nucleopolyhedrovirus and Epinotia aporema granulovirus, on A. gemmatalis (Lepidoptera: Noctuidae) larvae

The bean shoot borer Epinotia aporema Wals. (Lepidoptera: Tortricidae) and the velvet bean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) are key pests of soybean and other legume crops in South America. They are often found simultaneously in certain regions. A. gemmatalis nucleopolyhedrovirus (AgMNPV) is widely used to control A. gemmatalis. More recently, E. aporema granulovirus (EpapGV) has been characterized and evaluated as a bioinsecticide for E. aporema. In order to increase its potential use and to design optimized strategies for the management of lepidopteran pests, we evaluated the interaction between EpapGV and AgMNPV on third instar A. gemmatalis larvae. Larvae fed with 50 AgMNPV OBs/larva showed an increase in the mortality rates (from 42% to 81%) and a decrease in the median survival time (from 7.7 days to 5.7 days) when these OBs were mixed with 6000 EpapGV OBs/larva. When 300 AgMNPV OBs/larva were used alone or in combination with EpapGV OBs no changes in biological parameters were observed. No mortality was detected in A. gemmatalis larvae treated with EpapGV alone. In larvae fed with the viral mixtures, only AgMNPV DNA was detected by PCR. A. gemmatalis peritrophic membranes (PMs) examined by SDS–PAGE and scanning electron microscopy showed signs of damage. Notably, we found the presence of spheroidal bodies associated with damaged areas in the PMs of larvae fed with EpapGV but not in those that were given AgMNPV alone. These results show that EpapGV increases the viral potency of AgMNPV, and thus the insecticidal efficiency, suggesting that the use of formulations including both viruses might be a valuable tool for pest management.Instituto de BiotecnologíaFil: Biedma, Marina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Salvador, Ricardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Ferrelli, Maria Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Sciocco, Alicia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Romanowski, Víctor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Functional analysis of Spodoptera frugiperda nucleopolyhedrovirus late expression factors in Sf9 cells

We used transient expression assays to assess the function of the baculovirus Spodoptera frugiperda M nucleopolyhedrovirus (SfMNPV) homologs of Autographa californica MNPV (AcMNPV) factors involved in late gene expression (lefs), in the Sf9 insect cell-line, which is permissive for both viruses. It is well-established that nineteen AcMNPV lefs support optimal levels of activity from a late promoter-reporter gene cassette in this assay. A subgroup of SfMNPV lefs predicted to function in transcription-specific events substituted the corresponding AcMNPV lefs very efficiently. When all SfMNPV lefs were assayed, including replication lefs, activity was low, but addition of two AcMNPV lefs not encoded in SfMNPV genome, resulted in augmented reporter activity. SfMNPV IE-1 was able to activate an early promoter cis-linked to an hr-derived element from SfMNPV but not from AcMNPV. However, the level of early promoter activation with SfMNPV IE-1 was lower compared to AcMNPV IE-1.Instituto de Microbiología y Zoología AgrícolaFil: Berretta, Marcelo Facundo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Lopez, Maria Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina.Fil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Sciocco, Alicia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Romanowski, Victor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Characterization of a new Helicoverpa armigera nucleopolyhedrovirus variant causing epizootic on a previously unreported host, Helicoverpa gelotopoeon (Lepidoptera: Noctuidae)

This paper reports the first biological and molecular characterization of a nucleopolyhedrovirus isolated from the soybean and cotton pest Helicoverpa gelotopoeon. Studies were performed following a virus outbreak in a rearing facility and in wild H. gelotopoeon populations in Córdoba, Argentina. Host identity was corroborated by partial sequencing of the COI gene. Scanning electron microscope observations of purified OBs revealed their polyhedral morphology and an average diameter of 0.89 ± 0.14 lm. Ultrathin sections of infected larvae examined by transmission electron microscopy showed the intranuclear occurrence of polyhedra and virus particles in fat body cells. Nucleocapsids were singly enveloped. Phylogenetic analysis of lef-8, lef-9, polh, orf5/5b and hr3-orf62 viral sequences identified this new NPV isolate (hereafter HegeSNPV) as a variant of Helicoverpa armigera nucleopolyhedrovirus (HearNPV). Furthermore, HegeSNPV was closely related to the so-called ‘‘HzSNPV Group” within HearNPV, although having particular characteristics.Inst. de Microbiología y Zoología Agrícola IMyZAFil: Ferrelli, Maria Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Taibo, Catalina Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Centro de Investigación en Ciencias Veterinarias y Agronómicas. Servicio de Microscopía; ArgentinaFil: Fichetti, Patricia. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias. Departamento de Protección Vegetal; ArgentinaFil: Sciocco, Alicia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Arneodo Larochette, Joel Demian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Genetic variants in Argentinean isolates of Spodoptera frugiperda Multiple Nucleopolyhedrovirus

The fall armyworm, Spodoptera frugiperda (JE Smith) is a key pest in the Americas. Control strategies are mainly carried out by use of chemical insecticides and transgenic crops expressing Bacillus thuringiensis toxins. In the last years, resistance of S. frugiperda populations to transgenic corn was reported in different Latin American countries. The baculovirus Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) is a pathogenic agent for the fall armyworm and a potential alternative for its control in integrated pest management strategies. In this work, we analyze some characteristics of two baculovirus isolates collected from maize (SfMNPV-M) and cotton (SfMNPV-C) fields from Argentina. The isolates were compared by restriction enzymes patterns and the analysis reveals the presence of genotypic variants in the SfMNPV-M isolate. We confirmed a deletion by sequencing fragments encompassing egt gene and most part of its contiguous gene (orf A) in a SfMNVP-M genotypic variant. Additionally, we estimated the 50% lethal dose and median survival time of each isolate in bioassays with S. frugiperda larvae.Instituto de Microbiología y Zoología AgrícolaFil: Niz, José María. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Salvador, Ricardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Ferrelli, Maria Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Sciocco, Alicia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Romanowski, Víctor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Berretta, Marcelo Facundo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola e Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin