Baseline Susceptibility and Cross-Resistance of HearNPV in Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil.

The marked adoption of bioinsecticides in Brazilian agriculture in recent years is, at least partially, explained by the increasingly higher levels of insect pest resistance to synthetic insecticides. In particular, several baculovirus-based products have been registered in the last 5 years, including Helicoverpa armigera nucleopolyhedrovirus (HearNPV: Baculoviridae: Alphabaculovirus (Armigen®)). Understanding the susceptibility of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) to HearNPV is an important step toward development of robust Integrated Pest Management (IPM) and Insect Resistance Management programs (IRM) aimed at managing this serious insect pest. In this study, droplet feeding bioassays were used to characterize the baseline susceptibility to HearNPV (Armigen®) in H. armigera populations collected from major soybean and cotton-growing regions in Brazil. We defined and validated a diagnostic concentration for susceptibility monitoring of H. armigera populations to HearNPV. Additionally, cross-resistance between HearNPV and the insecticides flubendiamide and indoxacarb was evaluated by testing HearNPV in a susceptible strain and in resistant strains of H. armigera to these insecticides. A low interpopulation variation of H. armigera to HearNPV was detected. The LC50 values ranged from 1.5 × 105 to 1.1 × 106 occlusion bodies (OBs) per mL (7.3-fold variation). The mortality rate at the identified diagnostic concentration of 6.3 × 108 OBs/mL, based on the calculated LC99, ranged from 98.6 to 100% in populations of H. armigera collected from 2018 to 2020. No cross-resistance was detected between HearNPV and flubendiamide or indoxacarb. These results suggest that HearNPV (Armigen®) can be an effective tool in IPM and IRM programs to control H. armigera in Brazil.

Pathogenic Assessment of SfMNPV-Based Biopesticide on Spodoptera frugiperda (Lepidoptera: Noctuidae) Developing on Transgenic Soybean Expressing Cry1Ac Insecticidal Protein.

Pathogenic assessment of a baculovirus-based biopesticide containing Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV: Baculoviridae: Alphabaculovirus) infecting fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) is reported. In the bioassays, neonates were infected with different doses of SfMNPV applied on Cry1Ac Bt soybean and non-Bt soybean. Our findings indicated that S. frugiperda neonates did not survive at 10 d post infection or develop into adults on Bt and non-Bt soybean sprayed with the field recommended dose of SfMNPV. In contrast, a proportion of the infected neonates developed into adults when infected with lower doses of SfMNPV (50%, 25%, and 10% of field dose) in both Bt and non-Bt soybean. However, S. frugiperda neonates surviving infection at the lowest virus doses on both soybean varieties showed longer neonate-to-pupa and neonate-to-adult periods, lower larval and pupal weights, reduced fecundity, and increased population suppression. Nevertheless, more pronounced pathogenicity of SfMNPV infecting neonates of S. frugiperda were verified on larvae that developed on Bt soybean. These findings revealed that, beyond mortality, the biopesticide containing SfMNPV also causes significant sublethal pathogenic effects on neonates of S. frugiperda developing on Bt and non-Bt soybean and suggested an additive effect among SfMNPV and Cry1Ac insecticidal protein expressed in Bt soybean.

Differential insecticidal properties of Spodoptera frugiperda multiple nucleopolyhedrovirus isolates against corn-strain and rice-strain fall armyworm, and genomic analysis of three isolates.

The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a destructive crop pest native to North, Central, and South America that recently has spread to Africa and Asia. Isolates of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) have the potential to be developed as low-risk biopesticides for management of fall armyworm, and a commercially available formulation has been developed for control of fall armyworm in North and South America. In this study, the virulence (LC50 and LT50) of several SfMNPV isolates towards larvae of both corn-strain and rice-strain fall armyworm was assessed. Bioassays with corn-strain larvae revealed that the isolates could be organized into fast-killing (LT50 < 56 h post-infection) and slow-killing (LT50 > 68 h post-infection) groups. Rice-strain larvae exhibited narrower ranges of susceptibility to baculovirus infection and of survival times in bioassays with different isolates. Two SfMNPV isolates with rapid speeds of kill (SfMNPV-459 from Colombia and SfMNPV-1197 from Georgia, USA) along with an isolate that killed corn-strain at relatively low concentrations (SfMNPV-281 from Georgia) were selected for the complete determination of their genome sequences. The SfMNPV-1197 genome sequence shared high sequence identity with genomes of a Nicaraguan isolate, while SfMNPV-281 formed a separate clade with a USA and a Brazilian isolate in phylogenetic trees. The SfMNPV-459 sequence was more divergent with the lowest genome sequence identities in pairwise alignments with other sequenced SfMNPV genomes, and was not grouped reliably with either the 1197 clade or the 281 clade. SfMNPV-459 contained homologs of two ORFs that were unique to another Colombian isolate, but these isolates were not placed in the same clade in phylogenetic trees. This study identifies isolates with superior properties for control of fall armyworm and adds to our knowledge of the genetics of SfMNPV.

Insecticides, biologics and nematicides: Updates to IRAC's mode of action classification - a tool for resistance management.

Insecticide resistance has been and continues to be a significant problem for invertebrate pest control. As such, effective insecticide resistance management (IRM) is critical to maintain the efficacy of current and future insecticides. A technical group within CropLife International, the Insecticide Resistance Action Committee (IRAC) was established 35 years ago (1984) as an international association of crop protection companies that today spans the globe. IRAC's focus is on preserving the long-term utility of insect, mite, and most recently nematode control products through effective resistance management to promote sustainable agriculture and improved public health. A central task of IRAC has been the continual development and documentation of the Mode of Action (MoA) Classification scheme, which serves as an important tool for implementing IRM strategies focused on compound rotation / alternations. Updates to the IRAC MoA Classification scheme provide the latest information on the MoA of current and new insecticides and acaricides, and now includes information on biologics and nematicides. Details for these new changes and additions are reviewed herein.

A Novel Alphabaculovirus from the Soybean Looper, Chrysodeixis includens, that Produces Tetrahedral Occlusion Bodies and Encodes Two Copies of he65.

Isolates of the alphabaculovirus species, Chrysodeixis includens nucleopolyhedrovirus, have been identified that produce polyhedral occlusion bodies and infect larvae of the soybean looper, Chrysodeixis includens. In this study, we report the discovery and characterization of a novel C. includens-infecting alphabaculovirus, Chrysodeixis includens nucleopolyhedrovirus #1 (ChinNPV#1), that produces tetrahedral occlusion bodies. In bioassays against C. includens larvae, ChinNPV #1 exhibited a degree of pathogenicity that was similar to that of other ChinNPV isolates, but killed larvae more slowly. The host range of ChinNPV#1 was found to be very narrow, with no indication of infection occurring in larvae of Trichoplusia ni and six other noctuid species. The ChinNPV#1 genome sequence was determined to be 130,540 bp, with 126 open reading frames (ORFs) annotated but containing no homologous repeat (hr) regions. Phylogenetic analysis placed ChinNPV#1 in a clade with other Group II alphabaculoviruses from hosts of lepidopteran subfamily Plusiinae, including Chrysodeixis chalcites nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus. A unique feature of the ChinNPV#1 genome was the presence of two full-length copies of the he65 ORF. The results indicate that ChinNPV#1 is related to, but distinct from, other ChinNPV isolates.

No cross-resistance between ChinNPV and chemical insecticides in Chrysodeixis includens (Lepidoptera: Noctuidae).

Chrysodeixis includens nucleopolyhedrovirus (ChinNPV: Baculoviridae: Alphabaculovirus) is an active ingredient of a biological-based insecticide (Chrysogen®) recommended against soybean looper (SBL), Chrysodeixis includens (Walker, [1858]), in soybean in Brazil. We investigated if SBL strains resistant to chemical insecticides are cross-resistant to the baculovirus ChinNPV. In droplet feeding bioassays, SBL strains resistant to lambda-cyhalothrin and teflubenzuron showed equivalent susceptibility to ChinNPV as heterozygous and susceptible strains, indicating no cross-resistance between ChinNPV and chemical insecticides in SBL. Therefore, the ChinNPV is a valuable new "mode-of-action" tool for SBL resistance management in Brazil.

Field Studies on the Horizontal Transmission Potential by Voluntary and Involuntary Carriers of Helicoverpa armigera nucleopolyhedrovirus (Baculoviridae).

Horizontal transmission of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been found to occur through several pathways involving abiotic factors such as soil, wind, and rain, and biotic factors such as predators, parasitoids, and infected hosts. Previous studies examining horizontal transmission through certain biological carriers speculated they were likely not significant in increasing infection rates, however; these studies only focused on a relatively small number of arthropods present within a field setting. This study was conducted to evaluate the horizontal transmission potential of HearNPV by all potential biological carriers when applied as a foliar bioinsecticide or as virus-infected, nonmotile Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) larvae in a soybean field. Soybean plots were either sprayed with HearNPV or infested with late-stage HearNPV-infected larvae, and sample zones were sampled 3, 7, 10, 14, 17, and 21 days after the infestation, and analyzed for viral presence using PCR. We then identified HearNPV carriers through contamination from the application (involuntary) or through contact with a HearNPV-infected larva (voluntary). Both were confirmed through PCR analysis. Regardless of application technique, on average, HearNPV was capable of disseminating up to 61.0 m in 3 d after inoculation and was found within the sampled canopy 13-21 d after inoculation. Several arthropods were identified as novel carriers of HearNPV. Results from this study indicate that many novel HearNPV carriers are likely important in disseminating HearNPV.

Baseline Susceptibility of Spodoptera frugiperda (Lepidoptera: Noctuidae) to SfMNPV and Evaluation of Cross-Resistance to Major Insecticides and Bt Proteins.

The resistance evolution of Spodoptera frugiperda (J.E. Smith) to insecticides and Bt proteins along with the intensive crop production systems adopted in Brazil make it challenging to implement integrated pest management. The adoption of alternative methods to manage pests is fundamental to the implementation of favorable integrated pest management and insect resistance management. Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a valuable tool for S. frugiperda control. The characterization of the baseline susceptibility of S. frugiperda populations and cross-resistance involving SfMNPV and major insecticides and Bt proteins have not yet been conducted. The objective of this study was to characterize the baseline susceptibility of S. frugiperda populations from five Brazilian States to SfMNPV (Cartugen, AgBiTech, Fort Worth, TX). Possible cross-resistance to insecticides and Bt proteins among resistant S. frugiperda strains was also assessed. There were no differences in the susceptibility of the studied populations to SfMNPV. The estimated diagnostic concentration may be utilized in future monitoring studies to SfMNPV. The SfMNPV presented no cross-resistance to the chemical insecticides and to the Bt proteins tested. Our results provide evidence of the biological activity and high potential of SfMNPV as a distinct insecticidal mode of action for use in rotation with other tools. This biological insecticide is known to have a favorable toxicological and ecotoxicological profile and will be a valuable tool in insect resistance management and integrated pest management programs for control of S. frugiperda.

Baseline Susceptibility of Brazilian Populations of Chrysodeixis includens (Lepidoptera: Noctuidae) to C. includens Nucleopolyhedrovirus and Diagnostic Concentration for Resistance Monitoring.

The Chrysodeixis includens nucleopolyhedrovirus (ChinNPV: Baculoviridae: Alphabaculovirus) is a registered insecticide for the management of soybean looper, Chrysodeixis includens (Walker, [1858]) in Brazil. We conducted studies of baseline susceptibility of Brazilian populations of C. includens to the ChinNPV (Chrysogen, AgBiTech, Fort Worth, TX) as valuable knowledge in support of Integrated Pest Management and Insect Resistance Management programs. In bioassays, neonates were infected with different concentrations of ChinNPV using the droplet feeding bioassay method. Larvae were then transferred to artificial diet and mortality was assessed at 7 d. Results confirm that neonates from Brazilian populations of C. includens are susceptible to ChinNPV. Concentrations from 1.0 × 103 to 1.0 × 108 occlusion bodies (OBs) per ml caused mortality from 1.5 to 99%, respectively. The LC50 ranged from 1.4 × 105 to 7.7 × 105 OBs per ml for populations of C. includens (5.5-fold variation). Similar variation was detected for the LC90 which ranged from 1.6 × 107 to 7.7 × 107 OBs per ml (4.8-fold variation). Importantly, the field-collected populations showed equivalent susceptibility to the reference susceptible population. This indicates a low interpopulation variation in susceptibility of Brazilian populations of C. includens to ChinNPV, representing natural geographic variation and not variation caused by previous selection pressure. The candidate diagnostic concentration of 2.9 × 108 OBs per ml was estimated based on the pooled data and caused mortality ranging from 98.6 to 100%. This concentration will be used in proactive resistance monitoring programs. The Chrysogen will be a valuable tool as a new mode of action in C. includens resistance management in Brazil.

DIETARY SILVER NANOPARTICLES REDUCE FITNESS IN A BENEFICIAL, BUT NOT PEST, INSECT SPECIES.

Silver nanoparticles (AgNPs) have antimicrobial and insecticidal properties and they have been considered for their potential use as insecticides. While they do, indeed, kill some insects, two broader issues have not been considered in a critical way. First, reports of insect-lethal AgNPs are often based on simplistic methods that yield nanoparticles of nonuniform shapes and sizes, leaving questions about the precise treatments test insects experienced. Second, we do not know how AgNPs influence beneficial insects. This work addresses these issues. We assessed the influence of AgNPs on life history parameters of two agricultural pest insect species, Heliothis virescens (tobacco budworm) and Trichoplusia ni (cabbage looper) and a beneficial predatory insect species, Podisus maculiventris (spined soldier bug), all of which act in agroecosystems. Rearing the two pest species on standard media amended with AgNPs led to negligible influence on developmental times, pupal weights, and adult emergence, however, they led to retarded development, reductions in adult weight and fecundity, and increased mortality in the predator. These negative effects on the beneficial species, if also true for other beneficial insect species, would have substantial negative implications for continued development of AgNPs for insect pest management programs.

Introduction to the Use of Baculoviruses as Biological Insecticides.

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This section provides an overview of the baculovirus life cycle and use of baculoviruses as insecticidal agents. This chapter includes discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy. These viruses are used extensively for control of insect pests in a diverse range of agricultural and forest habitats.

Evaluation of the Insecticidal Efficacy of Wild Type and Recombinant Baculoviruses.

A considerable amount of work has been undertaken to genetically enhance the efficacy of baculovirus insecticides. Following construction of a genetically altered baculovirus, laboratory bioassays are used to quantify various parameters of insecticidal activity such as the median lethal concentration (or dose) required to kill 50 % of infected larvae (LC50 or LD50), median survival of larvae infected (ST50), and feeding damage incurred by infected larvae. In this chapter, protocols are described for a variety of bioassays and the corresponding data analyses for assessment of the insecticidal activity of baculovirus insecticides.

Unique synteny and alternate splicing of the chitin synthases in closely related heliothine moths.

Chitin is an extracellular biopolymer that contributes to the cuticular structural matrix in arthropods. As a consequence of its rigid structure, the chitinous cuticle must be shed and replaced to accommodate growth. Two chitin synthase genes that encode for chitin synthase A (ChSA), which produces cuticular exoskeleton, and chitin synthase B (ChSB), which produces peritrophic membrane, were characterized in the genomes of two heliothine moths: the corn earworm/cotton bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) and the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). In both moths, the two genes were arranged in tandem with the same orientation on the same strand with ChSB located 5' of ChSA. Sequence comparisons showed that the coding sequences were highly conserved with homologues from other species but that the tandem juxtaposed genomic arrangement of the two genes was unique in these insects. The mechanism that has led to this arrangement is unclear but is most likely a recent recombinational event. Transcript mapping of HzChSB and HzChSA in H. zea demonstrated that both transcripts were differentially spliced in various tissues and larval stages. The identification of the HzChSB-E12b alternate spliced transcript is the first report of alternate splicing for the ChSB group. The importance of this splice form is not clear because the protein produced would lack any enzymatic activity but retain the membrane insertion motifs. As for other insects, these genes provide an important target for potential control through RNAi but also provide a subject for broad scale genomic recombinational events.

Generation of a Transcriptome in a Model Lepidopteran Pest, Heliothis virescens, Using Multiple Sequencing Strategies for Profiling Midgut Gene Expression.

Heliothine pests such as the tobacco budworm, Heliothis virescens (F.), pose a significant threat to production of a variety of crops and ornamental plants and are models for developmental and physiological studies. The efforts to develop new control measures for H. virescens, as well as its use as a relevant biological model, are hampered by a lack of molecular resources. The present work demonstrates the utility of next-generation sequencing technologies for rapid molecular resource generation from this species for which lacks a sequenced genome. In order to amass a de novo transcriptome for this moth, transcript sequences generated from Illumina, Roche 454, and Sanger sequencing platforms were merged into a single de novo transcriptome assembly. This pooling strategy allowed a thorough sampling of transcripts produced under diverse environmental conditions, developmental stages, tissues, and infections with entomopathogens used for biological control, to provide the most complete transcriptome to date for this species. Over 138 million reads from the three platforms were assembled into the final set of 63,648 contigs. Of these, 29,978 had significant BLAST scores indicating orthologous relationships to transcripts of other insect species, with the top-hit species being the monarch butterfly (Danaus plexippus) and silkworm (Bombyx mori). Among identified H. virescens orthologs were immune effectors, signal transduction pathways, olfactory receptors, hormone biosynthetic pathways, peptide hormones and their receptors, digestive enzymes, and insecticide resistance enzymes. As an example, we demonstrate the utility of this transcriptomic resource to study gene expression profiling of larval midguts and detect transcripts of putative Bacillus thuringiensis (Bt) Cry toxin receptors. The substantial molecular resources described in this study will facilitate development of H. virescens as a relevant biological model for functional genomics and for new biological experimentation needed to develop efficient control efforts for this and related Noctuid pest moths.

Baculovirus replication induces the expression of heat shock proteins in vivo and in vitro.

A recent handful of studies have linked baculovirus infection with the induction of heat shock proteins, a highly conserved family of cytoprotective proteins. Here, we demonstrate baculovirus-stimulated upregulation of hsp70 transcription in the natural host, Helicoverpa zea. Larvae lethally infected with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) accumulated hsp70 transcripts throughout the 72-hour course of infection in the midgut, hemocytes, and fat body. While a maximal 17- or 15-fold induction of hsp70 was noted in the midgut and hemocytes, respectively, by 72 hours postinfection, the level of hsp70 transcription in the fat body of larvae was greater than two orders of magnitude higher than in mock-infected larvae. These results were largely mirrored in cultures of infected cells, and a potentiation effect was observed in cells that were both heat shocked and infected. In contrast, Spodoptera frugiperda multiple nucleopolyhedrovirus and ultraviolet-inactivated HzSNPV did not stimulate hsp70 transcription in these non-permissive larvae and in cell culture, respectively. Taken together, this report documents baculovirus-mediated upregulation of hsp70 in the host and demonstrates the requirement for productive infection for hsp70 induction in vitro and in vivo.

Determination and analysis of the genome sequence of Spodoptera littoralis multiple nucleopolyhedrovirus.

The Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV), a pathogen of the Egyptian cotton leaf worm S. littoralis, was subjected to sequencing of its entire DNA genome and bioassay analysis comparing its virulence to that of other baculoviruses. The annotated SpliMNPV genome of 137,998 bp was found to harbor 132 open reading frames and 15 homologous repeat regions. Four unique genes not present in SpltMNPV were identified, as were 14 genes that were absent or translocated by comparison. Bioassay analysis of experimentally infected Spodoptera frugiperda revealed an extended killing time for SpliMNPV as compared to S. frugiperda MNPV (SfMNPV), but a level of mortality similar to that caused by infection with SfMNPV and superior to that of Autographa californica MNPV (AcMNPV). Although extensive similarity was observed between the genome structure and predicted translation products of SpliMNPV and Spodoptera litura MNPV (SpltMNPV), genetic distances between isolates of SpliMNPV and SpltMNPV suggest that they are in fact different species of genus Alphabaculovirus.

Gut Transcription in Helicoverpa zea is Dynamically Altered in Response to Baculovirus Infection.

The Helicoverpa zea transcriptome was analyzed 24 h after H. zea larvae fed on artificial diet laced with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). Significant differential regulation of 1,139 putative genes (p < 0.05 T-test with Benjamini and Hochberg False Discovery Rate) was detected in the gut epithelial tissue; where 63% of these genes were down-regulated and 37% of genes were up-regulated compared to the mock-infected control. Genes that play important roles in digestive physiology were noted as being generally down-regulated. Among these were aminopeptidases, trypsin-like serine proteases, lipases, esterases and serine proteases. Genes related to the immune response reacted in a complex nature having peptidoglycan binding and viral antigen recognition proteins and antiviral pathway systems down-regulated, whereas antimicrobial peptides and prophenoloxidase were up-regulated. In general, detoxification genes, specifically cytochrome P450 and glutathione S-transferase were down-regulated as a result of infection. This report offers the first comparative transcriptomic study of H. zea compared to HzSNPV infected H. zea and provides further groundwork that will lead to a larger understanding of transcriptional perturbations associated with viral infection and the host response to the viral insult in what is likely the most heavily infected tissue in the insect.

Development of cell lines from the cactophagous insect: Cactoblastis cactorum (Lepidoptera: Pyralidae) and their susceptibility to three baculoviruses.

The unintentional introduction of the cactus moth, Cactoblastis cactorum, a successful biological control agent formerly employed in the control of invasive prickly pear cactus species (Opuntia spp.) in Australia, Hawaii, South Africa, and various Caribbean islands, has posed great concern as to the possible threat to native, endangered species of cactus in the southeastern USA as well as with the potential to cause a major infestation of commercial and agricultural cactus crops in Mexico. A number of control measures have been investigated with varying degrees of success including, field exploration for cactus moth-specific parasitoids, insecticides, fungal, bacterial, and nematode agents. Current tactics used by the USA-Mexico binational program to eradicate cactus moth from Mexico and mitigate its westward movement in the USA include host plant removal, the manual removal and destruction of egg sticks and infected cacti stems, and the Sterile Insect Technique. One other approach not taken until now is the development of a cactus moth cell line as a tool to facilitate the investigation of baculoviruses as an alternative biocontrol method for the cactus moth. Consequently, we established C. cactorum cell lines derived from adult ovarian tissue designated as BCIRL-Cc-AM and BCIRL-Cc-JG. The mean cell population doubling time was 204.3 and 112 h for BCIRL-Cc-AM and BCIRL-Cc-JG, respectively, with weekly medium change, while the doubling time was 176.6 and 192.6 h for BCIRL-Cc-AM and BCIRL-Cc-JG, respectively, with a daily change of medium. In addition, the daily versus weekly change in medium was reflected in the percentage viability with both cell lines showing higher levels with a daily medium change. Of the three baculoviruses tested, only the recombinant AcMNPV-hsp70Red and GmMNPV at a multiplicity of infection (MOI) of 1.0 were able to demonstrate significant production of extracellular virus (ECV) in each of the cell lines, whereas both cell lines were refractive to an HzSNPV challenge at an MOI of 10. In this study, we have demonstrated both the successful development of a C. cactorum cell line and its ability to support a complete baculovirus infection. The potential is also there to pursue further investigations to determine the susceptibility of the cactus moth cell line to other viruses. Additionally, the availability of a cactus moth cell line will facilitate the analysis of viruses prior to using the more expensive bioassay test. Finally, it is hoped with the knowledge presented here that baculoviruses may also be considered as an alternative biocontrol method for the cactus moth.

Iron levels change in larval Heliothis virescens tissues following baculovirus infection.

Inductively coupled plasma mass spectrometry and (59)Fe radiotracers were used to investigate changes in levels of Fe in the tissues of 4th instar Heliothis virescens larvae following infection with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) or with Autographa californica multiple nucleopolyhedrovirus. Baculovirus infection led to significant changes in hemolymph Fe levels late in infection. (24)Na radiotracer ingested by 4th instar larvae was rapidly cleared to nearly undetectable levels 6 h post-ingestion. In contrast, (59)Fe radiotracer fed to 4th instar larvae declined within the first few hours of ingestion and then remained constant at approximately 60% of the initial tracer activity. While Fe radiotracer levels among larval tissues changed, whole insect tracer levels did not decline from 6 to 60 h post-ingestion. Tissues from HzSNPV larvae had higher radiotracer levels in the hemolymph and midgut 36 and 60 h post-infection. The protein-bound/free ratio of (59)Fe was significantly higher in baculovirus infected hemolymph than in uninfected hemolymph at 60 h post-infection, indicating that Fe released from damaged cells is protein-bound. In both studies, hemolymph Fe levels were higher in HzSNPV infected larvae. This first study of tissue Fe levels during viral infection of an insect clearly demonstrates that Fe homeostasis is substantially disrupted.

Genetic variation and virulence of Autographa californica multiple nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus isolates.

To determine the genetic diversity within the baculovirus species Autographa calfornica multiple nucleopolyhedrovirus (AcMNPV; Baculoviridae: Alphabaculovirus), a PCR-based method was used to identify and classify baculoviruses found in virus samples from the lepidopteran host species A. californica, Autographa gamma, Trichoplusia ni, Rachiplusia ou, Anagrapha falcifera, Galleria mellonella, and Heliothis virescens. Alignment and phylogenetic inference from partial nucleotide sequences of three highly conserved genes (lef-8, lef-9, and polh) indicated that 45 of 74 samples contained isolates of AcMNPV, while six samples contained isolates of Rachiplusia ou multiple nucleopolyhedrovirus strain R1 (RoMNPV-R1) and 25 samples contained isolates of the species Trichoplusia ni single nucleopolyhedrovirus (TnSNPV; Alphabaculovirus). One sample from A. californica contained a previously undescribed NPV related to alphabaculoviruses of the armyworm genus Spodoptera. Data from PCR and sequence analysis of the ie-2 gene and a region containing ORF ac86 in samples from the AcMNPV and RoMNPV clades indicated a distinct group of viruses, mostly from G. mellonella, that are characterized by an unusual ie-2 gene previously found in the strain Plutella xylostella multiple nucleopolyhedrovirus CL3 (PlxyMNPV-CL3) and a large deletion within ac86 previously described in the AcMNPV isolate 1.2 and PlxyMNPV-CL3. PCR and sequence analysis of baculovirus repeated ORF (bro) genes revealed that the bro gene ac2 was split into two separate bro genes in some samples from the AcMNPV clade. Comparison of sequences in this region suggests that ac2 was formed by a deletion that fused the two novel bro genes together. In bioassays of a selection of isolates against T. ni, significant differences were observed in the insecticidal properties of individual isolates, but no trends were observed among the AcMNPV, TnSNPV, or RoMNPV groups of isolates. This study expands on what we know about the variation of AcMNPV, AcMNPV-like and TnSNPV viruses, provides novel information on the distinct groups in which AcMNPV isolates occur, and contributes to data useful for the registration, evaluation, and improvement of AcMNPV, AcMNPV-like, and TnSNPV isolates as biological control agents.