The Parapoynx stagnalis Nucleopolyhedrovirus (PastNPV), a Divergent Member of the Alphabaculovirus Group I Clade, Encodes a Homolog of Ran GTPase.

We report the analysis of the genome of a novel Alphabaculovirus, Parapoynx stagnalis nucleopolyhedrovirus isolate 473 (PastNPV-473), from cadavers of the rice case bearer, Parapoynx stagnalis Zeller (Lepidoptera: Crambidae), collected in rice fields in Kerala, India. High-throughput sequencing of DNA from PastNPV occlusion bodies and assembly of the data yielded a circular genome-length contig of 114,833 bp with 126 annotated opening reading frames (ORFs) and six homologous regions (hrs). Phylogenetic inference based on baculovirus core gene amino acid sequence alignments indicated that PastNPV is a member of the group I clade of viruses in genus Alphabaculovirus, but different phylogenetic methods yielded different results with respect to the placement of PastNPV and four similarly divergent alphabaculoviruses in the group I clade. Branch lengths and Kimura-2-parameter pairwise nucleotide distances indicated that PastNPV-473 cannot be classified in any of the currently listed species in genus Alphabaculovirus. A unique feature of the PastNPV genome was the presence of an ORF encoding a homolog of Ran GTPase, a regulator of nucleocytoplasmic trafficking. PastNPV appears to have acquired a homolog of Ran relatively recently from a lepidopteran host via horizontal gene transfer.

The complete genome sequence of an alphabaculovirus from the brown tussock moth, Olene mendosa Hübner, expands our knowledge of lymantriine baculovirus diversity and evolution.

The complete genome sequence was determined for an apparent alphabaculovirus isolated from larval cadavers of the brown tussock moth, Olene mendosa Hübner, collected during an epizootic in Coimbatore, India. The genome was determined to be a circular 142,291 bp molecule, and 147 ORFs and nine homologous regions were annotated for the sequence. Analysis of the sequence confirmed that this virus, Olene mendosa nucleopolyhedrovirus (OlmeNPV), was a member of genus Alphabaculovirus in family Baculoviridae. Phylogenies inferred from nucleotide and amino acid alignments indicated that OlmeNPV was part of a group of viruses that infect moths of genus Lymantria, suggesting that OlmeNPV may have shifted hosts from a Lymantria species to an ancestral Olene species at some point during its evolutionary history. OlmeNPV was most closely related to Lymantria xylina multiple nucleopolyhedrovirus isolate 5 (LyxyMNPV-5). The genomes of OlmeNPV and LyxyMNPV-5 were distinguished not only by differences in ORF content, but by a 27 kbp region of the genome that is inverted in LyxyMNPV-5 relative to OlmeNPV. Pairwise nucleotide distances between OlmeNPV and other Lymantria spp. alphabaculoviruses indicate that OlmeNPV represents a new baculovirus species.

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.

Pathology and genome sequence of a Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) isolate from Heilongjiang, China.

The pathogenicity and genome sequence of isolate LdMNPV-HrB of the gypsy moth alphabaculovirus, Lymantria dispar multiple nucleopolyhedrovirus from Harbin, Heilongjiang, China, were determined. A stock of this virus from one passage through the gypsy moth New Jersey Standard Strain (LdMNPV-HrB-NJSS) exhibited 6.2- to 11.9-fold greater pathogenicity against larvae from a Harbin colony of L. dispar asiatica than both Gypchek and a Massachusetts, USA LdMNPV isolate (LdMNPV-Ab-a624). Sequence determination and phylogenetic analysis of LdMNPV-HrB and LdMNPV-HrB-NJSS revealed that these isolates were most similar to other east Asian LdMNPV isolates with 98.8% genome sequence identity and formed a group with the east Asian LdMNPV isolates which was separate from groups of isolates from Russia, Europe, and USA.

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.

Complete Genome Sequence of an Alphabaculovirus from the Southern Armyworm, Spodoptera eridania.

We report the complete genome sequence of a baculovirus from the moth Spodoptera eridania, the southern armyworm. The genome sequence is 149,090 bp and exhibits the greatest degree of sequence similarity with genomes from alphabaculoviruses isolated from other moths of the genus Spodoptera.

The complete genome sequence of a third distinct baculovirus isolated from the true armyworm, Mythimna unipuncta, contains two copies of the lef-7 gene.

A baculovirus isolate from a USDA Forest Service collection was characterized by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species Mythimna unipuncta (true armyworm) and was renamed Mythimna unipuncta nucleopolyhedrovirus #7 (MyunNPV#7). The occlusion bodies (OBs) and virions exhibited a size and morphology typical for OBs produced by the species of genus Alphabaculovirus, with occlusion-derived virions consisting of 2-5 nucleocapsids within a single envelope. The MyunNPV#7 genome was determined to be 148,482 bp with a 48.58% G+C nucleotide distribution. A total of 159 ORFs of 150 bp or larger were annotated in the genome sequence, including the 38 core genes of family Baculoviridae. The genome contained six homologous repeat regions (hrs) consisting of multiple copies of a 34-bp imperfect palindrome. Phylogenetic inference from concatenated baculovirus core gene amino acid sequence alignments placed MyunNPV#7 with group II alphabaculoviruses isolated from other armyworm and cutworm host species of lepidopteran family Noctuidae. MyunNPV#7 could be distinguished from other viruses in this group on the basis of differences in gene content and order. Pairwise nucleotide distances suggested that MyunNPV#7 represents a distinct species in Alphabaculovirus. The MyunNPV#7 genome was found to contain two copies of the late expression factor-7 (lef-7) gene, a feature not reported for any other baculovirus genome to date. Both copies of lef-7 encoded an F-box domain, which is required for the function of LEF-7 in baculovirus DNA replication.

The Operophtera brumata Nucleopolyhedrovirus (OpbuNPV) Represents an Early, Divergent Lineage within Genus Alphabaculovirus.

Operophtera brumata nucleopolyhedrovirus (OpbuNPV) infects the larvae of the winter moth, Operophtera brumata. As part of an effort to explore the pesticidal potential of OpbuNPV, an isolate of this virus from Massachusetts (USA)-OpbuNPV-MA-was characterized by electron microscopy of OpbuNPV occlusion bodies (OBs) and by sequencing of the viral genome. The OBs of OpbuNPV-MA consisted of irregular polyhedra and contained virions consisting of a single rod-shaped nucleocapsid within each envelope. Presumptive cypovirus OBs were also detected in sections of the OB preparation. The OpbuNPV-MA genome assembly yielded a circular contig of 119,054 bp and was found to contain little genetic variation, with most polymorphisms occurring at a frequency of < 6%. A total of 130 open reading frames (ORFs) were annotated, including the 38 core genes of Baculoviridae, along with five homologous repeat (hr) regions. The results of BLASTp and phylogenetic analysis with selected ORFs indicated that OpbuNPV-MA is not closely related to other alphabaculoviruses. Phylogenies based on concatenated core gene amino acid sequence alignments placed OpbuNPV-MA on a basal branch lying outside other alphabaculovirus clades. These results indicate that OpbuNPV-MA represents a divergent baculovirus lineage that appeared early during the diversification of genus Alphabaculovirus.

The Complete Genome Sequence of a Second Distinct Betabaculovirus from the True Armyworm, Mythimna unipuncta.

The betabaculovirus originally called Pseudaletia (Mythimna) sp. granulovirus #8 (MyspGV#8) was examined by electron microscopy, host barcoding PCR, and determination of the nucleotide sequence of its genome. Scanning and transmission electron microscopy revealed that the occlusion bodies of MyspGV#8 possessed the characteristic size range and morphology of betabaculovirus granules. Barcoding PCR using cytochrome oxidase I primers with DNA from the MyspGV#8 collection sample confirmed that it had been isolated from the true armyworm, Mythimna unipuncta (Lepidoptera: Noctuidae) and therefore was renamed MyunGV#8. The MyunGV#8 genome was found to be 144,673 bp in size with a nucleotide distribution of 49.9% G+C, which was significantly smaller and more GC-rich than the genome of Pseudaletia unipuncta granulovirus H (PsunGV-H), another M. unipuncta betabaculovirus. A phylogeny based on concatenated baculovirus core gene amino acid sequence alignments placed MyunGV#8 in clade a of genus Betabaculovirus. Kimura-2-parameter nucleotide distances suggested that MyunGV#8 represents a virus species different and distinct from other species of Betabaculovirus. Among the 153 ORFs annotated in the MyunGV#8 genome, four ORFs appeared to have been obtained from or donated to the alphabaculovirus lineage represented by Leucania separata nucleopolyhedrovirus AH1 (LeseNPV-AH1) during co-infection of Mythimna sp. larvae. A set of 33 ORFs was identified that appears only in other clade a betabaculovirus isolates. This clade a-specific set includes an ORF that encodes a polypeptide sequence containing a CIDE_N domain, which is found in caspase-activated DNAse/DNA fragmentation factor (CAD/DFF) proteins. CAD/DFF proteins are involved in digesting DNA during apoptosis.

The Complete Genome Sequence of Plodia Interpunctella Granulovirus: Evidence for Horizontal Gene Transfer and Discovery of an Unusual Inhibitor-of-Apoptosis Gene.

The Indianmeal moth, Plodia interpunctella (Lepidoptera: Pyralidae), is a common pest of stored goods with a worldwide distribution. The complete genome sequence for a larval pathogen of this moth, the baculovirus Plodia interpunctella granulovirus (PiGV), was determined by next-generation sequencing. The PiGV genome was found to be 112, 536 bp in length with a 44.2% G+C nucleotide distribution. A total of 123 open reading frames (ORFs) and seven homologous regions (hrs) were identified and annotated. Phylogenetic inference using concatenated alignments of 36 baculovirus core genes placed PiGV in the "b" clade of viruses from genus Betabaculovirus with a branch length suggesting that PiGV represents a distinct betabaculovirus species. In addition to the baculovirus core genes and orthologues of other genes found in other betabaculovirus genomes, the PiGV genome sequence contained orthologues of the bidensovirus NS3 gene, as well as ORFs that occur in alphabaculoviruses but not betabaculoviruses. While PiGV contained an orthologue of inhibitor of apoptosis-5 (iap-5), an orthologue of inhibitor of apoptosis-3 (iap-3) was not present. Instead, the PiGV sequence contained an ORF (PiGV ORF81) encoding an IAP homologue with sequence similarity to insect cellular IAPs, but not to viral IAPs. Phylogenetic analysis of baculovirus and insect IAP amino acid sequences suggested that the baculovirus IAP-3 genes and the PiGV ORF81 IAP homologue represent different lineages arising from more than one acquisition event. The presence of genes from other sources in the PiGV genome highlights the extent to which baculovirus gene content is shaped by horizontal gene transfer.

Geographic isolates of Lymantria dispar multiple nucleopolyhedrovirus: Genome sequence analysis and pathogenicity against European and Asian gypsy moth strains.

Isolates of the baculovirus species Lymantria dispar multiple nucleopolyhedrovirus have been formulated and applied to suppress outbreaks of the gypsy moth, L. dispar. To evaluate the genetic diversity in this species at the genomic level, the genomes of three isolates from Massachusetts, USA (LdMNPV-Ab-a624), Spain (LdMNPV-3054), and Japan (LdMNPV-3041) were sequenced and compared with four previously determined LdMNPV genome sequences. The LdMNPV genome sequences were collinear and contained the same homologous repeats (hrs) and clusters of baculovirus repeat orf (bro) gene family members in the same relative positions in their genomes, although sequence identities in these regions were low. Of 146 non-bro ORFs annotated in the genome of the representative isolate LdMNPV 5-6, 135 ORFs were found in every other LdMNPV genome, including the 37 core genes of Baculoviridae and other genes conserved in genus Alphabaculovirus. Phylogenetic inference with an alignment of the core gene nucleotide sequences grouped isolates 3041 (Japan) and 2161 (Korea) separately from a cluster containing isolates from Europe, North America, and Russia. To examine phenotypic diversity, bioassays were carried out with a selection of isolates against neonate larvae from three European gypsy moth (Lymantria dispar dispar) and three Asian gypsy moth (Lymantria dispar asiatica and Lymantria dispar japonica) colonies. LdMNPV isolates 2161 (Korea), 3029 (Russia), and 3041 (Japan) exhibited a greater degree of pathogenicity against all L. dispar strains than LdMNPV from a sample of Gypchek. This study provides additional information on the genetic diversity of LdMNPV isolates and their activity against the Asian gypsy moth, a potential invasive pest of North American trees and forests.

Complete Genome Sequence of the Strain of Lymantria dispar Multiple Nucleopolyhedrovirus Found in the Gypsy Moth Biopesticide Virin-ENSh.

We report the genome sequence of an alphabaculovirus from the gypsy moth (Lymantria dispar) biopesticide Virin-ENSh. The genome sequence is 161,712 bp, and its structure and sequence similarity indicate that the virus used in Virin-ENSh is a strain of the species Lymantria dispar multiple nucleopolyhedrovirus.

Classification, genetic variation and pathogenicity of Lymantria dispar nucleopolyhedrovirus isolates from Asia, Europe, and North America.

Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) has been formulated and applied to control outbreaks of the gypsy moth, L. dispar. To classify and determine the degree of genetic variation among isolates of L. dispar NPVs from different parts of the range of the gypsy moth, partial sequences of the lef-8, lef-9, and polh genes were determined for Lymantria spp. virus samples from host populations throughout the world. Sequence analysis confirmed that all L. dispar virus samples tested contained isolates of the species Lymantria dispar multiple nucleopolyhedrovirus (Baculoviridae: Alphabaculovirus). Phylogenetic inference based on the lef-8 sequences indicated that the LdMNPV isolates formed two groups, one consisting primarily of isolates from Asia, and one consisting primarily of isolates from Europe and North America. The complete genome sequence was determined for an isolate from the Asian group, LdMNPV-2161 (S. Korea). The LdMNPV-2161 genome was 163,138bp in length, 2092bp larger than the previously determined genome of LdMNPV isolate 5-6 (CT, USA). The two genome sequences were co-linear, with an overall nucleotide sequence identity of 97.5% and some differences in ORF content. In droplet-feeding bioassays against neonate L. dispar larvae, isolates LdMNPV-3029 (Virin-ENSh/Russia) and LdMNPV-Ab-a624 (MA, USA) killed neonate larvae with an LC50 values that were 1.8- to 3.2-fold lower than a sample of Gypchek® (CT, USA) and isolates LdMNPV-3041 (Japan) and LdMNPV-2161. This study expands our knowledge about genetic variation among LdMNPV isolates and provides novel information on the distinct groups in which these NPVs occur.

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.

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.

Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens.

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus) obtained worldwide from larvae of Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes (lef-8, lef-9, and polh) indicated that 67 of these samples contained isolates of the H. zea-H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera, HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci (orf5/orf5b, hr3-orf62, orf26, orf79, orf124/orf117a, orf42, and a part of the region between hr2 and hr3) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea, the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC(50)s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.

Genetic and biological variation among nucleopolyhedrovirus isolates from the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

A PCR-based method was used to identify and distinguish among 40 uncharacterized nucleopolyhedrovirus (NPV) isolates from larvae of the moth Spodoptera frugiperda that were part of an insect virus collection. Phylogenetic analysis was carried out with sequences amplified from two strongly conserved loci (polh and lef-8) from the 40 isolates in the collection and from eight previously studied S. frugiperda NPV (SfMNPV) isolates. To further distinguish these isolates, analysis was also carried out with sequences from two less-conserved loci, hr4 and hr5. Phylogenetic inference from the sequence data could distinguish among several of the individual isolates and between different groups of isolates from Georgia (USA) and Colombia, South America. A stronger degree of bootstrap support for the phylogenetic trees was obtained with the hr4 and hr5 homologous repeat sequences. Sequencing and phylogenetic analysis detected a relatively high degree of larva-to-larva sequence divergence occurring among isolates of SfMNPV collected from the same field in Missouri, USA. Restriction endonuclease analysis of viral DNA from larvae infected with five isolates from Georgia, Missouri, Louisiana, Florida (USA), and Colombia allowed for further comparison with other previously reported isolates of SfMNPV. Bioassays with these five geographically distinct isolates detected minor differences in virulence. This study highlights the use of PCR to rapidly distinguish and characterize large numbers of historical baculovirus isolates from the same host using minimal quantities of material, and the use of sequences from homologous repeat regions to distinguish closely related isolates of the same NPV species.

Tracking the role of alternative prey in soybean aphid predation by Orius insidiosus: a molecular approach.

The soybean aphid, Aphis glycines (Hemiptera: Aphididae), is a pest of soybeans in Asia, and in recent years has caused extensive damage to soybeans in North America. Within these agroecosystems, generalist predators form an important component of the assemblage of natural enemies, and can exert significant pressure on prey populations. These food webs are complex and molecular gut-content analyses offer nondisruptive approaches for examining trophic linkages in the field. We describe the development of a molecular detection system to examine the feeding behaviour of Orius insidiosus (Hemiptera: Anthocoridae) upon soybean aphids, an alternative prey item, Neohydatothrips variabilis (Thysanoptera: Thripidae), and an intraguild prey species, Harmonia axyridis (Coleoptera: Coccinellidae). Specific primer pairs were designed to target prey and were used to examine key trophic connections within this soybean food web. In total, 32% of O. insidiosus were found to have preyed upon A. glycines, but disproportionately high consumption occurred early in the season, when aphid densities were low. The intensity of early season predation indicates that O. insidiosus are important biological control agents of A. glycines, although data suggest that N. variabilis constitute a significant proportion of the diet of these generalist predators. No Orius were found to contain DNA of H. axyridis, suggesting intraguild predation upon these important late-season predators during 2005 was low. In their entirety, these results implicate O. insidiosus as a valuable natural enemy of A. glycines in this soybean agroecosystem.

Prey preference and host suitability of the predatory and parasitoid carabid beetle, Lebia grandis, for several species of Leptinotarsa beetles.

Lebia grandis (Coleoptera: Carabidae), recorded as a parasitoid only on Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), is capable of parasitizing the false potato beetle, L. juncta, and also L. haldemani. Historical records show that L. decemlineata, while the only recorded host, was not present in much of the original range of L. grandis, and may not have been its host prior to its expansion into eastern North America, where L. juncta is endemic. Our laboratory comparisons suggest that L. juncta, the presumptive original host, best supports the development of the parasitoid larval L. grandis, based on 43.6% successful emergence of the adult carabid parasitoid, compared to 11.5% from the two other Leptinotarsa species. L. grandis adults accept eggs and larvae of all 3 Leptinotarsa species as adult food. Naive, newly-emerged adults show no preference when presented the 3 species of third-instar larvae, which they consume at a mean rate of 3.3 per day, a rate which does not differ significantly by sex, larval host, or weight at emergence. When presented with equal amounts by weight of the 3 species of Leptinotarsa eggs, such adults consume the equivalent of 23.0 L. decemlineata eggs per day, with consumption of L. juncta eggs 67% higher by weight than L. decemlineata consumption. Insight into the biotic and abiotic limitations on L. grandis should aid in determining its potential for suppression of Colorado potato beetle by biological control in diverse agroecosystems.