The Spruce Budworm Genome: Reconstructing the Evolutionary History of Antifreeze Proteins.

Insects have developed various adaptations to survive harsh winter conditions. Among freeze-intolerant species, some produce "antifreeze proteins" (AFPs) that bind to nascent ice crystals and inhibit further ice growth. Such is the case of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), a destructive North American conifer pest that can withstand temperatures below -30°C. Despite the potential importance of AFPs in the adaptive diversification of Choristoneura, genomic tools to explore their origins have until now been limited. Here we present a chromosome-scale genome assembly for C. fumiferana, which we used to conduct comparative genomic analyses aimed at reconstructing the evolutionary history of tortricid AFPs. The budworm genome features 16 genes homologous to previously reported C. fumiferana AFPs (CfAFPs), 15 of which map to a single region on chromosome 18. Fourteen of these were also detected in five congeneric species, indicating Choristoneura AFP diversification occurred before the speciation event that led to C. fumiferana. Although budworm AFPs were previously considered unique to the genus Choristoneura, a search for homologs targeting recently sequenced tortricid genomes identified seven CfAFP-like genes in the distantly related Notocelia uddmanniana. High structural similarity between Notocelia and Choristoneura AFPs suggests a common origin, despite the absence of homologs in three related tortricids. Interestingly, one Notocelia AFP formed the C-terminus of a "zonadhesin-like" protein, possibly representing the ancestral condition from which tortricid AFPs evolved. Future work should clarify the evolutionary path of AFPs between Notocelia and Choristoneura and assess the role of the "zonadhesin-like" protein as precursor of tortricid AFPs.

Opposing effects of mortality factors on progeny operational sex ratio may thwart adaptive manipulation of primary sex ratio.

Despite extensive research on mechanisms generating biases in sex ratios, the capacity of natural enemies to shift or further skew operational sex ratios following sex allocation and parental care remains largely unstudied in natural populations. Male cocoons of the sawfly Neodiprion abietis (Hymenoptera: Diprionidae) are consistently smaller than those of females, with very little overlap, and thus, we were able to use cocoon size to sex cocoons. We studied three consecutive cohorts of N. abietis in six forest stands to detect cocoon volume-associated biases in the attack of predators, pathogens, and parasitoids and examine how the combined effect of natural enemies shapes the realized operational sex ratio. Neodiprion abietis mortality during the cocoon stage was sex-biased, being 1.6 times greater for males than females. Greater net mortality in males occurred because male-biased mortality caused by a pteromalid parasitic wasp and a baculovirus was greater and more skewed than female-biased mortality caused by ichneumonid parasitic wasps. Variation in the susceptibility of each sex to each family of parasitoids was associated with differences in size and life histories of male and female hosts. A simulation based on the data indicated that shifts in the nature of differential mortality have different effects on the sex ratio and fitness of survivors. Because previous work has indicated that reduced host plant foliage quality induces female-biased mortality in this species, bottom-up and top-down factors acting on populations can affect operational sex ratios in similar or opposite ways. Shifts in ecological conditions therefore have the potential to alter progeny fitness and produce extreme sex ratio skews, even in the absence of unbalanced sex allocation. This would limit the capacity of females to anticipate the operational sex ratio and reliably predict the reproductive success of each gender at sex allocation.

Comparative genome sequence analysis of Choristoneura occidentalis Freeman and C. rosaceana Harris (Lepidoptera: Tortricidae) alphabaculoviruses.

The complete genome sequences of Choristoneura occidentalis and C. rosaceana nucleopolyhedroviruses (ChocNPV and ChroNPV, respectively) (Baculoviridae: Alphabaculovirus) were determined and compared with each other and with those of other baculoviruses, including the genome of the closely related C. fumiferana NPV (CfMNPV). The ChocNPV genome was 128,446 bp in length (1147 bp smaller than that of CfMNPV), had a G+C content of 50.1%, and contained 148 open reading frames (ORFs). In comparison, the ChroNPV genome was 129,052 bp in length, had a G+C content of 48.6% and contained 149 ORFs. ChocNPV and ChroNPV shared 144 ORFs in common, and had a 77% sequence identity with each other and 96.5% and 77.8% sequence identity, respectively, with CfMNPV. Five homologous regions (hrs), with sequence similarities to those of CfMNPV, were identified in ChocNPV, whereas the ChroNPV genome contained three hrs featuring up to 14 repeats. Both genomes encoded three inhibitors of apoptosis (IAP-1, IAP-2, and IAP-3), as reported for CfMNPV, and the ChocNPV IAP-3 gene represented the most divergent functional region of this genome relative to CfMNPV. Two ORFs were unique to ChocNPV, and four were unique to ChroNPV. ChroNPV ORF chronpv38 is a eukaryotic initiation factor 5 (eIF-5) homolog that has also been identified in the C. occidentalis granulovirus (ChocGV) and is believed to be the product of horizontal gene transfer from the host. Based on levels of sequence identity and phylogenetic analysis, both ChocNPV and ChroNPV fall within group I alphabaculoviruses, where ChocNPV appears to be more closely related to CfMNPV than does ChroNPV. Our analyses suggest that it may be appropriate to consider ChocNPV and CfMNPV as variants of the same virus species.

Characterization of the polydnaviral 'T. rostrale virus' (TrV) gene family: TrV1 expression inhibits in vitro cell proliferation.

Tranosema rostrale ichnovirus (TrIV) is a polydnavirus (PDV) transmitted by the endoparasitic wasp T. rostrale to its host Choristoneura fumiferana during oviposition. PDV genes are expressed in infected caterpillars, causing physiological disturbances that promote the survival of the developing endoparasite. The previously sequenced genome of TrIV contains ~86 genes organized in multigene families and distributed on multiple segments of circular dsDNA. Among these, the 'T. rostrale virus' (TrV) family comprises seven genes that are absent in other PDV genomes examined to date and whose function(s) remain(s) unknown. Here, we initiated a functional analysis of the TrV family using qPCR, transfection and RNAi approaches. TrV family genes were weakly expressed in wasp ovaries, but some displayed high transcript abundance in parasitized caterpillars. Whilst TrV1 was the most highly transcribed TrV gene in infected caterpillars, transcript levels for TrV5 and TrV6 were nearly undetectable, indicating that they may be pseudogenes. Temporal and tissue-specific patterns of transcript abundance were similar for all expressed TrV family genes, indicative of an apparent lack of difference in function or tissue specificity. Infection of Cf-203 and Sf-21 insect cells with TrIV led to a dose-dependent inhibition of cell proliferation with no sign of apoptosis. Whilst similar inhibition was observed following transfection of cells with a cloned genome segment carrying the TrV1 gene, RNA interference targeting TrV1 largely restored cell growth in TrIV-infected cells, indicating that TrV1 expression was responsible for the observed inhibition. We suggest that TrV genes may contribute to host developmental disruption by interfering with host-cell proliferation during parasitism.

Spread of a Gammabaculovirus within Larval Populations of Its Natural Balsam Fir Sawfly (Neodiprion abietis) Host Following Its Aerial Application.

Field trials and assessments of the balsam fir sawfly (Neodiprion abietis) nucleopolyhedrovirus (NeabNPV: Baculoviridae, Gammabaculovirus) against its natural host were conducted in July and August 2002 near Corner Brook, Newfoundland and Labrador, Canada, in naturally regenerated, precommercially thinned stands dominated by balsam fir (Abies balsamea). Two experimental blocks, each with its own untreated control, were established. The purpose of the Island Pond block was to examine the spread of NeabNPV from a 313-ha aerial treatment block out into adjacent populations of balsam fir sawflies. The purpose of the Old Man's Pond block (2,093 ha) was to determine whether NeabNPV could disperse into populations of balsam fir sawflies within a 200-m zone between spray swaths. NeabNPV was applied to treatment blocks by a Cessna 188B AgTruck aircraft equipped with MicronAir AU4000 rotary atomizers at an application rate equivalent to 1 × 108 NeabNPV occlusion bodies/ha in 2.5 L of 20% aqueous molasses. At Island Pond, NeabNPV infection increased with time following the spray, especially for individuals close to the treatment block, and infection rate decreased to a measured distance of 400 m from the treatment block. At Old Man's Pond, NeabNPV infection rose higher (80% vs. 15%) and sawfly densities declined more (84% vs. 60%) in the area between spray swaths than in the control block.

Transmission of a Gammabaculovirus within Cohorts of Balsam Fir Sawfly (Neodiprion abietis) Larvae.

Nucleopolyhedroviruses (NPV: Gammabaculovirus: Baculoviridae) of diprionid sawflies (Diprionidae: Hymenoptera) are highly host specific and only infect the midgut epithelium. While still alive, infected sawfly larvae excrete NPV-laden diarrhea that contaminates food sources. The diarrhea can then be consumed by conspecific larvae, resulting in rapid horizontal transmission of the virus. To better understand the efficacy of Gammabaculovirus-based biological control products, the horizontal spread of such a virus (NeabNPV) within cohorts of balsam fir sawfly (Neodiprion abietis) larvae was studied by introducing NeabNPV-treated larvae into single-cohort groups at densities similar to those observed during the increasing (field study) and peak (laboratory study) phases of an outbreak. In field studies (~200 N. abietis larvae/m² of balsam fir (Abies balsamea) foliage), NeabNPV-induced mortality increased positively in a density-dependent manner, from 23% (in control groups) to 51% with the addition of one first-instar NeabNPV-treated larva, to 84% with 10 first-instar-treated larvae. Mortality was 60% and 63% when one or 10 NeabNPV-treated third-instar larva(e), respectively, were introduced into groups. Slightly higher levels of NeabNPV-induced mortality occurring when NeabNPV-treated larvae were introduced into first- rather than third-instar cohorts suggests that early instars are more susceptible to the virus. In the laboratory (~1330 N. abietis larvae/ m² of foliage), NeabNPV-caused mortality increased from 20% in control groups to over 80% with the introduction of one, five or 10 NeabNPV-treated larvae into treatment groups of first-instar larvae.

Complete sequence, analysis and organization of the Orgyia leucostigma nucleopolyhedrovirus genome.

The complete genome of the Orgyia leucostigma nucleopolyhedrovirus (OrleNPV) isolated from the whitemarked tussock moth (Orgyia leucostigma, Lymantridae: Lepidoptera) was sequenced, analyzed, and compared to other baculovirus genomes. The size of the OrleNPV genome was 156,179 base pairs (bp) and had a G+C content of 39%. The genome encoded 135 putative open reading frames (ORFs), which occupied 79% of the entire genome sequence. Three inhibitor of apoptosis (ORFs 16, 43 and 63), and five baculovirus repeated ORFs (bro-a through bro-e) were interspersed in the OrleNPV genome. In addition to six direct repeat (drs), a common feature shared among most baculoviruses, OrleNPV genome contained three homologous regions (hrs) that are located in the latter half of the genome. The presence of an F-protein homologue and the results from phylogenetic analyses placed OrleNPV in the genus Alphabaculovirus, group II. Overall, OrleNPV appears to be most closely related to group II alphabaculoviruses Ectropis obliqua (EcobNPV), Apocheima cinerarium (ApciNPV), Euproctis pseudoconspersa (EupsNPV), and Clanis bilineata (ClbiNPV).

In vitro culture of Lambdina fiscellaria lugubrosa nucleopolyhedrovirus in heterologous cell lines.

Infections of two heterologous insect cell lines derived from Malacosoma disstria (Md108) and Choristoneura fumiferana (Cf70) by the Lambdina fiscellaria lugubrosa nucleopolyhedrovirus (LafiNPV-W) were characterized. Cytopathic effects characteristic of LafiNPV-W infection, including rounding of cells, nuclear hypertrophy, and occlusion body (OB) production, were observed in both cell lines. Budded virus titers were slightly higher in Md108 cells than Cf70 cells (5.8 x 10(7) versus 3.1 x 10(7) TCID(50) units mL(-1)). Viral replication kinetics and cytopathic effects induced by LafiNPV-W infection were very similar in both cell lines. Actin rearrangements and redistribution of heterochromatin and euchromatin were observed within 24 h post-inoculation (hpi), and large quantities of nucleocapsids and virions were observed by electron microscopy at 48 hpi in both cell lines. Cf70 cultures produced OBs with numerous embedded virions, while OBs in Md108 cultures contained few virions or were empty with nucleocapsids packed in the nucleoplasm between OBs. In bioassays against second instar L. fiscellaria lugubrosa, OBs derived from LafiNPV-W-infected Md108 cells induced significantly lower levels of mortality than OBs derived from LafiNPV-W-infected Cf70 cells or from infected L. fiscellaria fiscellaria larvae.

Application of 16S rDNA-DGGE and plate culture to characterization of bacterial communities associated with the sawfly, Acantholyda erythrocephala (Hymenoptera, Pamphiliidae).

Culture-based analysis was employed in parallel with PCR amplification of 16S rDNA, coupled with denaturing gradient gel electrophoresis (DGGE), to profile bacterial species associated with different developmental stages of the pine false webworm (PFW), Acantholyda erythrocephala, a sawfly pest responsible for incidents of severe defoliation in commercially important tree plantations in North America. Culture-based analysis revealed that Pseudomonas spp. along with Bacillus sphaericus and Arthrobacter sp. were the predominant components of the microflora of the internal organs and identified life-stage-specific associations including Photorhabdus temperata with egg and larval samples and a Janthinobacterium sp. with eonymphs. PCR-DGGE confirmed the predominance of Pseudomonas spp. and B. sphaericus in the majority of samples but did not detect Arthrobacter sp., P. temperate, or Janthinobacterium sp. In contrast, DGGE revealed the presence of a Chryseobacterium sp. as the predominant component of the PFW micoflora at all life stages, with the exception of adults. This species had been infrequently cultured, at low levels, from a limited number of samples and the existence of a possible relationship between this bacterium and the PFW had gone unnoticed using the culture-based approach. Our findings highlight the advantages of applying a dual approach to the study of microbe-insect associations and demonstrate that the benefits of one system can be used to overcome some of the limitations of the other.

An intracellular symbiont and other microbiota associated with field-collected populations of sawflies (Hymenoptera: Symphyta).

Six species of sawfly (Hymenoptera: Symphyta) from four taxonomic families (Agridae, Diprionidae, Pamphiliidae, and Tenthredinidae) were collected from locations across Canada and surveyed for their associated microbiota. Total DNA was extracted from individual insects, and polymerase chain reaction (PCR) was used to amplify the conserved 16S rRNA gene from microbiota. Denaturing gradient gel electrophoresis (DGGE) and restriction fragment length polymorphism (RFLP) were undertaken to separate bacterial clones associated with the host insect. Sequencing of the PCR-DGGE and PCR--RFLP products revealed a dominance of alpha- and gamma-Proteobacteria, with most sequences showing high similarity to bacteria previously identified from other insect species and environmental samples. Additionally, a strain of the bacterial endosymbiont Wolbachia and a Wolbachia bacteriophage were identified from the mountain ash sawfly (Pristiphora geniculata).

Molecular characterisation of a cypovirus isolated from the western spruce budworm Choristoneura occidentalis.

A novel cypovirus, assigned CoCPV, was isolated from natural populations of the western spruce budworm, Choristoneura occidentalis. The complete nucleotide sequences of genomic segments S2-S5 and S7-S10 were determined. Each segment contained a single open reading frame. Conserved motifs 5' (AGUUU......UUUGUGC) 3' were found at the ends of each segment. Analysis of S2, which encoded a putative RNA-dependent RNA polymerase protein, confirmed CoCPV belonged to the genus Cypovirus within the family Reoviridae. Further phylogenetic analysis using S10 (the polyhedrin gene) aligned this virus with species type-16, closely related to a cypovirus isolated from C. fumiferana.

Fluctuations in density of an outbreak species drive diversity cascades in food webs.

Patterns in food-web structure have frequently been examined in static food webs, but few studies have attempted to delineate patterns that materialize in food webs under nonequilibrium conditions. Here, using one of nature's classical nonequilibrium systems as the food-web database, we test the major assumptions of recent advances in food-web theory. We show that a complex web of interactions between insect herbivores and their natural enemies displays significant architectural flexibility over a large fluctuation in the natural abundance of the major herbivore, the spruce budworm (Choristoneura fumiferana). Importantly, this flexibility operates precisely in the manner predicted by recent foraging-based food-web theories: higher-order mobile generalists respond rapidly in time and space by converging on areas of increasing prey abundance. This "birdfeeder effect" operates such that increasing budworm densities correspond to a cascade of increasing diversity and food-web complexity. Thus, by integrating foraging theory with food-web ecology and analyzing a long-term, natural data set coupled with manipulative field experiments, we are able to show that food-web structure varies in a predictable manner. Furthermore, both recent food-web theory and longstanding foraging theory suggest that this very same food-web flexibility ought to be a potent stabilizing mechanism. Interestingly, we find that this food-web flexibility tends to be greater in heterogeneous than in homogeneous forest plots. Because our results provide a plausible mechanism for boreal forest effects on populations of forest insect pests, they have implications for forest and pest management practices.

In vivo replication kinetics and transcription patterns of the nucleopolyhedrovirus (NeabNPV) of the balsam fir sawfly, Neodiprion abietis.

DNA replication and transcription of NeabNPV, the nucleopolyhedrovirus (NPV) of the balsam fir sawfly, Neodiprion abietis (Hymenoptera: Diprionidae), in host larvae were investigated. NPV DNA replication kinetics and gene-expression patterns have been resolved only in lepidopteran cell-culture systems and in limited in vivo experiments with lepidopteran larvae. Furthermore, there are significant differences in pathologies caused by lepidopteran NPVs, which replicate in many tissues, and hymenopteran NPVs, known to replicate in midgut epithelium only. Despite the differences in host specificity and pathology, NeabNPV DNA replication kinetics were similar to those reported for lepidopteran NPVs. Maximal NeabNPV DNA synthesis was observed between 4 and 24 h post-inoculation (p.i.) but, in contrast to lepidopteran NPVs, synthesis continued at a lower rate up to 72 h p.i. Selected NeabNPV genes exhibited a cascade pattern of transcription similar to that of lepidopteran NPVs. RT-PCR products of the NeabNPV lef-1, lef-2 and dnapol transcripts were observed as early as 2 h p.i., whilst lef-8 and lef-9, encoding putative viral RNA polymerase subunits, were detected at 1 and 6 h p.i., respectively. Two structural late transcripts (gp41 and p74) were observed from 6 h p.i. The very late factor 1 (vlf-1) transcript, a transactivator of very late genes, was observed from 12 h p.i., but the very late transcript polh, encoding the major occlusion protein, polyhedrin, was observed from 24 h p.i. This study provides the first insight into DNA replication and gene expression of a non-lepidopteran baculovirus.

Ecosystem alteration modifies the relative strengths of bottom-up and top-down forces in a herbivore population.

1. Ecosystem alterations can affect the abundance, distribution and diversity of plants and animals, and thus potentially change the relative strength of bottom-up (the plant resource) and top-down (natural enemies) trophic forces acting on herbivore populations. 2. The hypothesis that alterations of the forest ecosystem associated with precommercial thinning have contributed to the increased severity of outbreaks of Neodiprion abietis (Harris), a sawfly defoliator, through the reduction of trophic forces acting on N. abietis larvae, was tested using exclusion techniques. 3. The relative contributions to N. abietis larval mortality of bottom-up and top-down forces both increased with increasing levels of defoliation and were both reduced by thinning. The reduction of bottom-up and top-down forces caused a 58% mean increase in N. abietis larval survival in thinned compared with untreated stands, which is less than would be expected by the sum of the effects of thinning on each source of mortality. Evidence indicates that the partly compensatory, partly additive nature of the mortality associated with trophic forces in the system under study is responsible for this discrepancy. 4. To our knowledge, this is the first study to show the impact of ecosystem alterations on the balance between bottom-up and top-down forces acting on an eruptive herbivore population along a gradient of host-plant defoliation, and how this can lead to increased outbreak severity. It is stressed that accurate estimates of the relative contributions of bottom-up and top-down forces to mortality cannot be obtained if the additive or compensatory nature of the mortality associated with these trophic forces is overlooked.

Sequence analysis and organization of the Neodiprion abietis nucleopolyhedrovirus genome.

Of 30 baculovirus genomes that have been sequenced to date, the only nonlepidopteran baculoviruses include the dipteran Culex nigripalpus nucleopolyhedrovirus and two hymenopteran nucleopolyhedroviruses that infect the sawflies Neodiprion lecontei (NeleNPV) and Neodiprion sertifer (NeseNPV). This study provides a complete sequence and genome analysis of the nucleopolyhedrovirus that infects the balsam fir sawfly Neodiprion abietis (Hymenoptera, Symphyta, Diprionidae). The N. abietis nucleopolyhedrovirus (NeabNPV) is 84,264 bp in size, with a G+C content of 33.5%, and contains 93 predicted open reading frames (ORFs). Eleven predicted ORFs are unique to this baculovirus, 10 ORFs have a putative sequence homologue in the NeleNPV genome but not the NeseNPV genome, and 1 ORF (neab53) has a putative sequence homologue in the NeseNPV genome but not the NeleNPV genome. Specific repeat sequences are coincident with major genome rearrangements that distinguish NeabNPV and NeleNPV. Genes associated with these repeat regions encode a common amino acid motif, suggesting that they are a family of repeated contiguous gene clusters. Lepidopteran baculoviruses, similarly, have a family of repeated genes called the bro gene family. However, there is no significant sequence similarity between the NeabNPV and bro genes. Homologues of early-expressed genes such as ie-1 and lef-3 were absent in NeabNPV, as they are in the previously sequenced hymenopteran baculoviruses. Analyses of ORF upstream sequences identified potential temporally distinct genes on the basis of putative promoter elements.

Genomic comparison of Neodiprion sertifer and Neodiprion lecontei nucleopolyhedroviruses and identification of potential hymenopteran baculovirus-specific open reading frames.

Genomic comparison of Neodiprion sertifer nucleopolyhedrovirus (NeseNPV) and Neodiprion lecontei nucleopolyhedrovirus (NeleNPV) showed that the hymenopteran baculoviruses had features in common and were distinct from other, fully sequenced lepidopteran and dipteran baculoviruses. Their genomes were small in size (86,462 and 81,755 bp, respectively), had low G+C contents (33.8 and 33.3 mol%, respectively) and contained fewer open reading frames (ORFs) (90 and 89, respectively) than other baculoviruses. They shared 69 ORFs (48.6% mean amino acid identity overall), 43 of which were previously identified baculovirus homologues. The remaining shared ORFs could be common to other baculoviruses, but low amino acid identities precluded identifying them as such. Some may also be unique to hymenopteran baculoviruses. These included a trypsin-like protease, a zinc-finger protein, regulator of chromosome condensation proteins, a densovirus capsid-like protein and a phosphotransferase. Structural analysis, the presence of conserved domains and phylogenetic studies suggested that some of these ORFs may be functional and could have been transferred horizontally from an insect host. ORFs found only in NeseNPV and NeleNPV may play a role in host specificity and/or tissue tropism, as hymenopteran baculoviruses are restricted to the midgut. The genomes were basically collinear, but contained non-syntenic regions (NSRs) with large numbers of repeats between their polyhedrin and dbp genes. They differed from each other in the number of ORFs and the G+C content of their NSRs and the presence of homologous regions in the NeseNPV genome. NeleNPV also had a short inversion relative to NeseNPV. NeseNPV contained 21 ORFs not found in NeleNPV and NeleNPV had 20 ORFs not found in NeseNPV.

Sequence and organization of the Neodiprion lecontei nucleopolyhedrovirus genome.

All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopolyhedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and characterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the redheaded pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content of only 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified by conserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity of homologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. The conserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA binding protein, late expression factor 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40, and p45, but lacks 21 others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k, odv-e25, protein kinase-1, fibroblast growth factor, and ubiquitin. The lack of identified baculovirus homologues may be due to difficulties in identification, differences in host-virus interactions, or other genes performing similar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, and phylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirus and granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteran baculoviruses may be necessary.