Impact of Biochar on Douglas-Fir Tussock Moth (Orgyia pseudotsugata Lepidoptera: Erebidae) Larvae Reared on Synthetic Die

The use of biochar as a soil amendment in forest ecosystems can be beneficial in the restoration of degraded soils. Forest insects such as the Douglas-fir tussock moth, Orgyia pseudotsugata (McDonnough) (Lepidoptera: Erebidae), may be exposed to biochar when the material is applied. Two experiments were conducted using biochar either (1) applied to the surface of the diet at three rates (0, 5, and 10 mg) or (2) incorporated into synthetic diet at four rates (0, 10, 20, and 40% volume/volume). The objective of both experiments was to determine if biochar on the surface or incorporated into a synthetic diet affected development and survival of O. pseudotsugata larvae. In both experiments, there was a significant decrease in estimated time to larval mortality in all biochar treatments compared to untreated controls. In the surface-applied biochar experiment, there was a significant difference in larval weight gain at day 12 between the control and 10 mg biochar treatments. In the experiment with biochar incorporated into the diet, mean larval weight at day 12 was highest in the low (10%) biochar treatment compared to all other treatments, although weight gain was only significantly different between the low- and high-concentration (40%) biochar treatments. Our results suggest that larvae, feeding on a low amount of biochar in the synthetic diet, may respond by engaging in compensatory feeding behavior. Fewer surviving larvae in the biochar treatment groups may contribute to the lack of significance found in the comparison of weight gain at day 24 in each experiment.

A Nymphalid-Infecting Group I Alphabaculovirus Isolated from the Major Passion Fruit Caterpillar Pest Dione juno juno (Lepidoptera: Nymphalidae)

Baculoviruses are capable of infecting a wide diversity of insect pests. In the 1990s, the Dione juno nucleopolyhedrovirus (DijuNPV) was isolated from larvae of the major passionfruit defoliator pest Dione juno juno (Nymphalidae) and described at ultrastructural and pathological levels. In this study, the complete genome sequence of DijuNPV was determined and analyzed. The circular genome presents 122,075 bp with a G + C content of 50.9%. DijuNPV is the first alphabaculovirus completely sequenced that was isolated from a nymphalid host and may represent a divergent species. It appeared closely related to Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and other Choristoneura-isolated group I alphabaculoviruses. We annotated 153 open reading frames (ORFs), including a set of 38 core genes, 26 ORFs identified as present in lepidopteran baculoviruses, 17 ORFs unique in baculovirus, and several auxiliary genes (e.g., bro, cathepsin, chitinase, iap-1, iap-2, and thymidylate kinase). The thymidylate kinase (tmk) gene was present fused to a dUTPase (dut) gene in other baculovirus genomes. DijuNPV likely lost the dut portion together with the iap-3 homolog. Overall, the genome sequencing of novel alphabaculoviruses enables a wide understanding of baculovirus evolution.

Baculovirus IAP1 induces caspase-dependent apoptosis in insect cells

Baculoviruses encode inhibitors of apoptosis (IAPs), which are classified into five groups, IAP1–5, based on their sequence homology. Most of the baculovirus IAPs with anti-apoptotic functions belong to the IAP3 group, with certain exceptions. The functional roles of IAPs from other groups during virus infection have not been well established. We have previously shown that Hyphantria cunea multiple nucleopolyhedrovirus (HycuMNPV) encodes three iap genes, hycu-iap1, hycu-iap2 and hycu-iap3, and that only Hycu-IAP3 has anti-apoptotic activity against actinomycin D-induced apoptosis of Spodoptera frugiperda Sf9 cells. In the present study, we demonstrate that transient expression of Hycu-IAP1 is capable of inducing apoptosis and/or stimulating caspase-3-like protease activity in various lepidopteran and dipteran cell lines. Transient-expression assay analysis also demonstrates that not only Hycu-IAP1 but also IAP1s from Autographa californica MNPV, Bombyx mori NPV and Orgyia pseudotsugata MNPV (OpMNPV) are capable of inducing apoptosis, and that apoptosis induced by Hycu-IAP1 is precluded by the functional anti-apoptotic baculovirus protein Hycu-IAP3. In HycuMNPV-infected Spilosoma imparilis (SpIm) cells and OpMNPV-infected Ld652Y cells, caspase-3-like protease activity is markedly stimulated during the late stages of infection, and the caspase-3-like protease activity stimulated in HycuMNPV-infected SpIm cells is repressed by RNA interference-mediated silencing of hycu-iap1. In addition, initiator caspase Bm-Dronc, the B. mori homologue of Dronc, is cleaved upon transfection of BM-N cells with a plasmid expressing Hycu-IAP1. These results provide the first evidence that baculovirus IAP1s act to induce caspase-dependent apoptosis, possibly by replacing the cellular IAP1 that prevents Dronc activation.

Molecular comparisons of alphabaculovirus-based products: Gypchek with Disparvirus (Lymantria dispar) and TM BioControl-1 with Virtuss (Orgyia pseudotsugata)

Gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), multicapsid nucleopolyhedrovirus (LdMNPV) has been registered as a microbial pest-control product in the United States (Gypchek®) and Canada (Disparvirus®). Similarly, Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough) (Lepidoptera: Lymantriidae), multicapsid nucleopolyhedrovirus (OpMNPV) is registered in the United States and Canada as TM BioControl-1® and a product derived from TM BioControl-1 (Virtuss®) is also registered in Canada. To determine changes that may have occurred in these products over time, we compared DNA from Gypchek with Disparvirus and DNA from TM BioControl-1 with Virtuss using restriction fragment length polymorphism (RFLP) analysis. Gypchek and Disparvirus showed the same RFLP banding patterns when viral genomic DNA was digested with BamH I, EcoR V, and Hind III and only a single band difference at approximately 1.6 kilobase (kb) when digested with Bgl II. TM BioControl-1 and Virtuss showed no differences in genomic DNA when digested with Bgl II, Sam I or Hind III. Twelve viral open reading frames (ORFs) were amplified from Gypchek and Disparvirus and nine from TM BioControl-1 and Virtuss by polymerase chain reactions (PCR). The amplified ORFs ranged from highly conserved (polyhedrin) to least conserved (vp91 capsid associated protein). The products were sequenced and the deduced protein products compared. Amino acid sequences deduced from the sequenced PCR products indicated that 8 of the 12 proteins were identical in the two LdMNPV products. The four proteins showing minor sequence variations were DNA polymerase, LEF-8, P74 envelope protein, and VP 91 capsid associated protein. No differences were detected in the protein products deduced from the nine sequenced ORFs from TM BioControl-1 and Virtuss. Comparative RFLP and protein phylogenetic analyses of Gypchek with Disparvirus and TM BioControl-1 with Virtuss revealed little difference between the respective LdMNPV and OpMNPV populations that make up these product pairs.

Host Insect Inhibitor-of-Apoptosis SfIAP Functionally Replaces Baculovirus IAP but Is Differentially Regulated by Its N-Terminal Leader

ABSTRACT The inhibitor-of-apoptosis (IAP) proteins encoded by baculoviruses bear a striking resemblance to the cellular IAP homologs of their invertebrate hosts. By virtue of the acquired selective advantage of blocking virus-induced apoptosis, baculoviruses may have captured cellular IAP genes that subsequently evolved for virus-specific objectives. To compare viral and host IAPs, we defined antiapoptotic properties of SfIAP, the principal cellular IAP of the lepidopteran host Spodoptera frugiperda. We report here that SfIAP prevented virus-induced apoptosis as well as viral Op-IAP3 (which is encoded by the Orgyia pseudotsugata nucleopolyhedrovirus) when overexpressed from the baculovirus genome. Like Op-IAP3, SfIAP blocked apoptosis at a step prior to caspase activation. Both of the baculovirus IAP repeats (BIRs) were required for SfIAP function. Moreover, deletion of the C-terminal RING motif generated a loss-of-function SfIAP that interacted and dominantly interfered with wild-type SfIAP. Like Op-IAP3, wild-type SfIAP formed intracellular homodimers, suggesting that oligomerization is a functional requirement for both cellular and viral IAPs. SfIAP possesses a ∼100-residue N-terminal leader domain, which is absent among all viral IAPs. Remarkably, deletion of the leader yielded a fully functional SfIAP with dramatically increased protein stability. Thus, the SfIAP leader contains an instability motif that may confer regulatory options for cellular IAPs that baculovirus IAPs have evolved to bypass for maximal stability and antiapoptotic potency. Our findings that SfIAP and viral IAPs have common motifs, share multiple biochemical properties including oligomerization, and act at the same step to block apoptosis support the hypothesis that baculoviral IAPs were derived by acquisition of host insect IAPs.

Virus loads in Douglas-fir tussock moth larvae infected with the Orgyia pseudotsugata nucleopolyhedrovirus

We studied Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) infections in larvae of the Douglas-fir tussock moth, Orgyia pseudotsugata McDunnough (Lepidoptera: Lymantriidae), to determine the quantity of OpMNPV particles that result in mortality. We observed a bimodal pattern of mortality in Douglas-fir tussock moth larvae that ingested diet plugs contaminated with 9.5 OpMNPV occlusion bodies. A mortality peak (80% of total mortality observed) occurred between day 5 and day 11 post ingestion, and a second, smaller mortality peak coincided with the onset of pupation. Virus loads, defined as the number of OpMNPV occlusion bodies in each sample of tested larval homogenate, were quantified using an indirect ELISA method. Virus loads that resulted in mortality were significantly greater than those quantified in larvae that were sacrificed during and after the peak mortality wave (P < 0.004 and P < 0.0001, Mann–Whitney U two-tailed rank test and SPSS®, respectively). This is the first known attempt to differentiate the quantity of virus produced during lethal infections from the virus loads in larvae that survive infection.