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Insect guts harbor diverse microbial assemblages that can be influenced by multiple factors, including gut physiology and interactions by the host with its environment. The Asian longhorned beetle (A. glabripennis; Cerambycidae: Lamiinae) is an invasive tree-killing insect that harbors a diverse consortium of fungal and bacterial gut associates that provision nutrients and facilitate lignocellulose digestion. The physicochemical conditions of the A. glabripennis gut and how these conditions may influence the microbial composition across gut regions are unknown. In this study, we used microsensors to measure in situ oxygen concentrations, pH, and redox potential along the length of the A. glabripennis larval gut from two North American populations. We then analyzed and compared bacterial and fungal gut communities of A. glabripennis within individual hosts along the length of the gut using 16S and ITS1 amplicon sequencing. The A. glabripennis midgut lumen was relatively anoxic (0.01kPa) with a pH gradient from 5.5 to 9, moving anterior to posterior. Redox potential was higher in the anterior midgut relative to posterior regions. No differences in physicochemistry were measured between the two populations of the beetle, but the two populations harbored different communities of bacteria and fungi. However, microbial composition of the A. glabripennis gut microbiota did not differ among gut regions despite physicochemical differences. Unlike other insect systems that have distinct gut compartmentalization and corresponding microbial assemblages, the A. glabripennis gut lacks dramatic morphological modifications, which may explain why discrete microbial community structures were not found along the digestive system.

There are a number of RNA virus pathogens that represent a serious threat to the health of managed honey bees (Apis mellifera). That some of these viruses are also found in the broader pollinator community suggests the wider environmental spread of these viruses, with the potential for a broader impact on ecosystems. Studies on the ecology and evolution of these viruses in the arthropod community as a whole may therefore provide important insights into these potential impacts. We examined managed A. mellifera colonies, nearby non-Apis hymenopteran pollinators, and other associated arthropods for the presence of five commonly occurring picorna-like RNA viruses of honey bees - black queen cell virus, deformed wing virus, Israeli acute paralysis virus, Kashmir bee virus and sacbrood virus. Notably, we observed their presence in several arthropod species. Additionally, detection of negative-strand RNA using strand-specific RT-PCR assays for deformed wing virus and Israeli acute paralysis virus suggests active replication of deformed wing virus in at least six non-Apis species and active replication of Israeli acute paralysis virus in one non-Apis species. Phylogenetic analysis of deformed wing virus also revealed that this virus is freely disseminating across the species sampled in this study. In sum, our study indicates that these viruses are not specific to the pollinator community and that other arthropod species have the potential to be involved in disease transmission in pollinator populations.

The peritrophic matrix (PM) lines the midgut of most insects, providing protection to the midgut epithelial cells while permitting passage of nutrients and water. Herein, we provide evidence that plant-mediated alteration of the PM contributes to the well-documented inhibition of fatal infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) of Heliothis virescens F. larvae fed cotton foliage. We examined the impact of the PM on pathogenesis using a viral construct expressing a reporter gene (AcMNPV-hsp70/lacZ) orally inoculated into larvae with either intact PMs or PMs disrupted by Trichoplusia ni granulovirus occlusion bodies containing enhancin, known to degrade insect intestinal mucin. Larvae possessing disrupted PMs displayed infection foci (lacZ signaling) earlier than those with intact PMs. We then examined PMs from larvae fed artificial diet or plant foliage using electron microscopy; foliage-fed larvae had significantly thicker PMs than diet-fed larvae. Moreover, mean PM width was inversely related to both the proportion of larvae with lacZ signaling at 18h post-inoculation and the final percentage mortality from virus. Thus, feeding on foliage altered PM structure, and these foliage-mediated changes reduced baculoviral efficacy. These data indicate that the PM is an important factor determining the success of an ingested pathogen in foliage-fed lepidopteran larvae.

Gypsy moth larvae become increasingly resistant to lethal infection by the Lymantria dispar M nucleopolyhedrovirus (LdMNPV) as they age within the fourth instar. Newly molted larvae are most sensitive to infection, mid-instars are least sensitive, and late-instars display intermediate sensitivity. This resistance occurs whether the virus is delivered orally or intrahemocoelically. The present study reveals a nearly identical pattern of resistance in third instar larvae. An LD48 dose of polyhedra for newly molted third instars produced 18%, 10%, 8%, 25%, and 24% mortalities in larvae to which virus was orally administered at 12, 24, 48, 72, and 96 hours post-molt (hpm), respectively, which is a 6-fold reduction in mortality between newly molted larvae and mid-instars. An LD44 dose of budded virus for newly molted third instars produced 33%, 23%, 17%, 31%, and 31% mortalities when injected into larvae that were 12, 24, 48, 72, and 96 hpm, respectively, which is a 2.6-fold reduction in mortality between newly molted larvae and mid-instars, indicating that approximately half of this resistance is midgut-based and half is systemically based. Doubling the viral dose did not overcome developmental resistance whether the virus was delivered orally or intrahemocoelically. In addition, time to death was significantly affected by the time post-molt at which the insect was inoculated with the virus. We suggest that intrastadial developmental resistance may affect both the ecology and management of the gypsy moth.

Interactions between insect herbivores and their pathogens can be modulated by host plants. Inter- and intraspecific differences in plant chemistry and structure can alter the susceptibility of insects to infection and the production and environmental persistence of pathogens. Whether plants can manipulate insect pathogens to act as "bodyguards" and increase their own fitness remains to be shown. Reduced insect performance owing to poor plant quality can enhance the susceptibility of an insect to disease while these same phytochemicals can also reduce the effectiveness of entomopathogens in killing the host. As we discuss here, plants have an important role in the evolution of insect-pathogen relationships and a tritrophic perspective should thus be incorporated into the study of insects and their pathogens.

We investigated intrastadial developmental resistance of the gypsy moth, Lymantria dispar , to its host-specific baculovirus, L. dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV). Susceptibility of the gypsy moth to LdMNPV decreased markedly as the insect aged within the fourth instar and this resistance was systemic because it could not be overcome by bypassing the midgut and injecting the virus directly into the hemocoel. An LD 88 dose of polyhedra delivered orally to newly molted fourth instars produced 74, 37, 29, 27, 38, and 60% mortalities in larvae that were orally inoculated at 12, 24, 48, 72, 96, or 120 h post-molt to the fourth instar, respectively. An LD 77 dose of budded virus delivered intrahemocoelically to newly molted fourth instars produced 84, 54, 29, 48, 59, and 46% mortalities in larvae that were injected at 12, 24, 48, 72, 96, or 120 h post-molt, respectively. Developmental resistance was also observed in fourth instars fed on oak foliage, no matter whether they were inoculated orally or intrahemocoelically, suggesting that intrastadial developmental resistance in gypsy moth larvae has a systemic component. Also, the host plant did not appear to affect systemic resistance, although it did increase midgut-based resistance. The degree of developmental resistance by intrahemocoelic inoculation was equivalent in oak- and diet-fed insects. In contrast, the degree of resistance by oral inoculation was much greater in oak- than in diet-fed insects, probably because of the combined effects of host-plant inhibition and increasing resistance to viral disease as the larvae aged within the instar.

Recombinant baculoviruses that express insect-specific toxins under the control of viral late gene promoters can kill susceptible lepidopteran hosts faster than the wild-type virus. In an effort to improve the efficacy of genetically engineered baculovirus pesticides, we have produced a recombinant that contains an insect specific toxin gene (AaIT) under the control of the promoter from an immediate early gene (ie1) ofAutographa californicanuclear polyhedrosis virus (AcNPV). AaIT protein was detected as early as 4 h postinfection in cultured insect cells infected with this recombinant. The protein continued to accumulate as the infection progressed and, even as late as 24 h postinfection, the amount of AaIT was about equal to that provided by a recombinant that expressed AaIT under the control of the very latep10promoter. Similar results were obtained with a recombinant that expressed a modified insect juvenile hormone esterase (JHE–KK) underie1control, confirming the ability of this promoter to provide early and abundant expression of gene products with potential pesticidal activity. Bioassays showed that theie1–AaIT recombinant killedHeliothis virescenslarvae faster than wild-type virus, but not faster than thep10–AaIT recombinant. However, larvae infected with theie1–AaIT recombinant were smaller than those infected with thep10–AaIT recombinant, suggesting that expression of AaIT earlier in infection enhanced the ability of the virus to reduce the feeding activity ofH. virescenslarvae.

Both wild-type and recombinant baculoviruses are becoming more attractive for the control of insect pests. Thus, there is an increased incentive to address and resolve logistical problems associated with large-scale production of these viruses. In this study, we have compared the potential of two insect cell lines, Tn5B1-4 and Sf21, for the production of polyhedra and compared the efficacy of both cell culture-derived and host-derived viruses by bioassay. The efficacy of both wild-type AcMNPV and AcAaIT, a recombinant baculovirus expressing an insect-specific scorpion toxin, were compared. Yields of polyhedra from Tn5B1-4 were sixfold higher than those from the cell line Sf21. Morphological analysis of polyhedra derived from cell culture showed greater variability in size relative to host-derived polyhedra. The maximum size of cell culture-derived polyhedra was over 1.5 times larger than that of insect-derived polyhedra. The efficacy of AcMNPV and AcAaIT derived from cell culture, or from amplification in larvae of Trichoplusia ni or Heliothis virescens , was compared by bioassay in H. virescens. There was a significant difference between the slopes for lethal time data for host-derived and cell culture-derived wild-type virus. Mortality occurred at a faster rate following infection with host-derived virus. No significant difference was seen for the recombinant virus AcAaIT. Lethal doses of cell- and host-derived polyhedra were not significantly different. The reasons for and implications of this for pest control are discussed. The data suggest that polyhedra production in larvae may be preferable to production in cell culture for the wild-type virus, but that this does not hold for recombinant viruses with enhanced speed of kill.

Genetically engineered viral insecticides have considerable potential as biological control agents. Through genetic manipulation, the wild-type baculovirus Autographa californica nuclear polyhedrosis virus (WT AcNPV) has been modified to encode for an insect-selective toxin derived from the venom of the scorpion Androctonus australis to produce the recombinant virus AcAaIT. Larvae of tobacco budworm (Heliothis virescens) infected with the baculovirus AcAaIT fell off the plant 5-11 h before death and were unable to climb back on the plant to continue feeding. These larvae were capable of feeding if placed oil diet or confined to a leaf with a clip cage. Larvae infected with WT AcNPV died on the plant. Larvae infected with AcAaIT consumed significantly less foliage than larvae infected with WT AcNPV or uninfected controls. Thus, because larvae infected with the recombinant AcAaIT fall off the plant prior to cessation of feeding, there is a greater reduction in feeding damage than expected from lethal times. In addition, because AcAaIT causes a knock-off effect, the absence of liquefied cadavers on plants following treatment with AcAaIT may be more desirable to growers and consumers relative to treatment with wild-type virus. The relevance of our results to field use of recombinant viruses in pest control is discussed.