Jasmonic acid-induced plant defenses delay caterpillar developmental resistance to a baculovirus: Slow-growth, high-mortality hypothesis in plant-insect-pathogen interactions.

Plants damaged by herbivore feeding can induce defensive responses that reduce herbivore growth. The slow-growth, high-mortality hypothesis postulates that these non-lethal plant defenses prolong the herbivore's period of susceptibility to natural enemies, such as predators and parasitoids. While many juvenile animals increase their disease resistance as they grow, direct tests of the slow-growth, high-mortality hypothesis in the context of plant-herbivore-pathogen interactions are lacking. Caterpillars increase their resistance to lethal baculoviruses as they develop within and across instars, a phenomenon termed developmental resistance. Progression of developmental resistance can occur through age-related increases in systemic immune functioning and/or midgut-based resistance. Here, we examined the slow-growth, high-mortality hypothesis in the context of developmental resistance of caterpillars to baculoviruses. Intra-stadial (within-instar) developmental resistance of the fall armyworm, Spodoptera frugiperda, to an oral inoculum of the baculovirus SfMNPV increased more rapidly with age when larvae were fed on non-induced foliage than foliage that was induced by jasmonic acid (a phytohormone that up-regulates plant anti-herbivore defenses). The degree of developmental resistance observed was attributable to larval weight at the time of virus inoculation. Thus, slower growth on induced plants prolonged the window of larval susceptibility to the baculovirus. Developmental resistance on induced and non-induced plants was absent when budded virus was injected intrahemocoelically bypassing the midgut, suggesting that developmental resistance was gut-based. Addition of fluorescent brightener, which weakens midgut-based resistance mechanisms to oral virus challenge, abolished developmental resistance. These results highlight the impact of plant defenses on herbivore growth rate and consequences for disease risk.

Deletion of v-chiA from a baculovirus reduces horizontal transmission in the field.

Nucleopolyhedroviruses (NPVs) can initiate devastating disease outbreaks in populations of defoliating Lepidoptera, a fact that has been exploited for the purposes of biological control of some pest insects. A key part of the horizontal transmission process of NPVs is the degradation of the larval integument by virus-coded proteins called chitinases, such as V-CHIA produced by the v-chiA genes. We used recombinant and naturally occurring strains of the Lymantria dispar NPV (LdMNPV) to test horizontal transmission in the field, release of virus from dead larvae under laboratory conditions, and cell lysis and virus release in cell culture. In the field, strains of LdMNPV lacking functional v-chiA genes showed reduced horizontal transmission compared to wild-type or repaired strains. These findings were mirrored by a marked reduction in released virus in laboratory tests and cell culture when the same strains were used to infect larvae or cells. Thus, this study tests the pivotal role of liquefaction and the v-chiA gene in field transmission for the first time and uses complementary laboratory data to provide a likely explanation for our findings.

Pathogenesis of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar and mechanisms of developmental resistance.

Lymantria dispar has a long historical association with the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), which is one of the primary population regulators of L. dispar in the field. However, host larvae exhibit strong developmental resistance to fatal infection by LdMNPV; the LD50 in newly moulted fourth instars is 18-fold lower than in the middle of the instar (48-72 h post-moult). Using a recombinant of LdMNPV expressing lacZ, we examined the key steps of pathogenesis in the host to explore mechanisms of developmental resistance. At the midgut level, we observed reduced primary midgut infections in mid-fourth instars, indicating increased sloughing of infected cells. Additional barriers were observed as the virus escaped the midgut. Mid-fourth instars had higher numbers of melanized foci of infection associated with the midgut, apoptotic tracheal epidermal cells and haemocytes, and reduced numbers of infected haemocytes later in infection. Our results show that the co-evolutionary relationship between L. dispar and LdMNPV has resulted in both midgut-based and systemic antiviral defences and that these defences are age-dependent within the instar. This age-related susceptibility may contribute to how the virus is maintained in nature and could influence management of L. dispar by using the virus.

Impact of viral enhancin genes on potency of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar following disruption of the peritrophic matrix.

Enhancins are metalloproteases found in many betabaculoviruses and several alphabaculoviruses, which enhance alphabaculovirus potency by degrading a protein component of the peritrophic matrix (PM), facilitating passage of virions through this structure. Earlier studies on betabaculovirus enhancins within heterologous systems suggested that enhancins facilitate virion binding to midgut cells. We compared the potency of wild-type Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) with that of single and double enhancin deletion viruses in L. dispar in the presence and absence of an intact PM. Compared to wild-type virus, the double enhancin deletion virus was 6-fold and 14-fold less potent, respectively, indicating that within this homologous system the LdMNPV enhancin genes have a function beyond PM degradation.

A gene for an extended phenotype.

Manipulation of host behavior by parasites and pathogens has been widely observed, but the basis for these behaviors has remained elusive. Gypsy moths infected by a baculovirus climb to the top of trees to die, liquefy, and "rain" virus on the foliage below to infect new hosts. The viral gene that manipulates climbing behavior of the host was identified, providing evidence of a genetic basis for the extended phenotype.

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus.

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar. L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD(50) in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48-72h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.

The Lymantria dispar nucleopolyhedrovirus enhancins are components of occlusion-derived virus.

Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two enhancin genes (E1 and E2) in the Lymantria dispar multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV enhancin genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of approximately 84 kDa and 90 kDa, respectively, on Western blots of extracts from L. dispar 652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the enhancin genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier.

The Lymantria dispar nucleopolyhedrovirus contains the capsid-associated p24 protein gene.

During the course of investigations on a wild-type strain of Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV), a region of the viral genome was analyzed and found to contain 697 bp that is lacking in the sequenced strain (5-6) of LdMNPV (Kuzio et al., Virology 253, 17-34, 1999). The sequenced strain of LdMNPV contains a mutation in the 25 K few polyhedra (FP) gene, and exhibits the phenotype of a FP mutant. The additional sequence was located at approximately 81.4 map units within the viral genome, and was found in 10 different wild-type LdMNPV genotypic variants analyzed. Since the additional sequence wasfound in all wild-type virus strains analyzed, this sequence should be included in the representative LdMNPV genome. Sequence analysis of the genomic region containing the additional sequences revealed the presence of a homologue of the Autographa californica MNPV capsid-associated p24 gene (ORF 129). This gene, absent in LdMNPV isolate 5-6, is also present in the Orgyia pseudotsugata MNPV, Bombyx mori NPV, Spodoptera exigua MNPV, S. litura MNPV, Mamestra configurata MNPV, Helicoverpa armigera SNPV, H. zea SNPV, Buzura suppressaria SNPV, Xestia c-nigrum granulovirus, Plutella xylostella GV, and Cydia pomonella GV.

N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth) cells using the LdMNPV expression system.

N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then used to infect Ld652Y cells, and the recombinant protein was harvested at 120 h postinfection. N-glycans were released from the purified recombinant human serum transferrin and derivatized with 2-aminopyridine; the glycan structures were analyzed by a two-dimensional HPLC and MALDI-TOF MS. Structures of 11 glycans (88.8% of total N-glycans) were elucidated. The glycan analysis revealed that the most abundant glycans were Man1-3(+/-Fucalpha6)GlcNAc2 (75.5%) and GlcNAcMan3(+/-Fucalpha6)GlcNAc2 (7.4%). There was only approximately 6% of high-mannose type glycans identified. Nearly half (49.8%) of the total N-glycans contained alpha(1,6)-fucosylation on the Asn-linked GlcNAc residue. However alpha(1,3)-fucosylation on the same GlcNAc, often found in N-glycans produced by other insects and insect cells, was not detected. Inclusion of fetal bovine serum in culture media had little effect on the N-glycan structures of the recombinant human serum transferrin obtained.