As insects develop from molt to molt, they become increasingly resistant to infection by baculoviruses (3, 13, 16, 21), but few studies of variation in susceptibility within an instar have been reported (for exceptions see 2, 4, 13]. For example, sensitivity to mortal infection in Lymantria dispar L. (gypsy moth) by L. dispar multiple nucleopolyhedrovirus (LdMNPV) drops dramatically in larvae challenged with virus in the middle of the third or fourth instar (at 24 to 72 h post-molt) (10, 11).
The gypsy moth is an exotic, invasive pest of forests and woody ornamentals in the eastern United States and Canada. LdMNPV is an effective, specific, microbial insecticide against this insect, but it is relatively expensive to produce enough virus to provide effective doses over large areas of forest because in vitro production remains problematic at this time (J. Slavicek, pers. comm.). Larval gypsy moths are most sensitive to lethal infection by LdMNPV immediately after molting (10, 11). They become most resistant in the middle of the instar, and regain some, but not all, of their initial sensitivity at the end of the instar. For example, delivery of a pulse of 325 occlusion bodies (OBs) per larva directly into the anterior midgut produced 88% mortality in newly molted fourth instars but only 29% and 27% mortality in larvae that were orally inoculated at 48 or 72 h post-molt to the fourth instar, respectively (11).
Reports of lepidopteran resistance to baculoviruses within an instar are usually midgut-based, resulting from sloughing of infected midgut cells before the virus has an opportunity to spread systemically (4, 23, 24). In these studies, intrahemocoelic inoculation of larvae produced equivalent mortality regardless of age post-molt within an instar (except for the final instar) (13), indicating that a systemic component to this resistance was ruled out. In contrast, gypsy moths also display developmental resistance to lethal intrahemocoelic inoculation of budded virus (BV) of LdMNPV (10, 11). For example, an LD77 dose of BV delivered intrahemocoelically to newly molted fourth instars produced 29% mortality in larvae that were injected at 48 h post-molt (11).
The objective of this study was to determine if systemic developmental resistance in gypsy moths to LdMNPV is generalizable to other insect viruses, such as Amsacta moorei entomopoxvirus (AMEV). AMEV was originally isolated from the red hairy caterpillar (Amsacta moorei Butler), an arctiid moth from Northern India (17) and was characterized by Granados and Roberts (9) and McCarthy et al. (15). AMEV is a member of the Entomopoxvirinae, a subfamily of the Poxviridae. While LdMNPV is host-specific, AMEV can infect semi-permissive hosts such as the saltmarsh caterpillar Estigmene acrea (Lepidoptera: Arctiidae) (17) and the gypsy moth. Gypsy moth is only considered susceptible to AMEV by intrahemocoelic challenge, unless larvae were fed high doses of virus in conjunction with an optical brightener (18). There may be other hosts permissive to AMEV, but very little is known about the host range of this and other entomopoxviruses (6). AMEV also replicates well in gypsy moth cell lines (5, 8). Despite our attempts to obtain E. acrea or A. moorei to test for developmental resistance within an instar for comparison to L. dispar, we were unable to find a source of these insects for study.
We chose AMEV for this study because similar to baculoviruses, entomopoxviruses (EPVs) are large, double-stranded DNA, insect viruses that can infect the gypsy moth systemically. Also similar to baculoviruses, EPVs initiate infection in midgut cells, but the mechanism of entry remains unknown; entry may occur via fusion with the plasma membrane or by receptor-mediated endocytosis (12). In contrast to baculoviruses, which replicate in the nucleus, EPVs replicate within discrete cyoplasmic foci (viroplasms) in the vicinity of the nucleus (9). Non-occluded, enveloped progeny virions (called intracellular virus or ICV) acquire a second envelope as they bud through the plasma membrane into the insect hemocoel (called extracellular virus or ECV) (8). Thus, EPVs differ from baculoviruses in that the non-occluded form of EPVs is phenotypically the same as the occluded form but are similar to NPVs in producing occlusions that vary considerably in size (5-20 μm in diameter) (1, 12).
To determine if systemic developmental resistance in gypsy moths to LdMNPV is generalizable to other DNA viruses, such as to AMEV, we challenged developmentally-staged cohorts of fourth instar gypsy moths with AMEV orally or intrahemocoelically and compared these results to the comparable time points within the fourth instar in gypsy moths to LdMNPV as reported previously (11).
Specificity of Developmental Resistance in Gypsy Moth (Lymantria dispar) to two DNA-Insect Viruses
- Received Date: 31 January 2009
- Accepted Date: 30 April 2009
Abstract: Gypsy moth (Lymantria dispar) larvae displayed marked developmental resistance within an instar to L. dispar M nucleopolyhedrovirus (LdMNPV) regardless of the route of infection (oral or intrahemocoelic) in a previous study, indicating that in gypsy moth, this resistance has a systemic component. In this study, gypsy moth larvae challenged with the Amsacta moorei entomopoxvirus (AMEV) showed developmental resistance within the fourth instar to oral, but not intrahemocoelic, inoculation. In general, gypsy moth is considered refractory to oral challenge with AMEV, but in this study, 43% mortality occurred in newly molted fourth instars fed a dose of 5×106 large spheroids of AMEV; large spheroids were found to be more infectious than small spheroids when separated by a sucrose gradient. Developmental resistance within the fourth instar was reflected by a 2-fold reduction in mortality (18%-21%) with 5×106 large spheroids in larvae orally challenged at 24, 48 or 72 h post-molt. Fourth instars were highly sensitive to intrahemocoelic challenge with AMEV; 1PFU produced approximately 80% mortality regardless of age within the instar. These results indicate that in gypsy moth, systemic developmental resistance may be specific to LdMNPV, reflecting a co-evolutionary relationship between the baculovirus and its host.