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Biological Control 40 (2007) 355–361 www.elsevier.com/locate/ybcon
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Michael J. Grove, Kelli Hoover ¤
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Intrastadial developmental resistance of third instar gypsy moths (Lymantria dispar L.) to L. dispar nucleopolyhedrovirus Department of Entomology, 501 ASI Building, The Pennsylvania State University, University Park, PA 16802, USA
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Received 10 August 2006; accepted 8 December 2006 Available online 15 December 2006
Abstract
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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 signiWcantly aVected by the time post-molt at which the insect was inoculated with the virus. We suggest that intrastadial developmental resistance may aVect both the ecology and management of the gypsy moth. © 2006 Elsevier Inc. All rights reserved.
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Keywords: Baculovirus; Developmental resistance; Innate immunity; Lymantria dispar; Lymantria dispar nucleopolyhedrovirus
1. Introduction
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Age-related changes in the susceptibility of insects to a variety of pathogens are well documented (Stairs, 1965; Evans, 1981; Teakle et al., 1986; Kirkpatrick et al., 1998). These changes may occur among life stages (i.e., egg, larva, pupa and adult), among larval instars, or within a given instar (reviewed by Tanada and Kaya, 1993). We refer to increasing resistance within an instar as intrastadial developmental resistance (IDR) (Hoover et al., 2002). The majority of studies of IDR have focused on interactions between larval Lepidoptera and baculoviruses. Baculoviruses are large, double-stranded, insect-speciWc DNA viruses, which have been used as biological *
Corresponding author. Tel.: +1 814 863 6369; fax: +1 814 865 3048. E-mail address:
[email protected] (K. Hoover).
1049-9644/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2006.12.003
control agents worldwide for many years. Baculovirus infections begin when susceptible insect larvae consume occlusion bodies called polyhedra, or granules, depending on the viral genus. In the nucleopolyhedroviruses (NPVs), the occlusions dissolve, releasing one or more virions (referred to as occlusion-derived-virus, or ODV) into the insect midgut. The virions infect columnar cells of the midgut epithelium, replicate in the nucleus of these cells, and produce a second viral phenotype, budded virus, which spreads infection throughout the host (Washburn et al., 2003). Late in infection, ODV are assembled and occluded in the nuclei, cells lyse, and the insect cuticle is eventually dissolved by the combined action of viral protease and chitinase activities, releasing particles of occluded virus into the environment (Granados and Williams, 1986; Engelhard et al., 1994; Federici, 1997; Funk et al., 1997; Volkman, 1997).
M.J. Grove, K. Hoover / Biological Control 40 (2007) 355–361
IDR, as in heliothine systems, this would permit us to narrow our focus to the most relevant resistance mechanism for improving the performance of LdMNPV as a biological control agent. 2. Materials and methods
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2.1. Insects
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Lymantria dispar egg masses of the New Jersey standard strain (NJSS) were obtained from the USDA-APHIS Insectary at Otis Air National Guard Base, MA and stored at 4– 5 °C until needed. Eight to ten egg masses were disinfected by soaking in 18.5% formaldehyde for 1 h, rinsing under running distilled water for 1 h, and drying in a biological containment hood for 30 min. Disinfected egg masses were divided into quarters by cutting them once along their long axis and again along their short axis. The resulting four egg mass pieces were placed individually into four sterile polystyrene Petri dishes, giving a total of 2–2.5 egg mass equivalents per dish. A small piece of artiWcial diet (Southland Products, Lake Village, AR) was also placed in each dish. Dishes were sealed with paraWlm, which was perforated with a #0 insect pin. Individual dishes were placed in a 25 °C growth chamber on four successive days. Neonates were transferred into 240 ml paper cups containing artiWcial diet with plastic lids (Sweetheart, Chicago, IL) at a density of 60–80 larvae/cup and reared in a growth chamber at 25 °C, 40% RH, 16:8 (L:D) h. When larvae became premolts to the third instar, they were removed from the growth chamber and moved into fresh, empty paper cups in a 4–5 °C refrigerator. Larvae were held for up to 4 days at 4 °C prior to molting. We have observed that chilling premolt third instar gypsy moths up to 4 days does not aVect their ability to molt or their permissiveness to viral infection (data not shown). Molting was initiated in chilled larvae by moving them to a 28 °C growth chamber. Newly molted larvae were placed individually in clear, plastic 30 ml cups with plastic lids (Comet Products, Chelmsford, MA) containing a 2 £ 2 £ 3 cm piece of artiWcial diet. Larvae were designated for injection at various times post-molt (e.g., 0, 12, 24, 48, 72, or 96 h §15 min post-molt, designated as 30, 312, ƒ, 396, respectively) (Engelhard and Volkman, 1995). In the case of third instar gypsy moths, 96 h postmolt larvae are pre-molts to the fourth instar (exhibiting head capsule slippage).
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Some degree of intrastadial developmental resistance (IDR) against lethal baculovirus infection has been reported for at least seven insect/NPV systems (Kobayashi et al., 1969; Merritt, 1977; Burgerjon et al., 1981; Teakle et al., 1986; Engelhard and Volkman, 1995; Washburn et al., 1995; Hoover et al., 2002). Although this appears to be a relatively small number of insect/virus combinations (baculoviruses are known to infect at least 500 species of insects (Miller, 1997)), it is more likely to reXect the state of current research, rather than the true frequency of this phenomenon. IDR has, to the best of our knowledge, been found in all insect/baculovirus systems in which it has been investigated. If IDR is indeed a widespread phenomenon, then understanding the underlying mechanism(s) will be important for fully exploiting the potential of baculoviruses as biological control agents. Four of the insect species studied thus far have been examined for IDR using both the natural, oral route of infection by polyhedra and the artiWcial, intrahemocoelic route of infection by BV, which bypasses the midgut. Three of these species—Heliothis punctigera Wallengren, Heliothis virescens F., and Trichoplusia ni Hübner—displayed IDR against oral but not intrahemocoelic inoculation at various times in the fourth (penultimate) instar (Teakle et al., 1986; Washburn et al., 1998). These data indicate that the principle mechanism of IDR in these species is midgut-, rather than systemically based. However, two of these species—H. punctigera and H. virescens—were tested in the Wfth (Wnal) instar. In these cases, both species displayed IDR against lethal intrahemocoelic as well as oral inoculation, indicating that a form of systemic resistance occurred in the last instar of these species (Teakle et al., 1986; Kirkpatrick et al., 1998). Kobayashi et al. (1969) reported that Bombyx mori (L.) larvae not only displayed strong IDR against oral inoculation, but also to some extent against intrahemacoelic inoculation of virus. However, the actual degree of systemic resistance of B. mori might not be directly comparable to other studies because these authors used occlusion-derived virus obtained by alkaline solubilization of polyhedra for intrahemacoelic injection rather than budded virus and cells within the hemocoel are quite refractory to occlusion derived virus (Volkman et al., 1976). In contrast, fourth (penultimate male/antepenultimate female) instar Lymantria dispar display a pattern of resistance to intrahemocoelic inoculation with budded virus that closely parallels the pattern of resistance to oral inoculation. In both cases, larvae are most sensitive to lethal infection in the 12 h immediately following ecdysis. By 24 h post-molt (hpm), larvae are signiWcantly less sensitive to infection. Sensitivity is least between 48 and 72 hpm, and increases to an intermediate level from 96 to 120 hpm (Hoover et al., 2002). Since baculoviral insecticidal (Gypchek) sprays are typically employed against earlier instars (seconds and sometimes thirds) of L. dispar, we investigated the signiWcance of the systemic component of IDR in third instars. If early instars relied less on systemic and more on midgut-based
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2.2. Viruses Polyhedral occlusion bodies (OBs) from the A21 (Slavicek et al., 1995) isolate of LdMNPV were ampliWed in gypsy moth larvae and provided to us as a stock suspension in distilled water by Dr. Suzanne Thiem (Michigan State University). OBs were maintained as a stock suspension in water at 4 °C for t4 years without loss of potency. For bioassays, aliquots of stock OBs were diluted under aseptic conditions in Wlter sterilized glycerol (EM Sciences,
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between the mandibles and gently inserted through the foregut into the anterior midgut to deliver a 1 l droplet of OBs suspended in 60% glycerol. For intrahemocoelic injections, a sharp needle was inserted through the abdominal body wall at the junction of either the second or third proleg to deliver 1 l of BV. Control larvae were inoculated with the same volume of carrier solution without virus. For all experiments, each larva was returned to its respective diet and observed daily over the next 21 days for mortality, molting, and pupation. Larvae that died within three days after injection were classiWed as non-viral, trauma-related deaths and removed from the experiment. Because newly molted third instars are small and delicate, non-viral mortality was highest (6–15%) in newly molted third instars regardless of inoculation method, but non-viral mortality at other ages post-molt was
