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Species of knotweeds, Reynoutria spp. Houtt. (Caryophyllales: Polygonaceae), including Japanese knotweed (R. japonica Houtt.), are among the most invasive and ecologically destructive plant species introduced to North America and Europe. The Kyushu strain of the psyllid Aphalara itadori Shinji (Hemiptera: Aphalaridae) has been approved as a biological control agent for release against Japanese knotweed in the United Kingdom, Canada, and the United States. However, recent reports from Canada suggest that both biotic and abiotic factors may limit its establishment. Therefore, we examined the potential role of predation by comparing open- versus closed-sleeve treatments, and climate mis-matches by collecting temperature data from release sites and performing climate suitability analyses using MaxEnt. Our results indicated that populations of the Kyushu strain could only be maintained in the field in closed-sleeve treatments, suggesting that predation is likely limiting the establishment success of this strain. In addition, we noted that daily maximum temperatures at our field sites might exceed documented developmental thresholds for this strain, and that MaxEnt species distribution modeling indicates no climate similarities between locations in eastern North America and Kyushu. Combined with previous results, our study suggests that the establishment of the Kyushu strain of A. itadori as a biological control agent for Japanese knotweed may be limited in eastern North America. We suggest that one strategy to increase the probability of establishment of the Kyushu strain could be to increase the number of release sites in an effort to find a more optimal niche with predator-free space.

Hybridization plays an important and underappreciated role in shaping the evolutionary trajectories of species. Following the introduction of a non-native organism to a novel habitat, hybridization with a native congener may affect the probability of establishment of the introduced species. In most documented cases of hybridization between a native and a non-native species, a mosaic hybrid zone is formed, with hybridization occurring heterogeneously across the landscape. In contrast, most naturally occurring hybrid zones are clinal in structure. Here, we report on a long-term microsatellite data set that monitored hybridization between the invasive winter moth, Operophtera brumata (Lepidoptera: Geometridae), and the native Bruce spanworm, O. bruceata, over a 12-year period. Our results document one of the first examples of the real-time formation and geographic settling of a clinal hybrid zone. In addition, by comparing one transect in Massachusetts where extreme winter cold temperatures have been hypothesized to restrict the distribution of winter moth, and one in coastal Connecticut, where winter temperatures are moderated by Long Island Sound, we found that the location of the hybrid zone appeared to be independent of environmental variables and maintained under a tension model wherein the stability of the hybrid zone was constrained by population density, reduced hybrid fitness, and low dispersal rates. Documenting the formation of a contemporary clinal hybrid zone may provide important insights into the factors that shaped other well-established hybrid zones.

Hemlock woolly adelgid (HWA; Adelges tsugae Annand (Hemiptera: Adelgidae)) is the cause of widespread mortality of Carolina and eastern hemlock (Tsuga caroliniana Engelmann and T. canadensis (L.) Carrière) throughout the eastern United States (U.S.). Since its arrival in the northeastern U.S., HWA has steadily invaded and established throughout eastern hemlock stands. However, in 2018, anecdotal evidence suggested a sharp, widespread HWA decline in the northeastern U.S. following above-average summer and autumn rainfall. To quantify this decline in HWA density and investigate its cause, we surveyed HWA density in hemlock stands from northern Massachusetts to southern Connecticut and analyzed HWA density and summer mortality in Pennsylvania. As native fungal entomopathogens are known to infect HWA in the northeastern U.S. and rainfall facilitates propagation and spread of fungi, we hypothesized high rainfall facilitates fungal infection of aestivating nymphs, leading to a decline in HWA density. We tested this hypothesis by applying a rain-simulation treatment to hemlock branches with existing HWA infestations in western MA. Our results indicate a regional-scale decline and subsequent rebound in HWA density that correlates with 2018 rainfall at each site. Experimental rain treatments resulted in higher proportions of aestivating nymphs with signs of mortality compared to controls. In conjunction with no evidence of increased mortality from extreme winter or summer temperatures, our results demonstrate an indirect relationship between high rainfall and regional HWA decline. This knowledge may lead to better prediction of HWA population dynamics.

Specialized natural enemies have long been used to implement the biological control of invasive insects. Although research tracking populations following biological control introductions has traditionally focused on the impact of the introduced agent, recent studies and reviews have reflected an appreciation of the complex interactions of the introduced specialist agents with native generalist natural enemies. These interactions can be neutral, antagonistic, or complementary. Here we studied the invasive defoliator winter moth (Operophtera brumata) in the Northeast USA to investigate the role of native, generalist pupal predators along with the introduced, host-specific parasitoid Cyzenis albicans. Prior research in Canada has shown that predation of winter moth pupae from native generalists increased after C. albicans was established as a biological control agent. To explain this phenomenon, the following hypotheses were suggested: (H

The density of insect herbivores is regulated by top-down factors (e.g., natural enemies), bottom-up effects (e.g., plant defenses against herbivory), or a combination of both. As such, understanding the relative importance of these factors can have important implications for the establishment of effective management options for invasive species. Here, we compared the relative importance of top-down and bottom-up factors on the abundance of hemlock woolly adelgid (HWA), Adelges tsugae. HWA is invasive in eastern North America, but its native range includes the Pacific Northwest of North America where it has co-evolved with western hemlock, Tsuga heterophylla. Eastern hemlock, Tsuga canadensis, can also be found planted in city and park settings in the Pacific Northwest and the presence of both host species allowed us to directly compare the importance of predators (top-down) and host plant resistance (bottom-up) on HWA abundance by placing mesh exclusion bags on branches of both species and monitoring HWA abundance over two years. We found no evidence for bottom-up control of HWA on western hemlock (a native host). HWA established more readily on that species than on eastern hemlock on which it is a major pest in eastern North America. We found strong evidence for top-down control in that both summer and winter-active predators significantly reduced HWA densities on the branches of both tree species where predators were allowed access. These findings support the validity of the biological control program for HWA, the goal of which is to reduce outbreak populations of HWA in eastern North America.

The recently described oak gall wasp Zapatella davisae Buffington & Melika (Hymenoptera: Cynipidae) has caused extensive damage and mortality to black oak trees, Quercus velutina L. (Fagales: Fagaceae), in coastal parts of New England, United States. Like many newly described and/or newly introduced species, it is unclear how long populations of Z. davisae have existed in this region. However, as this species forms galls on the woody-tissue of its host, it may be possible to obtain historical information about changes in its population size by examining the presence of galls in relation to annual growth nodes. Here, we explore the utility of this approach to determine population size changes in Z. davisae densities on Nantucket, Martha's Vineyard, and Cape Cod, Massachusetts, through dissection of black oak branches. In addition, we calculated parasitism rates during the years of study and obtained morphological and molecular identifications for the parasitoids associated with Z. davisae. Our results show significant changes in population sizes, with higher levels of parasitism at sites on Martha's Vineyard and Cape Cod compared to sites on Nantucket. In addition, morphological examinations, in combination with DNA sequencing, identified the associated parasitoids as five species in the genus Sycophila Walker (Hymenoptera: Eurytomidae). We comment that considerable morphological variation within several of these recovered species was observed, present the first record of males for a species from which only females have been described, and suggest that future work is required to clarify the species boundaries for this important parasitoid group.

Reconstructing the geographic origins of invasive species is critical for establishing effective management strategies. Frequently, molecular investigations are undertaken when the source population is not known, however; these analyses are constrained both by the amount of diversity present in the native region and by changes in the genetic background of the invading population following bottlenecks and/or hybridization events. Here we explore the geographical origins of the invasive winter moth (Operopthera brumata L.) that has caused widespread defoliation to forests, orchards, and crops in four discrete regions: Nova Scotia, British Columbia, Oregon, and the northeastern United States. It is not known whether these represent independent introductions to North America, or “stepping stone” spread among regions. Using a combination of Bayesian assignment and approximate Bayesian computation methods, we analyzed a population genetic dataset of 24 polymorphic microsatellite loci. We estimate that winter moth was introduced to North America on at least four occasions, with the Nova Scotian and British Columbian populations likely being introduced from France and Sweden, respectively; the Oregonian population likely being introduced from either the British Isles or northern Fennoscandia; and the population in the northeastern United States likely being introduced from somewhere in Central Europe. To our surprise, we found that hybridization has not played a large role in the establishment of winter moth populations even though previous reports have documented widespread hybridization between winter moth and a native congener. We discuss the impact of genetic bottlenecks on analyses meant to determine region of origin.

Winter moth, Operophtera brumata, native to Europe, invaded the northeastern United States in the late 1990s, where it caused widespread defoliation of forests and shade trees ranging from 2,266 to 36,360 ha/yr between 2003 and 2015 in Massachusetts. In 2005, we initiated a biological control effort based on the specialist tachinid parasitoid Cyzenis albicans, which had previously been introduced along with the generalist ichneumonid parasitoid Agrypon flaveolatum to control winter moth in Nova Scotia in the 1950s and British Columbia in the 1970s. Due to concerns of possible non-target impacts by A. flaveolatum, we focused entirely on the specialist C. albicans. Each year for 14 yr, we collected several thousand individuals of C. albicans from British Columbia and released them in widely spaced sites in the northeastern United States. As of 2020, we had established C. albicans at 41 of 44 sites from coastal Maine to southeastern Connecticut. By 2016, winter moth densities (pupae/m2 ) had declined from 100-500 to 0-10 pupae/m2 at six release sites at least 10 km apart and this was coincident with the onset of 10-40% parasitism. At one site in Wellesley, Massachusetts, the decline occurred in 2012 and winter moth densities have remained low for seven subsequent years. Defoliation in Massachusetts has been reduced to undetectable levels by aerial survey since 2016. DNA sequencing of the barcoding region of the mitochondrial gene CO1 confirmed that all C. albicans reared from winter moth matched the C. albicans collected from Vancouver Island and were distinct from parasitic flies (presumably a native species) reared from a native congener of winter moth, Bruce spanworm (O. bruceata). Successful establishment of C. albicans on winter moth represents a rare, if not the only, example of the biological control of a major forest defoliator that attacks a wide range of tree species anywhere in the world by the establishment of a single specialist natural enemy.

Winter moth, Operophtera brumata L. (Lepidoptera: Geometridae), causes widespread defoliation in both its native and introduced distributions. Invasive populations of winter moth are currently established in the United States and Canada, and pheromone-baited traps have been widely used to track its spread. Unfortunately, a native species, the Bruce spanworm, O. bruceata (Hulst), and O. bruceata × brumata hybrids respond to the same pheromone, complicating efforts to detect novel winter moth populations. Previously, differences in measurements of a part of the male genitalia called the uncus have been utilized to differentiate the species; however, the accuracy of these measurements has not been quantified using independent data. To establish morphological cutoffs and estimate the accuracy of uncus-based identifications, we compared morphological measurements and molecular identifications based on microsatellite genotyping. We find that there are significant differences in some uncus measurements, and that in general, uncus measurements have low type I error rates (i.e., the probability of having false positives for the presence of winter moth). However, uncus measurements had high type II error rates (i.e., the probability of having false negatives for the presence of winter moth). Our results show that uncus measurements can be useful for performing preliminary identifications to monitor the spread of winter moth, though for accurate monitoring, molecular methods are still required. As such, efforts to study the spread of winter moth into interior portions of North America should utilize a combination of pheromone trapping and uncus measurements, while maintaining vouchers for molecular identification.

To support efforts to manage and contain spotted lanternfly (SLF), Lycorma delicatula White (Hemiptera: Fulgoridae), research is being conducted to develop classical biological control methods. To date, two potential biocontrol agents from China have been identified: an egg parasitoid, Anastatus orientalis, and a nymphal parasitoid, Dryinus sinicus Olmi (Hymenoptera: Dryinidae). The research detailed here focuses on investigating the biology and rearing of A. orientalis to assess its potential efficacy in a biocontrol program and optimize its rearing. Female wasps lived significantly longer than male wasps (68 and 23 d, respectively) and females produced an average of 94 total progeny that successfully emerged as adults, with most progeny produced between weeks one and four of the females’ lives. The sex ratio of the progeny, with no re-mating, was initially highly female-biased but became progressively more male-biased, likely due to sperm depletion. There was no evidence of additional mortality to SLF eggs from wasp host feeding, but the data were highly variable and the sample size was small. There was high parasitoid emergence when oviposition conditions mimicked mid-September Beijing temperature and photoperiod; however, there was little emergence under 25°C and long-day conditions because most progeny entered a diapause. Storage of parasitized eggs in 5°C chill lowered parasitoid emergence rates. Lastly, there was no evidence that storing field-collected SLF egg masses in 5°C for 10 mo prior to parasitization affected parasitism rates. These findings inform our rearing protocol for A. orientalis and facilitate our testing of this species as a potential biological control agent for SLF.

Disease can affect biological invasions by acting as either a synergist or antagonist. Disease-mediated invasions have important implications for understanding the spread of invasive insects, which cost billions of dollars in damages annually. One such non-native, destructive insect is the winter moth, Operophtera brumata L. (Lepidoptera: Geometridae), which causes defoliation and mortality of deciduous trees in its introduced range. In the northeastern United States, winter moth populations overlap with a native congener, Bruce spanworm, Operophtera bruceata Hulst. Nucleopolyhedrovirus (NPV), appears to be an important natural enemy in Bruce spanworm and there is some evidence that the NPV infection found in winter moth in the northeastern U.S. may originate from Bruce spanworm. By sequencing two viral genes (the polyhedrin and p74 genes) from field-collected larvae of both species, we found that the winter moth virus (OpbuNPV) is distinct from the virus from Bruce spanworm (OpbrNPV). However, the two viruses do constitute a clade within the Alphabaculovirus Group 2 NPVs, indicating that they are more similar to each other than they are to other lepidopteran viruses, even other geometrid-derived NPVs. As far as we know, this is the first report of sequences from an NPV from Bruce spanworm. Results from cross infection trials suggest that cross infection is uncommon if it occurs at all. Our results show that these two closely related species have distinct viruses and, unlike previous suggestions, Bruce spanworm virus is not mediating the winter moth invasion.

We collected data on mortality of late-instar gypsy moth, Lymantria dispar (L.), from outbreak populations over 4 wk in June 2017 at 10 sites in the New England region of the United States, along with estimated rainfall at these sites. Deposition of airborne conidia of the fungal pathogen, Entomophaga maimaiga Humber, Shimazu & R.S. Soper, was measured at these same sites as well as at seven other locations in New England. We also quantified the geographical distribution of gypsy moth-caused defoliation in New England in 2017 and 2018 from Landsat imagery. Weekly mortality of gypsy moth larvae caused by E. maimaiga correlated with local deposition of conidia from the previous week, but not with rainfall. Mortality from this pathogen reached a peak during the last 2 wk of gypsy moth larval development and always exceeded that caused by LdNPV, the viral pathogen of gypsy moth that has long been associated with gypsy moth outbreaks, especially prior to 1989. Cotesia melanoscela (Ratzeburg) was by far the most abundant parasitoid recovered and caused an average of 12.6% cumulative parasitism, but varied widely among sites. Deposition of E. maimaiga conidia was highly correlated with percent land area defoliated by gypsy moths within distances of 1 and 2 km but was not significantly correlated with defoliation at distances greater than 2 km. This is the first study to relate deposition of airborne conidia of E. maimaiga to mortality of gypsy moths from that agent.

The emerald ash borer (EAB), Agrilus planipennis Fairmaire, a buprestid beetle native to Asia, has become a serious pest of ash trees (Fraxinus spp.) in North America since the early 2000s. Due to the impracticality of applying insecticides in natural forests, biocontrol is the most viable method to manage EAB in natural ecosystems. Here, we report the first evidence for the establishment and impact of Spathius galinae Belokobylskij & Strazenac, a larval parasitoid first released in North America in 2016 and 2017 at six mixed-hardwood forest sites, in Connecticut, New York, and Massachusetts. We also report current levels of abundance and parasitism of another introduced larval EAB parasitoid, Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae), released in 2015 and 2016 in these same sites. Spathius galinae was recovered at all release sites in 2018, and its density in sampled trees had increased 1.5- to 20-fold (relative to the first postrelease sample year), reaching a final density of 2.3–14.3 broods/m2 of phloem area and causing 13.1–49.2% marginal rate of parasitism at four of the six sites. In contrast, T. planipennisi was only recovered in 2018 at four of the six release sites, and both its density (0.1–2.3 broods/m2 of phloem area) and parasitism (0.1–5.6%) were lower than that of S. galinae throughout the study at the four sites where recoveries were made. Our data fill a critical gap in the development of a biocontrol-based EAB management plan to protect surviving ash trees capable of reaching maturity and producing replacement trees.

The winter moth, Operophtera brumata (L.) is an invasive forest and agricultural pest in North America that causes severe defoliation to a wide range of host species. This study examines the differential larval densities, development, and survival on seven host species in midcoast Maine: red oak (Quercus rubra L., Fagales: Fagaceae), apple (Malus domestica L., Rosales: Rosaceae) and crab apple (Malus sp. L., Rosales: Rosaceae), red maple (Acer rubrum L., Sapindales: Sapindaceae), pin cherry (Prunus pensylvanica L., Rosales: Rosaceae), white birch (Betula papyrifera L., Fagales: Betulaceae), wild lowbush blueberry (Vaccinium angustiflolium L., Ericales: Ericaceae), and highbush blueberry (Vaccinium corymbosum L., Ericales: Ericaceae). We also explore the degree of synchrony between selected host plants and larval hatch and its effect on survival. We found that densities, development, and survival were significantly greater on red oak (Quercus rubra) and apple (Malus sp.) than on all other target species and were lowest on pin cherry (Prunus pennsylvanica). We found low larval densities in open, wild lowbush blueberry fields; however, larvae successfully fed and developed on wild lowbush blueberry in a laboratory setting. This suggests that winter moth is a potential pest to wild lowbush blueberry in Maine if the outbreak expands to include areas with wild lowbush blueberry production.

The European winter moth,Operophtera brumata, is a non-native pest in the Northeastern USA causing defoliation of forest trees and crops such as apples and blueberries. This species is known to hybridize withO. bruceata, the Bruce spanworm, a native species across North America, although it is not known if there are hybrid generations beyond F1. To study winter moth population genetics and hybridization with Bruce spanworm, we developed two sets of genetic markers, single nucleotide polymorphisms (SNPs) and microsatellites, using genomic approaches. Both types of markers were validated using samples from the two species and their hybrids. We identified 1216 SNPs and 24 variable microsatellite loci. From them we developed a subset of 95 species-diagnostic SNPs and ten microsatellite loci that could be used for hybrid identification. We further validated the ten microsatellite loci by screening field collected samples of both species and putative hybrids. In addition to confirming the presence of F1 hybrids reported in previous studies, we found evidence for multi-generation asymmetric hybridization, as suggested by the occurrence of hybrid backcrosses with the winter month, but not with the Bruce spanworm. Laboratory crosses between winter moth females and Bruce spanworm males resulted in a higher proportion of viable eggs than the reciprocal cross, supporting this pattern. We discuss the possible roles of population demographics, sex chromosome genetic incompatibility, and bacterial symbionts as causes of this asymmetrical hybridization and the utility of the developed markers for future studies.

Insect populations are affected by density-dependent and density-independent factors, and knowing how these factors affect long-term population growth is critical to pest management. In this study, we experimentally manipulated densities of the hemlock woolly adelgid on eastern and western hemlock trees in the western USA to evaluate the effects of density and host species on hemlock woolly adelgid crawler colonization. We then followed development of hemlock woolly adelgid on each hemlock species. Settlement of crawlers was strongly density-dependent and consistent between host species. In addition, a period of hot days that coincided with the settlement of hemlock woolly adelgid crawlers put our experimental and naturally occurring populations into diapause during April. Diapause resulted in one generation that yr in our experimental population. Analyses of long-term air temperature records indicated that diapause-inducing temperatures in April similar to those observed in our experiment have occurred rarely since 1909 and the frequency of these events has not changed over time. Prior work suggests that hemlock woolly adelgid completes two generations per yr in the western USA with a diapause occurring in the summer. This typical life history reflects the long-term influence of regional average seasonal temperature patterns on development and the timing of diapause-inducing temperatures. However, the timing of unseasonal weather, such as the hot days observed in our experiment, occasionally changes life history trajectories from this normal pattern. Our results show that density-dependent and density-independent factors have strong effects on generational mortality and life history of hemlock woolly adelgid that are important to its population dynamics and management.

The success of invasive species is often thought to be due to release from natural enemies. This hypothesis assumes that species are regulated by top-down forces in their native range and are likely to be regulated by bottom-up forces in the invasive range. Neither of these assumptions has been consistently supported with insects, a group which includes many destructive invasive species. Winter moth (Operophtera brumata) is an invasive defoliator in North America that appears to be regulated by larval mortality. To assess whether regulation was caused by top-down or bottom-up forces, we sought to identify the main causes of larval mortality. We used observational and manipulative field and laboratory studies to demonstrate that larval mortality due to predation, parasitism, and disease were minimal. We measured the response of larval dispersal in the field to multiple aspects of foliar quality, including total phenolics, pH 10 oxidized phenolics, trichome density, total nitrogen, total carbon, and carbon-nitrogen ratio. Tree-level declines in density were driven by density-dependent dispersal of early instars. Late instar larvae dispersed at increased rates from previously damaged as compared to undamaged foliage, and in 2015 field larval dispersal rates were related to proportion of oxidative phenolics. We conclude that larval dispersal is the dominant source of density-dependent larval mortality, may be mediated by induced changes in foliar quality, and likely regulates population densities in New England. These findings suggest that winter moth population densities in New England are regulated by bottom-up forces, aligning with the natural enemy release hypothesis.

We describe approaches to addressing the perennial challenge of collecting a sufficient diversity of nontarget insects for host-specificity testing of candidate biocontrol agents of invasive wood-borers such as the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae). Multifunnel and intercept traps, retrofitted to maintain live insects and baited with cerambycid-specific pheromone lures, were deployed in diverse forests in southeastern Massachusetts. We collected 1,288 adult beetles comprising 56 species, mostly from the subfamilies targeted by the lures (Cerambycinae and Lamiinae). The type of trap and tree species in which the trap was hung did not seem to affect the species caught. Methods used to induce egg laying and techniques to rear cerambycid larvae are described. Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is the most common Asian longhorned beetle parasitoid in China; therefore, we conducted tests to determine whether cerambycids native to North America would be at risk should this species be released. D. helophoroides attacked all six species tested: Monochamus scutellatus (Say) (Coleoptera: Cerambycidae: Lamiinae), Monochamus notatus (Drury) (Coleoptera: Cerambycidae: Lamiinae), Apriona rugicollis Chevrolat (Coleoptera: Cerambycidae: Lamiinae), Enaphalodes rufulus (Haldeman) (Coleoptera: Cerambycidae: Cerambycinae), Graphisurus fasciatus (DeGeer) (Coleoptera: Cerambycidae: Lamiinae), and Neoclytus acuminatus (F.) (Coleoptera: Cerambycidae: Cerambycinae). Parasitism of native cerambycids was not statistically different from parasitism of Asian longhorned beetle, except for N. acuminatus, which is a considerably smaller species than Asian longhorned beetle, and for M. notatus and M. scutellatus, which attack pine trees rather than hardwood trees like Asian longhorned beetle and the other native species tested. Our testing indicates that many native cerambycids would be vulnerable to D. helophoroides and we conclude that D. helophoroides should not be considered for release as a biocontrol agent in North America.

Varying densities of the spring generation of the hemlock woolly adelgid were manipulated on 16 previously uninfested eastern hemlocks in an open-field plantation in Massachusetts. In contrast to experimentally created hemlock woolly adelgid populations in a forest, as reported previously, there was no evidence of density-dependent survival on a tree-wide basis in the plantation in the spring (progrediens) generation. There was, however, comparable density-dependent survival of settled crawlers and sexupara production when samples of the population were examined from branches with high density. Plantation hemlocks had 9.3 times more foliage and 10 times lower adelgid densities per cm than the forest hemlocks. These results show that density-dependent processes in the progrediens generation may only be evident when hemlock woolly adelgid density reaches a critical threshold. In the sistens generation that begins in midsummer, we counted a mean of 126 settled crawlers on marked branch on all 16 trees, but none of these adelgids survived the mid-summer aestivation phase, perhaps due to higher temperatures that were recorded in the plantation compared with a nearby hemlock forest, where 16% of the adelgids survived the aestivation phase. Whole tree counts of overwintering adelgids revealed that the adelgid populations had gone extinct on 13 out of the 16 trees. Mortality in the midsummer aestivation phase often exceeds overwintering mortality that has been widely thought to be the main factor that limits adelgid population growth and spread, particularly in northern states.

Delta- and bucket-style (Universal or Unitrap) traps baited with 1 standard survey lure and 1/3 and 3 lures were compared for their attractiveness and trapping efficiencies for gypsy moth, Lymantria dispar L. (Lepidoptera: Erebidae), males. With bucket traps, the numbers of males attracted to within 2 m of traps and the proportion of these actually captured were identical among the three doses although the percentage of attracted males actually captured in bucket traps was low, less than 15%. A three-lure delta trap attracted about 70% more males than traps with the two lower doses. Capture efficiencies were above 80% for 1/3- and one-lure traps and about 60% for traps baited with three lures. The number of males captured in delta traps was equivalent for the three doses although our observations also suggest that a delta trap baited with three lures drew males from a wider range than lower dose lures and therefore would be a more sensitive trap for detecting incipient populations. We also noted that males tended to arrive in clusters, suggesting that attraction over moderate distances requires periods when the wind direction is fairly constant. This observation coupled with the great variability in the direction of male arrival to the traps also suggests that important changes in the area of influence of the plume are driven in such forested areas by slower but greater changes in wind direction compared with open habitats.

The hemlock woolly adelgid (Adelges tsugae Annand) has decimated eastern hemlocks (Tsuga canadensis Carrière) in forests throughout the eastern United Sates, but its densities in central New England appear to have stabilized. To find out why, we infested 64 eastern hemlocks with varying densities of adelgid ovisacs in a typical eastern hemlock forest in western Massachusetts. We subsequently documented adelgid density, fecundity, and the amount of new growth on experimental trees over two consecutive years. We used a 2 by 2 randomized block design using previously and newly infested hemlocks that were either 1-m tall saplings or branches of mature trees. There was a density-dependent decline in the survival and fecundity of adelgid in both the spring and winter generations. This response was a function of both previous infestation by adelgid and current year's crawler density in the spring generation. Additionally, the production of sexuparae in the spring generation played a key role in the overall density-dependent survival of adelgid, suggesting that sexuparae production is strongly linked to developing crawler density.

Recent studies have identified a small number of individual eastern hemlock trees that demonstrate relative resistance to the introduced sap-feeding insect, the hemlock woolly adelgid. Using gas chromatography, we compared concentrations of twenty-two terpenoids in susceptible and relatively resistant trees, both in the forest and in propagated cuttings in a common-garden setting. Terpenoid concentrations were higher in twig tissue of resistant versus susceptible trees, across six sampling dates and at both sites. These changes may be particularly important because the hemlock woolly adelgid feeds on twig tissue. Because the common-garden cuttings were free of herbivores, the higher terpenoid concentrations are interpreted as a constitutive defense. Increased levels of monoterpenes and sesquiterpenes imply an overall increase in the input of carbon precursors to both terpenoid synthesis pathways.

Population cycles in forest Lepidoptera often result from recurring density-dependent epizootics of entomopathogens. While these systems are typically dominated by a single pathogen species, insects are often infected by multiple pathogens, yet little is known how pathogens interact to affect host dynamics. The apparent invasion of northeastern North America by the fungal entomopathogen Entomophaga maimaiga some time prior to 1989 provides a unique opportunity to evaluate such interactions. Prior to the arrival of E. maimaga, the oscillatory dynamics of host gypsy moth, Lymantria dispar, populations were apparently driven by epizootics of a nucleopolyhedrovirus. Subsequent to its emergence, E. maimaiga has caused extensive mortality in host populations, but little is known about how it has altered multigenerational dynamics of the gypsy moth and its virus. Here we compared demographic data collected in gypsy moth populations prior to vs. after E. maimaiga's invasion. We found that the recently invading fungal pathogen virtually always causes greater levels of mortality in hosts than does the virus, but fungal mortality is largely density independent. Moreover, the presence of the fungus has apparently not altered the gypsy moth-virus density-dependent interactions that were shown to drive periodic oscillations in hosts before the arrival of the fungus.

Hemlock woolly adelgid, Adelges tsugae, is an invasive pest of hemlock trees (Tsuga) in eastern North America. We used 14 microsatellites and mitochondrial COI sequences to assess its worldwide genetic structure and reconstruct its colonization history. The resulting information about its life cycle, biogeography and host specialization could help predict invasion by insect herbivores. We identified eight endemic lineages of hemlock adelgids in central China, western China, Ulleung Island (South Korea), western North America, and two each in Taiwan and Japan, with the Japanese lineages specializing on different Tsuga species. Adelgid life cycles varied at local and continental scales with different sexual, obligately asexual and facultatively asexual lineages. Adelgids in western North America exhibited very high microsatellite heterozygosity, which suggests ancient asexuality. The earliest lineages diverged in Asia during Pleistocene glacial periods, as estimated using approximate Bayesian computation. Colonization of western North America was estimated to have occurred prior to the last glacial period by adelgids directly ancestral to those in southern Japan, perhaps carried by birds. The modern invasion from southern Japan to eastern North America caused an extreme genetic bottleneck with just two closely related clones detected throughout the introduced range. Both colonization events to North America involved host shifts to unrelated hemlock species. These results suggest that genetic diversity, host specialization and host phylogeny are not predictive of adelgid invasion. Monitoring non-native sentinel host trees and focusing on invasion pathways might be more effective methods of preventing invasion than making predictions using species traits or evolutionary history. USDA NIFA Award [2007-35302-18097]; US Forest Service Northern and Southern Research Stations; Yale Institute for Biospheric Studies This project was supported by USDA NIFA Award 2007-35302-18097, the US Forest Service Northern and Southern Research Stations and the Yale Institute for Biospheric Studies. Andrea Gloria-Soria, Talbot Trotter and Sandy Liebhold provided comments on an early draft. We are grateful to Eric Maw who helped with laboratory work, to Darcy Nelson, with US Forest Service International Programs, who facilitated collecting logistics, and to our many colleagues who collected samples for this project. Public domain – authored by a U.S. government employee

Although biological invasions are of considerable concern to ecologists, relatively little attention has been paid to the potential for and consequences of indirect interactions between invasive species. Such interactions are generally thought to enhance invasives' spread and impact (i.e., the "invasional meltdown" hypothesis); however, exotic species might also act indirectly to slow the spread or blunt the impact of other invasives. On the east coast of the United States, the invasive hemlock woolly adelgid (Adelges tsugae, HWA) and elongate hemlock scale (Fiorinia externa, EHS) both feed on eastern hemlock (Tsuga canadensis). Of the two insects, HWA is considered far more damaging and disproportionately responsible for hemlock mortality. We describe research assessing the interaction between HWA and EHS, and the consequences of this interaction for eastern hemlock. We conducted an experiment in which uninfested hemlock branches were experimentally infested with herbivores in a 2 x 2 factorial design (either, both, or neither herbivore species). Over the 2.5-year course of the experiment, each herbivore's density was approximately 30% lower in mixed- vs. single-species treatments. Intriguingly, however, interspecific competition weakened rather than enhanced plant damage: growth was lower in the HWA-only treatment than in the HWA + EHS, EHS-only, or control treatments. Our results suggest that, for HWA-infested hemlocks, the benefit of co-occurring EHS infestations (reduced HWA density) may outweigh the cost (increased resource depletion).

Recent attention has focused on the harmful effects of introduced biological control agents on nontarget species. The parasitoid Compsilura concinnata is a notable example of such biological control gone wrong. Introduced in 1906 primarily for control of gypsy moth, Lymantria dispar, this tachinid fly now attacks more than 180 species of native Lepidoptera in North America. While it did not prevent outbreaks or spread of gypsy moth, we present reanalyzed historical data and experimental findings suggesting that parasitism by C. concinnata is the cause of the enigmatic near-extirpation of another of North America's most successful invaders, the browntail moth (Euproctis chrysorrhoea). From a range of approximately 160,000 km2 a century ago, browntail moth (BTM) populations currently exist only in two spatially restricted coastal enclaves, where they have persisted for decades. We experimentally established BTM populations within this area and found that they were largely free of mortality caused by C. concinnata. Experimental populations of BTM at inland sites outside of the currently occupied coastal enclaves were decimated by C. concinnata, a result consistent with our reanalysis of historical data on C. concinnata parasitism of the browntail moth. The role of C. concinnata in the disappearance of browntail moth outside these enclaves has not been reported before. Despite the beneficial role played by C. concinnata in reversing the browntail moth invasion, we do not advocate introduction of generalist biological control agents. Our findings illustrate that the impact of such organisms can be both unpredictable and far-reaching.

Winter moth, Operophtera brumata, originally from Europe, has invaded eastern Massachusetts causing widespread defoliation and damage to many deciduous tree species and a variety of crop plants in the infested area. We identified O. brumata nucleopolyhedrovirus (OpbuNPV) in winter moth larvae collected from field sites in Massachusetts by using PCR to amplify a 482 bp region of the baculovirus polyhedrin gene. Viral sequences were also detected in winter moth pupae that failed to emerge, suggesting that these insects may have died as a result of viral infection. This represents the first report of OpbuNPV in winter moth populations in the US.

Field and laboratory experiments were conducted to elucidate various factors influencing the temperature-dependent larval eclosion of winter moth, Operophtera brumata L, in New England. We found no difference in duration of the embryonic stage of eggs reared from larvae collected in Massachusetts (MA) and on Vancouver Island, British Columbia (BC), where winter temperatures are rarely below freezing. The number of growing degree days (GDD) required for larval eclosion declined with the number of days chilled in the laboratory and number of days below freezing in the field, confirming the findings of previous studies. Thus, eggs hatched with fewer GDD, when the spring came later than usual. Date of oviposition had no effect on date of hatch. Eggs laid by naturally occurring (feral) females hatched sooner, with lower GDD, than eggs from laboratory-reared females from MA and BC held on the same trees over the winter. South-facing eggs on the stems of trees hatched on average 1.6 days sooner than north-facing eggs. GDD calculated from bihourly measures of temperature were 15% greater than GDD estimates based on the average of daily maximum and minimum temperatures, as used by many GDD estimates made for online sources. Over two years, the mean GDD in °C for hatch of feral eggs was 176.53 ± 6.35 SE based on bihourly temperature measurements, a 1 January start date, and a 3.9°C developmental threshold. This value varied markedly, however, between the two years.

The hemlock woolly adelgid (Adelges tsugae Annand) is an invasive species causing high mortality of eastern hemlock (Tsuga canadensis (L) Carriere) in the forests of eastern North America. Recent findings revealed that sapling eastern hemlocks artificially infested with hemlock woolly adelgid in a plantation setting responded to the insect with an array of induced resin volatile changes. Here we determine if eastern hemlocks growing beneath a forest canopy respond to hemlock woolly adelgid infestation with the same patterns of constitutive and inducible volatile resin production as those plantation specimens. We inoculated previously uninfested branches of mature and immature hemlocks in a central New England forest with hemlock woolly adelgid. We then sampled twig tissue of infested and uninfested trees in late spring, early summer, and mid-autumn, after known intervals of adelgid activity when an induced response might be expected. We identified and quantified resin volatiles by gas chromatography-mass spectrometry. Constitutive levels of total monoterpenoids, sesquiterpenoids, and combined resin volatiles were all several-fold more abundant in forest trees than those previously measured in a plantation setting, pointing to further study of the influence of site factors on hemlock volatile production. Hemlock woolly adelgid infestation induced an array of changes in eastern hemlock's volatile profile, including many-fold increases in benzyl alcohol and methyl salicylate accumulation. Despite differences in constitutive concentrations of volatiles between the two sites, our findings verify that hemlock woolly adelgid elicits patterns of resin volatile induction in forest-grown eastern hemlocks quite similar to those previously observed in plantation grown trees.

As a measure of cold hardiness, we tested the supercooling points or freezing temperatures of individual hemlock woolly adelgids ( Adelges tsugae Annand) collected from 15 locations across the north to south range of the adelgid in eastern North America at different times during two winters. Adelgids from the northern interior locations with USDA hardiness zones of 5B–6B had lower supercooling points than adelgids from more southern or more coastal locations (zones 7A and 6B), where minimum winter temperatures were higher. Supercooling points reached a minimum in February in northern but not in southern locations. Laboratory experiments demonstrated that adelgids exposed to colder temperatures (−12 °C) had lower supercooling points after 3 d and adelgids held at 10 °C had higher supercooling points than did adelgids held at 2 °C for the same period. Extending these periods to 7 d produced no further change in supercooling points. Adelgids at northern sites had much lower supercooling points in February 2015 following at least 10 d of much colder weather than adelgids from those same sites in February 2016 following much warmer weather. The induction of cold hardiness produced much year-to-year variation in cold hardiness, especially in northern sites, in addition to concurrently and previously demonstrated genetic differences in cold hardiness between northern and southern adelgid populations. Consequently, the cold temperatures required to kill hemlock woolly adelgids will vary year to year and week to week based on exposure to prior temperatures.

Eastern hemlock (Tsuga canadensis) is in rapid decline because of infestation by the invasive hemlock woolly adelgid (Adelges tsugae; ‘HWA’) and, to a lesser extent, the invasive elongate hemlock scale (Fiorinia externa; ‘EHS’). For many conifers, induced oleoresin-based defenses play a central role in their response to herbivorous insects; however, it is unknown whether eastern hemlock mobilizes these inducible defenses. We conducted a study to determine if feeding by HWA or EHS induced changes in the volatile resin compounds of eastern hemlock. Young trees were experimentally infested for 3 years with HWA, EHS, or neither insect. Twig and needle resin volatiles were identified and quantified by gas chromatography/mass spectrometry. We observed a suite of changes in eastern hemlock’s volatile profile markedly different from the largely terpenoid-based defense response of similar conifers. Overall, both insects produced a similar effect: most twig volatiles decreased slightly, while most needle volatiles increased slightly. Only HWA feeding led to elevated levels of methyl salicylate, a signal for systemic acquired resistance in many plants, and benzyl alcohol, a strong antimicrobial and aphid deterrent. Green leaf volatiles, often induced in wounded plants, were increased by both insects, but more strongly by EHS. The array of phytochemical changes we observed may reflect manipulation of the tree’s biochemistry by HWA, or simply the absence of functional defenses against piercing-sucking insects due to the lack of evolutionary contact with these species. Our findings verify that HWA and EHS both induce changes in eastern hemlock’s resin chemistry, and represent the first important step toward understanding the effects of inducible chemical defenses on hemlock susceptibility to these exotic pests.

Range expansion by native and exotic species will continue to be a major component of global change. Anticipating the potential effects of changes in species distributions requires models capable of forecasting population spread across realistic, heterogeneous landscapes and subject to spatiotemporal variability in habitat suitability. Several decades of theory and model development, as well as increased computing power and availability of fine-resolution GIS data, now make such models possible. Still unanswered, however, is the question of how well this new generation of dynamic models will anticipate range expansion. Here we develop a spatially explicit stochastic model that combines dynamic dispersal and population processes with fine-resolution maps characterizing spatiotemporal heterogeneity in climate and habitat to model range expansion of the hemlock woolly adelgid (HWA; Adelges tsugae). We parameterize this model using multiyear data sets describing population and dispersal dynamics of HWA and apply it to eastern North America over a 57-year period (1951-2008). To evaluate the model, the observed pattern of spread of HWA during this same period was compared to model predictions. Our model predicts considerable heterogeneity in the risk of HWA invasion across space and through time, and it suggests that spatiotemporal variation in winter temperature, rather than hemlock abundance, exerts a primary control on the spread of HWA. Although the simulations generally matched the observed current extent of the invasion of HWA and patterns of anisotropic spread, it did not correctly predict when HWA was observed to arrive in different geographic regions. We attribute differences between the modeled and observed dynamics to an inability to capture the timing and direction of long-distance dispersal events that substantially affected the ensuing pattern of spread.

We tested different pheromone-baited traps for surveying winter moth, Operophtera brumata (L.) (Lepidoptera: Geometridae), populations in eastern North America. We compared male catch at Pherocon 1C sticky traps with various large capacity traps and showed that Universal Moth traps with white bottoms caught more winter moths than any other trap type. We ran the experiment on Cape Cod, MA, where we caught only winter moth, and in western Massachusetts, where we caught only Bruce spanworm, Operophtera bruceata (Hulst) (Lepidoptera: Geometridae), a congener of winter moth native to North America that uses the same pheromone compound [(Z,Z,Z)-1,3,6,9-nonadecatetraene] and is difficult to distinguish from adult male winter moths. With Bruce spanworm, the Pherocon 1C sticky traps caught by far the most moths. We tested an isomer of the pheromone [(E,Z,Z)-1,3,6,9-nonadecatetraene] that previous work had suggested would inhibit captures of Bruce spanworm but not winter moths. We found that the different doses and placements of the isomer suppressed captures of both species to a similar degree. We are thus doubtful that we can use the isomer to trap winter moths without also catching Bruce spanworm. Pheromone-baited survey traps will catch both species.

The study of ecological boundaries and their dynamics is of fundamental importance to much of ecology, biogeography, and evolution. Over the past two decades, boundary analysis (of which wombling is a subfield) has received considerable research attention, resulting in multiple approaches for the quantification of ecological boundaries. Nonetheless, few methods have been developed that can simultaneously (1) analyze spatially homogenized data sets (i.e., areal data in the form of polygons rather than point-reference data); (2) account for spatial structure in these data and uncertainty associated with them; and (3) objectively assign probabilities to boundaries once detected. Here we describe the application of a Bayesian hierarchical framework for boundary detection developed in public health, which addresses these issues but which has seen limited application in ecology. As examples, we analyze simulated spread data and the historic pattern of spread of an invasive species, the hemlock woolly adelgid (Adelges tsugae), using county-level summaries of the year of first reported infestation and several covariates potentially important to influencing the observed spread dynamics. Bayesian areal wombling is a promising approach for analyzing ecological boundaries and dynamics related to changes in the distributions of native and invasive species.

The winter moth, Operophtera brumata (Lepidoptera: Geometridae), is an early-season defoliator that attacks a wide variety of hardwoods and, in some cases, conifers. The insect is native to Europe but has become established in at least three areas of North America including southeastern New England. The female-produced sex attractant pheromone of the winter moth was identified as (3Z,6Z,9Z)-1,3,6,9-nonadecatetraene (1), which also attracts a native congener, the Bruce spanworm, Operophtera bruceata . Dissection, or (for certainty) DNA molecular testing, is required to differentiate between males of the two species. Thus, a trapping method that is selective for winter moth would be desirable. A geometric isomer of the pheromone, (3E,6Z,9Z)-1,3,6,9-nonadecatetraene (2), can reportedly inhibit attraction of Bruce spanworm to traps without affecting winter moth catch, but use of the pheromone and inhibitor together has not been optimized, nor has the synthesis of the inhibitor. This paper presents two new syntheses of the inhibitor (3E,6Z,9Z)-1,3,6,9-nonadecatetraene based on the intermediate (3Z,6Z)-3,6-hexadecadien-1-ol (4), which has also been utilized in the synthesis of the pheromone. The syntheses combine traditional acetylenic chemistry and Wittig olefination reactions. In one approach, 2 was synthesized in 80% purity (20% being pheromone 1), and in the second, tetraene 2 of 96% purity (and free of 1) was produced in 25% overall yield from dienol 4. The last method benefitted from a refined TEMPO-mediated PhI(OAc)(2) oxidation of 4 and a two-carbon homologation of the corresponding aldehyde 7.

Monitoring programs increasingly are used to document the spread of invasive species in the hope of detecting and eradicating low-density infestations before they become established. However, interobserver variation in the detection and correct identification of low-density populations of invasive species remains largely unexplored. In this study, we compare the abilities of volunteer and experienced individuals to detect low-density populations of an actively spreading invasive species, and we explore how interobserver variation can bias estimates of the proportion of sites infested derived from occupancy models that allow for both false negative and false positive (misclassification) errors. We found that experienced individuals detected small infestations at sites where volunteers failed to find infestations. However, occupancy models erroneously suggested that experienced observers had a higher probability of falsely detecting the species as present than did volunteers. This unexpected finding is an artifact of the modeling framework and results from a failure of volunteers to detect low-density infestations rather than from false positive errors by experienced observers. Our findings reveal a potential issue with site occupancy models that can arise when volunteer and experienced observers are used together in surveys.

Scant attention has been paid to invasive species whose range and abundance has decreased after an initial range expansion. One such species is the browntail moth Euproctis chrysorrhoea L, which was discovered in the eastern United States in 1897. Its range expanded until 1914; after 1915, however, its range contracted and now it persists in only two isolated coastal locations. Although a biological control agent has been implicated in this range collapse, cold inland winter temperatures may also help to restrict browntail moth populations. We surveyed coastal versus inland habitats in Maine and Massachusetts for browntail moth overwintering mortality and larval density per web. We also performed an experiment assessing these same variables in coastal versus inland habitats on different host plant species and at different initial larval densities. We also analyzed temperature records to assess whether winter temperatures correlated with changes in the invasive range. Overwintering mortality was lower in coastal populations for both the experimental populations and in the Maine field survey. Experimental populations in Cape Cod coastal areas also had lower rates of fall mortality and higher larval densities, suggesting that coastal areas are better year-round habitats than inland areas. There were no consistent differences between coastal and inland populations in their response to larval density or host plant, although overall survival in both areas was higher at low initial larval densities and affected by host identity. There was also no difference in two measures of the coldest winter temperatures during browntail moth's expansion and contraction. Our results show that climate affects browntail moth, but suggest that winter temperatures cannot explain both the rapid expansion and subsequent collapse of this pest.

In a laboratory study, we tested the feeding preferences of three coccinellid predators of hemlock woolly adelgid, Adelges tsugae Annand, an introduced pest of hemlock in the eastern United States. The species tested were Sasajiscymnus tsugae Sasaji & McClure (formerly Pseudoscymnus tsugae) from Japan, Scymnus ningshanensis Yu & Yao from China, and Harmonia axyridis (Pallas), a generalist species introduced from Asia that is currently widespread in eastern hemlock, Tsuga canadensis Carriere, forests. We measured the feeding preference of each beetle species when given the choice of A. tsugae and either 1) Pineus strobi (Hartig) on Pinus strobus L.; 2) Adelges laricis Vallot on Larix decidua Mill.; 3) Adelges cooleyi (Gillette) on Pseudotsuga menziesii (Mirb.) Franco; or 3) Paraprociphilus tessellatus (Fitch) on Alnus serrulata (Ait.) Willd. We evaluated beetle preference for adults, nymphs, and eggs of each prey species. Generally, when adult or nymphal prey stages were compared, S. tsugae preferred A. tsugae adults to P. strobi, A. cooleyi, A. laricis, and P. tessellatus. S. ningshanensis showed less preference between adelgid species, but it did not prefer P. tessellatus nymphs. When preferences for adelgid eggs were assayed, S. tsugae and S. ningshanensis showed no preference between A. tsugae and A. cooleyi or P. strobi, but S. tsugae did prefer A. tsugae to A. laricis. Larvae of S. tsugae were unable to survive on P. tessellatus nymphs. H. axyridis adults readily consumed both A. tsugae and P. tessellatus, but H. axyridis larvae did not complete their life cycle on A. tsugae. Our host range tests suggest that S. ningshanensis and S. tsugae may feed on several species of Adelgidae and that A. tsugae is often preferred.

Invertebrate pathologists have multiple definitions for the terms pathogenicity and virulence, and these definitions vary across disciplines that focus on host-pathogen interactions. We surveyed various literatures, including plant pathology, invertebrate pathology, evolutionary biology, and medicine, and found most define pathogenicity as the broader term, which incorporates virulence. Virulence is seen as the severity of disease manifestation that can only be measured in infected individuals. These definitions readily apply to both lethal and non-lethal diseases. Invertebrate pathologists commonly use dose-response bioassays to estimate LD(50) or LC(50) (dose or concentration needed to kill 50% of hosts exposed). These bioassays measure pathogenicity if the bioassay includes a transmission component, and measure virulence if the bioassay is measured in infected individuals only. Another common bioassay estimate is LT(50) (median time to death of infected hosts), which is a measure of virulence as long as survivors are not included in its calculation.

We used the nuclear polyhedrosis virus of the gypsy moth, Lymantria dispar, to investigate whether the timing of transmission influences the evolution of virulence. In theory, early transmission should favour rapid replication and increase virulence, while late transmission should favour slower replication and reduce virulence. We tested this prediction by subjecting one set of 10 virus lineages to early transmission (Early viruses) and another set to late transmission (Late viruses). Each lineage of virus underwent nine cycles of transmission. Virulence assays on these lineages indicated that viruses transmitted early were significantly more lethal than those transmitted late. Increased exploitation of the host appears to come at a cost, however. While Early viruses initially produced more progeny, Late viruses were ultimately more productive over the entire duration of the infection. These results illustrate fitness trade-offs associated with the evolution of virulence and indicate that milder viruses can obtain a numerical advantage when mild and harmful strains tend to infect separate hosts.

The gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus was genetically engineered for nonpersistence by removal of the gene coding for polyhedrin production and stabilized using a coocclusion process. A beta-galactosidase marker gene was inserted into the genetically engineered virus (LdGEV) so that infected larvae could be tested for its presence using a colorimetric assay. In 1993, LdGEV-infected gypsy moths were released in a forested plot in Massachusetts to test for spread and persistence. A similar forested plot 2 km away served as a control. For 3 years (1993-1995), gypsy moths were established in the two plots in Massachusetts to serve as test and control populations. Each week, larvae were collected from both plots. These field-collected larvae were reared individually, checked for mortality, and then tested for the presence of beta-galactosidase. Other gypsy moth larvae were confined on LdGEV-contaminated foliage for 1 week and then treated as the field-collected larvae. The LdGEV was sought in bark, litter, and soil samples collected from each plot. To verify the presence of the LdGEV, polymerase chain reaction, slot blot DNA hybridization, and restriction enzyme analysis were also used on larval samples. Field-collected larvae infected with the engineered virus were recovered in the release plot in 1993, but not in subsequent years; no field-collected larvae from the control plot contained the engineered virus. Larvae confined on LdGEV-contaminated foliage were killed by the virus. No LdGEV was recovered from bark, litter, or soil samples from either of the plots.

Polymerase chain reaction (PCR) technology was employed to detect baculovirus DNA sequences from viral occlusion bodies (OB) contaminating the surface of gypsy moth eggs. The level of sensitivity of the technique was as low as 5 viral genome copies and DNA from 1 OB equivalent. Thirty minutes of shortwave UV irradiation of eggs contaminated with 8.4 × 10 4 OBs prevented amplification of viral DNA sequences from OBs on the egg surface. These methods are important for providing a better understanding of gypsy moth virus epizootiology as well as for the examination of insect eggs for the persistence of baculovirus gene sequences inside the egg or on the egg surface. In addition, these methods can be easily modified for monitoring the persistence of genetically engineered baculoviruses in insect populations as well as the fate of genes that these viruses might carry.

DNA hybridization assays were used to detect the presence of viral DNA in gypsy moth (Lymantria dispar L.) larvae collected weekly from high density populations or reared from field-collected egg masses. DNA was extracted from larvae, bound to nitrocellulose filters, and hybridized with digoxigenin-labeled L. dispar NPV (LdNPV) DNA probes. The virus incidence determined from DNA hybridization assays was compared with that determined with conventional microscopic examination of larvae for polyhedral inclusion bodies. Among neonates reared from field-collected egg masses, average mortality from LdNPV (15.4%) within 10 d after hatch was not significantly different from the percentage of extracts containing LdNPV DNA (14.8%) found among larvae frozen 5 d after hatch before any mortality occurred. Field-collected larvae were split into two groups: half were frozen immediately and probed for LdNPV DNA and the other half were reared on artificial diet. The proportion containing LdNPV DNA closely approximated the proportion that died within 6 d of collection, but the proportion that died within 13 d of collection was underestimated.