Environmental DNA assays for Laricobius beetles (Coleoptera: Derodontidae), biocontrol agents of the hemlock woolly adelgid in North America.

The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), is an invasive pest causing significant ecological and economic damage to certain hemlock tree (Tsuga (Endlicher) Carrière, Pinales:Pinaceae) species. In response to this invasive threat, biological control strategies have been implemented, introducing natural predators such as Laricobius nigrinus Fender (Coleoptera: Derodontidae) and, more recently, Laricobius osakensis Montgomery and Shiyake (Coleoptera: Derodontidae), as specialist predators against A. tsugae. However, the genetic and morphological similarities between L. osakensis and both L. nigrinus and the native beetle, Laricobius rubidus LeConte (Coleoptera: Derodontidae), pose challenges in their identification. Effective monitoring of released predators is integral to evaluating the success of biological control measures. Environmental DNA (eDNA) holds potential for various detection applications, including species monitoring. In this study, we developed specific primers and probes targeting the mitochondrial cytochrome oxidase 1 gene sequences, achieving high specificity despite their 95% sequence similarity. With an optimal annealing temperature of 60 °C, our tools effectively differentiated L. osakensis from the other 2 beetles and demonstrated eDNA detection sensitivity down to 2 copies/µl. This research underscores the potential of precise molecular tools for advancing biological control and biodiversity assessment against invasive threats like A. tsugae.

Observation of key phenological stages of hemlock woolly adelgid (Hemiptera: Adelgidae): using citizen science as a tool to inform research and management.

Increasing efficiency of data gathering at the landscape scale on the growing number of pests and pathogens threatening forests worldwide has potential to improve management outcomes. Citizen science is expanding, with growing support and utility in environmental and conservation fields. We present a case study showing how citizen science observations can be used to inform research and management of a devastating forest pest. Hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), was introduced to eastern North America, leading to decline and mortality of eastern [Tsuga canadensis (L.) Carrière] (Pinales: Pinaceae) and Carolina hemlock (Tsuga caroliniana Engelmann) trees. Management activities, most notably biological control, rely on observations of A. tsugae phenology to inform the timing of releases and monitoring surveys of their highly synchronized specialist predators. In this article, we outline a citizen science program and report phenological observations on A. tsugae. Additionally, we report data comparing A. tsugae estivation break in Virginia (VA) and New York (NY) State, revealing that estivation break is synchronized between NY and VA. This observation is supported by 6 years of citizen scientist observations, showing similar patterns throughout NY, with egg laying shown to be much more variable than estivation break.

Hymenopteran Parasitoids of Leucotaraxis argenticollis (Diptera: Chamaemyiidae) and Leucotaraxis piniperda: Implications for Biological Control of Hemlock Woolly Adelgid (Hemiptera: Adelgidae).

The hemlock woolly adelgid (Adelges tsugae (Annand)) is a serious invasive pest of hemlock trees in eastern North America. Multiple biological control agents have been the focus of research aimed at pest management and conserving hemlock communities. Three promising A. tsugae specialist predators are the beetle Laricobius nigrinus (Fender) (Coleoptera: Derodontidae) and flies in the genus Leucotaraxis (Diptera: Chamaemyiidae), Leucotaraxis argenticollis (Zetterstedt), and Leucotaraxis piniperda (Malloch). However, these flies are vulnerable to parasitism by wasps in the genera Pachyneuron (Walker) (Hymenoptera: Pteromalidae) and Melanips (Walker) (Hymenoptera: Figitidae). This study explores parasitoid wasp interactions with these Leucotaraxis species in their native western North American range and potential impacts on the biological control program in the East. Leucotaraxis, La. nigrinus, and parasitoid emergences were observed from adelgid-infested foliage collected from Washington State and British Columbia in 2018, 2019, and 2020. Undescribed species of Pachyneuron and Melanips emerged from puparia as solitary parasitoids. Parasitoid emergence was positively correlated with Leucotaraxis emergence. Percent parasitism increased between February and July, with the months of June and July experiencing higher parasitoid emergence than Leucotaraxis. Differences in emergence patterns suggest that Pachyneuron may be more closely associated with Le. argenticollis as a host, and that Melanips may be associated with Le. piniperda. High parasitism in Leucotaraxis had no effect on La. nigrinus larval abundance, whereas the combined emergence of parasitoids and Leucotaraxis was positively correlated with La. nigrinus. This suggests that there is limited competition among these predators.

Biological Control of Hemlock Woolly Adelgid: Implications of Adult Emergence Patterns of Two Leucopis spp. (Diptera: Chamaemyiidae) and Laricobius nigrinus (Coleoptera: Derodontidae) Larval Drop.

The hemlock woolly adelgid (Hemiptera: Adelgidae Adelges tsugae Annand) poses a serious threat to hemlocks in eastern North America, and ongoing research is focused on the identification and development of biological controls to protect and manage hemlock resources. Three predators native to the Pacific Northwest of North America that have been the focus of much research are Leucopis argenticollis (Zetterstedt), Leucopis piniperda (Malloch) (Diptera: Chamaemyiidae), and Laricobius nigrinus (Fender) (Coleoptera: Derodontidae). This study addresses the knowledge gap of adult Leucopis spp. emergence patterns, with comparisons to the timing of larval La. nigrinus drop for pupation. Adult Leucopis spp. emergence was observed in the lab from field-collected, adelgid-infested foliage from Washington state in 2019 and 2020. Adult Leucopis spp. were collected daily as they emerged from foliage collections and identified to species using morphological features; a subset was validated using DNA barcoding. Accumulated heating degree days were calculated to compare a standardized emergence timing across collections made at different locations and temperature regimes. The abundance of the two Leucopis spp. and of the combined Leucopis spp. and La. nigrinus varied among sites and years, and no species was consistently more abundant than the other. Evaluations of seasonal emergence trends of the three species determine the predator complex behaves in a temporally stratified and predictable way. Emergence of adult Le. argenticollis was observed first, followed by La. nigrinus larval drop, with Le. piniperda emerging at the end of larval drop, and finally a second emergence of Le. argenticollis.

Relating Aerial Deposition of Entomophaga maimaiga Conidia (Zoopagomycota: Entomophthorales) to Mortality of Gypsy Moth (Lepidoptera: Erebidae) Larvae and Nearby Defoliation.

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.

Phytophagous larvae occurring in Central and Southeastern European oak forests as a potential host of Entomophaga maimaiga (Entomophthorales: Entomophthoraceae) - A field study.

We evaluated the presence and impact of Entomophaga maimaiga on both target and non-target phytophagous larvae. All six study plots, with low gypsy moth population density, were situated in Central and Southeastern European oak forests and E. maimaiga had previously been reported from these plots. Totally, 45 of 4,045 (1.13%) collected non-target larvae died due to fungal infections. No non-target insect specimen was infected by E.maimaiga, although the presence of the pathogen could not be fully excluded in three cadavers. Out of 1,780L.dispar larvae collected, 15individuals (0.84%) were infected by E.maimaiga.

Symbiont Spillover from Invasive to Native Woodwasps.

Hosts and their associated microbes are being increasingly introduced around the world, which can lead to novel host/microbe associations via new sympatries. Woodwasps (Hymenoptera: Siricidae) are able to utilize wood for its nutrients due to obligate mutualistic associations with white rot fungi in the genus Amylostereum and when invasive woodwasps are introduced to new areas, their symbionts accompany them. There is increasing evidence that woodwasp-fungus associations previously believed to be highly specific are actually flexible. We show that in North America, both Urocerus albicornis and Urocerus cressoni, which develop in trees in the Pinaceae, usually use Amylostereum chailletii but sometimes carry an Amylostereum areolatum strain putatively introduced to North America by the invasive woodwasp Sirex noctilio. Symbiont spillover from invasive to native hosts is a source of new host/introduced symbiont associations that could result in changes in microbes and host fitness with the potential to impact communities.

Modification of a Pollen Trap Design To Capture Airborne Conidia of Entomophaga maimaiga and Detection of Conidia by Quantitative PCR.

The goal of this study was to develop effective and practical field sampling methods for quantification of aerial deposition of airborne conidia of Entomophaga maimaiga over space and time. This important fungal pathogen is a major cause of larval death in invasive gypsy moth (Lymantria dispar) populations in the United States. Airborne conidia of this pathogen are relatively large (similar in size to pollen), with unusual characteristics, and require specialized methods for collection and quantification. Initially, dry sampling (settling of spores from the air onto a dry surface) was used to confirm the detectability of E. maimaiga at field sites with L. dispar deaths caused by E. maimaiga, using quantitative PCR (qPCR) methods. We then measured the signal degradation of conidial DNA on dry surfaces under field conditions, ultimately rejecting dry sampling as a reliable method due to rapid DNA degradation. We modified a chamber-style trap commonly used in palynology to capture settling spores in buffer. We tested this wet-trapping method in a large-scale (137-km) spore-trapping survey across gypsy moth outbreak regions in Pennsylvania undergoing epizootics, in the summer of 2016. Using 4-day collection periods during the period of late instar and pupal development, we detected variable amounts of target DNA settling from the air. The amounts declined over the season and with distance from the nearest defoliated area, indicating airborne spore dispersal from outbreak areas.IMPORTANCE We report on a method for trapping and quantifying airborne spores of Entomophaga maimaiga, an important fungal pathogen affecting gypsy moth (Lymantria dispar) populations. This method can be used to track dispersal of E. maimaiga from epizootic areas and ultimately to provide critical understanding of the spatial dynamics of gypsy moth-pathogen interactions.