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Background: Flight can drastically enhance dispersal capacity and is a key trait defining the potential of exotic insect species to spread and invade new habitats. The phytophagous European spongy moths (ESM, Lymantria dispar dispar) and Asian spongy moths (ASM; a multi-species group represented here by L. d. asiatica and L. d. japonica), are globally invasive species that vary in adult female flight capability-female ASM are typically flight capable, whereas female ESM are typically flightless. Genetic markers of flight capability would supply a powerful tool for flight profiling of these species at any intercepted life stage. To assess the functional complexity of spongy moth flight and to identify potential markers of flight capability, we used multiple genetic approaches aimed at capturing complementary signals of putative flight-relevant genetic divergence between ESM and ASM: reduced representation genome-wide association studies, whole genome sequence comparisons, and developmental transcriptomics. We then judged the candidacy of flight-associated genes through functional analyses aimed at addressing the proximate demands of flight and salient features of the ecological context of spongy moth flight evolution., Results: Candidate gene sets were typically non-overlapping across different genetic approaches, with only nine gene annotations shared between any pair of approaches. We detected an array of flight-relevant functional themes across gene sets that collectively suggest divergence in flight capability between European and Asian spongy moth lineages has coincided with evolutionary differentiation in multiple aspects of flight development, execution, and surrounding life history. Overall, our results indicate that spongy moth flight evolution has shaped or been influenced by a large and functionally broad network of traits., Conclusions: Our study identified a suite of flight-associated genes in spongy moths suited to exploration of the genetic architecture and evolution of flight, or validation for flight profiling purposes. This work illustrates how complementary genetic approaches combined with phenotypically targeted functional analyses can help to characterize genetically complex traits., (© 2024. His Majesty the King in Right of Canada as represented by the Minister of Natural Resources and Julien Prunier, Richard Hamelin, Francois Oliver Hebert, Roger C. Levesque, Ilga Porth. Parts of this work were authored by US Federal Government authors and are not under copyright protection in the US; foreign copyright protection may apply.)

The adelgids (Adelgidae) are a small family of sap-feeding insects, which, together with true aphids (Aphididae) and phylloxerans (Phylloxeridae), make up the infraorder Aphidomorpha. Some adelgid species are highly destructive to forest ecosystems such as Adelges tsugae, Adelges piceae, Adelges laricis, Pineus pini, and Pineus boerneri. Despite this, there are no high-quality genomic resources for adelgids, hindering advanced genomic analyses within Adelgidae and among Aphidomorpha. Here, we used PacBio continuous long-read and Illumina RNA-sequencing to construct a high-quality draft genome assembly for the Cooley spruce gall adelgid, Adelges cooleyi (Gillette), a gall-forming species endemic to North America. The assembled genome is 270.2 Mb in total size and has scaffold and contig N50 statistics of 14.87 and 7.18 Mb, respectively. There are 24,967 predicted coding sequences, and the assembly completeness is estimated at 98.1 and 99.6% with core BUSCO gene sets of Arthropoda and Hemiptera, respectively. Phylogenomic analysis using the A. cooleyi genome, 3 publicly available adelgid transcriptomes, 4 phylloxera transcriptomes, the Daktulosphaira vitifoliae (grape phylloxera) genome, 4 aphid genomes, and 2 outgroup coccoid genomes fully resolves adelgids and phylloxerans as sister taxa. The mitochondrial genome is 24 kb, among the largest in insects sampled to date, with 39.4% composed of noncoding regions. This genome assembly is currently the only genome-scale, annotated assembly for adelgids and will be a valuable resource for understanding the ecology and evolution of Aphidomorpha., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Theodor Hartigs aphid and scale insect type specimens have been presumed lost or destroyed for the last 140 years. Here we document their discovery at the Bavarian State Collection of Zoology (Zoologische Staatssammlung Mnchen, ZSM), in Munich, Germany. These specimens include primary types for 24 aphid, three adelgid, and two armored scale insect species named by Hartig between 1834 and 1851, as well as other specimens of unknown importance.

The European winter moth, Operophtera brumata L. (Lepidoptera: Geometridae), was accidentally introduced to North America on at least 4 separate occasions, where it has been hybridizing with the native Bruce spanworm, O. bruceata Hulst, at rates up to 10% per year. Both species are known to respond to the same sex pheromones and to produce viable offspring, but whether they differ in the seasonal timing of their mating flights is unknown. Therefore, we collected adult male moths weekly along 2 transects in the northeastern United States and genotyped individuals using polymorphic microsatellite markers as males of these 2 species cannot be differentiated morphologically. Along each transect, we then estimated the cumulative proportions (i.e., the number of individuals out of the total collected) of each species on each calendar day. Our results indicate that there are significant differences between the species regarding their seasonal timing of flight, and these allochronic differences likely are acting to promote reproductive isolation between these 2 species. Lastly, our results suggest that the later flight observed by winter moth compared to Bruce spanworm may be limiting its inland spread in the northeastern United States because of increased exposure to extreme winter events., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Some introduced species cause severe damage, although the majority have little impact. Robust predictions of which species are most likely to cause substantial impacts could focus efforts to mitigate those impacts or prevent certain invasions entirely. Introduced herbivorous insects can reduce crop yield, fundamentally alter natural and managed forest ecosystems, and are unique among invasive species in that they require certain host plants to succeed. Recent studies have demonstrated that understanding the evolutionary history of introduced herbivores and their host plants can provide robust predictions of impact. Specifically, divergence times between hosts in the native and introduced ranges of a nonnative insect can be used to predict the potential impact of the insect should it establish in a novel ecosystem. However, divergence time estimates vary among published phylogenetic datasets, making it crucial to understand if and how the choice of phylogeny affects prediction of impact. Here, we tested the robustness of impact prediction to variation in host phylogeny by using insects that feed on conifers and predicting the likelihood of high impact using four different published phylogenies. Our analyses ranked 62 insects that are not established in North America and 47 North American conifer species according to overall risk and vulnerability, respectively. We found that results were robust to the choice of phylogeny. Although published vascular plant phylogenies continue to be refined, our analysis indicates that those differences are not substantial enough to alter the predictions of invader impact. Our results can assist in focusing biosecurity programs for conifer pests and can be more generally applied to nonnative insects and their potential hosts by prioritizing surveillance for those insects most likely to be damaging invaders., (© 2022 The Ecological Society of America. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

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., (© 2022 John Wiley & Sons Ltd.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Many plant-sap-feeding insects have maintained a single, obligate, nutritional symbiont over the long history of their lineage. This senior symbiont may be joined by one or more junior symbionts that compensate for gaps in function incurred through genome-degradative forces. Adelgids are sap-sucking insects that feed solely on conifer trees and follow complex life cycles in which the diet fluctuates in nutrient levels. Adelgids are unusual in that both senior and junior symbionts appear to have been replaced repeatedly over their evolutionary history. Genomes can provide clues to understanding symbiont replacements, but only the dual symbionts of hemlock adelgids have been examined thus far. Here, we sequence and compare genomes of four additional dual-symbiont pairs in adelgids. We show that these symbionts are nutritional partners originating from diverse bacterial lineages and exhibiting wide variation in general genome characteristics. Although dual symbionts cooperate to produce nutrients, the balance of contributions varies widely across pairs, and total genome contents reflect a range of ages and degrees of degradation. Most symbionts appear to be in transitional states of genome reduction. Our findings support a hypothesis of periodic symbiont turnover driven by fluctuating selection for nutritional provisioning related to gains and losses of complex life cycles in their hosts., (© 2021. The Author(s).)

A new genus of Chamaemyiidae (Diptera: Lauxanioidea) is described, namely Leucotaraxis gen. nov. (type species Leucopis atrifacies Aldrich; other included species Leucotaraxis argenticollis (Zetterstedt), comb. nov., Leucotaraxis piniperda (Malloch), comb. nov., and Leucotaraxis sepiola sp. nov.). These species are predators of Adelgidae (Hemiptera) infesting Pinaceae. Leucotaraxis argenticollis is Holarctic, while the other three species are Nearctic. The phylogeny of Leucotaraxis with other representatives of Chamaemyiidae was elucidated using mitochondrial and nuclear DNA sequences and the genus was found to be monophyletic. Egg and puparial stages are discussed or described and illustrated for all species except Leucotaraxis sepiola. A key is provided to all species of Chamaemyiidae known to attack Pinaceae-infesting Sternorrhyncha, an annotated list of these taxa is provided, and a habitus photograph is provided for each genus with such species. In addition, a lectotype is designated for Leucopis olivacea Meijere, and it is synonymized under Neoleucopis obscura (Haliday), syn. nov.

The southern pine beetle , Dendroctonus frontalis , is a native pest of pine trees that has recently expanded its range into the northeastern United States. Understanding its colonization, dispersal, and connectivity will be critical for mitigating negative economic and ecological impacts in the newly invaded areas. Characterization of spatial-genetic structure can contribute to this; however, previous studies have reached different conclusions about regional population genetic structure, with one study reporting a weak east-west pattern, and the most recent reporting an absence of structure. Here we systematically assessed several explanations for the absence of spatial-genetic structure. To do this, we developed nine new microsatellite markers and combined them with an existing 24-locus data matrix for the same individuals. We then reanalyzed this full dataset alongside datasets in which certain loci were omitted with the goal of creating more favorable signal to noise ratios. We also partitioned the data based on the sex of D. frontalis individuals, and then employed a broad suite of genotypic clustering and isolation-by-distance (IBD) analyses. We found that neither inadequate information content in the molecular marker set, nor unfavorable signal-to-noise ratio, nor insensitivity of the analytical approaches could explain the absence of structure. Regardless of dataset composition, there was little evidence for clusters ( i.e ., distinct geo-genetic groups) or clines ( i.e. , gradients of increasing allele frequency differences over larger geographic distances), with one exception: significant IBD was repeatedly detected using an individual-based measure of relatedness whenever datasets included males (but not for female-only datasets). This is strongly indicative of broad-scale female-biased dispersal, which has not previously been reported for D. frontalis , in part owing to logistical limitations of direct approaches (e.g., capture-mark-recapture). Weak spatial-genetic structure suggests long-distance connectivity and that gene flow is high, but additional research is needed to understand range expansion and outbreak dynamics in this species using alternate approaches., Competing Interests: The authors declare there are no competing interests., (©2021 Garrick et al.)

Reconstructing the geographic origins of non-native species is important for studying the factors that influence invasion success, however; these analyses can be constrained by the amount of diversity present in the native and invaded regions, 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 Nova Scotia, British Columbia, Oregon, and the northeastern United States. It is not known whether these represent independent introductions to North America, or a "stepping stone" spread among regions. Using a combination of Bayesian assignment and approximate Bayesian computation methods, we analysed a population genetic data set of 24 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 probably being introduced from France and Sweden, respectively; the Oregonian population probably being introduced from either the British Isles or northern Fennoscandia; and the population in the northeastern United States probably being introduced from somewhere in Central Europe. We discuss the impact of genetic bottlenecks on analyses meant to determine region of origin., (© 2021 John Wiley & Sons Ltd.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Two species of silver fly, Leucopis argenticollis (Zetterstedt) and Leucopis piniperda (Malloch) (Diptera: Chamaemyiidae), from the Pacific Northwest region of North America have been identified as potential biological control agents of hemlock woolly adelgid (Hemiptera: Adelgidae: Adelges tsugae Annand) in eastern North America. The two predators are collectively synchronized with A. tsugae development. To determine whether adult emergence of the two species of silver fly are also synchronized with one another, we collected adult Leucopis which emerged from A. tsugae-infested western hemlock [Pinaceae: Tsuga heterophylla (Raf.) Sarg.] from four sites in the Pacific Northwest over a 29-d period. Specimens were collected twice daily in the laboratory and identified to species using DNA barcoding. The study found that more adult Leucopis were collected in the evening than the morning. Additionally, the daily emergences of adults over the 29-d sampling period exhibited sinusoidal-like fluctuations of peak abundance of each species, lending evidence to a pattern of temporal partitioning. This pattern could have logistical implications for their use as biological control agents in eastern North America, namely the need to release both species for maximum efficacy in decreasing A. tsugae populations., (Published by Oxford University Press on behalf of Entomological Society of America 2020.)

The hemlock woolly adelgid (Hemiptera: Adelgidae: Adelges tsugae Annand) is an invasive insect, introduced from Japan to eastern North America, where it causes decline and death of hemlock trees. There is a closely related lineage of A. tsugae native to western North America. To inform classical biological control of A. tsugae in the eastern USA, the density and phenology of three native western adelgid specialist predators, Leucopis argenticollis (Zetterstedt), Le. piniperda (Malloch) (Diptera: Chamaemyiidae), and Laricobius nigrinus Fender (Coleoptera: Derodontidae), were quantified in the Pacific Northwest. Infested branches were collected from western hemlock (Pinaceae: Tsuga heterophylla (Raf.) Sarg.) at four sites around the Puget Sound, Washington and three sites in Oregon. Immature Leucopis were identified to species using DNA barcodes. Leucopis argenticollis was roughly twice as abundant as Le. piniperda. Laricobius nigrinus larvae were more abundant than the two species of Leucopis during the egg stage of the first adelgid generation, but Leucopis were present as feeding larvae during the second adelgid generation when La. nigrinus was aestivating in the soil, resulting in Leucopis being more abundant than La. nigrinus across the entire sampling period. Adelges tsugae and La. nigrinus densities were not correlated, while A. tsugae and Leucopis spp. densities were positively correlated. Leucopis spp. and La. nigrinus densities were negatively correlated. These results support the complementary use of La. nigrinus and the two Leucopis species for biological control of A. tsugae in the eastern USA, and point to the need for further investigation of spatial and temporal niche partitioning among the three predator species.

Numerous studies have shown that the genetic diversity of species inhabiting temperate regions has been shaped by changes in their distributions during the Quaternary climatic oscillations. For some species, the genetic distinctness of isolated populations is maintained during secondary contact, while for others, admixture is frequently observed. For the winter moth ( Operophtera brumata ), an important defoliator of oak forests across Europe and northern Africa, we previously determined that contemporary populations correspond to genetic diversity obtained during the last glacial maximum (LGM) through the use of refugia in the Iberian and Aegean peninsulas, and to a lesser extent the Caucasus region. Missing from this sampling were populations from the Italian peninsula and from North Africa, both regions known to have played important roles as glacial refugia for other species. Therefore, we genotyped field-collected winter moth individuals from southern Italy and northwestern Tunisia-the latter a region where severe oak forest defoliation by winter moth has recently been reported-using polymorphic microsatellite. We reconstructed the genetic relationships of these populations in comparison to moths previously sampled from the Iberian and Aegean peninsulas, the Caucasus region, and western Europe using genetic distance, Bayesian clustering, and approximate Bayesian computation (ABC) methods. Our results indicate that both the southern Italian and the Tunisian populations are genetically distinct from other sampled populations, and likely originated in their respective refugium during the LGM after diverging from a population that eventually settled in the Iberian refugium. These suggest that winter moth populations persisted in at least five Mediterranean LGM refugia. Finally, we comment that outbreaks by winter moth in northwestern Tunisia are not the result of a recent introduction of a nonnative species, but rather are most likely due to land use or environmental changes., Competing Interests: None declared., (© 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

A long-standing goal of invasion biology is to identify factors driving highly variable impacts of non-native species. Although hypotheses exist that emphasize the role of evolutionary history (e.g., enemy release hypothesis & defense-free space hypothesis), predicting the impact of non-native herbivorous insects has eluded scientists for over a century.Using a census of all 58 non-native conifer-specialist insects in North America, we quantified the contribution of over 25 factors that could affect the impact they have on their novel hosts, including insect traits (fecundity, voltinism, native range, etc.), host traits (shade tolerance, growth rate, wood density, etc.), and evolutionary relationships (between native and novel hosts and insects).We discovered that divergence times between native and novel hosts, the shade and drought tolerance of the novel host, and the presence of a coevolved congener on a shared host, were more predictive of impact than the traits of the invading insect. These factors built upon each other to strengthen our ability to predict the risk of a non-native insect becoming invasive. This research is the first to empirically support historically assumed hypotheses about the importance of evolutionary history as a major driver of impact of non-native herbivorous insects.Our novel, integrated model predicts whether a non-native insect not yet present in North America will have a one in 6.5 to a one in 2,858 chance of causing widespread mortality of a conifer species if established ( R 2  = 0.91) Synthesis and applications . With this advancement, the risk to other conifer host species and regions can be assessed, and regulatory and pest management efforts can be more efficiently prioritized., Competing Interests: The authors declare no conflict of interest., (© 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

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 Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Bacterial endosymbionts of sap-sucking insects provide their host with a number of beneficial qualities, including the supply of nutrition, defense against parasitoids, and protection from heat stress. Damage to these bacterial associates can therefore have a negative impact on the fitness of their insect host. We evaluated observational and experimental factors regarding the nonnative hemlock woolly adelgid (Adelges tsugae Annand) (Hemiptera: Adelgidae) to help understand the roles of its three recently identified symbionts, including under heat stress conditions. The prevalence of A. tsugae's facultative symbiont (Serratia symbiotica) was examined at different spatial scales to determine how variable infection rates are for this symbiont. There was no significant difference found in infection rates between adelgids on a tree, within a plot, or within a state. However, significantly more adelgids in Georgia (95%) had S. symbiotica compared to those in New York (68%). Microsatellite genotyping of the adelgids found that this difference was most likely not the result of a second introduction of A. tsugae into eastern North America. Comparison of S. symbiotica proportions between first and fourth instars showed that symbiont absence did not affect the ability of A. tsugae to survive aestivation. Evaluations of symbiont densities within each adelgid found that when S. symbiotica was absent, the density of obligate symbionts was significantly higher. Exposure to heat stress (32.5 °C) was not consistently correlated with changes in symbiont densities over a 4-d period. Overall, we have shown that symbiont prevalence and densities vary within the broad population of A. tsugae in eastern North America, with potentially significant effects upon the ecology of this important pest., (© 2017 Institute of Zoology, Chinese Academy of Sciences.)

Nutritional bacterial symbionts enhance the diets of sap-feeding insects with amino acids and vitamins missing from their diets. In many lineages, an ancestral senior symbiont is joined by a younger junior symbiont. To date, an emergent pattern is that senior symbionts supply a majority of amino acids, and junior symbionts supply a minority. Similar to other hemipterans, adelgids harbor obligate symbionts, but have higher diversity of bacterial associates, suggesting a history of symbiont turnover. The metabolic roles of dual symbionts in adelgids and their contributions to the consortium are largely unexplored. Here, we investigate the symbionts of Adelges tsugae, the hemlock woolly adelgid (HWA), an invasive species introduced from Japan to the eastern United States, where it kills hemlock trees. The response of hemlocks to HWA feeding has aspects of a defensive reaction against pathogens, and some have speculated that symbionts may be involved. We sequenced the genomes of "Ca. Annandia adelgestsuga" and "Ca. Pseudomonas adelgestsugas" symbionts to detail their metabolic capabilities, infer ages of relationship, and search for effectors of plant defenses. We also tested the relationship of "Ca. Annandia" to symbionts of other insects. We find that both symbionts provide nutrients, but in more balanced proportions than dual symbionts of other hemipterans. The lesser contributions of the senior "Ca. Annandia" support our hypothesis for symbiont replacements in adelgids. Phylogenomic results were ambiguous regarding the position of "Ca. Annandia". We found no obvious effectors of plant defenses related to insect virulence, but hypothetical proteins in symbionts are unknown players.

Sap-sucking insects typically engage in obligate relationships with symbiotic bacteria that play nutritional roles in synthesizing nutrients unavailable or in scarce supply from the plant-sap diets of their hosts. Adelgids are sap-sucking insects with complex life cycles that involve alternation between conifer tree species. While all adelgid species feed on spruce during the sexual phase of their life cycle, each adelgid species belongs to a major lineage that feeds on a distinct genus of conifers as their alternate host. Previous work on adelgid symbionts had discovered pairs of symbionts within each host species, and unusual diversity across the insect family, but left several open questions regarding the status of bacterial associates. Here, we explored the consistency of symbionts within and across adelgid lineages, and sought evidence for facultative vs. obligate symbiont status. Representative species were surveyed for symbionts using 16 S ribosomal DNA gene sequencing, confirming that different symbiont pairs were consistently present within each major adelgid lineage. Several approaches were used to establish whether symbionts exhibited characteristics of long-term, obligate mutualists. Patterns of symbiont presence across adelgid species and diversification with host insects suggested obligate relationships. Fluorescent in situ hybridization and electron microscopy localized symbionts to bacteriocyte cells within the bacteriome of each species (with one previously known exception), and detection of symbionts in eggs indicated their vertical transmission. Common characteristics of long-term obligate symbionts, such as nucleotide compositional bias and pleomorphic symbiont cell shape were also observed. Superimposing microbial symbionts on the adelgid phylogeny revealed a dynamic pattern of symbiont gains and losses over a relatively short period of time compared to other symbionts associated with sap-sucking insects, with each adelgid species possessing an older, "senior" symbiont and a younger "junior" symbiont. A hypothesis relating adelgid life cycles to relaxed constraints on symbionts is proposed, with the degradation of senior symbionts and repeated acquisition of more junior symbionts creating opportunities for repeated colonization of new alternate-conifer hosts by adelgids.

Changes in climate conditions, particularly during the Quaternary climatic oscillations, have long been recognized to be important for shaping patterns of species diversity. For species residing in the western Palearctic, two commonly observed genetic patterns resulting from these cycles are as follows: (1) that the numbers and distributions of genetic lineages correspond with the use of geographically distinct glacial refugia and (2) that southern populations are generally more diverse than northern populations (the "southern richness, northern purity" paradigm). To determine whether these patterns hold true for the widespread pest species the winter moth ( Operophtera brumata ), we genotyped 699 individual winter moths collected from 15 Eurasian countries with 24 polymorphic microsatellite loci. We find strong evidence for the presence of two major genetic clusters that diverged ~18 to ~22 ka, with evidence that secondary contact (i.e., hybridization) resumed ~ 5 ka along a well-established hybrid zone in Central Europe. This pattern supports the hypothesis that contemporary populations descend from populations that resided in distinct glacial refugia. However, unlike many previous studies of postglacial recolonization, we found no evidence for the "southern richness, northern purity" paradigm. We also find evidence for ongoing gene flow between populations in adjacent Eurasian countries, suggesting that long-distance dispersal plays an important part in shaping winter moth genetic diversity. In addition, we find that this gene flow is predominantly in a west-to-east direction, suggesting that recently debated reports of cyclical outbreaks of winter moth spreading from east to west across Europe are not the result of dispersal.

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., (© 2016 John Wiley & Sons Ltd.)

The predatory species Laricobius nigrinus (Fender) and Laricobius osakensis (Shiyake and Montgomery) (Coleoptera: Derodontidae) have been released for biological control of hemlock woolly adelgid (Adelges tsugae; Hemiptera: Adelgidae) in eastern North America. L. osakensis is native to Japan, whereas L. nigrinus is endemic to the Pacific Northwest of the United States and Canada. After release, L. nigrinus was found to hybridize with the native eastern species, Laricobius rubidus (LeConte). The purpose of this study is to observe prey location behaviors of these three Laricobius species and L. nigrinus × L. rubidus (Ln × Lr) hybrids. Olfactometer bioassays were used to test response to host odors of adelgid-infested eastern hemlock, uninfested eastern hemlock, and uninfested eastern white pine. Predators reacted in the olfactometer more quickly when adelgid-infested foliage was included as a choice. L. nigrinus preferred infested eastern hemlock over uninfested eastern white pine, and L. rubidus preferred uninfested eastern white pine over uninfested eastern hemlock. Laricobius hybrids did not show a preference for foliage types known to be primary adelgid hosts (eastern hemlock and eastern white pine). Unequal preference by species of Laricobius for host trees of different adelgid prey could therefore be maintaining Laricobius species barriers despite hybridization. L. osakensis for this study were reared in the laboratory, whereas other species in this study were collected from the field, yet still were attracted to infested and uninfested eastern hemlock. This species also responded most quickly in the olfactometer, which is encouraging for successful biological control with this species., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A taxonomic and nomenclatural Catalogue of the adelgids (Hemiptera: Adelgidae) is presented. Six family-group names are listed, five being synonyms of Adelgidae. Twenty-two genus-group names, of which nine are subjectively valid and in use, are presented with their type species, etymology, and grammatical gender. One hundred and six species-group names are listed, of which 70 are considered subjectively valid.

Knowledge of intraspecific variation in symbioses may aid in understanding the ecology of widespread insects in different parts of their range. We investigated bacterial symbionts of Adelges tsugae, a pest of hemlocks in eastern North America introduced from Asia. Amplification, cloning, and sequencing of bacterial 16S rDNA, in situ hybridizations, and electron microscopy revealed that A. tsugae harbours up to five bacterial phylotypes, according to population. Three Gammaproteobacteria species are maternally transmitted. The first, designated 'Ca. Pseudomonas adelgestsugas' resides in the haemocoel, and was detected in all populations except Taiwan. The second phylotype, 'Ca. Serratia symbiotica', resides in bacteriocytes of populations on Tsuga sieboldii in Japan and in E. North America. The third phylotype, designated 'Ca. Annandia adelgestsuga', clustered within a lineage of several insect endosymbionts that included Buchnera aphidicola. It was detected in bacteriocytes in all populations, and in salivary glands of first instars. Two Betaproteobacteria phylotypes were detected in some Japanese T. sieboldii and eastern North America populations, and were observed only in salivary glands with no evidence of maternal transmission. Our results support the ideas that symbiont gain and loss has been volatile in adelgids, and that symbionts may help to trace the source of invasive species., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

This article documents the addition of 238 microsatellite marker loci and 72 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Adelges tsugae, Artemisia tridentata, Astroides calycularis, Azorella selago, Botryllus schlosseri, Botrylloides violaceus, Cardiocrinum cordatum var. glehnii, Campylopterus curvipennis, Colocasia esculenta, Cynomys ludovicianus, Cynomys leucurus, Cynomys gunnisoni, Epinephelus coioides, Eunicella singularis, Gammarus pulex, Homoeosoma nebulella, Hyla squirella, Lateolabrax japonicus, Mastomys erythroleucus, Pararge aegeria, Pardosa sierra, Phoenicopterus ruber ruber and Silene latifolia. These loci were cross-tested on the following species: Adelges abietis, Adelges cooleyi, Adelges piceae, Pineus pini, Pineus strobi, Tubastrea micrantha, three other Tubastrea species, Botrylloides fuscus, Botrylloides simodensis, Campylopterus hemileucurus, Campylopterus rufus, Campylopterus largipennis, Campylopterus villaviscensio, Phaethornis longuemareus, Florisuga mellivora, Lampornis amethystinus, Amazilia cyanocephala, Archilochus colubris, Epinephelus lanceolatus, Epinephelus fuscoguttatus, Symbiodinium temperate-A clade, Gammarus fossarum, Gammarus roeselii, Dikerogammarus villosus and Limnomysis benedeni. This article also documents the addition of 72 sequencing primer pairs and 52 allele specific primers for Neophocaena phocaenoides., (© 2009 Blackwell Publishing Ltd.)

The Adelgidae form a small group of insects in the Aphidoidea. They are cyclically parthenogenetic with host alternating, multiple-generation complex life cycles and are restricted to certain host genera in the Pinaceae. Species that host alternate always have Picea as the primary host where sexual reproduction and gall formation occur, and another genus in the Pinaceae as the secondary host where a series of parthenogenetic generations are produced. Other species that do not host alternate complete their entire life cycle on one host and only reproduce parthenogenetically. We studied relationships within Adelgidae using DNA sequences from the mitochondrial COI, COII, and cytb genes, and the nuclear EF1alpha gene. Analysis of the combined data resulted in a well-resolved phylogeny in which the major adelgid clades correspond neatly to their association with secondary host genera. Specialization on each secondary host genus occurred only once and was followed by diversification on the host genus. Molecular dating of divergence times in the Adelgidae suggest that diversification among host genera occurred in the Late Cretaceous and Early Tertiary when the Pinaceae genera were diverging. It is not clear, however, whether the Adelgidae and Pinaceae co-diversified because the relationships among the Pinaceae genera are not fully resolved. We discuss implications for adelgid taxonomy, life cycle evolution, and evolution of the interaction between adelgids and their host plants.

The Adelgidae form a small clade of insects within the Aphidoidea (Hemiptera) that includes some of the most destructive introduced pest species threatening North American forest ecosystems. Despite their importance, little is known about their evolutionary history and their taxonomy remains unresolved. Adelgids are cyclically parthenogenetic and exhibit multigeneration complex life cycles. They can be holocyclic, with a sexual generation and host alternation, or anholocyclic, entirely asexual and without host alternation. We discuss adelgid behavior and ecology, emphasizing plant-insect interactions, and we explore ways that the biogeographic history of their host plants may have affected adelgid phylogeny and evolution of adelgid life cycles. Finally, we highlight several areas in which additional research into speciation, population genetics, multitrophic interactions, and life-history evolution would improve our understanding of adelgid biology and evolution.

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.

Behavioral choice tests comprise one of the most commonly used experimental designs in ecology. However a critical assumption of these assays, that the outcome is independent of the number of choices, has not been tested explicitly. We developed a methodology for testing this assumption, and discuss how it can be incorporated into experimental design. The model with which we performed this test consisted of an insect herbivore, the gypsy moth Lymantria dispar L., feeding on a clonal host plant, Populus. We established a dose-response feeding gradient by amending leaves of a single age class with defined concentrations of a diterpene, isopimaric acid, that exhibits feeding deterrent properties. We selected various concentrations that elicited different levels of feeding for subsequent tests in which we modified the number of choices. A sample size of 30 assay units per test generated statistically significant separations in two-way choice tests, yielded statistically significant but somewhat inconsistent results when four concentrations were offered, and failed to provide complete separation when five concentrations were offered. Other factors associated with the number of choices that affected results included specific combinations of doses, physical arrangement of choices, and total consumption per assay unit. We used our results to develop procedures for estimating the sample sizes needed to compare a specified number of choices. We based these methods on power considerations, the requirements for data transformation and inclusion of covariates. We develop a general approach for estimating the number of replicates needed to support a particular number of choices for a test organism, and discuss factors to be considered when relating this approach to various types of behavioral choice assays.

We examined the effects of various wounding treatments and genotypic variation on induced resistance in Populus (Salicales: Salicaceae) against herbivory by the gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae). Second-instar larvae grew and consumed less on leaves from induced than non-induced trees. Likewise, larvae preferred leaf disks from non-induced trees. Among induction treatments, gypsy moth feeding had the strongest and most consistent effect in behavioral choice tests. Mechanical wounding of leaves and mechanical wounding plus application of gypsy moth regurgitant had intermediate effects, while application of jasmonic acid had the weakest overall effect. Under no-choice conditions, there were no consistent trends across clones in the ability of various treatments to elicit plant responses affecting the herbivore. Levels of constitutive and inducible resistance to herbivory varied significantly among 12 Populus clones. Larvae grew up to 30-fold more, and consumed up to 250-fold more on the most suitable than the least suitable clone. Prior feeding by gypsy moths reduced larval feeding up to 71.4% on the most highly inducible clone, but it had little or no effect for the least inducible clones. There was no evidence for a relationship between levels of inducible and constitutive resistance, or between inducible resistance and phylogenetic relatedness among clones. We discuss implications for the ecology and evolution of plant-insect interactions and the management of insect pests.