Genome assembly of the southern pine beetle (Dendroctonus frontalis Zimmerman) reveals the origins of gene content reduction in Dendroctonus.

Dendroctonus frontalis, also known as southern pine beetle (SPB), represents the most damaging forest pest in the southeastern United States. Strategies to predict, monitor and suppress SPB outbreaks have had limited success. Genomic data are critical to inform on pest biology and to identify molecular targets to develop improved management approaches. Here, we produced a chromosome-level genome assembly of SPB using long-read sequencing data. Synteny analyses confirmed the conservation of the core coleopteran Stevens elements and validated the bona fide SPB X chromosome. Transcriptomic data were used to obtain 39,588 transcripts corresponding to 13,354 putative protein-coding loci. Comparative analyses of gene content across 14 beetle and 3 other insects revealed several losses of conserved genes in the Dendroctonus clade and gene gains in SPB and Dendroctonus that were enriched for loci encoding membrane proteins and extracellular matrix proteins. While lineage-specific gene losses contributed to the gene content reduction observed in Dendroctonus, we also showed that widespread misannotation of transposable elements represents a major cause of the apparent gene expansion in several non-Dendroctonus species. Our findings uncovered distinctive features of the SPB gene complement and disentangled the role of biological and annotation-related factors contributing to gene content variation across beetles.

Presence of the causal agent of laurel wilt disease in sassafras-associated insects.

Laurel wilt disease (LWD) is a lethal vascular wilt caused by an exotic ambrosia beetle-fungal complex, the redbay ambrosia beetle, Xyleborus glabratus Eichhoff, and its nutritional symbiont, Harringtonia lauricola (Harr., Fraedrich & Aghayeva) de Beer & Procter. LWD is responsible for the widespread mortality of redbay, Persea borbonia (L.) Spreng., devastating coastal forests in the southeast United States. More recently, LWD is causing mortality of understory sassafras, Sassafras albidum (Nutt.) Nees, in deciduous forests in Kentucky, USA; the biology, epidemiology, and long-term impacts of LWD in deciduous forests are unclear. All North American lauraceous species evaluated have shown susceptibility, and numerous additional ambrosia beetles have demonstrated vector potential, but no studies to date have assessed the presence of H. lauricola in other insects associated with LWD-infected sassafras. We sampled infected sassafras from the leading edge of the LWD range and collected insect associates to evaluate phoretic and internal presence of H. lauricola. We recorded 118 individuals of 38 morphospecies emerging; most were Coleoptera. Of the 48 specimens evaluated for H. lauricola, none tested positive for phoretic presence, but internal presence was evident in the granulate ambrosia beetle, Xylosandrus crassiusculus Motschulsky, and in a hidden snout weevil, Apteromechus ferratus Say. This is the first report of H. lauricola associated with a non-ambrosia beetle and expands our understanding of the vector potential of additional insect species while confirming the role of the granulate ambrosia beetle. These findings contribute to our understanding of LWD epidemiology in sassafras hosts from more northerly latitudes.

Spatial Distribution and Retention in Loblolly Pine Seedlings of Exogenous dsRNAs Applied through Roots.

Exogenously applied double-stranded RNA (dsRNA) can induce potent host specific gene knockdown and mortality in insects. The deployment of RNA-interference (RNAi) technologies for pest suppression is gaining traction in both agriculture and horticulture, but its implementation in forest systems is lagging. While numerous forest pests have demonstrated susceptibility to RNAi mediated gene silencing, including the southern pine beetle (SPB), Dendroctonus frontalis, multiple barriers stand between laboratory screening and real-world deployment. One such barrier is dsRNA delivery. One possible delivery method is through host plants, but an understanding of exogenous dsRNA movement through plant tissues is essential. Therefore, we sought to understand the translocation and persistence of dsRNAs designed for SPB throughout woody plant tissues after hydroponic exposure. Loblolly pine, Pinus taeda, seedlings were exposed to dsRNAs as a root soak, followed by destructive sampling. Total RNA was extracted from different tissue types including root, stem, crown, needle, and meristem, after which gel electrophoresis confirmed the recovery of the exogenous dsRNAs, which were further verified using Sanger sequencing. Both techniques confirmed the presence of the exogenously applied target dsRNAs in each tissue type after 1, 3, 5, and 7 d of dsRNA exposure. These findings suggest that root drench applications of exogenous dsRNAs could provide a viable delivery route for RNAi technology designed to combat tree feeding pests.

Feasibility of Systemically Applied dsRNAs for Pest-Specific RNAi-Induced Gene Silencing in White Oak.

The efficacy of double-stranded RNA (dsRNA) in inducing host specific gene knockdown and mortality has been demonstrated in a multitude of insects and dsRNAs are being integrated for pest suppression in a variety of agricultural and horticultural crops. However, less attention has been applied to their use in forest settings, despite the demonstrated susceptibility of multiple forest pests to RNAi. Prior to implementation for forest pest suppression, characterization of the specificity, efficacy, and behavior of dsRNAs in the environment is essential. Therefore, we investigated the translocation and retention of exogenously applied dsRNA in an economically and ecologically significant hardwood tree when applied hydroponically. White oak (Quercus alba, L.) seedlings were exposed to dsRNAs as a root soak, and at 1, 3, 5, and 7 days post-exposure were destructively sampled, divided into stem and leaf tissue, and the RNA extracted. Gel electrophoresis was used to visualize the presence of exogenous dsRNA in treated seedling material and Sanger sequencing was used to further verify recovery of treatment dsRNAs. Both techniques confirmed the presence of the exogenously applied dsRNAs in each tissue type at each sample interval, demonstrating successful uptake and translocation of dsRNAs through white oak tissues. Our findings support root uptake as a viable delivery method for dsRNAs in hardwood seedlings, which could provide single tree protection from selected tree feeding pests or pathogens.

Validation of reference genes for quantitative PCR in the forest pest, Ips calligraphus.

The six-spined ips, Ips calligraphus, is a North American bark beetle that can exploit most eastern North American Pinus species and can cause mortality. Biotic and abiotic disturbances weaken trees, creating breeding substrate that promotes rapid population growth. Management historically relied on silvicultural practices, but as forests become increasingly stressed, innovative management is needed. Manipulation of the cellular RNA interference (RNAi) pathway to induce gene silencing is an emerging means of insect suppression, and is effective for some bark beetles. Quantitative PCR (qPCR) is a powerful tool for analysis of gene expression, and is essential for examining RNAi. To compare gene expression among individuals, stably expressed reference genes must be validated for qPCR. We evaluated six candidate reference genes (18s, 16s, 28s, ef1a, cad, coi) for stability under biotic (beetle sex, developmental stage, and host plant), and abiotic (temperature, photoperiod, and dsRNA exposure) conditions. We used the comprehensive RefFinder tool to compare stability rankings across four algorithms. These algorithms identified 18s, 16s, and 28s as the most stably expressed. Overall, 16s and 28s were selected as reference genes due to their stability and moderate expression levels, and can be used for I. calligraphus gene expression studies using qPCR, including those evaluating RNAi.

Oral Ingestion of Bacterially Expressed dsRNA Can Silence Genes and Cause Mortality in a Highly Invasive, Tree-Killing Pest, the Emerald Ash Borer.

RNA interference (RNAi) is a naturally occurring process inhibiting gene expression, and recent advances in our understanding of the mechanism have allowed its development as a tool against insect pests. A major challenge for deployment in the field is the development of convenient and efficient methods for production of double stranded RNA (dsRNA). We assessed the potential for deploying bacterially produced dsRNA as a bio-pesticide against an invasive forest pest, the emerald ash borer (EAB). EAB feeds on the cambial tissue of ash trees (Fraxinus spp.), causing rapid death. EAB has killed millions of trees in North America since its discovery in 2002, prompting the need for innovative management strategies. In our study, bacterial expression and synthesis of dsRNA were performed with E. coli strain HT115 using the L4440 expression vector. EAB-specific dsRNAs (shi and hsp) over-expressed in E. coli were toxic to neonate EAB after oral administration, successfully triggering gene silencing and subsequent mortality; however, a non-specific dsRNA control was not included. Our results suggest that ingestion of transformed E. coli expressing dsRNAs can induce an RNAi response in EAB. To our knowledge, this is the first example of an effective RNAi response induced by feeding dsRNA-expressing bacteria in a forest pest.

RNA interference and validation of reference genes for gene expression analyses using qPCR in southern pine beetle, Dendroctonus frontalis.

RNA interference (RNAi) is a highly specific gene-silencing mechanism that can cause rapid insect mortality when essential genes are targeted. RNAi is being developed as a tool for integrated pest management of some crop pests. Here we focus on an aggressive forest pest that kills extensive tracts of pine forests, the southern pine beetle (SPB), Dendroctonus frontalis. We sought to identify reference genes for quantitative real-time PCR (qPCR) and validate RNAi responses in SPB by mortality and gene silencing analysis. Using an adult beetle feeding bioassay for oral ingestion of dsRNA, we measured the expression and demonstrated knockdown of target genes as well as insect mortality after ingestion of target genes. Our study validates reference genes for expression analyses and demonstrates highly effective RNAi responses in SPB, with RNAi response to some target dsRNAs causing 100% beetle mortality after ingestion.

Transcriptome sequencing for identification of diapause-associated genes in fall webworm, Hyphantria cunea Drury.

Fall webworm, Hyphantria cunea Drury (Lepidoptera: Arctiidae) is extremely adaptable and highly invasive in China as a defoliator of ornamental and forest trees. Both voltinism and diapause strategies of fall webworm in China are variable, and this variability contributes to it invasiveness. Little is known about molecular regulation of diapause in fall webworm. To gain insight into possible mechanisms of diapause induction, high-throughput RNA-seq data were generated from non-diapause pupae (NDP) and diapause pupae (DP). A total of 58,151 unigenes were assembled and researched against nine public databases. In total, 29,013 up-regulated and 3451 down-regulated unigenes were differentially expressed by DP when compared with those of NDP. Genes encoding proteins such as UDP-glycosyl transferase (UGT), cytochrome P450 and Hsp70 were predicted to be involved in diapause. Moreover, GO function and KEGG pathway enrichments were performed on all differentially expressed genes (DEGs) and showed that cell cycle and insulin signaling pathways may be related to the diapause of the fall webworm. This study provides valuable information about the fall webworm transcriptome for future gene function research, especially as it relates to diapause.

Identification of highly effective target genes for RNAi-mediated control of emerald ash borer, Agrilus planipennis.

Recent study has shown that RNA interference (RNAi) is efficient in emerald ash borer (EAB), Agrilus planipennis, and that ingestion of double-stranded RNA (dsRNA) targeting specific genes causes gene silencing and mortality in neonates. Here, we report on the identification of highly effective target genes for RNAi-mediated control of EAB. We screened 13 candidate genes in neonate larvae and selected the most effective target genes for further investigation, including their effect on EAB adults and on a non-target organism, Tribolium castaneum. The two most efficient target genes selected, hsp (heat shock 70-kDa protein cognate 3) and shi (shibire), caused up to 90% mortality of larvae and adults. In EAB eggs, larvae, and adults, the hsp is expressed at higher levels when compared to that of shi. Ingestion of dsHSP and dsSHI caused mortality in both neonate larvae and adults. Administration of a mixture of both dsRNAs worked better than either dsRNA by itself. In contrast, injection of EAB.dsHSP and EAB.dsSHI did not cause mortality in T. castaneum. Thus, the two genes identified cause high mortality in the EAB with no apparent phenotype effects in a non-target organism, the red flour beetle, and could be used in RNAi-mediated control of this invasive pest.

Development of RNAi method for screening candidate genes to control emerald ash borer, Agrilus planipennis.

The ingestion of double-strand RNAs (dsRNA) targeting essential genes in an insect could cause mortality. Based on this principle, a new generation of insect control methods using RNA interference (RNAi) are being developed. In this work, we developed a bioassay for oral delivery of dsRNA to an invasive forest and urban tree pest, the emerald ash borer (EAB, Agrilus planipennis). EAB feeds and develops beneath the bark, killing trees rapidly. This behavior, coupled with the lack of a reliable artificial diet for rearing larvae and adults, make them difficult to study. We found that dsRNA is transported and processed to siRNAs by EAB larvae within 72 h after ingestion. Also, feeding neonate larvae with IAP (inhibitor of apoptosis) or COP (COPI coatomer, ß subunit) dsRNA silenced their target genes and caused mortality. Both an increase in the concentration of dsRNA fed and sequential feeding of two different dsRNAs increased mortality. Here we provide evidence for successful RNAi in EAB, and demonstrate the development of a rapid and effective bioassay for oral delivery of dsRNA to screen additional genes.

Canopy vegetation influences ant (Hymenoptera: Formicidae) communities in headwater stream riparian zones of central Appalachia.

In the eastern United States, eastern hemlock Tusga canadensis (L.) Carriere forests are threatened by the invasive hemlock woolly adelgid, Adelges tsugae, a pest that is causing widespread hemlock mortality. Eastern hemlock is an essential component of forested communities. Adelgid-induced hemlock mortality is causing a shift in forest composition and structure, altering ecosystem function and thereby influencing the arthropod community. Using pitfall traps at three sites, we monitored ground-dwelling arthropods at 30-d intervals in hemlock-dominated and deciduous-dominated forests in central Appalachia over 2 yr. Here, we focus on the ant community (Hymenoptera: Formicidae) collected in the summer months. Ants form a ubiquitous and integral component of the invertebrate community, functioning at various trophic levels as predators, herbivores, and omnivores, and fulfilling important roles in forest ecosystems. We found no difference in overall ant abundance between hemlock-dominated and deciduous-dominated forests but did detect significant differences in the genera Prenolepis between forest types (P < 0.01) and Aphaenogaster across study locations (P = 0.02). Three genera were unique to deciduous forests; one was unique to hemlock forests. Not surprisingly, total formicids and several genera demonstrated temporal differences in abundance, with greater numbers captured in July than in August. As hemlock woolly adelgid-induced mortality of eastern hemlock becomes more pervasive, changes in forest composition and structure are imminent, accompanied by shifts in hemlock associates.

Microclimatic variation within sleeve cages used in ecological studies.

Sleeve cages for enclosing or excluding arthropods are essential components of field studies evaluating trophic interactions. Microclimatic variation in sleeve cages was evaluated to characterize its potential effects on subsequent long-term experiments. Two sleeve cage materials, polyester and nylon, and two cage sizes, 400 and 6000 cm(2), were tested on eastern hemlock, Tsuga canadensis (L.) Carrière. Temperature and relative humidity inside and outside cages, and the cost and durability of the cage materials, were compared. Long-term effects of the sleeve cages were observed by measuring new growth on T. canadensis branches. The ultimate goal was to identify a material that minimizes bag-induced microclimatic variation. Bagged branches whose microclimates mimic those of surrounding unbagged branches should have minimal effects on plant growth and may prove ideal venues for assessing herbivore and predator behavior under natural conditions. No differences were found in temperature or humidity between caging materials. Small cages had higher average temperatures than large cages, especially in the winter, but this difference was confounded by the fact that small cages were positioned higher in trees than large cages. Differences in plant growth were detected. Eastern hemlock branches enclosed within polyester cages produced fewer new growth tips than uncaged controls. Both polyester and nylon cages reduced the length of new shoot growth relative to uncaged branches. In spite of higher costs, nylon cages were superior to polyester with respect to durability and ease of handling.

Potential fecundity of a highly invasive gall maker, Dryocosmus kuriphilus (Hymenoptera: Cynipidae).

Fecundity is a key factor in modulating population growth rate, and is of particular significance when considering the invasiveness of introduced species. In insects, fecundity is affected by body size, age, and nutrition. We investigated the potential fecundity of the invasive Asian chestnut gall wasp Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), an introduced parthenogenetic gall former of Asian origin and a global pest of chestnut (Castanea spp.), to better understand its invasiveness. We compared ovarian, egg, and body metrics of adult wasps of different age. We evaluated insect weight, body length, mesosomal and metasomal lengths and widths, hind femur length, number of eggs, and size of eggs in wasps from four age cohorts. Adult weight and metasomal width were positively correlated with number of eggs. Egg load decreased with wasp age, and egg size initially increased before decreasing. Our findings suggest that adult D. kuriphilus, previously reported as proovigenic, may be resorping eggs in the absence of suitable hosts, and reallocating nutritive resources for body maintenance and egg quality to increase fitness, implicating a plasticity in its reproductive strategy. D. kuriphilus may be able to vary its potential fecundity in response to nutrition and host availability, thus increasing its invasiveness.

Biomechanical Properties of Hemlocks: A Novel Approach to Evaluating Physical Barriers of the Plant-Insect Interface and Resistance to a Phloem-Feeding Herbivore.

Micromechanical properties that help mediate herbivore access may be particularly important when considering herbivorous insects that feed with piercing-sucking stylets. We used microindentation to quantify the micromechanical properties of hemlock, Tsuga spp., to quantify the hardness of the feeding site of the invasive hemlock woolly adelgid, Adelges tsugae. We measured hardness of the hemlock leaf cushion, the stylet insertion point of the adelgid, across four seasons in a 1 y period for four hemlock species growing in a common garden, including eastern, western, mountain, and northern Japanese hemlocks. Leaf cushion hardness was highest in the fall and winter and lowest in summer for all species. Northern Japanese hemlock had relatively greater hardness than the remaining species. Our data contributes an additional perspective to the existing framework within which greater susceptibility and subsequent mortality of eastern hemlocks is observed. The potential application of microindentation to understanding the nature and relevance of plant mechanical defenses in plant-herbivore interactions is also demonstrated and highlighted.

Arboreal spiders in eastern hemlock.

Eastern hemlock [Tsuga canadensis (L.) Carrière] is a foundation species in forests of eastern North America that plays a key role in ecosystem function. It is highly susceptible to the exotic invasive hemlock woolly adelgid (Adelges tsugae Annand), which is causing widespread hemlock mortality. We surveyed the spider communities of eastern hemlock and deciduous canopies over 2 yr, collecting over 4,000 spiders from 21 families. We found that eastern hemlock canopies harbored a more abundant, rich, and diverse spider community than did deciduous canopies. Five spider families were present in our hemlock collections that were absent from the deciduous collections, including Mysmenidae, Theridiosomatidae, Mimetidae, Lycosidae, and Agelenidae. In hemlock canopies there were 4× the number of web builders, consisting primarily of the Tetragnathidae and Araneidae, than active hunters, consisting primarily of the Anyphaenidae and the Salticidae. Ours is the first in depth study of the spider community in eastern hemlock. Spider abundance in hemlock canopies suggest that they may play a role regulating herbivore populations, and could possibly affect the invasive hemlock woolly adelgid, either through direct consumption of the adelgids themselves or through interactions with classical biological control agents.

Chestnut species and jasmonic acid treatment influence development and community interactions of galls produced by the Asian chestnut gall wasp, Dryocosmus kuriphilus.

Jasmonic acid (JA) is a plant-signaling hormone involved in defenses against insects and pathogens as well as the regulation of nutrient partitioning. Gall wasps (Hymenoptera: Cynipidae) induce the formation of galls on their host plants, which house immature wasps and provide them with nutrition and protection. The goal of this study was to investigate the effects of JA application on gall development and defenses. Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) galls on American chestnut, Castanea dentata (Marsh.) Borkhausen (Fagales: Fagaceae), and Chinese chestnut, C. mollissima Blume, were treated with JA or a JA- inhibitor, diethyldithiocarbamic acid (DIECA), to determine the effects of these treatments on gall characteristics and defenses. Chinese chestnut galls treated with JA had greater volume and dry weight, thicker sclerenchyma layers, and fewer external fungal lesions compared with controls. Galls from both chestnut species treated with JA contained a lower proportion of empty chambers, and elevated tannin levels compared with controls. The effects of DIECA on galls were generally opposite from those of JA. American chestnut galls treated with DIECA had lower dry weight and fewer feeding punctures caused by the lesser chestnut weevil compared with controls. Galls from both chestnut species that were treated with DIECA were smaller and had more external fungal lesions compared with controls. Compared to American chestnut galls, Chinese chestnut galls had increased parasitism rates and fewer gall wasps. This study is the first to investigate the effects of JA on an insect gall, and indicates that JA treatments benefit gall wasps by increasing gall size and defenses.

Gall structure affects ecological associations of Dryocosmus kuriphilus (Hymenoptera: Cynipidae).

Gall wasps (Hymenoptera: Cynipidae) induce structures (galls) on their host plants that house developing wasps and provide them with protection from natural enemies. The Asian chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu, is an invasive pest that is destructive to chestnut (Castanea spp.). An improved understanding of the interactions among D. kuriphilus, its host, and its natural enemies is critical for the development of effective management strategies against this pest. The objective of our study was to evaluate the D. kuriphilus community interactions, and relate these interactions to variations among gall traits. Galls were collected from four locations throughout the eastern United States from May (gall initiation) through August (after gall wasp emergence), and January. Gall characteristics (volume, weight, and schlerenchyma layer thickness), gall inhabitants (D. kuriphilus, parasitoids, and chamber fungi), and other community associates (insect herbivores and lesions thought to be caused by endophytes) were evaluated and correlated using canonical correlation analyses. The primary mortality factors for D. kuriphilus were parasitism, gall chamber-invading fungi, and failure of adult gall wasps to emerge. Larger gall size and thicker schlerenchyma layers surrounding the larval chambers were negatively correlated with parasitoids and chamber fungi, indicating these gall traits are important defenses. External fungal lesions and insect herbivory were positively correlated with the absence of D. kuriphilus within galls. This study provides support for the protective role of cynipid galls for the gall inducer, identifies specific gall traits that influence gall wasp mortality, and improves our knowledge of D. kuriphilus ecology in North America.

Web orientation and prey resources for web-building spiders in eastern hemlock.

We examined the arthropod community on eastern hemlock, Tsuga canadensis (L.) Carr, in the context of its role in providing potential prey items for hemlock-associated web-weaving spiders. Using sticky traps simulating spider webs, we evaluated what prey items are available to web-weaving spiders in eastern hemlock based on web orientation (horizontal versus vertical) and cardinal direction. We found that the overwhelming majority (>70%) of prey items available to spiders in hemlock canopies were Diptera. Psocoptera, Hymenoptera, and Hemiptera comprised most of the remaining potential prey. A significant direction × orientation interaction, and greater trap capture in some direction-orientation combinations, suggests that spiders might locate their webs in eastern hemlock canopies for thermoregulatory purposes, ultimately optimizing prey capture. We also evaluated these findings in the context of hemlock infestation by the invasive hemlock woolly adelgid, Adelges tsugae Annand. The adelgid is a sedentary insect with a mobile crawler stage that provides a readily available, easily obtained food source for predators in hemlock canopies. However, an abundance of alternative prey will affect within canopy spider distribution and the potential intensity with which spiders consume these prey. Understanding the response of spiders to potential prey availability is essential to understanding the trophic interactions involving these predators and their potential for influencing herbivore populations.