Different Bacillus thuringiensis (Bt) strains produce a broad variety of pore-forming toxins (PFTs) that show toxicity against insects and other invertebrates. Some of these insecticidal PFT proteins have been used successfully worldwide to control diverse insect crop pests. There are several studies focused on describing the mechanism of action of these toxins that have helped to improve their performance and to cope with the resistance evolved by different insects against some of these proteins. However, crucial information that is still missing is the structure of pores formed by some of these PFTs, such as the three-domain crystal (Cry) proteins, which are the most commercially used Bt toxins in the biological control of insect pests. In recent years, progress has been made on the identification of the structural changes that certain Bt insecticidal PFT proteins undergo upon membrane insertion. In this review, we describe the models that have been proposed for the membrane insertion of Cry toxins. We also review the recently published structures of the vegetative insecticidal proteins (Vips; e.g. Vip3) and the insecticidal toxin complex (Tc) in the membrane-inserted state. Although different Bt PFTs show different primary sequences, there are some similarities in the three-dimensional structures of Vips and Cry proteins. In addition, all PFTs described here must undergo major structural rearrangements to pass from a soluble form to a membrane-inserted state. It is proposed that, despite their structural differences, all PFTs undergo major structural rearrangements producing an extended α-helix, which plays a fundamental role in perforating their target membrane, resulting in the formation of the membrane pore required for their insecticidal activity.

The entomopathogenic fungus Beauveria bassiana is cosmopolitan and known to infect a variety of sap-sucking pests like aphids, mealybugs, and scales in the order of Hemiptera. In Fall 2017, spotted lanternfly (SLF) adults killed by the fungal entomopathogen B. bassiana were found in Berks County, Pennsylvania. In 2018-2020 we collected SLF and nearby non-target insects killed by Beauveria spp. from 18 field sites in southeastern Pennsylvania. We identified 159 Beauveria isolates from SLF and six isolates from non-targets. Five isolates of B. bassiana and one isolate of B. brongniartii were identified from the non-targets. Based on sequence data from the nuclear B locus (Bloc) intergenic region, all the isolates from SLF were identified as B. bassiana, but there were 20 different strains within this species, grouped into two clades. Three B. bassiana strains (A, B, and L) were found in most field sites and were the most prevalent. Representative isolates for these three strains were used in laboratory bioassays and were compared to a commercial B. bassiana strain (GHA). Strain B was inferior to A, L, and GHA against nymphs; strains A and L had greater efficacy than B and GHA against adults. We also quantified conidial production on SLF cadavers. This paper discusses the diversity of these B. bassiana strains in SLF populations and implications for biological control of this abundant invasive.

The Asian citrus psyllid, Diaphorina citri, vectors the bacterial causative agent of citrus greening disease, which has severely impacted citrus production on a global scale. As the current repeated application of chemical insecticides is unsustainable for management of this insect and subsequent protection of groves, we investigated the potential use of the bacteria-derived pesticidal protein, Cry1Ba1, when delivered via transgenic citrus plants. Having demonstrated transformation of the Indian curry leaf tree, Bergera koenigii, for Cry1Ba1 expression for use as a trap plant, we produced transgenic plants of Duncan grapefruit, Citrus paridisi, Valencia sweet orange, Citrus sinensis, and Carrizo citrange, C. sinensis x Poncirus trifoliata, for expression of Cry1Ba1. The presence of the cry1ba1 gene, and cry1ba1 transcription were confirmed. Western blot detection of Cry1Ba1 was confirmed in most cases. When compared to those from wild-type plants, leaf discs from transgenic Duncan and Valencia expressing Cry1Ba1 exhibited a “delayed senescence” phenotype, similar to observations made for transgenic B. koenigii. In bioassays, significant reductions in the survival of adult psyllids were noted on transgenic B. koenigii and Valencia sweet orange plants expressing Cry1Ba1, but not on transgenic Duncan grapefruit or Carrizo citrange. In contrast to psyllids fed on wild type plants, the gut epithelium of psyllids fed on transgenic plants was damaged, consistent with the mode of action of Cry1Ba1. These results indicate that the transgenic expression of a bacterial pesticidal protein in B. koenigii and Valencia sweet orange offers a viable option for management of D. citri, that may contribute to solutions that counter citrus greening disease.

Diabrotica undecimpunctata is a multivoltine polyphagous beetle species that has long been documented as a significant agricultural pest throughout its native range in North America. This beetle can vector bacterial and viral plant pathogens that result in major losses to crops such as cucumber and soybean. Many countries outside the Americas treat D. undecimpunctata as a species of quarantine importance, while in the USA only the subspecies D. u. duodecimnotata is subject to quarantine, to prevent introduction from Mexico. Identification of D. undecimpunctata on the basis of morphology alone can be complicated given the use of conflicting characters in the description of some subspecific taxa. To better understand relationships among D. undecimpunctata subspecies and other related species, we sequenced mitochondrial cytochrome oxidase 1 (CO1) and nuclear internal transcribed spacer 2 (ITS2) DNA from individuals in different subspecific taxa and across different parts of the species range using museum samples and interceptions. When our data were combined with publicly available Diabrotica data, no pattern of divergence consistent with the currently recognized subspecific designations was found. In addition, we compared phylogenetic patterns in CO1 data from the congener D. virgifera to demonstrate the utility of mitochondrial data in resolving subspecies. From the CO1 data, a diagnostic real-time PCR assay was developed that could successfully identify all haplotypes within the large D. undecimpunctata clade for use in surveys and identification at ports of entry. These findings underscore the need to resolve molecular and morphological datasets into cogent, lineage-based groupings. Such efforts will provide an evolutionary context for the study of agriculturally important attributes of Diabrotica such as host preferences, xenobiotic metabolism, and natural and anthropogenic patterns of dispersal.

The spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae), an invasive planthopper discovered in Pennsylvania, U.S. in 2014, has spread to many surrounding states despite quarantines and control efforts, and further spread is anticipated. A classical (importation) biological control program would contribute to the long-term management of L. delicatula in the eastern U.S. In its native range of China, Anastatus orientalis (Hymenoptera: Eupelmidae), an egg parasitoid, causes significant mortality. Anastatus orientalis consists of multiple haplotypes that differ in important biological parameters. To delineate the physiological host range of A. orientalis Haplotype C, we completed no-choice and choice testing. No-choice testing of non-target eggs from 36 insect species spanning six orders and 18 families showed that physiologically this haplotype of A. orientalis can develop in a variety of host species eggs from the families Coreidae, Fulgoridae, Pentatomidae, and Saturniidae. Ten of the 16 species that were attacked in the no-choice tests were also attacked in the choice tests. The production of progeny on non-target egg masses was significantly lower than on the controls (L. delicatula egg masses run simultaneously) in the no-choice and choice tests. For the non-target species that were attacked and resulted in female wasp progeny, these females were able to produce their own progeny at the same rate as control females that were reared from the L. delicatula eggs. Larger host eggs corresponded to an increased female-biased sex ratio of the progeny, suggesting that gravid females select them for fertilized eggs. Results from these studies suggest that A. orientalis Haplotype C prefers to parasitize L. delicatula egg masses but is capable of developing in some non-target species.

Spotted lanternfly (SLF) (Lycorma delicatula (White)), an invasive planthopper discovered in Pennsylvania, USA in 2014, continues to spread and is now present in 14 states with substantial infestations present in seven states. Population projections using adult SLF trapping or visual counts are not reliable due to the transient, migratory behavior of the adults which make population forecasts difficult. Another approach to population monitoring is utilization of the stationary egg mass stage, but counting small cryptic egg masses throughout the canopy of large trees in dense woodlots is arduous and prone to error. After several field seasons testing various trapping configurations and materials, we have identified an efficient, simple, low-cost trap termed a ‘lamp shade trap’ that is attached to the lower trunk area of an SLF host tree. SLF females readily enter the trap and lay eggs on the thin, flexible trap surface. A vertical trap orientation was superior, and the most productive woodlots yielded an average of 47 and 54 egg masses per trap, and several traps had over 100 egg masses. There were 1,943 egg masses tallied from 105 traps placed at six locations in two states. Egg mass counts in the area above and below the traps and on nearby control trees yielded very few egg masses in comparison. Selection of trees 15 to 20 cm in diameter for trap placement is most efficient, yielding good egg mass abundance while minimizing the amount of trap material used. The lamp shade trap has potential as an effective tool to identify SLF in new areas, gauge SLF population levels in woodlots and can also be used to collect and monitor egg masses for research purposes.

The grass-cutting ant Atta vollenweideri is well suited for studies examining the negative effect leaf-cutting ants have on livestock production in South American grasslands because they forage on the same plants as cattle. This study investigated the impact of A. vollenweideri on livestock production in Argentinean rangelands. First, we assessed A. vollenweideri herbivory rates and its economic injury level (EIL). Second, using satellite imagery in a region covering 15,000 ha, we estimated the percentage of this area that surpassed the calculated EIL. Results showed that A. vollenweideri consumed approximately 276 kg of dry plant weight/ha/year, foraging mostly on grasses (70%). Additionally, ants cut 25% of herbs and 5% of trees. In summer and autumn, ants consumed more grasses, while in winter and spring, herbs and trees were also significantly cut. Ants consumed 7% of the forage demand needed to raise a calf according to the management regime applied by farmers. Our calculated EIL (5.85 nests/ha) falls in the range of previous studies. Colonies were absent in 93.6% of the surveyed area, while their density was below the EIL in 6.2% of the area. A. vollenweideri populations surpassed the EIL in only 0.2% of the area, which corresponds to 2.6% of the locations holding colonies. These results question the perception that Atta leaf-cutting ants are a pest of livestock production. Although ants consume a small percentage of cattle’s forage demand, evidence that ants and cattle are competing in the few cases in which density surpasses the EIL is arguable. First, grass-cutting ants are capable of consuming herbs and trees in addition to the grasses on which cattle mostly feed. Second, there is no evidence indicating that both are cutting the same plant portions when preferences overlap. Third, evidence suggests that ants are not displaced under high-pressure grazing regimes by cattle. In the countries where A. vollenweideri is present, decision makers have promulgated several acts making its control mandatory. It is time to revisit the pest status of A. vollenweideri and include the use of EIL as a control criterion.

We examined amino acid appetite in the omnivorous house cricket (Acheta domesticus), a common model organism for both research and teaching. Our first experiment addressed the hypothesis that house crickets can discriminate between sucrose and essential amino acids (EAA), and that preference for the latter would be affected by prior feeding experience. To test this hypothesis, we compared feeding responses of juvenile and adult crickets following pre-feeding with sucrose or an essential amino acid mixture, predicting that sucrose-only pre-feeding would enhance subsequent intake of amino acids in a two-choice preference test. Based on previous studies, we also predicted that amino acid consumption would be enhanced in females compared to males, and in mated compared to virgin females. Hence we compared responses in male and female last instar nymphs, adult males, virgin females, mated females, and mated females allowed to lay eggs. The second experiment examined how extended periods of essential amino acid deprivation (48 h to 6 days) affected appetite for these nutrients in adult male and female insects. Finally, we examined growth and survival of juvenile and adult crickets fed a holidic diet lacking all amino acids and protein. Our results demonstrated that house crickets can distinguish EAA from sucrose and that consumption of the former is enhanced following sucrose-only pre-feeding. We also found sex and developmental differences, with juvenile and virgin females showing a greater preference for EAA than juvenile or adult males. Contrary to expectation, mated females preferred sucrose over EAA both prior to and after egg laying. We also found that the crickets of both sexes increased their intake of EAA when exposed to longer periods of deprivation, indicating that they engage in compensatory feeding on these nutrients. Finally, as expected we found that growth was severely limited in juveniles fed a diet lacking all amino acids, but adults and many juveniles survived for 30 days on this diet.

Rhodnius prolixus, a major vector of Chagas disease, may be considered the model upon which the foundations of insect physiology and biochemistry were built. It is an obligate blood feeder in which the blood meal triggers growth, development and reproduction. The blood meal also triggers a post-prandial diuresis to maintain osmotic homeostasis. In R. prolixus, as with other insects, the Malpighian tubules play a critical role in this diuresis, and much has been learned about diuresis in R. prolixus, and in other model insects. But the post-genomic era has brought new insights, identifying functions quite apart from diuresis for Malpighian tubules. Indeed, microarrays, transcriptomes, and proteomics have revealed the major roles that Malpighian tubules play in immunity, detoxification, pesticide resistance, and in tolerance to overall stress. This is particularly relevant to R. prolixus since gorging on blood creates several challenges in addition to osmotic balance. Xenobiotics may be present in the blood or toxins may be produced by metabolism of blood; and these must be neutralized and excreted. These processes have not been well described at the molecular level for Malpighian tubules of R. prolixus. This paper will review the involvement of Malpighian tubules in immunity and detoxification, identifying new aspects for Malpighian tubule physiology of R. prolixus by virtue of a transcriptome analysis. The transcriptome analysis indicates the potential of Malpighian tubules of R. prolixus to mount a robust innate immune response, and to contribute to antioxidant production and heme detoxification.

IntroductionTemperature fluctuations are important for the distribution and survival of insects. Rapid hardening, a type of phenotypic plasticity, is an adaptation that can help individuals better tolerate lethal temperatures because of earlier exposure to a sublethal but stressful temperature. Nutrition and sex are also known to influence a species ability to tolerate thermal stress. This study determined the effects of larval diet, adult diet, sex and hardening on the thermal tolerance of Ceratitis cosyra (Walker) (Diptera: Tephritidae) at lower and upper lethal temperatures.MethodsLarvae were raised on either an 8% torula yeast (high) or a 1% torula yeast (low) larval diet and then introduced to one of three dietary regimes as adults for thermal tolerance and hardening assays: no adult diet, sugar only, or sugar and hydrolysed yeast diet. Flies of known weight were then either heat- or cold-hardened for 2 hours before being exposed to a potentially lethal high or low temperature, respectively.ResultsBoth nutrition and hardening as well as their interaction affected C. cosyra tolerance of stressful temperatures. However, this interaction was dependent on the type of stress, with nutrient restriction and possible adult dietary compensation resulting in improved cold temperature resistance only.DiscussionThe ability of the insect to both compensate for a low protein larval diet and undergo rapid cold hardening after a brief exposure to sublethal cold temperatures even when both the larva and the subsequent adult fed on low protein diets indicates that C. cosyra have a better chance of survival in environments with extreme temperature variability, particularly at low temperatures. However, there appears to be limitations to the ability of C. cosyra to cold harden and the species may be more at risk from long term chronic effects than from any exposure to acute thermal stress.

Spotted lanternfly (SLF) (Lycorma delicatula (White)), an invasive planthopper discovered in Pennsylvania, U.S.A. in 2014, feeds for approximately six months by sucking phloem sap from trunks and limbs of tree of heaven, Ailanthus altissima, along with several native trees and woody vines. Basal trunk sprays of dinotefuran, a systemic neonicotinoid insecticide, are commonly used to reduce SLF densities and spread. Information on dinotefuran persistence and within-tree distribution can help identify optimal timing of annual basal trunk sprays, facilitating efficient use of available resources. We applied dinotefuran to 20 uninfested A. altissima trees in early April then periodically sampled foliage to monitor insecticide residues. Foliar dinotefuran residues averaged (± SE) 7.8 ± 1.1 and 6.3 ± 1.2 in July and August, respectively, then dropped significantly to 2.6 ± 0.5 ppm in September. In a second study, 20 A. altissima trees were similarly treated with dinotefuran basal trunk sprays in early June. Trees were felled to collect foliage and phloem from branches and the trunk in either mid-July or September. Foliar residues averaged 12.7 ± 1.3 and 14.6 ± 2.2 ppm in July and September, respectively. For trees felled in July, residues were detected in phloem collected from below the spray line on trunks of seven trees and above the spray line on three trees, averaging 8.6 ± 4.4 and 7.4 ± 2.9 ppm, respectively. In trees felled in September, phloem from below spray lines of seven trees averaged 3.7 ± 1.3 ppm but dinotefuran was not detected in phloem from above the spray line on any trees. Dinotefuran was not detected in phloem sampled from any branches in either July or September. Results suggest dinotefuran basal trunk sprays applied between late May and mid June should persist long enough to effectively control SLF late instars and adults.

IntroductionInsecticidal RNAi is a targeted pest insect population control measure. The specificity of insecticidal RNAi can theoretically be enhanced by using symbiotic bacteria with a narrow host range to deliver RNAi, an approach termed symbiont-mediated RNAi (SMR), a technology we have previously demonstrated in the globally-invasive pest species Western Flower Thrips (WFT).MethodsHere we examine distribution of the two predominant bacterial symbionts of WFT, BFo1 and BFo2, among genome-sequenced insects. Moreover, we have challenged two non-target insect species with both bacterial species, namely the pollinating European bumblebee, Bombus terrestris, and an insect predator of WFT, the pirate bug Orius laevigatus. ResultsOur data indicate a very limited distribution of either symbiont among insects other than WFT. Moreover, whereas BFo1 could establish itself in both bees and pirate bugs, albeit with no significant effects on insect fitness, BFo2 was unable to persist in either species.DiscussionIn terms of biosafety, these data, together with its more specific growth requirements, vindicate the choice of BFo2 for delivery of RNAi and precision pest management of WFT.

IntroductionSocial organisms, including honey bees (Apis mellifera L.), have defense mechanisms to control the multiplication and transmission of parasites and pathogens within their colonies. Self-grooming, a mechanism of behavioral immunity, seems to contribute to restrain the population growth of the ectoparasitic mite Varroa destructor in honey bee colonies. Because V. destructor is the most damaging parasite of honey bees, breeding them for resistance against the mite is a high priority of the beekeeping industry.MethodsA bidirectional breeding program to select honey bee colonies with low and high V. destructor population growth (LVG and HVG, respectively) was conducted. Having high and low lines of bees allowed the study of genetic mechanisms underlying self-grooming behavior between the extreme genotypes. Worker bees were classified into two categories: ‘light groomers’ and ‘intense groomers’. The brains of bees from the different categories (LVG-intense, LVG-light, HVG-intense, and HVG-light) were used for gene expression and viral quantification analyses. Differentially expressed genes (DEGs) associated with the LVG and HVG lines were identified.ResultsFour odorant-binding proteins and a gustatory receptor were identified as differentially expressed genes. A functional enrichment analysis showed 19 enriched pathways from a list of 219 down-regulated DEGs in HVG bees, including the Kyoto Encyclopedia of Genes and Genomes (KEGG) term of oxidative phosphorylation. Additionally, bees from the LVG line showed lower levels of Apis rhabdovirus 1 and 2, Varroa destructor virus -1 (VDV-1/DWV-B), and Deformed wing virus-A (DWV-A) compared to bees of the HVG line. The difference in expression of odorant-binding protein genes and a gustatory receptor between bee lines suggests a possible link between them and the perception of irritants to trigger rapid self-grooming instances that require the activation of energy metabolic pathways.DiscussionThese results provide new insights on the molecular mechanisms involved in honey bee grooming behavior. Differences in viral levels in the brains of LVG and HVG bees showed the importance of investigating the pathogenicity and potential impacts of neurotropic viruses on behavioral immunity. The results of this study advance the understanding of a trait used for selective breeding, self-grooming, and the potential of using genomic assisted selection to improve breeding programs.

BackgroundCulex pipiens and Aedes albopictus are closely related to human life, and transmit a variety of viruses, causing serious harm to human health. Cytochrome c oxidase I (COI) gene has been selected as a marker gene for studying phylogeny and molecular evolution of species and is also an effective molecular marker for studying the evolutionary mechanism and systematic reconstruction of diptera insects.MethodsA loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of Cx. pipiens and Ae. albopictus were first described in this study. The experimental results were verified by real-time PCR.ResultsOur study showed the lower limit of sample concentration that can be detected by LAMP method is 0.5 pg/μl within 20 min for Cx. pipiens, and 1 pg/μl within 20 min for Ae. albopictus, which were more sensitive than PCR method. Validation tests with field samples showed LAMP method had good specificity and sensitivity and could identify the target species quickly and accurately.ConclusionThe LAMP method developed in this study allowed the rapid and sensitive detection of Cx. pipiens and Ae. albopictus, which will be expected to be used for mass screening in batches of the field.

IntroductionBiophysical approaches validated against haplotype and trap catch patterns have modeled the migratory trajectory of fall armyworms at a semi-continental scale, from their natal origins in Texas or Florida through much of the United States east of the Rocky Mountains. However, unexplained variation in the validation analysis was present, and misalignments between the simulated movement patterns of fall armyworm populations and the haplotype ratios at several locations, especially in the northeastern US and Canada, have been reported.MethodsUsing an expanded dataset extending into Canada, we assess the consistency of haplotype patterns that relate overwintered origins of fall armyworm populations to hypothesized dispersal trajectories in North America and compare the geographic distribution of these patterns with previous model projections.Results and discussionWe confirm the general accuracy of previous modeling efforts, except for late in the season where our data suggests a higher proportion of Texas populations invading the northeast, extending into eastern Canada. We delineate geographic limits to the range of both overwintering populations and show that substantial intermixing of the Texas and Florida migrants routinely occurs north of South Carolina. We discuss annual variation to these migratory trajectories and test the hypothesis that the Appalachian Mountains influence geographic patterns of haplotypes. We discuss how these results may limit gene flow between the Texas and Florida natal populations and limit the hereditary consequences of interbreeding between these populations.

The invasive planthopper, spotted lanternfly (SLF), Lycorma delicatula (White) (Hemiptera: Fulgoridae), feeds on a broad range of plants including species of economic importance such as grape. Although SLF feeds on wild and cultivated grape, the effect of grapevines on the insect’s life history traits is unknown. This study examined the effect of cultivated Concord grapevines (Vitis labrusca) and the insect’s preferred host tree of heaven (TOH), Ailanthus altissima, on SLF development, survival, reproduction, and body mass. Newly emerged nymphs were allowed to feed on either TOH, Concord grapevines or a mixed diet of Concord grapevines plus TOH through adulthood until death. Development, mortality, and oviposition of paired adults were tracked daily to calculate the SLF rate of development, survival, and reproduction among treatments. When feeding exclusively on Concord grapevines, SLF was able to develop and reproduce but had higher mortality, slower development, and produced fewer eggs. SLF fed on the mixed diet of grapevines plus TOH exhibited faster nymphal development, laid more eggs, and had higher body mass compared with those fed only on grape or TOH. SLF had greater survival when fed on either the mixed diet or on TOH alone. We conclude that Concord grapevines are a poor-quality host for SLF, but when combined with TOH, SLF fitness increases above that of feeding on TOH alone. This study supports the elimination of TOH as a part of SLF vineyard management practices.

The selection of honeybee strains resistant to the ectoparasitic mite Varroa destructor is generally considered as one of the most sustainable ways of coping with this major bee parasite. Thus, breeding efforts increasingly focus on resistance parameters in addition to common beekeeping traits like honey yield and gentleness. In every breeding effort, the success strongly depends on the quantifiability and heritability of the traits accounted. To find the most suitable traits among the manifold variants to assess Varroa resistance, it is necessary to evaluate how easily a trait can be measured (i.e., testing effort) in relation to the underlying heritability (i.e., expected transfer to the following generation). Various possible selection traits are described as beneficial for colony survival in the presence of Varroa destructor and therefore are measured in breeding stocks around the globe. Two of them in particular, suppressed mite reproduction (SMR, sensu lato any reproductive failure of mother mites) and recapping of already sealed brood cells have recently gained increasing attention among the breeders because they closely resemble resistance mechanisms of some Varroa-surviving honeybee populations. However, it was still unknown whether the genetic background of the trait is sufficient for targeted selection. We therefore investigated the heritabilities and genetic correlations for SMR and REC, distinguishing between recapping of infested cells (RECinf) and all cells (RECall), on an extensive dataset of Buckfast and Carniolan stock in Germany. With an accessible h² of 0.18 and 0.44 for SMR and an accessible h² of 0.44 and 0.40 for RECinf, both traits turned out to be very promising for further selection in the Buckfast and Carnica breeding population, respectively.

Lycorma delicatula (Hemiptera: Fulgoridae), the spotted lanternfly, native to China, invaded and established in the northeast U.S. in 2014. Since this time, populations have grown and spread rapidly, and invasion bridgeheads have been detected in mid-western states (i.e., Indiana in 2021). This invasive pest presents a significant threat to Californian agriculture. Therefore, a proactive classical biological control program using Anastatus orientalis (Hymenoptera: Eupelmidae), a L. delicatula egg parasitoid native to China, was initiated in anticipation of eventual establishment of L. delicatula in California. In support of this proactive approach, the potential host range of A. orientalis was investigated. Eggs of 34 insect species either native or non-native to the southwestern U.S. were assessed for suitability for parasitism and development of A. orientalis. Of the native species tested, 10, 13, and one were Hemiptera, Lepidoptera, and Mantodea, respectively. Of the non-native species, eight Hemiptera and two Lepidoptera were evaluated. Host range tests conducted in a quarantine facility, exposed individually mated A. orientalis females (Haplotype C) to non-target and target (i.e., L. delicatula) eggs in sequential no-choice and static choice experiments to determine suitability for parasitization and development. Additionally, the sex ratio, fertility, and size of offspring obtained from non-target and target eggs were evaluated. Results of host range testing indicated that A. orientalis is likely polyphagous and can successfully parasitize and develop in host species belonging to at least two different orders (i.e., Hemiptera, Lepidoptera) and seven families (Coreidae, Erebidae, Fulgoridae, Lasiocampidae, Pentatomidae, Saturniidae and Sphingidae). Prospects for use of A. orientalis as a classical biological control agent of L. delicatula in the southwestern U.S. are discussed.

The northern giant hornet, Vespa mandarinia (Hymenoptera: Vespidae), was detected for the first time in North America in 2019. Four nests have since been located and removed in northwestern Washington State as part of an extensive survey and eradication program. This recent introduction into North America has prompted new research on the biology and ecology of V. mandarinia to help inform management strategies. In its native range, V. mandarinia is known to prey on a variety of insects including the economically important honey bee species Apis cerana and Apis mellifera. Although A. cerana has developed defense mechanisms against attack by V. mandarinia, A. mellifera have no such defenses and an entire hive can be quickly destroyed by only a few hornets. In North America the hornet has been observed foraging on paper wasps (Polistes dominula) and honey bees, but little else is known about prey use in its novel range. To address this knowledge gap, we employed a DNA metabarcoding approach to characterize species detected in larval feces collected from 3 of the 4 Washington V. mandarinia nests found to date. Sequences were recovered for 56 species across fourteen orders, of which 36 species were likely prey items and 20 were suspected inquilines. The most frequently detected species were other social Hymenoptera, with Dolichovespula maculata, P. dominula, and A. mellifera present in most samples. All of the species detected, except for A. mellifera, represent new prey records for V. mandarinia, with eight families of insects newly associated with giant hornets. These results suggest that V. mandarinia in Washington preys on an assortment of insects similar to those documented in its native range, and that this new invader has readily incorporated novel species into its foraging and diet.

Verson’s glands are segmental pairs of dermal glands attached to the epidermis in lepidopteran larvae. They produce macromolecules during intermolt period and empty them during each molt. Morphological, histochemical, developmental, and protein analysis studies have been conducted to determine the functions of Verson’s glands. However, the exact role of Verson’s glands remains unclear. In our previous study, a strain of transgenic fall armyworm, Spdoptera frugiperda expressing green fluorescence protein (GFP) and Systemic RNA interference defective protein 1 (SID1) from Caenorhabditis elegans was established to improve RNA interference (RNAi) efficiency. Unexpectedly, we found that GFP fluorescence was significantly brighter in Verson’s glands than in other tissues. Also, RNAi efficiency improved more in Verson’s glands than in other tissues. We took advantage of improved RNAi efficiency to explore the function of Verson’s glands. RNA-seq analysis revealed that genes highly expressed in Verson’s glands code for cuticular proteins, molting fluid proteins, hemolymph proteins, and antimicrobial peptides. Injection of dsRNA targeting essential genes, inhibitor of apoptosis (IAP), Actin, and vacuolar-type ATPase (VATPase) interfered with Verson’s glands growth. These results revealed that Verson’s glands may contribute to hemolymph, cuticle, molting fluid, and immune response during molting. This study also provide useful tools for future research in identifying the physiological role of Verson’s glands in lepidopteran insects.

Host range assessment for emerging invasive insects is a vital step toward fully defining the issues the insect may pose. Spotted lanternfly (SLF) is an invasive species that is rapidly expanding its presence in the United States. The primary hosts facilitating this spread are tree of heaven, a plant from SLF’s native range, and the economically important winegrape. Black walnut is also implicated as an important and common host plant. This study investigated the survival and development of SLF on diets that included a variety of crop host plants in the presence or absence of tree of heaven. The following plant species, ‘Honeycrisp’ apple, ‘Reliance’ peach, silver maple, and tree of heaven were paired with winegrape or black walnut throughout the study. SLF had strong development and high survival on a diet of winegrape alone, and winegrape or black walnut paired with tree of heaven. Survival parameters were reduced with all other plant pairings. In particular, SLF in the winegrape and peach diet treatment did not develop past the third nymphal instar. A second experiment evaluated the survival of early and late instar nymphs and adult SLF life stages on three specialty crops – ‘Cascade’ hops, muscadine grapes, and kiwifruit over a two-week period. Nymphs survived longer than adults, with survival of first and second instar nymphs on hops not differing from the control tree of heaven treatment. The adult stage survived best on kiwi and muscadine grape. Our results show tree of heaven and winegrape were the only single plant diets evaluated that are sufficient for complete SLF development, while other host plants may require additional host or hosts of sufficient nutritional quality for SLF survival.

Emerald ash borer (EAB), Agrilus plannipenis Fairmaire, is an invasive insect accidentally introduced to North America from Asia that attacks and kills ash trees (Fraxinus spp.). A common control strategy in urban centers has been the injection of systemic insecticides into mature trees, which can be costly at large scales. This study investigated whether treating a subset of a susceptible urban ash population could confer associational protection to untreated trees; i.e. improving or maintaining crown health of the latter. We selected approximately 100 mature ash trees along city streets in each of 12 sites in central and southeastern Minnesota in 2017. Each site had low but growing infestations of EAB such that canopy decline was not yet widespread. We treated 50% of trees with emamectin benzoate in eight sites and 50% of trees in four sites with azadirachtin in site-wide spatial gradients, such that the remaining 50% of trees at all sites were left untreated. Crown health of all trees was monitored for five years (2017 to 2021). Across all sites, we noted an overall maintenance or increase in crown health of both treated and untreated trees, while groups of untreated reference trees approximately three km distant from each site to monitor general tree health and EAB pressure declined quickly. These results suggested that protective benefits were conferred by treated trees to untreated trees within sites. Quantifying the spatial scale of canopy preservation of untreated trees within sites proved challenging due to the lack of variation in crown condition between treated and untreated trees. In two of the twelve sites treated with emamectin benzoate, we noted statistical evidence of improvements in crown condition of untreated trees when located within 100m of treated trees. Treating a subset of a susceptible ash population may aid in preserving untreated trees and provides a basis for developing a more cost-effective and environmentally favorable treatment regimen against EAB.

The spotted lanternfly is an invasive pest for which we lack individual movement data due in part to the difficulty posed by individual identification. We developed a computer‐aided method to identify individual adult spotted lanternfly using wing spot patterns from photos processed in the software I3S and demonstrated the method’s accuracy with lab and field validations. Based on 176 individuals in the lab, we showed that digitizing the spots of one wing allowed a 100% reliable individual identification. The errors due to user input and the variation in the angle of the image were largely negligible compared to inter-individual variations. We applied this method in the context of a mark-recapture experiment to assess the feasibility of this method in the field. We initially identified a total of 84 unique spotted lanternflies, 31 of which were recaptured after four hours along with 49 new individuals. We established that the analysis of recaptures can possibly be automated based on scores and may not require systematic visual pairwise comparison. The demonstration of the effectiveness of this method on relatively small sample sizes makes it a promising tool for field experimentation as well as lab manipulations. Once validated on larger datasets and in different contexts, it will provide ample opportunity to collect useful data on spotted lanternfly ecology that can greatly inform management.