Genetics of flight in spongy moths (Lymantria dispar ssp.): functionally integrated profiling of a complex invasive trait.

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.

Is there hybridization between 2 species of the same genus in sympatry?-The genetic relationships between Anoplophora glabripennis, Anoplophora chinensis, and putative hybrids.

Anoplophora glabripennis (Asian longhorn beetle, ALB) and Anoplophora chinensis (Citrus longhorn beetle, CLB) are native forest pests in China; they have become important international quarantine pests. They are found using the same Salix aureo-pendula host tree of Cixi, Zhejiang province, China. On this host tree, we collected additional beetles that appeared to be morphologically intermediate between ALB and CLB. By using a stereoscope, we observed that there were several bumps on the base of the elytra, which was inconsistent with ALB, which typically has a smooth elytral base, but was more like CLB, which has numerous short tubercles on the elytral base. Given their sympatry and intermediate morphology, we hypothesized that these may represent ALB × CLB hybrids. We studied the genomic profiles for 46 samples (ALB, CLB, and putative hybrids) using genotyping-by-sequencing (GBS) providing a reduced representation of the entire genome. Employing principal component analyses on the 163 GBS-derived single nucleotide polymorphism data, we found putative hybrids tightly clustered with ALB, but genetically distinct from the CLB individuals. Therefore, our initial hybrid hypothesis was not supported by genomic data. Further, while mating experiments between adult ALB and CLB were successful in 4 separate years (2017, 2018, 2020, and 2021), and oviposition behavior was observed, no progeny was produced. Having employed population genomic analysis and biological hybridization experiments, we conclude that the putative hybrids represent newly discovered morphological variants within ALB. Our approach further confirmed the advantage of genome-wide information for Anoplophora species assignment in certain ambiguous classification cases.

The potential climatic range of spotted lanternfly may be broader than previously predicted.

Spotted lanternfly (Lycorma delicatula White) is an invasive planthopper that was introduced to the United States from Asia and readily spreads via human aided means. Three geographically separated populations in the United States (NJ, PA, and WV) were collected and used to assess the effects of fluctuating thermal regimes that included temperatures above or below the upper (Tmax) and lower (Tmin) developmental thresholds, respectively, on nymphal survival and development, and to determine if there was within- and among-population variation in hatch timing and temperature responses of nymphs. Nymphs exposed to temperatures > Tmax and

Estivation and Postestivation Development of Hemlock Woolly Adelgid (Adelges tsugae) (Hemiptera: Adelgidae) at Different Temperatures.

Hemlock woolly adelgid (Adelges tsugae) is the most important pest of hemlocks in the eastern United States, where it completes three generations a year. We investigated the impact of temperature (8, 12, 16, and 20°C) on the estivation and postaestivation stages of the A. tsugae sistens generation. Temperature significantly impacted development and survival of this generation. The highest mortality occurred at the coolest temperature (8°C). Adelges tsugae developed rapidly as the temperature increased and optimum temperatures for development ranged between 17 and 22°C for the different instars. The estimated lower temperature threshold was 0°C for second instar nymphs and 3 -5°C for the other instars and the preoviposition period. Estivating first-instar sistentes resumed development (as evidenced by segments becoming visible) after 40-100 d at the constant temperatures (fastest at 16°C) then required only 105 degree-days (DD) for 50% of the individuals to molt. Subsequent instars developed rapidly (another 470 DD total to reach adult), and oviposition began at ~623 DD from the time the first instars resumed development. This study provides valuable data required to develop an annual phenology model for A. tsugae which will assist in timing monitoring and control treatments.

Effects of Dietary Iron Quantities and Sources on Lymantria dispar (Lepidoptera: Erebidae) Survival and Development.

Lymantria dispar L. is a serious invasive defoliator of broadleaf trees in both North America and Eurasia. When rearing L. dispar for research, the artificial diet must have a source of iron that is bioavailable and in the quantity needed to ensure normal development and survival. The standard iron supplement in L. dispar diet, amorphous ferric phosphate, is no longer commercially available, which motivated studies on alternative iron sources and concentrations. The responses of three L. dispar populations (including an L. dispar asiatica Vnukovskij population) to seven different iron doses were determined over two generations. The response to eight iron compounds and no added iron was also assessed over two generations for an L. dispar dispar population. The optimal levels of iron were 100, 150, and 198 mg/l of available iron for the New Jersey Standard Strain, a near-wild West Virginia population, and a near-wild Russian Far East population, respectively. All three populations showed symptoms of insufficient iron when reared on the lowest dose of iron and signs of excess iron at the highest two doses. All of the alternate iron sources evaluated provided adequate available iron, although three sources had some issues that may not make them the best options. These results reveal differences in nutritional requirements among different populations of L. dispar and verify the existence of viable replacement sources of iron for the standard amorphous ferric phosphate long used in L. dispar rearing.

Effects of Temperature on Anoplophora chinensis (Coleoptera: Cerambycidae) Larvae and Pupae.

Anoplophora chinensis (Forster) is a xylophagous invasive cerambycid whose larvae feed on the lower bole and exposed roots of many tree species in orchard, urban, and forested habitats. Larval survival and development of A. chinensis from Italy and China were evaluated at eight constant temperatures (5, 10, 15, 20, 25, 30, 35, and 40°C). Development was slow or did not occur at temperatures ≤10 and >35°C. The TMin for the first six instars and the pupa was <10°C, while TMin for the higher instars was closer to 12°C. The ultimate instar for both populations was insensitive to temperature. When the TMax thresholds could be estimated they were between 31 and 41°C. Temperature also influenced larval weight gain; larvae held at 25-30°C generally weighed the most from the fourth instar on. The number of degree days for 50% of the population to molt to the next instar increased with increasing instar for both populations up through the sixth instar. Anoplophora chinensis may use wood moisture content in conjunction with temperature as a cue as to when to pupate. These responses of A. chinensis to temperature can be used for developing phenological models to predict timing of stages for management or eradication efforts.

The impact of host plant species on instar duration and body weight of nymphal Lycorma delicatula.

The spotted lanternfly, Lycorma delicatula (White) (Hemiptera: Fulgoridae), is an invasive species of planthopper that was introduced to North America and is a threat to multiple industries. Nymphs and egg masses were collected to assess each instar's rate of development at a constant temperature of 25°C on the following hosts: Ailanthus altissima (Miller) (Sapindales: Simaroubaceae), Vitis labrusca (L.) (Vitales: Vitaceae), Salix babylonica (L.) (Malpighiales: Salicaceae), Acer rubrum (L.) (Sapindales: Sapindaceae), Celastrus orbiculata (Thunberg) (Celastrales: Celastraceae), Ocimum basilicum (L.) (Lamiales: Lamiaceae), and Rosa multiflora (Thunberg) (Rosales: Rosaceae). Host plant species was found to have a significant effect on developmental time for nymphs in the first through third instars, as well as on nymphal survival. Nymphs failed to develop through the second instar on O. basilicum and the third and fourth instars on A. rubrum. Host plant species also had a significant effect on the mean weight of nymphs in the first, second, and fourth instars (but not in the third instar), and on the hind tibia length and forewing width of adult nymphs. This variability in L. delicatula developmental time by host plant species can potentially impact phenology models, which should be updated to reflect these new insights. Rearing practices should also be refined to account for host plant influences on the physiology of L. delicatula.

Hybridization Potential of Two Invasive Asian Longhorn Beetles.

The Asian longhorned beetle (ALB), Anoplophora glabripennis (Motschulsky) and citrus longhorned beetle (CLB), Anoplophora chinensis (Förster) (both Coleoptera: Cerambycidae: Lamiinae), are high-risk invasive pests that attack various healthy hardwood trees. These two species share some similar host plants and overlapping distributions in large parts of their native ranges in China and the Korean peninsula as well as similar reproductive behaviors. The original Anoplophora malasiaca (Thomson) occurs in Japan and has been synonymized as CLB (hereafter referred to JCLB). In this study, a 30-min behavioral observation of paired adults, followed by a four-week exposure to host bolts, showed that ALB could not successfully cross with CLB. Mating was observed between female CLB and male ALB but not between female ALB and male CLB, no laid eggs hatched. JCLB males successfully crossed with ALB females to produce viable eggs although the overall percentage of hatched eggs was lower than those from conspecific mating pairs. However, ALB males could not successfully cross with JCLB females. CLB and JCLB mated and produced viable hybrid offspring and the hybrid F1 offspring eggs were fertile. These results suggest an asymmetrical hybridization between ALB and JCLB, and that both CLB and JCLB might be considered as two subspecies with different hybridization potential with congeneric ALB. Given their potential impacts on ecosystems and many economically important tree hosts, invasion of these geographically isolated species (ALB and JCLB) or distant subspecies (CLB and JCLB) into the same region may facilitate potential hybridization, which could be a potential concern for the management of these two globally important invasive forest pests. Further studies are needed to determine if fertile hybrid offspring are capable of breeding continually or backcrossing with parental offspring successfully.

Comparison of the Hatch of Newly Laid Lycorma delicatula (Hemiptera: Fulgoridae) Eggs From the United States After Exposure to Different Temperatures and Durations of Low Temperature.

Comparisons were made of the effects of temperature and duration of low temperature on hatch of newly laid egg masses of the invasive spotted lanternfly, Lycorma delicatula (White). Egg masses were collected in mid-October 2019 and estimated to be less than 14 d old. There was a significant positive nonlinear relationship between temperature and developmental rate (1/d) for eggs held at constant temperatures. The lower threshold for egg development was estimated as 7.39°C. Eggs held at constant 10, 15, and 20°C were estimated to require 635, 715, and 849 DD7.39, respectively, to develop. Egg hatch was variable, egg hatch rates were highest (58.4%) when held at a constant 15°C, though high rates (52.7%) were also obtained when eggs were held for 84 d at 10°C, then moved to 25°C. Almost all eggs enter diapause since very few eggs were able hatch when moved to 25°C after 7 d of chill at either 5 or 10°C. Chilling at 5 or 10°C increased percentage egg hatch as the duration in chill increased up to ~100 d and eggs held at 10°C were able to complete some or all the post-diapause development before being moved to 25°C. All egg masses were held at constant 16:8 (L:D) photoperiod and 65%RH. Our data suggest that temperature is the driving factor for diapause termination in spotted lanternfly, but other abiotic factors should be investigated. These identified developmental temperature threshold and degree day requirements for egg hatch will improve predictive distribution and phenological models.

Effects of Temperature on Development and Survival of Nymphal Lycorma delicatula (Hemiptera: Fulgoridae).

Lycorma delicatula (White), an invasive planthopper originally from Asia, is an emerging pest in North America. It is important to understand its phenology in order to determine its potential range in the United States. Lycorma delicatula nymphs were reared on Ailanthus altissima (Miller) (Sapindales: Simaroubaceae) at each of the following constant temperatures: 5, 10, 15, 20, 25, 30, 35, and 40°C. The time spent in each instar and survival was recorded. Developmental rate increased with temperature from 15 to 30°C for all instars, then declined again at higher temperatures. Nymphal survival was lower at 35°C than between 15 and 30°C for all instars, and first instars placed at 5, 10, and 40°C all died without molting. This suggests that L. delicatula survival and development may be affected in the Southern United States by high temperatures and developmental delays will occur under cool spring conditions. The lower developmental threshold was found to be 13.00 ± 0.42°C for first instars, 12.43 ± 2.09°C for second instars, 8.48 ± 2.99°C for third instars, and 6.29 ± 2.12°C for fourth instars. The degree-day (DD) requirement for nymphs in the previous instar to complete development to reach the second instar, third instar, fourth instar, and adult was 166.61, 208.75, 410.49, and 620.07 DD (base varied), respectively. These results provide key data to support the development of phenology models and help identify the potential range of L. delicatula in North America.

Pathology and genome sequence of a Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) isolate from Heilongjiang, China.

The pathogenicity and genome sequence of isolate LdMNPV-HrB of the gypsy moth alphabaculovirus, Lymantria dispar multiple nucleopolyhedrovirus from Harbin, Heilongjiang, China, were determined. A stock of this virus from one passage through the gypsy moth New Jersey Standard Strain (LdMNPV-HrB-NJSS) exhibited 6.2- to 11.9-fold greater pathogenicity against larvae from a Harbin colony of L. dispar asiatica than both Gypchek and a Massachusetts, USA LdMNPV isolate (LdMNPV-Ab-a624). Sequence determination and phylogenetic analysis of LdMNPV-HrB and LdMNPV-HrB-NJSS revealed that these isolates were most similar to other east Asian LdMNPV isolates with 98.8% genome sequence identity and formed a group with the east Asian LdMNPV isolates which was separate from groups of isolates from Russia, Europe, and USA.

Assessing Niche Shifts and Conservatism by Comparing the Native and Post-Invasion Niches of Major Forest Invasive Species.

Invasive species experience biotic and abiotic conditions that may (or may not) resemble their native environment. We explored the methodology of determining climatic niches and compared the native and post-invasion niches of four invasive forest pests to determine if these species experienced shifts or changes in their new climatic niches. We used environmental principle components analysis (PCA-env) method to quantify climatic niche shifts, expansions, and temporal changes. Furthermore, we assessed the effect of variable selection in the delineation and comparison of niche space. We found that variable selection influenced the delineation and overlap of each niche, whereas the subset of climatic variables selected from the first two PCA-env axes explained more variance in environmental conditions than the complete set of climatic variables for all four species. Most focal species showed climatic niche shifts in their invasive range and had not yet fully occupied the available niche within the invaded range. Our species varied the proportion of niche overlap between the native and invasive ranges. By comparing native and invasive niches, we can help predict a species' potential range expansion and invasion potential. Our results can guide monitoring and help inform management of these and other invasive species.

Comparison of Survival and Development of Gypsy Moth Lymantria dispar L. (Lepidoptera: Erebidae) Populations from Different Geographic Areas on North American Conifers.

Host utilization information is critical to managers for estimating the hosts at risk and potential geographic range for gypsy moths from different geographic origins. In this study, the development and survival of gypsy moths from all three subspecies on 13 North American conifers and three broadleaf hosts were compared. There was variation in the ability of gypsy moth larvae from different geographic origins to utilize (survive and develop on) key North American conifers. However, that variation was not consistent within gypsy moth subspecies, but instead was more consistent with populations from different origins being preadapted to utilize different hosts and having different biologic traits. Some Asian populations developed and survived well on some conifers while populations from Europe and North America gained weight faster and/or survived better than some Asian populations. Although development was slower and survival poorer on several of the conifers, first instar larvae were able to utilize conifers unless the needles were tough or feeding deterrents were present. Host phenology was also critical since the early instars fed preferentially on new foliage or buds. Gypsy moth larvae can utilize many hosts, so this makes it a very adaptable invasive species that warrants taking measures to prevent its spread.

Assessing the Potential Distribution of Asian Gypsy Moth in Canada: A Comparison of Two Methodological Approaches.

Gypsy moth (Lymantria dispar L.) is one of the world's worst hardwood defoliating invasive alien species. It is currently spreading across North America, damaging forest ecosystems and posing a significant economic threat. Two subspecies L. d. asiatica and L. d. japonica, collectively referred to as Asian gypsy moth (AGM) are of special concern as they have traits that make them better invaders than their European counterpart (e.g. flight capability of females). We assessed the potential distribution of AGM in Canada using two presence-only species distribution models, Maximum Entropy (MaxEnt) and Genetic Algorithm for Rule-set Prediction (GARP). In addition, we mapped AGM potential future distribution under two climate change scenarios (A1B and A2) while implementing dispersal constraints using the cellular automation model MigClim. MaxEnt had higher AUC, pAUC and sensitivity scores (0.82/1.40/1.00) when compared to GARP (0.70/1.26/0.9), indicating better discrimination of suitable versus unsuitable areas for AGM. The models indicated that suitable conditions for AGM were present in the provinces of British Columbia, Ontario, Quebec, Nova Scotia and New Brunswick. The human influence index was the variable found to contribute the most in predicting the distribution of AGM. These model results can be used to identify areas at risk for this pest, to inform strategic and tactical pest management decisions.

Mating Behavior and Reproductive Biology of Emerald Ash Borer (Coleoptera: Buprestidae) and Two of Its Native Congeners, the Twolined Chestnut Borer and the Bronze Birch Borer.

We studied the mating behavior and reproductive biology of three members of the genus Agrilus: the bronze birch borer, Agrilus anxius Gory; the twolined chestnut borer, Agrilus bilineatus (Weber); and the emerald ash borer, Agrilus planipennis Fairmaire. All three species share a highly stereotyped mating behavior. However, the copulation duration of A. planipennis was 90% longer than that of its two congeners. Female reproductive tracts of the three species were anatomically similar, as were the spermatophores. Within the spermatophores, sperm were single in A. anxius and A. bilineatus, while in A. planipennis, sperm were bundled in groups of approximately 20 in a hyaline sheath. We found that field-caught A. anxius and A. bilineatus had higher rates of female insemination than A. planipennis. In additional studies with A. planipennis and A. anxius, we found that mating duration was related to mating success, and fecundity for A. planipennis, but not for A. anxius. For both A. planipennis and A. anxius, the spermatophore was passed to the female toward the end of the copulatory period. Sperm were found in the spermatheca immediately after copulation ended in A. planipennis and 30 min after copulation ended in A. anxius. We present possible explanations for these differences.

A Phenology Model for Asian Gypsy Moth Egg Hatch.

Phenology models are useful tools in pest management interventions, biosecurity operations targeting alien invaders, and answering questions regarding the potential for range expansion/shift. The Gypsy Moth Life Stage model (GLS) has been used to predict the invasive range of the North American gypsy moth (Lymantria dispar dispar Linnaeus [Lepidoptera: Erebidae]) in North America and New Zealand. It has been used to examine the role of supra-optimal temperatures in range expansion/stasis/retraction. However, GLS has also been used where the target organism is the Asian subspecies L. d. asiatica Vnukovskij, despite observed differences between the predominant phenotypes of the two subspecies in the temperature requirements for egg hatch and the absence of egg phenology model parameters specific to the Asian phenotype. Here we describe the results of temperature and exposure duration on the timing of Asian gypsy moth egg hatch, and we present phenology model parameters for the Asian phenotype. Sum of squared differences (observed minus predicted day of median egg hatch) was reduced from 7,818 d2 (North American parameters) to 178 d2. Days of simulated median egg hatch differed from the observed days by 0-7 d (x¯=0.2; SD=3.1). The pattern of simulated egg hatch closely mimicked the irregular pattern of observed egg hatch from the temperature regimes of our experiment. Egg hatch is arguably the most important life cycle event in gypsy moth population suppression/eradication interventions and in estimating their potential invasive range. The model parameters described here produce accurate predictions of Asian gypsy moth egg hatch.

Effects of Temperature on First Instar Lymantria (Lepidoptera: Erebidae) Survival and Development With and Without Food.

Lymantria dispar L. and Lymantria monacha (L.) are Eurasian pests that have the potential for accidental introduction via trade into other world areas. Establishment of first instars of Lymantria depends on larvae surviving long enough to disperse and finding suitable hosts. The survival and development of newly hatched Lymantria larvae from nine geographic populations at seven temperatures (1-30°C) held without food, with summer foliage of a preferred or conifer host was determined. There was considerable variation both within and among the Lymantria populations in the survival of larvae at different temperatures when held with and without food. Without food survival declined from about a month at 5°C to a few days at 30°C, following a typical enzymatic kinetic rate function. At 1°C larval survival was less than at 5°C likely because the larvae were susceptible to freezing. Larvae from the one L. monacha population fed and gained weight on the summer foliage, particularly on the conifer, at 10-15°C but < 20% survived for 14 d at 20-30°C. The newly hatched L. dispar larvae from all eight populations fed (at 10-30°C) and developed (at 15-30°C) on the summer foliage of one or both of the hosts. This suggests that they may be able to find adequate food for establishment even if hatch is not synchronous with bud break in the invaded habitat. Survival on the conifer was highest for one Chinese and two European populations of L. dispar, suggesting the ability to utilize conifers is population and not subspecies specific.

Factors That Influence Flight Propensity in Anoplophora glabripennis (Coleoptera: Cerambycidae).

The effects of mating status, sex, beetle age, host quality, temperature, and wind speed on the propensity of Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) to take flight were evaluated using a free flight test in the laboratory. Time to initiate flight, the angle of flight, and flight capability (when beetles were enticed to take flight) were also evaluated. Host quality, mating status, beetle age, sex, temperature, and the interactions between one or more of these were all found to be significant predictors of flight in A. glabripennis in one or more of the experiments. Female flight propensity peaked at sexual maturity and declined thereafter. Both sexes had a higher propensity to take flight from a stem section of dry host material than from a fresh one. Most (78%) males flew at least once during the four mating status/ages tested from a fresh host stem section, while only 43% of the females flew at least once after chewing an oviposition pit. Flight propensity and distance flown increased with temperature and there was no voluntary flight at 15°C. Flight propensity did not increase with wind speeds 0.0-1.0 m/s, but no ascending flight was observed at 0.5 or 1.0 m/s. Time to flight initiation did not vary with the factors evaluated. Implications these results could have on the success of eradication programs are discussed. Specifically, what factors increase the propensity of mated females to disperse, effectively expanding the infestation zone.

A Variable-Instar Climate-Driven Individual Beetle-Based Phenology Model for the Invasive Asian Longhorned Beetle (Coleoptera: Cerambycidae).

Efforts to manage and eradicate invasive species can benefit from an improved understanding of the physiology, biology, and behavior of the target species, and ongoing efforts to eradicate the Asian longhorned beetle (Anoplophora glabripennis Motschulsky) highlight the roles this information may play. Here, we present a climate-driven phenology model for A. glabripennis that provides simulated life-tables for populations of individual beetles under variable climatic conditions that takes into account the variable number of instars beetles may undergo as larvae. Phenology parameters in the model are based on a synthesis of published data and studies of A. glabripennis, and the model output was evaluated using a laboratory-reared population maintained under varying temperatures mimicking those typical of Central Park in New York City. The model was stable under variations in population size, simulation length, and the Julian dates used to initiate individual beetles within the population. Comparison of model results with previously published field-based phenology studies in native and invasive populations indicates both this new phenology model, and the previously published heating-degree-day model show good agreement in the prediction of the beginning of the flight season for adults. However, the phenology model described here avoids underpredicting the cumulative emergence of adults through the season, in addition to providing tables of life stages and estimations of voltinism for local populations. This information can play a key role in evaluating risk by predicting the potential for population growth, and may facilitate the optimization of management and eradication efforts.