Diverse Host Plants of the First Instars of the Invasive Lycorma delicatula: Insights from eDNA Metabarcoding.

Identification of host plants of the invasive spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae), has been the focus of many studies. While the adults and late nymphs are relatively easy to observe on plants and to use for molecular gut-content analysis, studying the early instars is more challenging. This study is the continuation of our ongoing efforts to determine the host range for each developmental stage of L. delicatula. In the present study, we focused exclusively on the first nymphal instars, and we used a novel approach, utilizing "bulk" DNA extracts for DNA metabarcoding of nymphal gut contents, to identify all the detectable plants that the nymphs had ingested prior to being collected. We were able to obtain high-quality amplicons (up to 406 bp) of a portion of the rbcL gene and detect 27 unique ingested plant species belonging to 17 families. Both native and introduced plants with the prevalence of trees and grasses were present among the ingested plants. We also identified 13 novel host plants that have not been previously reported for L. delicatula on the U.S. territory. The results from our study have important applications for developing effective programs on early monitoring of invasive L. delicatula.

Environmental and Spatial Predictors of the Distribution Patterns of the Host-Seeking Black Fly, Simulium jenningsi (Diptera: Simuliidae).

Black flies are nuisance pests as adults, yet they are best managed in the larval stage in flowing waters. As a result, more effort is put into understanding the distribution of the immature life stages than the blood-seeking females that form nuisance. The seemingly localized nature of Simulium jenningsi Malloch (Diptera: Simuliidae) pest problems in western Maryland offered a study system to investigate the spatial and environmental correlates to their severity. Collections of adult black flies were taken at 260 sites within a 2,000 km2 region centered on Washington County, Maryland, during June, July, and August of 2 yr. Average S. jenningsi counts were greater in the June of both years compared to July and August. Although S. jenningsi was found at the majority of sampling sites, higher fly counts were significantly clustered in the southern portion of the county where the majority of resident complaints originated. A generalized linear mixed-model (GLMM) approach was used to determine the correlates to S. jenningsi abundance. The highest performing model showed a negative relationship of S. jenningsi counts with the amount of surrounding impervious surface, distance to the riffles along the confluence of the Shenandoah and Potomac Rivers, distance to the closest body of flowing water, and light intensity, as well as a positive relationship with elevation and air temperature. The results suggest S. jenningsi females are not readily found in urban environments in this study region, and the most relevant monitoring locations for S. jenningsi may be outside of human population centers.

Choosing an Effective PCR-Based Approach for Diet Analysis of Insect Herbivores: A Systematic Review.

Identification of ingested plant species using polymerase chain reaction (PCR)-based methods is an increasingly useful yet challenging approach to accurately determine the diet composition of insect herbivores and thus their trophic interactions. A typical process of detection of DNA of ingested plants involves the choice of a DNA extraction method, a genomic target region, and/or the best approach for an accurate plant species identification. The wide range of available techniques makes the choice of the most appropriate method for an accurately and timely identification of ingested plants from insect guts difficult. In our study, we reviewed the commonly used PCR-based approaches in studies published from 1977 to 2019, to provide researchers with the information on the tools which have been shown to be effective for obtaining and identifying ingested plants. Our results showed that among five insect orders used in the retrieved studies Coleoptera and Hemiptera were prevalent (33 and 28% of all the records, respectively). In 79% of the studies a DNA barcoding approach was employed. In a substantial number of studies Qiagen DNA extraction kits and CTAB protocol were used (43 and 23%, respectively). Of all records, 65% used a single locus as a targeted plant DNA fragment; trnL, rbcL, and ITS regions were the most frequently used loci. Sequencing was the dominant type of among DNA verification approaches (70% of all records). This review provides important information on the availability of successfully used PCR-based approaches to identify ingested plant DNA in insect guts, and suggests potential directions for future studies on plant-insect trophic interactions.

Response of Five Miscanthus sinensis Cultivars to Grasshopper Herbivory: Implications for Monitoring of Invasive Grasses in Protected Areas.

Introduced grasses can aggressively expand their range and invade native habitats, including protected areas. Miscanthus sinensis is an introduced ornamental grass with 100+ cultivars of various invasive potential. Previous studies have demonstrated that the invasive potential of M. sinensis cultivars may be linked to seed viability, and some of the physiological traits, such as growth rate. Little is known, however, about whether these traits are associated with response of M. sinensis to insect herbivory, and whether plant tolerance and resistance to herbivory vary among its cultivars; which, in turn, can contribute to the invasive potential of some of M. sinensis cultivars. To address this issue, in our study we explored the response of five cultivars of M. sinensis to herbivory by Melanoplus grasshoppers. We demonstrated that plant responses varied among the cultivars during a season; all the cultivars, but "Zebrinus", demonstrated a significant increase in plant tolerance by the end of the growing season regardless of the amount of sustained leaf damage. Different patterns in plant responses from "solid green" and "striped/spotted" varieties were recorded, with the lowest plant resistance detected for "Autumn Anthem" in the cage experiment. Our results have important applications for monitoring low-risk invaders in protected areas, as well as for biotic resistance of native communities to invasive grasses.

Detecting Ingested Host Plant DNA in Potato Leafhopper (Hemiptera: Cicadellidae): Potential Use of Molecular Markers for Gut Content Analysis.

Detection of host plant DNA from sap-feeding insects can be challenging due to potential low concentration of ingested plant DNA. Although a few previous studies have demonstrated the possibility of detecting various fragments of plant DNA from some sap-feeders, there are no protocols available for potato leafhopper, Empoasca fabae (Harris) (Hemiptera: Cicadellidae), a significant agricultural pest. In this study we focused on optimizing a DNA-based method for host plant identification of E. fabae and investigating the longevity of the ingested plant DNA as one of the potential applications of the protocol. We largely utilized and modified our previously developed PCR-based method for detecting host plant DNA from grasshopper and the spotted lanternfly gut contents. We have demonstrated that the trnL (UAA) gene can be successfully utilized for detecting ingested host plant DNA from E. fabae and determining plant DNA longevity. The developed protocol is a relatively quick and low-cost method for detecting plant DNA from E. fabae. It has a number of important applications-from determining host plants and dispersal of E. fabae to developing effective pest management strategies.

Perceptions and Responses of Residents to the Nuisance Black Fly Simulium jenningsi (Diptera: Simuliidae) in the Mid-Atlantic United States.

Current management practices of nuisance black flies are conducted on an area-wide level and rely on the support of the public to implement programs. In Maryland, a vocal group of residents campaigned their representatives to begin a management program for the black fly Simulium jenningsi Malloch. To determine how residents in Maryland and its surrounding states perceived the severity of black fly nuisance, we deployed surveys online and in-person on the ways their outdoor activities were impacted and the preventive methods used to mitigate nuisance. Online respondents, those with children, and those who had lived in the region for a shorter amount of time were more likely to report black flies as 'extremely annoying'. Quality-of-life concerns stemming from black fly swarms were primarily related to avoiding outdoor exercise and recreation. The majority of respondents used at least one method of personal protection against black fly annoyance, but satisfaction with any method was low. Methods used by respondents included the removal of standing water and rotting vegetation from their properties, indicating a lack of knowledge about black fly breeding habitats. The results contextualized the needs of residents in future management and topics for outreach efforts to address misconceptions about black fly biology. This study offers an example of the application of social science methodology in understanding the needs of stakeholders in area-wide pest management.

Use of Molecular Gut Content Analysis to Decipher the Range of Food Plants of the Invasive Spotted Lanternfly, Lycorma delicatula.

Spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae), is an introduced highly invasive insect pest in the US that poses a significant risk to forestry and agriculture. Assessing and predicting plant usage of the lanternfly has been challenging, and little is known regarding the lanternfly nymph association with its host plants. In this study, we focused on: (a) providing a protocol for using molecular markers for food plant identification of L. delicatula; (b) determining whether the ingested plant DNA corresponds with DNA of the plants from which the lanternfly was collected; and, (c) investigating the spectrum of ingested plants. We utilized gut contents of third and fourth instar nymphs that were collected from multiple plants; we isolated ingested plant DNA and identified consumed plants. We demonstrated that (a) up to 534 bp of the rbcL gene from ingested plants can be detected in L. delicatula guts, (b) ingested plants in ~93% of the nymphs did not correspond with the plants from which the nymphs were collected, and (c) both introduced and native plants, as well as woody and non-woody plants, were ingested. This information will aid effective the monitoring and management of the lanternfly, as well as predict the lanternfly host plants with range expansion.

External morphology and developmental changes of tarsal tips and mouthparts of the invasive spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae).

External structures of insects contribute to the ability of herbivores to select and feed on their host plants. The invasive spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae) is an economically important and polyphagous insect pest in the eastern US. The lanternfly causes substantial damage to many woody plants by sucking phloem sap, reducing photosynthesis, causing weeping wounds, and creating conditions for sooty mold. Lanternfly nymphs switch host plants during their development. However, little is known about relationship between the lanternfly and its plant hosts, and particularly about morphological adaptations of the lanternfly to host plant usage at each developmental stage of the pest. In this study, we focused on assessing changes in morphology of (a) the lanternfly mouthparts (stylets and labium), and (b) the lanternfly tarsal tips (arolia and tarsal claws) at each developmental stage. Our study revealed several developmental patterns among which the presence of the indentations on mandibular stylets in late instars and adults, as well as the exponential growth of the labium and stylet length, and the tarsal claw dispersal during the lanternfly development. Our findings are critical for investigating and predicting the lanternfly host range, and the lanternfly dispersal to new host trees at each developmental stage.

Short-Term Physiological Response of a Native Hawaiian Plant, Hibiscus arnottianus, to Injury by the Exotic Leafhopper, Sophonia orientalis (Hemiptera: Cicadellidae).

Sophonia orientalis (Matsumura), also known as the two-spotted leafhopper, is a widespread exotic pest of many economically important crop plants and ornamental plants in Hawaii. Sophonia orientalis is highly polyphagous and is a major threat to some of the native endemic plants. Despite the successful establishment in Hawaii, interactions of S. orientalis with its host plants remain poorly understood. Previous studies primarily focused on distribution, parasitism, and oviposition of S. orientalis in Hawaii, whereas plant physiological responses to the leafhopper's injury, and, specifically, gas exchange rates in plants, have not yet been described. In this study, we examined a short-term physiological response of a native Hawaiian plant, Hibiscus arnottianus (A. Gray), to injury by S. orientalis. We also explored whether Camellia sinensis (L.) Kuntze, a native host plant of S. orientalis in Asia, exhibits a similar response. We found that H. arnottianus plants demonstrated a rapid (2-d) physiological response to injury accompanied by 40% reduction in rate of photosynthesis and 42% reduction in rate of transpiration, whereas C. sinensis did not exhibit any reduction in gas exchange rates. We did not record any changes in plant chlorophyll levels after plant injury in either species. Our results suggest that H. arnottianus is responding to the leafhopper feeding with a generalized wound response predicted for novel plant-insect herbivore associations. We discuss potential future directions for studies which might focus on host plant responses to S. orientalis in its native versus introduced range.

Degree Day Requirements for Kudzu Bug (Hemiptera: Plataspidae), a Pest of Soybeans.

Understanding the phenology of a new potential pest is fundamental for the development of a management program. Megacopta cribraria Fabricius (Hemiptera: Plataspidae), kudzu bug, is a pest of soybeans first detected in the United States in 2009 and in Maryland in 2013. We observed the phenology of kudzu bug life stages in Maryland, created a Celsius degree-day (CDD) model for development, and characterized the difference between microhabitat and ambient temperatures of both kudzu, Pueraria montana (Lour.) Merr. (Fabales: Fabaceae) and soybeans, Glycine max (L.) Merrill (Fabales: Fabaceae). In 2014, low population numbers yielded limited resolution from field phenology observations. We observed kudzu bug populations persisting within Maryland; but between 2013 and 2016, populations were low compared to populations in the southeastern United States. Based on the degree-day model, kudzu bug eggs require 80 CDD at a minimum temperature of 14°C to hatch. Nymphs require 545 CDD with a minimum temperature of 16°C for development. The CDD model matches field observations when factoring a biofix date of April 1 and a minimum preoviposition period of 17 d. The model suggests two full generations per year in Maryland. Standard air temperature monitors do not affect model predictions for pest management, as microhabitat temperature differences did not show a clear trend between kudzu and soybeans. Ultimately, producers can predict the timing of kudzu bug life stages with the CDD model for the use of timing management plans in soybean fields.

Cold Tolerance of Megacopta cribraria (Hemiptera: Plataspidae): An Invasive Pest of Soybeans.

Kudzu bug, Megacopta cribraria Fabricius (Hemiptera: Plataspidae), first discovered in the United States in 2009, is an invasive pest of soybeans. From 2013 to 2016, Maryland has been the northern limit of its distribution in the United States. We sought to determine the physiological cold temperature limits, timing of movement to overwintering locations, and to characterize overwintering microhabitat temperature. We measured supercooling point (SCP) on three populations from distinct USDA plant hardiness zones in Maryland and Virginia between October and December of 2015. The average SCP across all sample months and populations was -12.6°C and no consistent trend of month or population location were observed. Additionally, we assessed the lower lethal temperature to kill 50% of the population (LLT50) at the same population locations in October and November 2015. The average LLT50 over both months and all three population locations was -5.1°C. Again, no consistent trend based on population location was observed but we did find a modest depression in the LLT50 values between October and November. We observed that kudzu bug overwinters in leaf litter and begins to move into the litter in late November to early December. Leaf litter moderates day to night temperature differences and was warmer than ambient temperature by an average of 0.7°C. Evidence suggests that the cold tolerance of the kudzu bug limits its distribution north of Maryland.

Contrasting Role of Temperature in Structuring Regional Patterns of Invasive and Native Pestilential Stink Bugs.

OBJECTIVES: Assessment and identification of spatial structures in the distribution and abundance of invasive species is important for unraveling the underlying ecological processes. The invasive agricultural insect pest Halyomorpha halys that causes severe economic losses in the United States is currently expanding both within United States and across Europe. We examined the drivers of H. halys invasion by characterizing the distribution and abundance patterns of H. halys and native stink bugs (Chinavia hilaris and Euschistus servus) across eight different spatial scales. We then quantified the interactive and individual influences of temperature, and measures of resource availability and distance from source populations, and their relevant spatial scales. We used Moran's Eigenvector Maps based on Gabriel graph framework to quantify spatial relationships among the soybean fields in mid-Atlantic Unites States surveyed for stink bugs. FINDINGS: Results from the multi-spatial scale, multivariate analyses showed that temperature and its interaction with resource availability and distance from source populations structures the patterns in H. halys at very broad spatial scale. H. halys abundance decreased with increasing average June temperature and distance from source population. H. halys were not recorded at fields with average June temperature higher than 23.5°C. In parts with suitable climate, high H. halys abundance was positively associated with percentage developed open area and percentage deciduous forests at 250m scale. Broad scale patterns in native stink bugs were positively associated with increasing forest cover and, in contrast to the invasive H. halys, increasing mean July temperature. Our results identify the contrasting role of temperature in structuring regional patterns in H. halys and native stink bugs, while demonstrating its interaction with resource availability and distance from source populations for structuring H. halys patterns. CONCLUSION: These results help predicting the pest potential of H. halys and vulnerability of agricultural systems at various regions, given the climatic conditions, and its interaction with resource availability and distance from source populations. Monitoring and control efforts within parts of the United States and Europe with more suitable climate could focus in areas of peri-urban developments with deciduous forests and other host plants, along with efforts to reduce propagule pressure.

Spatiotemporal Dynamics of the Invasive Halyomorpha halys (Hemiptera: Pentatomidae) in and Between Adjacent Corn and Soybean Fields.

Knowledge on movement and spatial patterns of insect pest populations among preferred hosts aids in the development of effective pest management strategies. In this study, we quantified the spatiotemporal dynamics of the invasive brown marmorated stink bug, Halyomorpha halys (Stål 1855), in relation to field corn, Zea mays L., and soybean, Glycine max (L.), crop phenology. We also examined the potential role of corn as a source of stink bugs in adjacent soybean. The highest density of stink bugs in each crop coincided with blister to milk-dough stages in corn (R2-R3/R4), and beginning seed to full seed (R5-R6) stages in soybean. In entire fields of adjacent corn and soybean, H. halys was found in very low density (<0.5/m(2)) or absent beyond 25 m from the field edge. Inverse distance weighted interpolations of H. halys densities suggest potential dispersal of H. halys, particularly adults and large nymphs, from corn into soybean, coinciding with the end of dough stage in corn and beginning of soybean seed development stage. These findings have important implications for managing H. halys through location and timing of scouting efforts, consideration of crop arrangement, and decisions on management interventions. Repeated scouting of field corn to assess H. halys densities, particularly from blister stage onwards, could inform decisions on management interventions for preventing or mitigating H. halys colonization into soybean. Where H. halys is an economic problem, reducing the extent of boundary shared between corn and soybean could reduce dispersal into soybean.

Climate Change and Phenology: Empoasca fabae (Hemiptera: Cicadellidae) Migration and Severity of Impact.

Climate change can benefit individual species, but when pest species are enhanced by warmer temperatures agricultural productivity may be placed at greater risk. We analyzed the effects of temperature anomaly on arrival date and infestation severity of potato leafhopper, Empoasca fabae Harris, a classic new world long distance migrant, and a significant pest in several agricultural crops. We compiled E. fabae arrival dates and infestation severity data at different states in USA from existing literature reviews and agricultural extension records from 1951-2012, and examined the influence of temperature anomalies at each target state or overwintering range on the date of arrival and severity of infestation. Average E. fabae arrival date at different states reveal a clear trend along the south-north axis, with earliest arrival closest to the overwintering range. E. fabae arrival has advanced by 10 days over the last 62 years. E. fabae arrived earlier in warmer years in relation to each target state level temperature anomaly (3.0 days / °C increase in temperature anomaly). Increased temperature had a significant and positive effect on the severity of infestation, and arrival date had a marginal negative effect on severity. These relationships suggest that continued warming could advance the time of E. fabae colonization and increase their impact on affected crops.

Adjacent habitat influence on stink bug (Hemiptera: Pentatomidae) densities and the associated damage at field corn and soybean edges.

The local dispersal of polyphagous, mobile insects within agricultural systems impacts pest management. In the mid-Atlantic region of the United States, stink bugs, especially the invasive Halyomorpha halys (Stål 1855), contribute to economic losses across a range of cropping systems. Here, we characterized the density of stink bugs along the field edges of field corn and soybean at different study sites. Specifically, we examined the influence of adjacent managed and natural habitats on the density of stink bugs in corn and soybean fields at different distances along transects from the field edge. We also quantified damage to corn grain, and to soybean pods and seeds, and measured yield in relation to the observed stink bug densities at different distances from field edge. Highest density of stink bugs was limited to the edge of both corn and soybean fields. Fields adjacent to wooded, crop and building habitats harbored higher densities of stink bugs than those adjacent to open habitats. Damage to corn kernels and to soybean pods and seeds increased with stink bug density in plots and was highest at the field edges. Stink bug density was also negatively associated with yield per plant in soybean. The spatial pattern of stink bugs in both corn and soybeans, with significant edge effects, suggests the use of pest management strategies for crop placement in the landscape, as well as spatially targeted pest suppression within fields.

Development of a biopolymer nanoparticle-based method of oral toxicity testing in aquatic invertebrates.

Aquatic toxicity testing generally focuses on the water absorption/dermal route of exposure to potential toxic chemicals, while much less work has been done on the oral route of exposure. This is due in part to the difficulties of applying traditional oral toxicity testing to aquatic environments, including the tendency for test chemicals to dissolve into water. The use of biopolymer nanoparticles to encapsulate test chemicals onto food to prevent dissolution is one solution presented herein. The biopolymers zein and chitosan were explored for their previously known nanoparticle-forming abilities. Nanoparticles containing the test chemical rhodamine B were formed, applied as films to coat food, and then fed to the test organism, the freshwater amphipod Hyalella azteca. In feeding trials both zein and chitosan nanoparticles showed a significantly lower release rate of rhodamine B into water than food dyed with rhodamine B without biopolymer nanoparticles. Zein nanoparticles also showed better retention ability than chitosan nanoparticles. Both kinds of nanoparticles showed no significant effect on the survival, growth, or feeding behavior of H. azteca. Thus these biopolymers may be an effective system to encapsulate and deliver chemicals to aquatic invertebrates without interfering with common toxicity assessment endpoints like survival and growth.

Host suitability and gas exchange response of grapevines to potato leafhopper (Hemiptera: Cicadellidae).

Although potato leafhopper, Empoasca fabae (Harris) (Hemiptera: Cicadellidae), is highly polyphagous, classic host studies do not recognize grapevines (Vitis spp.), as suitable hosts. Recently, injury has been reported and reproduction documented within grape vineyards, suggesting a host expansion for the leafhopper. To document this apparent expansion in host use, we determined whether grape plants were suitable hosts for potato leafhopper reproduction, measured the consequence of feeding injury on gas exchange rates of grape leaves, and compared the susceptibility to feeding injury among cultivars. We found that potato leafhopper adults survived equally well on grape (Vitis vinifera L.), alfalfa (Medicago sativa L.), and fava bean (Vicia faba L.). The total number of offspring was greater on fava bean but did not differ between alfalfa and grape. Injury to grapevines was assessed by measuring gas exchange responses of leaves in field cages and in greenhouse tests. We found marginally significant declines in photosynthesis and transpiration rates in the field (9.6 and 13.2%, respectively), and much stronger effects in greenhouse tests (ranging between 22 and 52%). Our results verify that Vitis is a suitable host, and that potato leafhopper is capable of injuring its gas exchange physiology. We discuss possible explanations for the host expansion, and its potential to damage commercial grapevines.

Exposure and nontarget effects of transgenic Bt corn debris in streams.

Corn (Zea mays L.) transformed with a gene from the bacterium Bacillus thuringiensis (Bt) comprises 49% of all corn in the United States. The input of senesced corn tissue expressing the Bt gene may impact stream-inhabiting invertebrates that process plant debris, especially trichopteran species related to the target group of lepidopteran pests. Our goal was to assess risk associated with transgenic corn debris entering streams. First, we show the input of corn tissue after harvest was extended over months in a stream. Second, using laboratory bioassays based on European corn borer [Ostrinia nubilalis (Hübner)], we found no bioactivity of Cry1Ab protein in senesced corn tissue after 2 wk of exposure to terrestrial or aquatic environments. Third, we show that Bt near-isolines modify growth and survivorship of some species of invertebrates. Of the four nontarget invertebrate species fed Bt near-isolines, growth of two closely related trichopterans was not negatively affected, whereas a tipulid crane fly exhibited reduced growth rates, and an isopod exhibited reduced growth and survivorship on the Cry1Ab near-isoline but not on the stacked Cry1Ab + Cry3Bb1 near-isoline. Because of lack of evidence of bioactivity of Bt after 2 wk and because of lack of nontarget effects on the stacked near-isoline, we suggest that tissue-mediated differences, and not the presence of the Cry1Ab protein, caused the different responses among the species. Overall, our results provide evidence that adverse effects to aquatic nontarget shredders involve complex interactions arising from plant genetics and environment that cannot be ascribed to the presence of Cry1Ab proteins.

Evidence for divergent selection between the molecular forms of Anopheles gambiae: role of predation.

BACKGROUND: The molecular forms of Anopheles gambiae are undergoing speciation. They are characterized by a strong assortative mating and they display partial habitat segregation. The M form is mostly found in flooded/irrigated areas whereas the S form dominates in the surrounding areas, but the ecological factors that shape this habitat segregation are not known. Resource competition has been demonstrated between species undergoing divergent selection, but resource competition is not the only factor that can lead to divergence. RESULTS: In a field experiment using transplantation of first instar larvae, we evaluated the role of larval predators in mediating habitat segregation between the forms. We found a significant difference in the ability of the molecular forms to exploit the different larval sites conditioned on the presence of predators. In absence of predation, the molecular forms outcompeted each other in their respective natural habitats however, the developmental success of the M form was significantly higher than that of the S form in both habitats under predator pressure. CONCLUSION: Our results provide the first empirical evidence for specific adaptive differences between the molecular forms and stress the role of larval predation as one of the mechanisms contributing to their divergence.