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.

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.

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.

Effect of Temperature and Humidity on the Seasonal Phenology of Drosophila suzukii (Diptera: Drosophilidae) in Wisconsin.

Drosophila suzukii (Matsumura) is an invasive pest of soft-skinned fruits that has caused significant economic damage worldwide. In this study, we focused on the seasonal abundance of D. suzukii during the early years of establishment in Wisconsin. We explored the seasonal patterns of summer and winter morphs, their reproductive output, and the effect temperature and humidity may have on their seasonal phenology. The seasonal abundance of D. suzukii during 2 yr (2014-2015) revealed that flies were detected in Wisconsin from early July to late December, with winter morphs being trapped from August through December. The adult populations trapped spanned 1 mo longer in 2015 than in 2014. The peak proportional abundance of D. suzukii in 2015 was recorded in August which was about 2 mo earlier than that in 2014. The combined factor [maximum temperature and maximum humidity] explained the most amount of variation in D. suzukii abundance consistently across the 2 yr in Wisconsin. We did not find significant differences in the fat content, number of mature eggs, proportion of females with immature eggs, or proportion of mated females between summer morph females at the beginning, summer and winter morph females during the middle, or winter morph females at the end of the collecting season in 2015. Our results build on the body of work providing a better understanding of the D. suzukii-overwintering abilities and strengthen the importance of early crop risk assessment and targeted control strategies.

Should I Eat or Should I Go? Acridid Grasshoppers and Their Novel Host Plants: Potential for Biotic Resistance.

Novel, non-coevolved associations between introduced plants and native insect herbivores may lead to changes in trophic interactions in native communities, as well as to substantial economic problems. Although some studies in invasion ecology demonstrated that native herbivores can preferentially feed on introduced plants and therefore contribute to the biotic resistance of native communities to plant invasions, the role of acridid grasshoppers as native generalist insect herbivores is largely overlooked. This systematic review aimed to identify patterns of grasshopper feeding preferences for native versus introduced plants and, consequently, a potential of grasshoppers to provide biotic resistance of native communities. The analysis of 63 records of feeding preference trials for 28 North-American grasshopper species (retrieved from 2146 studies published during 1967⁻2017) has demonstrated a preference of grasshoppers for introduced host plants, and identified 12 preferred introduced plants with high or middle invasive ranks. A significant effect of the life stage (p < 0.001), but not the experimental environment, plant material, and measurements, on grasshopper preferences for introduced plants was also detected. Overall, results suggest a potential of acridid grasshoppers to contribute to the biotic resistance of native communities. The review also provides methodological recommendations for future experimental studies on grasshopper-host plant interactions.

Multistate Comparison of Attractants and the Impact of Fruit Development Stage on Trapping Drosophila suzukii (Diptera: Drosophilidae) in Raspberry and Blueberry.

Spotted-wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae), is an invasive pest of soft-skinned fruits across the globe. Effective monitoring is necessary to manage this pest, but suitable attractants are still being identified. In this study, we combined lures with fermenting liquid baits to improve D. suzukii trapping specificity and attractiveness. We also measured the efficiency and specificity of baits/lures during different times of the season; the reproductive status of females among baits/lures; and the effects of locations and crop type on these response variables. We developed a metric that combined mating status and fat content to determine differences in types of females attracted. Lures utilizing yeast and sugar-based volatiles trapped the most D. suzukii. The addition of a commercial lure to yeast and sugar-based lures increased catches in most locations, but was also the least specific to D. suzukii. Apple juice-based chemical lures tended to be most specific to D. suzukii, while lures comprised of a singular attractant tended to trap more D. suzukii with a higher reproductive potential than combinations of attractants. Trap catch and lure specificity was lower during fruit development than fruit ripening. While catch amounts varied by geographic location and crop type, attractants performed similarly relative to each other in each location and crop. Based on the metrics in this study, the yeast and sugar-based attractants were the most effective lures. However, further work is needed to improve early season monitoring, elucidate the effects of physiological status on bait attraction, and understand how abiotic factors influence bait attraction.

Isolating Spermathecae and Determining Mating Status of Drosophila suzukii: A Protocol for Tissue Dissection and Its Applications.

The spotted wing drosophila, Drosophila suzukii (Diptera: Drosophilidae), is an emerging invasive pest, which attacks a wide variety of fruits and berries. Although previous studies have focused on different aspects of D. suzukii reproductive biology, there are no protocols available for determining the mating status of D. suzukii females and drosophilids in general. In this study, a step-by-step protocol for tissue dissection, isolating spermathecae, and determining the mating status of females was developed specifically for D. suzukii. This protocol is an effective and relatively quick method for determining female mating status. It has important applications from exploring reproductive output of D. suzukii females to understanding the biology of D. suzukii winter morph, which presumably plays the main role in the overwintering of this invasive species. We demonstrated applicability of this protocol for both field collected flies and flies reared in the lab, including fly specimens stored on a long-term basis.

An empirical review: Characteristics of plant microsatellite markers that confer higher levels of genetic variation.

During microsatellite marker development, researchers must choose from a pool of possible primer pairs to further test in their species of interest. In many cases, the goal is maximizing detectable levels of genetic variation. To guide researchers and determine which markers are associated with higher levels of genetic variation, we conducted a literature review based on 6782 genomic microsatellite markers published from 1997-2012. We examined relationships between heterozygosity (H e or H o) or allele number (A) with the following marker characteristics: repeat type, motif length, motif region, repeat frequency, and microsatellite size. Variation across taxonomic groups was also analyzed. There were significant differences between imperfect and perfect repeat types in A and H e. Dinucleotide motifs exhibited significantly higher A, H e, and H o than most other motifs. Repeat frequency and motif region were positively correlated with A, H e, and H o, but correlations with microsatellite size were minimal. Higher taxonomic groups were disproportionately represented in the literature and showed little consistency. In conclusion, researchers should carefully consider marker characteristics so they can be tailored to the desired application. If researchers aim to target high genetic variation, dinucleotide motif lengths with large repeat frequencies may be best.

Plant DNA detection from grasshopper guts: A step-by-step protocol, from tissue preparation to obtaining plant DNA sequences.

PREMISE OF THE STUDY: A PCR-based method of identifying ingested plant DNA in gut contents of Melanoplus grasshoppers was developed. Although previous investigations have focused on a variety of insects, there are no protocols available for plant DNA detection developed for grasshoppers, agricultural pests that significantly influence plant community composition. • METHODS AND RESULTS: The developed protocol successfully used the noncoding region of the chloroplast trnL (UAA) gene and was tested in several feeding experiments. Plant DNA was obtained at seven time points post-ingestion from whole guts and separate gut sections, and was detectable up to 12 h post-ingestion in nymphs and 22 h post-ingestion in adult grasshoppers. • CONCLUSIONS: The proposed protocol is an effective, relatively quick, and low-cost method of detecting plant DNA from the grasshopper gut and its different sections. This has important applications, from exploring plant "movement" during food consumption, to detecting plant-insect interactions.

Micro-spatial distribution of two sibling periwinkle species across the intertidal indicates hybrdization.

Populations of periwinkles Littorina saxatilis (Olivi 1792) and L. arcana Hannaford Ellis, 1978 are well suited for microevolutionary studies, being at the same time closely related and intraspecifically diverse. The divergence between these two sibling species, sympatric over large parts of their distribution areas, is small, the only morphological difference being the pallial gland complex structure in females. Molecular identification is possible with the use of a RAPD nuclear marker (cloned A2.8 DNA fragment) typical for L. arcana. However, in some individuals from sympatric populations molecular and morphological criteria suggest conflicting species affiliation, which may be explained either by hybridization or by shared ancestral polymorphism. We tested the hybridization hypotheses examining the micro-spatial distribution of these two species across the intertidal zone in two distant sites at the Barents Sea. We found that (a) the frequency of putative hybrids in sympatric populations was proportional to the frequency of L. arcana; (b) L. saxatilis bearing A2.8 DNA fragment were almost absent in the lower part of the intertidal zone, where L. arcana was absent too; (c) there was a close positive correlation between the distribution of potential parent molluscs and putative hybrids. Moreover, logistic regression models showed a good agreement between the distribution of putative hybrid frequencies and that of parental species frequencies. All our observations taken together support the hypothesis of hybridization between L. saxatilis and L. arcana. Elucidating the mechanisms that support the species status of these sympatric populations is necessary.