Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
Entomology
Non Technical Summary
The invasive spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae) is an emerging, highly invasive insect pest of fruit crops and trees in the eastern US. It poses a significant economic threat to many woody tree species in MD including native and economically-important trees and woody plants. Nymphs and adults cause substantial damage by sucking phloem sap and subsequently reducing photosynthesis, causing weeping wounds, and creating conditions for sooty mold. Lanternfly nymphs switch host plants during their development. However, little is known about the relationship between the lanternfly and its tree hosts. Information is particularly needed regarding consumption of the host plants associated with lanternfly feeding behavior. Our objectives include (1) identifying host plant utilization at different nymphal stages of L. delicatula through detection of plant host DNA within insect gut contents, (2) determining the longevity of the plant DNA in the gut contents of L. delicatula at different nymphal stages, and (3) investigating feeding behavior of different nymphal stages of L. delicatula on multiple host plants. For this study, we will largely utilize the next-gen DNA sequencing technology which will allow us to accurately determine which host plants the spotted lanternfly utilizes for feeding at each nymphal stage.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goal of this study is to explore and predict lanternfly host plant usage using next-gen DNA sequencing technology. This is especially important for two reasons: 1) as a sap-feeder, it is difficult to detect host use by simply observing the insect on a plant, and 2) young nymphs are able to feed on a much wider host range than older nymphs and adults, and host determination of early instars is important for pest management.Our specific objectives are:Objective 1. To utilize next-gen technology to identify host plant utilization at different nymphal stages of L. delicatula through detection of plant host DNA within insect gut contents.For this objective, we will specifically focus on determining the host use of wild nymphs in relation to a local plant community. Our current findings of plant DNA detection from the gut contents of the lanternfly nymphs (Avanesyan and Lamp, manuscript is in preparation) suggest that while the observed lanternfly nymphs actively move on a host plant, they may not utilize it for feeding. A substantial number of the nymphs (~90%) we have analyzed (collected during summer 2018) showed the ingested DNA from a host plant other than the plant from which the nymphs were collected. In our current work we use Sanger Sequencing technology which allows us to obtain only ingested DNA from one plant species. In this proposed study, using Next-Gen Sequencing technology, we will be able to obtain DNA from all the plants consumed prior the insect collection.Objective 2. To determine the longevity of the plant DNA in the gut contents of L. delicatula at different nymphal stages. For this objective, we will use one host plant and will focus on how long the DNA from this host plant can be detected in the gut contents of different nymphal stages of the spotted lanternfly. Our previous work on plant DNA detection from the potato leafhopper, also a sap-feeder (Avanesyan and Lamp, in review), demonstrated that the plant DNA can be detected up to 2 hours post ingestion. Given a larger size of the lanternfly compared to that of the potato leafhopper, as well as different sizes among the lanternfly nymphs, we expect the plant DNA to be detected during a longer time period. This information is critical in prediction of the time needed for lanternfly to switch host plants and migrate to another host tree.Objective 3. To investigate feeding behavior of different nymphal stages of L. delicatula on multiple host plants.For this objective (based on the findings from Objective 2), we will use at least 2 different host plants and focus on how long the DNA from different host plants can be detected in the lanternfly gut contents. Given polyphagous behavior of early nymphal instars, we are interested in exploring how fast the nymphs would switch the host plants, how long they would feed on each host plant, and how quickly the DNA from each ingested host plant degrades in the lanternfly gut contents. This information will serve as an important tool for monitoring, collection, and prediction the lanternfly migration to a new host plant.
Project Methods
This one-year project will be conducted at the Department of Entomology, University of Maryland, and will include a laboratory study, as well as several collection trips to Pennsylvania. During this year we will (a) utilize next-gen technology to identify host plant utilization of L. delicatula at different nymphal stages through detection of plant host DNA within insect gut contents (Objective 1); (b) determine the longevity of the plant DNA in the gut contents of L. delicatula at different nymphal stages (Objective 2); and (c) investigate feeding behavior of different nymphal stages of L. delicatula on multiple host plants (Objective 3).The proposed study will include laboratory work, collection trips (at three time points to ensure the collection of 1st-4th instar nymphs), and setting up feeding trials in the field. The lanternfly is currently under quarantine within Pennsylvania, so all the feeding trials will be conducted at a field site in PA, and all of the laboratory studies planned during this year will use dead specimens, preserved in the field either with ethyl alcohol or by freezing before we re-enter Maryland. In addition, because of the annual life cycle of the insect, we have already collected a number of specimens for our studies to commence in the spring. Another option for our experiments is to get APHIS approval four using our departmental BL2 Lab for caged studies. We are seeking approval at this time.Objective 1. To utilize next-gen technology to identify host plant utilization of L. delicatula at different nymphal stages through detection of plant host DNA within insect gut contents.We will use 1st-4th instar nymphs of L. delicatula. We will collect the nymphs at three time points in PA (the field sites are to be determined) during summer 2020. Each time point will be chosen to ensure the collection of all the early, middle, and late-instar nymphs: the collections will be scheduled tentatively in May, June, and July. Following our previous collection procedures (Avanesyan and Lamp, in prep), the nymphs and leaf samples of the reference plant species from a local plant community (which are present at a field site) will be immediately dry-frozen and transported to the laboratory.For this proposed study, we will largely utilize our previously developed PCR-based method for detecting a non-coding region of plant chloroplast gene (trnL intron) from grasshopper gut contents (Avanesyan 2014, Avanesyan and Culley 2015). We have modified this protocol and we have successfully applied it in our previous study on plant DNA detection from the potato leafhopper gut contents (Avanesyan and Lamp, in review), as well as in our current work on molecular gut content analysis of L. delicatula using Sanger sequencing technology. In this proposed study, in addition to the trnL gene, we will also utilize a coding region of the chloroplast DNA, rbcL gene: our current findings suggest that this targeted gene provides a good species resolution when we identify the host plant of L. delicatula nymphs. Using such a combination of a conserved coding region and a more rapidly evolving non-coding region has been suggested as a good option for valid plant identification (Kress et al., 2009).Following this approach, we will isolate parts of the chloroplast DNA, trnL and rbcl regions, from ingested host plant DNA within gut contents in each insect individual. We will then obtain a set of plant DNA sequences isolated from each insect individual using meta-barcoding Next-Generation Sequencing method ("Amplicon-EZ" service) at GENEWIZ (GENEWIZ Inc., South Plainfield, NJ); this Next-Generation Sequencing approach will allow us to obtain sequences from multiple ingested plant species. Host plant species identity will be determined using BLAST engine in the National Center for Biotechnology Information (NCBI) GenBank database (http://www.ncbi.nlm.nih.gov/genbank/).During Spring 2020, we will first apply this approach to our previously collected lanternfly nymphs (currently stored in the laboratory) to optimize amplification of the targeted plant DNA regions. We will then apply this approach to all the lanternfly nymphs (from 1st to 4th instars) collected during Summer 2020, and we will identify host plants for each developmental stage.Objective 2. To determine the longevity of the plant DNA in the gut contents of L. delicatula at different nymphal stages. To determine how long the plant DNA can be detectable in the lanternfly gut contents, a separate feeding experiment will be conducted in the field, during Summer 2020. In this experiment, one host plant, such as a maple, vine, or tree-of-heaven sprout (to accommodate feeding of all the early, middle, and late-instar nymphs) will be selected at a field site in PA. About 32 nymphs of each developmental stage (possibly at different time points) will be confined on one host plant using a fabric enclosure until the feeding is observed. The lanternfly nymphs will be allowed to feed on the enclosed host plant for about 2 hours (not exceeding 30 min of the post feeding time). After feeding, the host plant will be carefully removed from the enclosure and the lanternfly nymphs (~4 at a time) will be frozen at - 20° C at eight time intervals post ingestion (PI): 0 min PI, 30 min PI, 1 hour PI, 2 hours PI, 4 hours PI, 6 hours PI, 10 hours PI, and 24 hours PI. The insects will be given access to water or sugar solution before freezing. Plant DNA will be then extracted from each retrieved insect following the procedure described above for Objective 1. The presence of DNA will be verified by both gel electrophoresis and sequencing.Objective 3. To investigate feeding behavior of different nymphal stages of L. delicatula on multiple host plantsTo investigate feeding behavior of different nymphal stages of L. delicatula on multiple host plants, another separate experiment, similar to that for objective 2, will be conducted during Summer 2020. For this experiment, three appropriate host plants will be selected at a field site within a "jumping" distance of the nymph (~0.5m). All the nymphs will be collected at the same field site and starved before confinement. First, about 30 nymphs (at each developmental stage) will be first confined on one appropriate host plant (host plant #1); the nymphs will be allowed to feed on the plant until the first feeding break. Once feeding break is observed (~50% of nymphs are expected to actively feed) the nymphs will be immediately transfer to another enclosure which is set up around a different host plant (host plant #2). The nymphs will be allowed to feed on host plant #2, and once the feeding break is recorded the nymphs will be transferred to host plant #3. After feeding on host plant #3, all the nymphs will be dry-frozen immediately and transported to the laboratory for DNA analysis. The rest of the nymphs (from all three host plants), for which active feeding is not observed, will be also dry-frozen and transported to the laboratory for verification of the presence of plant DNA in their gut contents.