Project : USDA ARS

ARS Home » Southeast Area » Gainesville, Florida » Center for Medical, Agricultural and Veterinary Entomology » Insect Behavior and Biocontrol Research » Research » Research Project #439207

Research Project: Improved Biologically-Based Methods for Management of Native and Invasive Crop Insect Pests

Location: Insect Behavior and Biocontrol Research

2023 Annual Report

Objectives
Objective 1: Develop genetically modified (GM) strains by transposon and CRISPR/Cas-mediated transgenesis for improved SIT in fruit fly pests, and new DNA delivery systems to apply this technology to a wider range of insect, including emerging pest species (e.g., caribfly, mexfly, medfly, spotted-winged drosophila). Objective 2: Develop strains of moths transinfected with Wolbachia that produce males with strong cytoplasmic incompatibility for use in the Incompatible Insect Technique (IIT) to reduce pest populations of fall armyworm and corn earworm. Objective 3: Develop CRISPR gene editing in pest moths (e.g., Indian meal moth, fall armyworm, gypsy moth) that target genes critical for acquired biopesticide resistance using both whole insects and cultured insect cells. Objective 4: Develop improved surveillance and detection methods for hidden and invasive pests (e.g., red palm and citrus root weevil, Asian long-horned beetle, Asian citrus psyllid, and stored product insect pests) that incorporate automated collection, processing, and analysis of insect acoustic signals and behavioral activity. Objective 5: Develop improved surveillance of invasive and outbreak insect pests (e.g., corn silk flies and kudzu bug) using visual-cue traps, and improve strategies for detection and prediction of such dispersing pests by understanding the role of visual and other stimuli affecting specific behaviors. Objective 6: Improve area-wide landscape management tactics by developing conservation biological control strategies to mitigate pest populations and attract or support natural enemies (e.g., against fall armyworm). Objective 7: Combine genetic methods with air-transport and climate modeling to describe and predict the distribution and behavior of agricultural pests to facilitate the mitigation of migratory source populations and to identify locations at high risk for infestations by invasive species such as fall armyworm, corn silk fly, soybean looper, Old World bollworm, and corn earworm.

Approach
Research conducted by the Behavior and Biocontrol Research Unit at the Center for Medical, Agricultural and Veterinary Entomology has historically been focused on the development of novel technologies that improve the cost-efficiency of traditional pest control strategies as well as provide environmentally benign alternatives to the use of chemical pesticides. The goals are to improve crop productivity while reducing the environmental impact and costs of pest management. The proposed research integrates different levels of biology that range from the genetic modification of pest insects to generate novel and improved variations of Sterile Insect Technique (SIT) strategies, the manipulation of pest endosymbionts to develop Insect Incompatibility Technique (IIT) strategies, the optimization of acoustic, olfactory, and visual cues to improve pest surveillance and disrupt pest behavior, the application of climate and air transport models to project pest distribution and migration patterns, and the development of landscape strategies for sustainable mitigation of pest populations. This multidisciplinary structure encourages innovation and facilitates synergism between projects. Anticipated accomplishments will initially apply to the control of high priority invasive fruit flies, beetles, psyllids, moths, and corn silk flies through new biologically based methods for pest control, improved capability to monitor pests, and better projections of pest movements to more effectively target the time and place of treatments. The impact will be higher productivity at lower cost for domestic agriculture and new and improved tools to detect and control emerging native and invasive pests.

Progress Report
Progress was made on project objectives which fall under National Program 304, Component 3, Insects and Mites with activities focusing on; Problem 3A, early detection prediction and monitoring of beneficial and pest arthropods; Problem 3B, developing new or improved management tools and knowledge to control arthropod pests; Problem 3C, integrating management strategies to control arthropod pests; Component 4, Problem 4A, protection of postharvest commodities, quarantine and methyl bromide alternatives and Problem 4B, improving and developing postharvest and quarantine treatments. Under Objective 1, we developed and characterized DNA constructs for the genetic modification of important fruit fly pests (Anastrepha suspensa (Caribfly), Anastrepha ludens (Mexfly), and spotted wing Drosophila (D. suzukii) as a basis for more efficient Sterile Insect Technique strategies of pest control. Specifically, functional quantitative RT-PCR testing of the D. melanogaster CG14427 and nullo gene orthologs in D. suzukii have verified embryonic expression in this species. Transformation vectors for new transgenic tet-Transactivator lines (CG14427/nullo-tTA) were created for A. ludens, with germ-line transformation experiments in progress (Objective 1.1). The wampa dynein subunit and Prosalpha6T genes from both A. suspensa and D. suzukii were verified for adult male sperm-specific transcription by use of quantitative RT-PCR in both species. CRISPR/Cas9 gene-editing to create null mutations for wampa in D. suzukii has succeeded and gene-edited lines are currently being evaluated. CRISPR/Cas9 gene-editing has been initiated for As-wampa in A. suspensa and Ds-Prosalpha6T in D. suzukii (Objective 1.2). Putative transformant lines having a vector plasmid integration for dominant temperature sensitive (DTS) female-specific lethality in A. suspensa have been isolated and are currently being inbred for transgene homozygosity required to evaluate female-specific lethality at 29oC (Objective 1.3). No progress was made on Objectives 2 and 3 due to retirement of a research scientist. For Objective 4, prototypes of acoustic devices for use as detection, trapping, and mating disruption systems continue to be developed, optimized, and field tested with significant enhancement of signal detection and noise filtration. Efficacy of monitoring and optimization was conducted in the field with the invasive, spotted lanternfly (Lycorma delicatula) and for determination of mortality caused by entomopathogenic fungi on the red palm weevil (Rhynchophorus ferrugineus). In addition, programming tools were developed capable of statistically distinguishing between male and female vibrational communication calls of the Asian citrus psyllid. This may lead to improved understanding of mating behavior on citrus trees with high psyllid populations. For Objective 5, laboratory tests were conducted using a range of protein-associated baits with two species of corn silk flies. Several of these baits or compounds resulted in strong attraction of flies in the laboratory. Field studies were conducted in maize and results indicated that revealed several lures were highly attractive for corn silk fly species, however differences were noted in response between species of fly attracted. Field trials were also conducted with different colored traps and differences were noted between species for optimal color for attraction. Laboratory studies are on-going relating to differences in fly response and species to sex pheromone extracts and these are being further examined. Laboratory assays are being utilized for evaluation of attraction responses of kudzu bugs to visual and olfactory attractants. For Objective 6, we made significant progress in studies on Florida natural enemies to important insect pests. Widespread predators of the fall armyworm, Spodoptera frugiperda were identified and included the earwig (Labidura riparia) and striped lynx spider (Oxyopes salticus). Initial efforts to colonize these species were successful and the evaluation of carbohydrates to enhance colony health is on-going. Laboratory observations showed that adult earwigs can attack and feed on eggs through 4th stage fall armyworm larvae; spiders were observed feeding on early-stage larvae and eggs. For Objective 7, an international network of collaborators was organized to provide fall armyworm specimens from Africa, South America, Mexico, and the Caribbean. Genetic analysis of these specimens revealed evidence of population structure and helped define the direction and limits of fall armyworm migration on a hemispheric level. The genetic data provides a database of genetic variation that allow a reanalysis of past data to better understand fall armyworm migration and distributions in the United States.

Accomplishments
1. New genetic constructs to improve gene editing methods in fruit fly pest species. Fruit fly pest species are an important agricultural problem that would benefit from improvements in nonchemical means of control, including sterile insect technique (SIT). A scientist in Gainesville, Florida, demonstrated that the wampa and Prosalpha6T genes were verified as good targets for adult male sperm-specific transcription. These will provide alternative genomic target sequences for CRISPR/Cas9 gene-editing that can result in male sterility in drosophilid and tephritid fruit fly pest species through this precision-guided SIT method. The promoter sequences of these genes could also be used for sperm-specific fluorescent protein marking (to identify females mated to field released males) and to drive conditional lethality systems for male sterility and female lethality. These constructs will facilitate the development of more efficient SIT methods.

2. Identification of genes in fruit fly pests as potential targets for induced male sterility for sterile insect technique (SIT). A major problem with SIT strategies is the difficulty in causing the desired sterility without compromise of the mating performance of the released population which is critical for dissemination. One solution would be a genetic method that specifically causes sterility in the targeted gender. A scientist in Gainesville, Florida, discovered that the Prosalpha6T gene exhibits sperm-specific expression in spotted wing Drosophila adult males, where it replaces the expression of the non-sex-specific Prosalpha6 isoform in testes and is therefore a potential target for male sterility. However, in the Caribbean fruit fly Prosalpha6T expression is not detected and, while Prosalpha6 expression persists in testes and the rest of the body tissues. This indicates that the Prosalpha6T isoform may not exist in the Caribbean fruit fly and possibly other tephritid species, and that Prosalpha6 is most likely not a useful target for male sterility in these flies since it may be essential for viability in these species.

3. Insecticide resistance in fall armyworm. In addition to field crops, fall armyworm are capable of significant damage to turf. Populations of fall armyworm were unusually high in pastures and lawns in the mid-south and southeastern U.S. during summers of 2021 and 2022. One possible explanation is the development of insecticide resistance. A scientist in Gainesville, Florida, tested populations from Louisiana and Florida using an insecticide contact larval bioassay. Results indicated differences in insecticide resistance with a population collected from turf grass (Florida) being highly resistant to several pyrethroid insecticides, while a population collected from pasture grass (Louisiana) was moderately resistant. Growers and homeowners who use chemical control to manage fall armyworm in pastures or lawns need to use insecticides with different modes of action to slow the development of resistance.

4. Mapping the distribution of the egg parasitoid Telenomus remus in Florida. Fall armyworm is a serious pest of sweet corn, and nearly 50,000 acres are grown in southcentral and southeastern Florida. The egg parasitoid Telenomus remus is an important natural control agent of this pest and has been documented in several northcentral and central locations in Florida since 2009. A scientist in Gainesville, Florida, determined the distribution of T. remus throughout Florida using sentinel egg masses and found a new population in southwest Florida (Immokalee). While this is the most southernly location so far for the distribution of this important egg parasitoid, it has yet to be found in the sweet corn belt of western Palm Beach County. Once present in the sweet corn belt, it can serve as an addition tool for mitigation of fall armyworm populations.

5. Temporal assortative mating of fall armyworm strains. Fall armyworm as a species contains two host strains that feed either on corn and sorghum (C-strain), or pasture and turfgrasses and rice (R-strain). These two strains, however, inhabit the same locations and are considered sympatric. A scientist in Gainesville, Florida, with the collaboration of scientists at Texas A&M University, determined that there are significant behavioral differences between the populations. In trapping studies, male moths captured early in scotophase (before solar midnight) were 95% C-strain moths whereas moths captured late in scotophase were 89.5% R-strain. This clear separation in behavior is evidence of assortative mating which in likely a mechanism maintaining separation of the two strains in sympatric habitats.

6. Using fungi to control the red palm weevil. The most devasting pest of palm trees globally is the red palm weevil, in part because infested trees can harbour weevils undetected until trees are dying. Control generally relies on synthetic insecticides that are not sustainable, negatively impacting both the environment and biological diversity. A scientist in Gainesville, Florida, led an international collaboration to use acoustic monitoring of the decline of insect activity inside trees after various treatments. Acoustic bursts from weevils in date palm plantations were reduced within 2-3 months after treatment with entomopathogenic nematodes and fungi, aluminum phosphide fumigation, and insecticidal spray treatments. The insecticides and entomopathogenic treatments provided the most effective treatments in heavily infested plantations. This demonstrates the utility of acoustic monitoring in treatment assessments for this weevil, as well as demonstrating efficacy of new and ecologically friendly management tools for this devastating pest.

7. Developing attractant lures of cornsilk fly surveillance. One of the primary pests of fresh market sweet corn produced in Florida is a group of flies known as corn silk flies due to their preference to oviposit on corn silk. Upon emergence, larvae feed on developing corn kernels ultimately causing sufficient damage to result in rejection at packing houses and heavy economic losses. As members of Tephritoidea, these flies feed on protein and studies were conducted to evaluate attraction of flies to volatiles related to protein foods. A scientist in Gainesville, Florida, evaluated the attraction of two cornsilk fly species to various food sources, commercial lures and volatile chemicals were evaluated under laboratory and field conditions to identify those with the most potential for use in a surveillance system. Differences observed in responses between species indicated that optimal lure formulations for each species should be comprised of different formulations. Effective lures combined with visual traps can improve surveillance and guide targeted control efforts to protect the crop.

8. The migratory origins of fall armyworm infestations in Arizona. Fall armyworm is a significant agricultural pest native to the Western Hemisphere that has become a global problem with its discovery in most of the Eastern Hemisphere. It causes substantial economic losses in corn and can be a significant problem in many other crops. The inability of fall armyworm to survive freezing winters limits permanent populations to areas south of 30°N latitude, with known overwintering locations that include southern Florida, southern Texas, and portions of Mexico. Infestations are routinely found in agricultural areas as far west as southern Arizona. Scientists in Gainesville, Florida, in collaboration with colleagues from three universities used genetic techniques to identify possible migratory pathways in the American southwest.

9. The phylogeny and population biology of fall armyworm populations that differ in host use. The threat of invasive species is increasing with the expansion of global trade and habitat disruption. A recent example is the establishment of fall armyworm, a noctuid moth native to the Americas, into most of the Eastern Hemisphere with projections of significant economic losses on a global scale. The species has traditionally been subdivided into two populations that differ in their propensity to use different plant hosts, a phenotype with clear relevance for identifying crops at risk. However, inconsistencies in the genetic and phenotypic descriptions of these “host strains” has led to controversy about their composition and even existence. In this study, a scientist in Gainesville, Florida, used phylogenetic analysis to uncover genetic differentiation between populations that further supports the existence of the host strains and provided evidence that they are subject to different selection pressures. Genetic comparisons of moths collected from multiple locations revealed significant differences in the presence of different molecular markers that indicate that additionally there are regional variations in host strain behavior. The data confirm that the host strains are a fundamental characteristic of the species that needs to be taken into consideration when developing monitoring and mitigation strategies.

U.S. DEPARTMENT OF AGRICULTURE

Insect Behavior and Biocontrol Research: Gainesville, FL