Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Entomology and Nematology
Non Technical Summary
Insect pests cause substantial damage to agricultural food security and global trade worldwide. The State of Florida has one of the most diverse agricultural production bases in the United States, and as such is challenged by many insect pests from the Asian citrus psyllid that has decimated citrus production our state to the many invasive fruit flies that have wide host ranges and can affect many commodities. The effects of these invasive insect pests are felt by growers through both direct damage to fresh fruits and vegetables, and also through export restrictions that may impede the transportation and sale of crops from an infested area even if those crops themselves are not damaged. For example, the outbreak of Oriental fruit fly in South Florida in 2015-2016 led to a direct cost of >$4 million to growers with additional economic impacts >$10 million due to multiple factors including: crops that had to be destroyed because they were infested, the loss of the ability to ship undamaged commodities outside the quarantine zone, additional treatment costs to existing fields, and losses from the decision to not plant annual crops due to the uncertainty of when the infestation would be eradicated (Alvarez et al. 2017). A much smaller incursion of Oriental fruit fly also occurred in South Florida in 2018 and caused much concern before it was eradicated. Given Florida's variety of climates from subtropical in the south to moderately temperate in the north along with the traffic in international visitors and importation of goods and commodities, our state faces tremendous pressure from invasive insect pests, including economically important fruit flies. Beyond their critical threat to food security and Florida's agricultural economy, some insects in our state can also vector pathogens causing debilitating human diseases. For example, the well-established invasive mosquito Aedes aegypti is common in many areas of Florida where it is a major nuisance because it is an aggressive daytime biter with immature stages developing in small water sources around houses (e.g., gutters, old tires, plant pots, or even a discarded bottle cap). Beyond its nuisance status, Ae. aegypti is the primary vector of several viruses including yellow fever, dengue, chikungunya, and Zika that can produce serious disease and pathology. Dengue virus has cropped up with small pockets of local transmission in Florida several times in the past two decades, and there was local transmission of Zika in Miami in 2016. The impact of the Zika outbreak in Miami was certainly less than in other locations in the Caribbean and Latin America in 2016 due to the relatively few cases of local transmission that occurred (Lee et al. 2017). However, there was an enormous impact from the costs of controlling a large urban vector population of Ae. aegypti, plus substantial societal impact due to stress on pregnant mothers and their families in the outbreak locations and throughout the greater Miami area, as well as substantial losses to local businesses in the affected areas of Miami, and both a decrease in tourism and negative perceptions of our state as a vacation destination in general (Page et al. 2017). Clearly we must be able to control these insect pests of agricultural and medical importance and chemical insecticides have played the main role in doing so. However, because of regulation, development of resistance to common chemical pesticide classes, and environmental and human health concerns, there is increasing demand for the replacement of the intensive use of these chemicals by environmentally friendly, cost effective and sustainable methods within integrated pest management approaches. The sterile insect technique (SIT hereafter) is one such biologically based tool for integrative pest management that has proven to be both effective and economically advantageous for the management of numerous species of agriculturally important pest insects, and is emerging as an important tool for integrative management of disease-vectoring mosquitoes including Ae. aegypti. Here, I propose developing a series of new approaches that could improve the efficacy and cost-effectiveness of SIT for both agricultural pest flies and mosquitoes.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Goals / Objectives
Most insects face times of the year when reproduction or development are suppressed due to a lack of resources or unfavorable environmental conditions. Dormancy responses have evolved to mitigate the stresses of these unfavorable times and to synchronize insect life cycles with favorable periods. Dormancy responses can include both pre-programmed, hormonally mediated diapause, and also quiescence induced directly by the environment (e.g., low temperatures, drought, lack of hosts, etc.). Quiescence is a state of developmental slowdown that can occur in any life stage. In contrast, diapause is a stage-specific developmental arrest that can be either facultative (determined by token stimuli) or obligate (occurring regardless of prevailing environmental conditions). Many univoltine pest species have an obligatory diapause that synchronizes them with resource availability. For such univoltine insect pest species, sterile insect technique (SIT) and augmentative natural enemy control have not been either practical or possible due to obligatory diapause responses that prevent or interfere with continuous mass rearing. Examples include the European cherry fruit fly, apple maggot fly, Chinese citrus fruit fly, Russian melon fly, and processionary moths. Although obligatory diapause has been a major roadblock to developing biological control programs for many pests, current research suggests that there are approaches that can potentially disrupt obligatory diapause and facilitate mass rearing. Four approaches that appear particularly promising for circumventing the challenges of obligate diapause include: 1) simple environmental manipulations, such as thermal shock, 2) chemical or hormonal treatments, such as application of organic solvents, 3) choosing geographical populations without diapause or artificial selection for non-diapausing strains within populations, and 4) genetic modification by mutagenesis or transgenesis of critical genes for diapause. Successfully circumventing obligate diapause with any of these approaches, or a combination thereof, would provide new opportunities for effective mass rearing of important pest species. Beyond dormancy there is a spectrum of responses that may increase the stress tolerance of insects, and many of these responses could be exploited for mass rearing and biological control. For example, rapid cold-hardening responses can be induced in active insects. Similarly, variations in the storage environment (for example, via altering oxygen levels or temperatures) can significantly improve low temperature tolerance. Although insects stored at low temperatures are likely in a quiescent or dormant state, there remain opportunities to increase the duration and reduce the impacts of low temperature exposure via manipulations of storage conditions or physiological states.While obligate diapause is an obstacle in some cases, two aspects of dormancy and other physiological tolerance responses can be effectively exploited to improve the efficacy of biological control programs. 1) Dormancy can be used to stockpile mass-reared insects and to time the supply of biological control agents to coincide with seasonal demand for release. The ability to synchronize the supply of control agents with demand is critical for the growing biological control industry. Furthermore, an enhanced understanding of dormancy responses could improve phenological models for both pest species and beneficial insects. Inducing dormant states or other physiological-conditioning treatments opens up new opportunities for either enhancing classical cryopreservation of embryos (in liquid nitrogen) or developing new methods for long-term cold storage of other life stages, such as larvae or pupae. Development of such techniques from an organismal or biochemical perspective could make it feasible to maintain strains over the long term without compromising the genetic integrity of those strains, while avoiding the efforts and costs involved in continuous rearing. This ability to maintain stocks without continuous rearing is especially important when considering the rapid accumulation of mutant and transgenic strains in entomological research laboratories. 2) Increased stress tolerance is often a hallmark of dormancy, a feature that could be exploited in biological control applications. The efficacy of biological control, including sterile insect programs and natural enemy releases, is affected by the quality of insects released into the field. Poor performance of insects used in field releases can be a product of stresses experienced at multiple points during the production, marking, irradiation, shipping, and release process. The ability to specifically induce dormant states, including either diapause or quiescence, could potentially reduce the above stresses, thereby improving the performance of individuals in field releases. For example, some diapausing insects are known to be resistant to low-level irradiation. Perhaps diapause could be exploited to reduce off-target irradiation damage, outside of germ-line genomic DNA, and improve the performance of sterile insects. Similarly, insects are frequently exposed to mechanical disturbance, hypoxia, and thermal stress during shipping, stresses that may be mitigated by inducing dormant states prior to shipping. Thus there is a substantial opportunity to use knowledge of dormancy (both diapause and other stress-driven responses) to improve biological control, especially through the sterile insect technique. OBJECTIVES Identify genetic and physiological mechanisms generating natural occurring variation in dormancy induction and the timing of dormancy termination in the tephritid fly pest Rhagoletis pomonella. The expected outcome is use this knowledge for downstream manipulations to control the induction and timing of dormancy to facilitate economical mass-rearing and stockpiling of insects possible in R. pomonella. We also expect this work to extend to other economically important Rhagoletis species and additional tephritid pest species with an obligate diapause like the Chinese citrus fruit fly B. minax.2. Apply and test existing SIT irradiation protocols developed for other mosquitoes to Florida-derived populations of Ae. aegypti to yield male sterility while maintaining male performance, specifically male longevity, dispersal, and mating competitiveness. The expected outcome will be to develop protocols for laboratory mass rearing, irradiation, and process quality control metrics for use in field releases of sterile male Ae. aegypti by Florida mosquito control districts and to identify treatment and handling protocols that may improve male performance by reducing stress on insects across the SIT pipeline. This current project will lay the critical groundwork in developing SIT for Ae. Aegypti mosquitoes. In future work, my group will apply dormancy manipulation approaches developed for other insect pests to mosquito SIT.
Project Methods
?Objective 1. Identify genetic and physiological mechanisms generating naturally occurring variation in dormancy induction and the timing of dormancy termination in the tephritid fly pest Rhagoletis pomonella.O1A. Respirometry and Phenotyping. Early-emerging insects collected from apple fruits and late-emerging insects collected from hawthorn fruits will be reared under standard Rhagoletis lab conditions. After 4 mos. of chilling, diapausing flies will be returned to 21oC and individuals tracked respirometrically. We will sample multiple sets of individuals at different time points during diapause: 24h after return to 21oC, each week 1-4 weeks after return, and 24h & 48h after diapause termination. Individuals from each fly host population at each time point will be snap-frozen for endocrinology and cell cycle analyses, or have their brains dissected and homogenized in trizol, and frozen, for RNAseq and peptide work. Carcasses will be kept for DNA extraction and genotyping.O1B. Developmental Physiology of Wnt/β-catenin Signaling and the Cell Cycle. We will quantify the activity of several members of the Wnt/β-catenin signaling pathway including Wnt1, β-catenin, GSK3, and c-Myc using QrtPCR for insects during the transition to diapause termination. In addition, we will perform Western blotting for Wnt1 & β-catenin using commercially available Drosophila antibodies. We will also screen other commercially available antibodies for Wnt/β-catenin signaling pathway members.O1C. Endocrinology and Biogenic Amines. Hormone extraction and quantification from apple maggot flies will follow standard methods, including separation and quantification/identification on HPLC-MS. We will use pools of 5 flies and generate 10 replicate pool measures of ecdysteroids and JHs at each developmental point. First, we will sample flies from the time series described above. We will pool groups of 10 similarly staged individuals together for extraction and quantification of biogenic amines via HPLC-MS using standard methods.O1D. Transcriptomics. We will analyze gene expression during diapause maintenance and termination for from each Rhagoletis host race. Brains will be dissected out of flies phenotyped for diapause status using respirometry, homogenized individually, and stored at -80oC until analysis. Individual homogenized brains will then be pooled into 6-8 replicate pools of 10 brains ea. for each fly host population. RNA will be extracted using the Ambion Ribopure kit, libraries constructed, and RNAseq will be performed by sequencing on an Illumina HiSeq instrument, as we have previously done for flies.O1E. We will investigate the genetics of eclosion time using a genome wide association approach. First, we will perform whole genome sequencing on individual adult flies (n = 30 individuals ea.) reared directly from field collected apple and hawthorn fruit. Individuals will be evenly sampled from their eclosion distributions and by sex for sequencing. Second, we will conduct a bulk segregant analysis of the earliest and latest eclosing fractions of adults (n = 50-100 ea.). These flies will be genotyped by pool-seq to test for allele frequency differences associated with diapause timing. Third, we will compare patterns in the bulk segregant study and physiology functional assays above.Whole genome sequencing of 450 bp insert libraries will be carried out on an Illumina HiSeq 4000 for the geographic survey and by pool-seq for samples of early and late eclosing insects in the bulk segregant studies. Paired end sequences will be aligned to the R. pomonella reference genome with the BWA-MEM algorithm. We will identify variant sites using HaplotypeCaller in the Genome Analysis Toolkit (GATK v. 3.5). Population level allele frequencies and individual genotypes will be estimated via a hierarchical Bayesian model.Objective 2. Apply and test existing SIT irradiation protocols developed for other insects to Florida-derived populations of Ae. aegypti to yield male sterility while maintaining male performance, specifically male longevity, dispersal, and mating competitiveness. O2A. Mass Rearing Parameters. We have already established field-collected colonies from two partner mosquito control districts, Anastasia Mosquito Control District and Lee County Mosquito Control district. Colonies will be evaluated for rearing parameters including: egg-adult viability, growth, and egg-adult development time.O2B. Dose-Response Experiments: Treating males with radiation doses that are too high, or treating at the wrong stage of development, can induce off-target damage that leads to male performance loss, ultimately decreasing efficacy of sterile insects to control wild populations. Doses will range from 0-100 Gy in 10 Gy increments to determine a dose for 100% male sterility that maintains male performance. Delivered doses will be verified for each sample using Gafchromic V2 radiation-sensitive film read in our lab.O2C. Small-Cage Mating Trials: Male sterility of both the St. Augustine and Lee County strains at each dose will be assessed by mating groups of 50 irradiated virgin males to 50 non-irradiated virgin females in 30 x 30 x 30 cm cages. Three days after trials begin, females will be blood-fed and allowed to oviposit on seed germination paper partially submerged in water inside oviposition cups in adult cages. Egg paper will be removed daily for 7 days and inspected. Presence of larvae and numbers of hatched and unhatched eggs will be counted after exposing eggs to deoxygenated water.O2D. Field Mark-Release-Recapture (MRR) Experiments: We will perform field releases of sterile male Ae. aegypti at sites in Lee County and St. Augustine to estimate sterile male dispersal, sterile male residence time in the field, and quantify field Ae. aegypti population densities to estimate numbers needed for future suppressive field releases. Note that no special permitting is needed for radiation-based SIT when working with local strains.We will do MRR at two different times for Ae. aegypti populations in each mosquito district, early in the spring when population densities are low (e.g., March), and late spring/early summer as population densities increase (e.g., May). MRR will allow us to both estimate the standing mosquito population and assess dispersal and longevity of sterile males in the field. We will release 10,000 irradiated male mosquitoes marked with DayGlo fluorescent dust. Recapture will be monitored daily with an array of 24 BG Sentinel traps placed in transects.O2E. Biweekly releases for field suppression will include releasing 10,000 sterile male Ae. aegypti of the appropriate local strain along roads running across our field sites early and late each week. Mosquitoes will be released from containers at several points along roads in the focal neighborhood, probably on Mondays and Thursdays to yield 10,000 mosquitoes/week for our ~3-5 hectare treatment site.Control site: Field suppression of Ae. aegypti in our target site will be assessed by comparing population densities with a similar 3-5 hectare area of suburban neighborhood as a control site between 1.5-2 km away.