Recipient Organization
UNIVERSITY OF TEXAS AT ARLINGTON
400 S CORN ST
ARLINGTON,TX 76019
Performing Department
(N/A)
Non Technical Summary
Maximizing food production is essential to prevent food scarcity, but as global temperatures rise, food scarcity will increase. Shifts in temperature will alter insect pest abundances, with increased temperature potentially also causing shifts in disease transmission rates to heat stressed crops. Furthermore, insect thermal tolerance may be altered by the local availability of their environmentally acquired bacterial symbionts, making these insect hosts better suited for changing environments. Unlike maternally transmitted symbionts, insect participants in the bug-Caballeronia symbiosis utilize numerous symbiont species. This allows host insects to utilize novel microbes that are better suited for the local environment due to the high genetic mutation rates found in environmental bacteria. This work focuses on understanding how thermally tolerant Caballeronia (bacterial) strains can contribute to shifts in host vectoring capacity. Using the agricultural pest Anasa tristis, and sole vector of the phytopathogenic lineage of Serratia marcescens, I will 1) determine whether Caballeronia strains with varying thermal optima differentially alter host thermal tolerance, 2) determine whether environmental temperature affects Serratia titer in A. tristis, and 3) assess whether temperature influences disease transmission rates to cucurbits.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The primary goal of this research is to determine whether increased temperature (thermal stress) contributes to shifts in pathogenic Serratia titer and host insect (Anasa tristis) pathogenicity.The planned objecties are:Confirm whether symbiont thermal tolerance influences Anasa tristis' performance, under sustained thermal stress, similar to the effects observed in L. phyllopusDetermine whether environmental temperature affects A. tristis' vector capacity (i.e., Serratia titer after exposure)Assess whether temperature influences phytopathogenic Serratia transmission by A. tristis to cucurbit plants and between insects
Project Methods
Objective 1Following established Gerardo Lab protocols, I will maintain colonies of A. tristis on cucurbit plants in mesh cages. Eggs will be collected and surface sterilized prior to use in experiments. Upon molting to second instar, nymphs will be provided with an overnight culture of one of four different Caballeronia symbionts for 24 hours. After the infections, 30 third instar nymphs for each of the five infection groups will be reared to adulthood at 24, 30, and 36°C. I will record the number of insects that survive to adulthood, adult insect weight, and development time from third instar to adult. I will perform qPCR, using total host DNA from each surviving adult, with each sample run in triplicate, to approximate Caballeronia titer. Data will be analyzed using linear mixed effect models, with temperature and symbiont strain as fixed effects.Objective 2First, I will evaluate the in vitro thermal optima of phytopathogenic Serratia strains already available in the Gerardo lab. I will dilute overnight cultures to 0.1 OD (optical density), and grow them in a plate reader for 48 hours to find the optimal growth rate for each Serratia strain across temperatures ranging from 20 - 40°C. Then, insects will be reared and infected with symbiont strains following the procedure in Obj 1. I will use the symbiont strains conferring the highest and lowest host performance at higher temperatures (evaluated in the previous objective). If the insect is to be infected with Serratia, after the 24 hr symbiont inoculation period, hosts will be transferred to zucchini fruit for 24 hr before subsequently being provided with Serratia for a second inoculation phase.Insects will be infected following a factorial design, using two Caballeronia strains with either high or low thermal optima paired in combination with two Serratia strains with either high or low thermal optima. Finally, two aposymbiotic control groups will be included that are only infected with Serratia. After the infection period, 70 third instar nymphs for each of the six treatment groups (four-symbiont/Serratia combinations and two-Serratia only groups) and three temperatures (28, 32, and 36°C) will be reared to fourth instar (20 insects per treatment), fifth instar (20) and adulthood (30). I will collect data on adult host fitness and their symbiont titer as above. I will also use qPCR to quantify Serratia titer within hosts across developmental stages. Data will be analyzed using linear mixed effect models, with temperature, symbiont strain, and Serratia strain as fixed effects.Objective 3Insects will be infected with their symbionts and Serratia using the same procedures as above. Once infected, insects from all three treatments will be divided into three temperature groups where they will be reared at 28, 32, and 36°C (n = 30 per symbiont treatment x temperature, for 270 individuals total). Fourth instar hosts will be placed in pairs onto healthy one-month old squash plants, where they will be restricted to the bottom leaves of the plants within mesh rearing cages to feed for 2 weeks. Rearing cages will be incubated in Percival chambers at appropriate temperatures for each treatment group. After the inoculation phase, insects will be removed and sacrificed for quantification of Caballeronia and Serratia, as in the previous objectives.After insects are removed, leaf surfaces that touched the Serratia infected insects will be swabbed and subsequently applied to a bacterial growth media to determine whether the phytopathogen was excreted onto the plant surface by visualizing fluorescent bacterial colonies. Additionally, after the Serratia infected insects are removed, aposymbiotic juvenile A. tristis will be added to the top leaves (never touched by the Serratia infected insects). These nymphs will remain on the plants for an additional 10 days. Afterwards, these insects will be removed, surface sterilized with ethanol, homogenized, and then plated on bacterial growth media to determine whether Serratia was ingested by the nymphs by visualizing fluorescent bacterial colonies. Finally, the plants will remain in temperature-controlled chambers for six additional weeks for disease symptoms to progress before extracting DNA from leaf, root, and stem tissues. The presence of Serratia will be evaluated through conventional PCR using Serratia specific primers to confirm pathogen transmission.Collected data will be used in linear mixed-effect models to evaluate how Serratia titer across temperatures is associated with different levels of disease transmission to seedlings and subsequent acquisition by the next generation of insects.