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 diseasetransmission rates to heat stressed crops. Furthermore, insect thermal tolerance may be altered by the local availability of theirenvironmentally acquired bacterial symbionts, making these insect hosts better suited for changing environments. Unlikematernally transmitted symbionts, insect participants in the bug-Caballeronia symbiosis utilize numerous symbiont species. Thisallows host insects to utilize novel microbes that are better suited for the local environment due to the high genetic mutationrates 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 thephytopathogenic lineage of Serratia marcescens, I will 1) determine whether Caballeronia strains with varying thermal optimadifferentially alter host thermal tolerance, 2) determine whether environmental temperature affects Serratia titer in A. tristis, and3) 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 inpathogenic Serratia titer and host insect (Anasa tristis) pathogenicity.The planned objecties are:1. Confirm whether symbiont thermal tolerance influences Anasa tristis' performance, under sustained thermal stress,similar to the effects observed in Leptoglossus phyllopus2. Determine whether environmental temperature affects A. tristis' vector capacity (i.e., Serratia titer after exposure)3. Assess whether temperature influences phytopathogenic Serratia transmission by A. tristis to cucurbit plants andbetween insects
Project Methods
Obj 1. As the effects of climate change continue to worsen, accounting for how shifts in environmental pressures (i.e., temperature, humidity, etc.) will alter pathogen transmission is essential for ensuring global food security. In my doctoral research, I studied the effects of temperature on L. phyllopus. Leveraging this understanding, I will first perform a similar, but smaller scale experiment, to evaluate A. tristis' response to thermal stress and how different symbiont strains affect host performance. I hypothesize that A. tristis will have the same response to temperature as L. phyllopus. I will use Caballeronia strains LZ003, LZ019, LZ008, and TF1N1, which I had used previously in L. phyllopus thermal experiments (Stillson et al, unpublished). These strains are ideal as we know their impact on thermal performance in another host (L. phyllopus), and A. tristis receives equal fitness benefits from L. phyllopus-derived and A. tristis-derived Caballeronia strains (data unpublished), likely because there is minimal host-symbiont specialization. Along with these strains, I will include an aposymbiotic control group to evaluate how the hosts respond to thermal stress without a symbiont. Following established Gerardo Lab protocols (19, 30, 38), 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 the Caballeronia symbiont 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.Obj 2. Under standard laboratory rearing conditions, ~28°C, the Serratia titer in A. tristis declines when hosts are colonized with Caballeronia symbionts. I hypothesize that when a host is provided a symbiont with a low thermal optimum and reared at temperatures above 32°C, the symbiont titer will decrease and the Serratia titer will increase. Implications of this would be that at higher temperatures in the field, the vectoring capability of A. tristis may increase as the Caballeronia symbiont titer declines. First, I will evaluate the in vitro thermal tolerance 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.Obj 3. The Anasa-Serratia transmission model is assumed to follow a classic host to vector disease cycle (26), with all previous work conducted before the identification of the Caballeronia symbiont in A. tristis. Studies verified A. tristis vectors Serratia, but the symbiont status is typically unknown, as some vectors could have been aposymbiotic. This would impact host fitness and likely the Serratia titer and vectoring capabilities of the host. This may also significantly change the host's vectoring ability, as Serratia titers decrease when the host is provided a Caballeronia symbiont (38). I hypothesize that elevated environmental temperatures will cause the symbiont titer to decrease in hosts possessing symbiont strains with lower thermal optima. This will cause Serratia titers to increase, leading to increased disease transmission to cucurbits. Leveraging the Gerardo Lab's preliminary transmission work to determine whether the likelihood of Serratia transmission to squash is impacted by shifts in vector capacity due to temperature or symbiont effects (Obj 2). Individuals will be infected with either 1) no Caballeronia symbiont (control), 2) a low thermal optima Caballeronia strain (from Obj 2), and 3) a high thermal optima Caballeronia strain (from Obj 2). Due to logistical constraints, all treatments groups will be provided with the same Serratia strain, selected based on outcomes of Obj 2, but it will be fluorescently marked for easier identification. Insects 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 andsacrificed for quantification of Caballeronia and Serratia, as above. 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 ofSerratia will be evaluated through conventional PCR using Serratia specific primers (46) 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.