Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
Entomology
Non Technical Summary
Termites are major structural pests that cause an estimated $40 billion USD in damage a year worldwide. Damage is caused when termite colonies, which can consist of as many as tens to hundreds of thousands of individuals, infest and consume wooden parts of structures. Because colonies live underground and consume wood from the inside out, infestations generally remain unnoticed until significant damage has already occurred. Therefore, control efforts are primarily focused on preventing new infestations. Synthetic termiticides are the most common and cost-effective form of control and are poured in liquid form around at-risk structures to create a long-lasting barrier that kills any termite passing through it. However, synthetic termiticides are also harmful to beneficial soil invertebrates such as earthworms and springtails. In addition, improper application can lead to groundwater contamination, harming aquatic invertebrates and fish. To develop alternative termite control strategies that are sustainable and environmentally friendly, it is necessary to target specific aspects of termite biology, thereby removing the threat posed to other organisms.One possibility is to take advantage of the unique caste systems present in termites. The majority of a termite colony consists of workers and soldiers: workers are physically weak and responsible for a variety of tasks, including nest construction, foraging, and brood care, while soldiers are physically strong and responsible for defending the colony from intruders. Previous work by our lab has revealed that soldiers may also play a role in managing stress of their nestmates. Workers that are continuously exposed to scent cues from a competitor species will die within one to two days. Death occurs even without physical contact between the worker and any competitors, and combined with the erratic behavior displayed by workers under these conditions implies that it is a result of stress alone. If a soldier is present alongside these workers, however, the workers will not die. Essentially, if a worker knows that a soldier is nearby, it will remain relatively free of stress because it is aware that the soldier will fight off any physical threats that may appear. Soldiers are always present at the periphery of the colony and are therefore always available to provide this "peace of mind" to workers, while themselves appearing to be highly resistant to stress. Disrupting this relationship of stress management between workers and soldiers, or artificially inducing stress in workers could represent feasible routes of termite-specific control.The goal of this project is to investigate the phenomenon of termite "peace of mind" so as to better understand its viability as a target for alternative termite control methods. First, the brains of termite workers and soldier will be directly examined and compared to establish how they differ structurally, with consideration for how these differences may relate to the response of each caste to stress. Next, the stress experienced by termites exposed to competitors will be directly quantified by measuring the levels of specific stress hormones present in the brain at different time points and under different conditions. Finally, the regions of the brain that respond stress will be visualized and compared between workers and soldiers. Together, this project will provide a detailed, comprehensive view of how termite workers and soldiers experience stress alongside a deeper understanding of the role that soldiers play in alleviating stress in workers. This work will establish a rationale while also forming the foundation for future alternative forms of termite control that seek to either disrupt the relationship of stress management between workers and soldiers, or artificially induce stress in workers to induce death of the colony.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The major goal of this project is to investigate how competitor-induced stress leads to changes in neurochemistry and neurophysiology in different termite castes. The following objectives will be carried out:investigate differences in brain structure between workers and soldiersidentify specific neurochemicals responding to competitor-induced stress in workers and soldiersidentify regions in the brain that respond, chemically, to competitor-induced stress
Project Methods
Objective 1. Categorize differences in brain structure between Reticulitermes flavipes castesReticulitermes flavipes worker and soldier brains will be dissected, then immunostained and fixed as follows: 1 hr in 2% paraformaldehyde (PFA), 3 reps of 15 min in PBS mixed with 10% Triton X-100 (PBST), 1 hr in 5% goat serum, 16-24 hrs in primary antibody nc82, 3 reps of 15 min in PBST, 16-24 hrs in secondary antibody Alexa Fluor goat anti-mouse 546, 3 reps of 15 min in PBST, 4 hrs in 4% PFA, 3 reps of 15 min in PBST. Brains are then placed on a poly-lysine-treated cover slip and submerged in the following for 5 mins each: 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol, 100% ethanol, 100% ethanol, 100% ethanol, 100% xylene, 100% xylene, 100% xylene. Finally, DPX is used to adhere the cover slip to a microscope slide. After drying for 2 days, slides are imaged using a Leica SP8 DLS confocal microscope. All brains are imaged with identical magnification and speed settings, and confocal image stacks are captured with 1 micron intervals. A minimum of 50 brains will be imaged for each caste.Template brain generation and brain comparison are conducted using the Computational Morphometry Toolkit software package (CMTK; https://www.nitrc.org/projects/cmtk/). CMTK's iterative shape averaging tool will be used to generate worker and soldier template brains using the 10 most symmetrical brains imaged for each respective caste (determined manually). Worker and soldier template brains will then be average using the same tool into a worker-soldier intercaste template brain. All 50 brains from each caste will be compared to the intercaste template using CMTK's registration, reformatx, and warp functions. The outputs of this analysis, in the form of Jacobian maps, will be compared between workers and soldiers using CMTK's ttest function, which computes a pixel-wise unpaired two-tailed t-test between two given groups of images. P-values less than 0.05 will be interpreted as significant.Objective 2. Identify neurochemicals responding to competitor-induced stress in Reticulitermes flavipes workers and soldiersHPLC samples will be collected using the competitor-induced stress assay. A group of 20 R. flavipes termites will be placed in a 3.5-cm diameter Petri dish with narrow slits cut into the wall, which will then be placed inside of a larger 5.5-cm diameter Petri dish containing 40 termites. The inner group will consist of either 20 workers or 19 workers and 1 soldier, while the outer group will consist of 39 workers and 1 soldier from either the same colony as the inner group, or the competitor species Reticulitermes virginicus. Inner group termites will be collected at the following time points: pre-setup, 10 min, 30 min, and 12 hrs post-setup. Collected termites will be snap frozen with liquid nitrogen, decapitated, and stored at -80°C. Samples will be shipped to Dr. Colin Brent at the USDA's Arid Land Agricultural Research Center in Maricopa, Arizona. Dr. Brent will carry out HPLC analysis using standard procedures.The results of HPLC analysis will identify biogenic amines that are elevated in termites exposed to competitor-induced stress. The roles of these amines in the competitor-induced stress response will be validated using RNAi. Genes corresponding to biosynthesis of these amines or coding for receptors of these amines will be selected from the annotated R. flavipes genome possessed by our lab. dsRNA will be synthesized using a commercially available kit and delivered orally. The competitor-induced stress assay will be conducted as described above using either dsRNA-treated termites or untreated control termites in the inner group. Mortality will be recorded at 12-, 24-, 48-, and 72-hours post-setup. Mortality in RNAi-treated groups will be compared across time points and between treated and untreated groups using a two-way ANOVA. P-values less than 0.05 will be interpreted as significant.Objective 3. Identify brain regions responding to competitor-induced stress in Reticulitermes flavipes workers and soldiersThe competitor-induced stress assay will be conducted as described in Objective 2. Inner group termites will be collected at the same time points and their brains will be dissected and prepared for visualization. Immunostaining will follow the process described in Objective 1, with the addition of an antibody targeting one of the biogenic amines identified as responding to competitor-induced stress, which will be used alongside nc82. Images of brains will be captured as described in Objective 1 and template brains will be generated using biogenic amine staining patterns. Comparison of biogenic amine staining patterns between worker and soldier termites will be conducted using CMTK, as described in Objective 1. P-values less than 0.05 will be interpreted as significant.Efforts - Efforts expected are: undergraduate student mentoring, teaching university courses as a guest lecturer and/or teaching assistant, and participation in outreach programs at local schools and community centers.Evaluations - Progress towards project completion will be evaluated at annual graduate committee meetings. The primary mentor, Dr. Xuguo Zhou, will perform additional evaluation in the form of annual reviews required by the University of Kentucky's entomology department for all graduate students.