THE ROLE OF CHEMICAL ESPIONAGE BETWEEN HERBIVORES: INVESTIGATING HOW EAVESDROPPING OF CHEMICAL SIGNALS BY ANASA TRISTIS INFLUENCES PERFORMANCE AND SURVIVAL

In this project, there are three primary goals. The goals are the following:Determine the relationship between A. vittatum damage and the amount of vittatalactone released.Determine how the presence of vittatalactone and other A. vittatum damage-specific semi chemicals influence A. tristis choice.Determine how previous damage by A. vittatum affects the performance and survival of A. tristis across multiple varieties.How goals will be accomplished Goal 1 - Investigating the relationship between A. vittatum damage and the amount of vittatalactone releasedTo understand the relationship between striped cucumber beetle damage and pheromone released we will need to quantify volatile headspace emissions of active A. vittatum damage in the lab. In groups of 1, 5, 10, and 50, male A. vittatum adult beetles will be starved for 4 hours and added to 1-gallon glass jars for 72 hours. Groups will either receive a plant at the two true-leaf stage or no plant using C. pepo var. Zephyr Summer Squash. Air purified through activated charcoal and humidified through a distilled water bubbler will be pushed into 1-gallon jars at 0.5 mL/min. Outflow air of each jar will pass through an Orbo ® filter packed with activated charcoal filters to collect volatiles. Overhead full spectrum light shown through a diffuser will be on an 18:6 LD cycle to simulate natural conditions. Following the allowed time, filters will then be eluted with 500uL of MeOAc and transferred to Agilent 2mL glass vials fitted with 250µL glass inserts. All vials will then be stored at -80 Celsius until analyzed. Beetles will be returned to rearing cages and plants will be assessed for percent damage using LeafByte26 taking the average damage of both leaves. Volatile samples will be analyzed using GC-MS and verified using pure vittatalactone standards received from27, 28, 18. We will collect between six and twelve replications for each treatment group. Quantification of vittatalactone and other major volatile emissions will be analyzed using Agilent Quant ® GC-MS software (2021). Concentrations of quantified volatiles will be modeled using R and Rstudio (Ver. 4.0.3; R Core Team, 2020). and compared using parametric measures such as ANOVAGoal 2 - Investigating how the presence of vittatalactone and other A. vittatum damage-specific semiochemicals influence A. tristis choiceTo understand how the presence of vittatalactone and other A. vittatum damage-specific volatiles influence A. tristis we will measure choices of gravid female A. tristis adults using a Y-tube olfactometry. To measure A. tristis choices, a glass Y-tube will be held at a 15-degree angle and set inside a blacked-out cardboard box with full spectrum diffused light entering from the roof. Air purified through activated charcoal, humidified through a distilled water bubbler, and then split up to six channels, will be pushed into the 1-gallon jars at 0.5 mL/min29, 30. Outflow air will be vented through an exhaust hose in the lab. Lab-reared gravid female A. tristis will be carefully loaded into the bottom of the Y-tube and given a maximum of four minutes to make a choice. Choices will be determined once the insect passes an opaque line marked about one inch past the left and left arm of the Y-tube. All glassware will be then sterilized with 70% ethanol following each run and then washed with hot water and soap and baked at 200 degrees Fahrenheit every six trials. Airflow lines will be switched every other run. All choices will be recorded on video using a Sony a6000 model mirrorless camera. For conditions with plants, we will use C. pepo var. Zephyr Summer Squash plants at the two true-leaf stage grown. For conditions with plants and herbivores, ten A. vittatum or six A. tristis adults will be starved for 4 hours and then added to a 1-gallon jar. Insects will actively feed on these plants for 72 hours. Lastly, for conditions with plants and synthetic pheromone, 110ng of synthetic pheromone suspended in hexane will be added to a rubber septum following natural emission of ~11ng/h/beetle (Morris et al. 2005). Preferences will be interpreted using Pearson's chi-squared test of independence. All statistical analyses were conducted using R and RStudio (Ver. 4.2.1; R Core Team, 2022). The results of this experiment will determine what how A. vittatum volatiles are attractive to A. tristis and if there are synergies between certain volatile combinations.Goal 3 - Investigating how previous damage by A. vittatum affect the performance and survival of A. tristis across multiple varietiesTo investigate potential tradeoffs associated with eavesdropping we will perform the choice tests, no choice tests, and no choice field tests using gravid female adult A. tristis and then record the performance and survival of offspring. For all experiments, we will use a simple 3x2 factorial design to investigate how the presence or absence of previous damage by either A. vittatum or A. tristis on either a variety A. tristis normally performs well on (C. pepo var. Zephyr Summer Squash)31 or a variety they normally perform poor on (C. lanatus var. Crimson Sweet) 31 affects the performance and survival of offspring A. tristis. For the choice test, single gravid female A. tristis will be placed in a small cage with a plant of each treatment for 24 hours. To manipulate damage, A. vittatum males in groups of ten and A. tristis adults in groups of six will be starved for 4 hours prior to feeding per plant. Following the allowed time, the number of A. tristis egg clutches will be counted per plant. Preferences will be interpreted using Pearson's chi-squared test of independence. For no-choice experiments, a single gravid female A. tristis will be assigned one of the six treatment conditions and given 72 hours to feed and oviposit eggs. Following the allowed time, the number of egg clutches will be counted. Plants will then be left in growth chambers until A. tristis eggs hatch. A. tristis nymphs will then be allowed 10 days to grow and developed. Average nymph weight and mortality will be recorded for each trial. No choice experiments will then be replicated in open cage experiments using the Homer C. Johnson Vegetable Research Farm, Freeville New York, USA. Large pop-up cages will be placed in a randomized block design over potted 4 plants of each treatment. In cages, 5 gravid female A. tristis will be placed inside each cage and allowed feed and oviposit for 1 week. After the allowed time, cages will be opened for 1 week and then sealed again for another week. After this time, A. tristis adults and offspring will be assessed for parasitism.