Trigeminal Mediation of Mammalian Aversion to Insect Chemical Defense Compounds Paige M. Richards, Annalyn M. Welp, Deirdre R. Craven and Wayne L. Silver Department of Biology, Wake Forest University, Winston-Salem, NC Background One way that insects protect their territory or avoid predation is by releasing defensive chemicals. A wide variety of insects use the same chemicals for this purpose. We hypothesize that defensive chemicals used by multiple insect orders target the mammalian trigeminal system and elicit chemesthesis. In the current study, we have begun to test some of these compounds to determine if they irritate a mammalian predator (rat). We determined whether the chemicals activate the trigeminal nerve by recording from the ethmoid branch while perfusing stimuli through the rat’s nasal cavity. As trigeminal nerve activation depresses respiratory frequency, we also monitored respiration in the rats both before and after stimulus presentation. We then determined if rats are behaviorally averse to these compounds at concentrations released by insects. This project is funded by the Center for Molecular Communication and Signaling at Wake Forest University. Insect Images are taken from the Encylopedia of Life (eol.org). Either Public Domain or Creative Commons Licensing photographs by (in order) : Pavla Tochorova, Ivo Antusek, Richard Bartz, California Academy of Science (2 photos), Phil Myers, Adam Opiola FIGURE 1. Insect defense chemicals activate the mammalian trigeminal nerve. FIGURE 2. Rats are behaviorally averse to defense chemicals at concentrations insects release. A. Image of a rat in the plexiglass arena for the behavioral aversion paradigm. Either an irritant or water is placed in one corner with water in all other corners. The time the rat spends in each corner (or zone) during a 10 minute period is determined using Noldus Ethovision. Rats were habituated to the arena for three days prior to testing. Sample tracking for a rat in an arena containing (B) AITC in the bottom right corner or (C) water in every corner.. C.B. Summary We have identified several insect allomones that activate the rat trigeminal nerve resulting in respiratory depression. Several of these compounds also elicit behavioral aversion in rats at biologically relevant concentrations. One tested allomone (tetradecane) did not elicit trigeminal responses nor did it cause behavioral aversion even at the highest concentration possible. Three compounds – formic acid, 6-methyl-5-hepten-2- one and trans-2-hexen-1-ol did not elicit behavioral aversion at relevant concentrations even though they activated the trigeminal nerve when tested. D. Mean time spent by rats in a designated quadrant (either containing irritant or water) vs. mean time spent in each of the other quadrants (all containing only water). Asterisks indicate statistical significance (p<0.05). n=6-8 Experimental Set-Up for Delivering Fluids to the Rat Nasal Cavity and recording neural and respiratory responses Future Directions Conduct calcium image studies on rat trigeminal ganglion cultures. Determine which sensory receptors these defense compounds may be activating using physiological and molecular techniques. 2-Heptanone (25 mM) A. mellifera O. fasciatus trans-2- Hexenal (10 mM) Stimulus Representative Nerve Recording Representative Respiratory Trace Representative Releasing Insect Stimulus Representative Nerve Recording Representative Respiratory Trace Representative Releasing Insect Stimulus Representative Nerve Recording Representative Respiratory Trace Representative Releasing Insect N/A Ringer’s (saline) Benzoquinone (75 mM) B. explodens 6-methyl-5- hepten-2-one (20 mM) L. fuliginosis trans-2-Hexen- 1-ol (100 mM) P. apterus Benzaldehyde (50 mM) S. sipylus Tetradecane (1.5 g/mL bolus) M. gulosa Formic Acid (50 mM) F. rufa