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1. Limonene (LIM) Eugenol (EUG) Nicotine (NIC) Ally Isothiocyanate (AITC) Amyl Acetate (AMA) α-Terpineol (TER) Toluene (TOL) Cinna- maldehyde (CIN) # of sucrose only flies total # of flies feeding Cyclo- hexanone (CY) # of test irritant flies total # of flies feeding PI = A Behavioral Assay using Drosophila to Test for Chemesthetic Irritants Activating TRPA1 Channels Wayne L. Silver, Matthew W. Greene, Paige M. Roe, Erik C. Johnson Department of Biology, Wake Forest University Winston-Salem, NC, USA silver@wfu.edu Conclusions There were significant differences in the ability of Painless and wild type flies to avoid most but not all of chemical irritants tested. Thus, the feeding assay designed by Al-Anzi et al. (2006), appears to be a useful tool for testing for the avoidance of chemical irritants. We are currently testing rescued flies to confirm this. Al-Anzi et al. (2006) and the current study suggest that Painless is involved in detecting chemical irritants. However, Sokabe et al.(2008) reported that Painless expressed in HEK cells is not activated by irritants such as AITC. Perhaps Painless is not a direct receptor for these compounds and interaction with accessory proteins, the formation of heteromeric channels and/or splice variants may be responsible for the in vivo response. Future experiments will test these possibilities. Introduction Chemesthesis is the sense of irritation produced by chemicals. In mammals, the trigeminal nerve is a mediator for chemesthesis in the head and face.  Chemical irritants stimulate the trigeminal nerve, although the mechanism of stimulation for many irritants is unknown. The transient receptor potential (TRP) family constitutes a large variety of polymodal receptors which respond to various noxious stimuli including irritating chemicals. The TRPA1 channel in mammals responds to numerous irritants, e.g. allyl isothiocyanate (AITC), the active ingredient in mustard oil and wasabi (Karashima et al, 2007).  Drosophila express at least three TRPA1 channels; dTRPA1, Pyrexia, and Painless (Sokabe et al., 2008). The painless gene expressed in gustatory receptor neurons is an evolutionary homolog of mammalian TRPA1 and plays a role in the flies’ ability to avoid AITC (Al-Anzi et al, 2006). We are using a Drosophila feeding assay to examine what other known trigeminal irritants might also stimulate these TRPA1 channels. Rat multiunit trigeminal nerve responses to irritants tested 1% sucrose 1% sucrose + irritant Combination of blue and red solutions Starved flies 96 well plate with lid Acetic Acid (ACA) Figure 1. Male flies were starved in 2% agar vials for 24 hours. They were then used in one hour feeding preferences tests in complete darkness. After completion of the test the abdomens of the flies were scored for feeding preference based on their color. (from Al-Anzi et al., 2006) Materials & Methods The feeding assay is described by Al-Anzi et al. (2006) ( Figure 1). Two groups of 50 male D. melanogaster were placed into separate vials containing a 2% agar solution. The flies were then starved in these vials for a 24 hour period. After the 24 hour starvation period, 50 flies were placed on one of two 96-well plates (see fig. 1) with lids that served as the test arena. For one plate each well contained either 2% agar + 1% sucrose + reddyeor 2% agar +1% sucrose + bluedye + 5 mM of a chemical irritant. The other well plate contained the same compounds except the placement of the dyes was reversed. We tested 11 known trigeminal nerve irritants. The flies were left in the feeding assay for one hour in complete darkness. After an hour, the flies were anesthetized and scored for feeding preference based on the color of their abdomens. Each red and blue abdomen was counted as 1 fly for its respective stimulus; purple abdomens were counted as ½ of a fly for each stimulus. Flies with clear abdomens were counted as 0. A Preference Index was calculated as the fraction of flies that had eaten the test stimulus, minus the fraction of flies that had eaten the control sucrose solution. That is: A PI close to +1 indicates that the flies were attracted to the test material while -1 indicates an avoidance. 0.1 AITC* AMA* EUG* LIM* TER* CIN* NIC* BEN* ACA TOL CYC 0 -0.1 -0.2 Literature Cited Al-Anzi B, Tracey DW Jr., Benzer S. (2006) Repsonse of Drosophila to Wasabi Is Mediated by painless, the Fly Homolog of Mammalian TRPA1/ANKTM1. Current Biology. 16:1034-1040. Karashima Y, Damann N, Prenen J, Talavera K, Segal A, Voets T, Nilius B. (2007) Bimodal action of menthol on the transient receptor protential channel TRPA1. Neuroscience. 27:9874-84. Sokabe T, Tsujiuchi S, Kadowaki T, Tominaga M. (2008) Drosophila Painless is a Ca2+-Requiring Channels Activated by Noxiuos Heat. Journal of Neuroscience. 28:9929-9938. Acknowledgement We thank Dr. Dan Tracey at Duke University for supplying the Painless flies. Preference Index -0.3 Benzaldehyde (BEN) -0.4 Wild Type -0.5 Painless -0.6 Figure 2. Wild type flies avoided all of the compounds tested. Painless flies avoided AITC, EUG, BEN, AMA, TER, LIM, and CIN significantly less than did the wild type flies. There was no difference in responses to CYC, ACA, and TOL. Painless flies avoided NIC significantly more than did the wild type flies. (Student’s T-test *P< 0.05, n=6, error bars = standard error) -0.7