environmental acclimatization. Then flies were transferred to 18uC and 32uC incubator for 15 min. After tapping the flies down to the bottom, we counted the number of flies that climb more than 10 cm from the bottom in 1 min. Three trials were performed for each genotype and repeated with 3 different batches. All mutant strains used in our TPB assays showed similar climbing rates as the wild-type. A horizontal locomotor activity test was done as described previously [37]. It showed that cyp6a17D173 mutant flies are normal in the horizontal locomotor activity. It has been reported that the mushroom bodies are involved in suppressing hyperactivity of flies [38]. We found that test flies of the same genotype placed at high and low temperatures show different locomotor activity at the beginning of the trial but results in the same distribution profiles at the end of the assay. This indicates that the difference in the initial locomotor activity does not affect the temperature preference behavior in the standard assay conditions that allow time for test flies to choose and settle at their preferred temperature. Construction of transgenic flies cyp6a17-RNAi construct was generated as described [39]. cyp6a17-RNAi flies contain two copies of a 222 bp fragment of cyp6a17 cDNA. PCR primers used to create the target region for cyp6a17 knockdown were 59- TGCTCTAGACAGCTTGTAC- GATCCAAA-39 and 59- TGCTCTAGAAATCATTTCGCTT- TTCCT-39. A PCR fragment was cloned into pWIZ vector. To construct UAS-cyp6a17, 1.5 kb fragment of a cyp6a17 cDNA obtained from the Drosophila Genome Research Center was cloned into pUAST. A cyp6a17-Gal4 construct was generated using pCaSper vector and 1,201 bp sequence upstream from the transcription start site of the cyp6a17 gene. cyp6a17SG-Gal4 was inserted at 78A1. Unlike cyp6a17MB, Gal4 expression in the mushroom bodies was strongly repressed, thus showing a preferential expression in the surface glia. To make a cyp6a17 genomic rescue construct, a 4.2 kb SacI-digested genomic fragment containing about 1.2 kb upstream from the transcription start site and about 1.5 kb downstream from the transcription stop site, was cloned into pCaSper vector. Transgenic lines were generated in the w1118 background using standard germline transformation techniques. Relative quantitative RT-PCR 4–6 day old adult flies were collected and frozen immediately in liquid nitrogen. Heads were removed from bodies by vortex- mixing and sorting through a chilled sieve. Total RNA was extracted from heads with a Trizol (Invitrogen) and reverse transcribed using the AMV reverse transcriptase system (Pro- mega). RT-PCR was performed with the following primers: for cyp6a17, 59-GTTGTTACTGGCGCTAATCGT-39 and 59-TT- CCATCACAACCTGCTCCA-39; for cyp6a22, 59-TTCGGG- AAACTGTGAAGCAG-39 and 59-GATAAAAACATTTGATT- GTGT-39; for cyp6a23, 59-TCGCTGTTACTAACGTTGAT-39 and 59-TTTCCATGACGACCTGCTCCA-39; for gapdh1, 59- ACCGTCGACGGTCCCTCT-39 and 59-GTGTAGCCCAGG- ATTCCCT-39. Real-time PCR was performed with iCycler iQ (Bio-Rad) using the KAPA SYBR FAST Bio-Rad iCycler qPCR kit (Kapa Biosystems). The level of gapdh1 mRNA was measured as an internal control for the RNA amount in each sample. Data References cyp6a17 Is Required for TPB in Drosophila processing was performed with Microsoft Excel. All quantitative analysis were done more than three times. Immunohistochemistry Adult brains expressing transgenic cyp6a17-Gal4.UAS- mCD8.GFP were removed from head capsules and fixed in 4% paraformaldehyde in PBS for 30 min, and rinsed in PBS containing 0.5% Triton X-100. For staining with anti-FasII (Hybridoma Bank), adult brains were fixed for 30 min in 4% paraformaldehyde in PBS containing 0.5% Triton X-100. Fixed samples were washed for 10 min three times and incubated with 5% normal goat serum. Antibody dilution was 1:50 for mouse anti-FasII and 1:250 for goat anti-mouse Alexa Fluor 568 (Molecular Probe). Confocal analysis was performed on a Zeiss LSM710 microscope. Confocal stacks were processed with ZEN 2009 Light Edition and Adobe Photoshop. Supporting Information Figure S1 Effects on cyp6a17 mutation on mushroom body morphology. Mushroom body morphology in cyp6a17D173 mutant. Mushroom body axons are visualized using FASII antisera. Compared with wild type (A), no significant defects were found in mutant (B). Doral is up. (PDF) Figure S2 Effects of cyp6a17 RNAi and tissue distribu- tion of cyp6a17 expression. (A) Reduction of the expression level by MB247.cyp6a17-RNAi. gapdh1 was used as internal control. The number of tests: N = 4. Two asterisks, P,0.001. (B) Transcriptional profiles of cyp6a17 in the head and body assayed by real-time PCR. The histogram shows that cyp6a17 mRNA is slightly more abundant in the head compared with the body. gapdh1 was used as internal control. The number of tests: N = 4. Two asterisks, P,0.001. All data are means, and the error bars indicate s.e.m. (PDF) Figure S3 The expression pattern of cyp6a17SG-Gal4. cyp6a17 reporter expression by cyp6a17SG-Gal4.UAS-mCD8:GFP (green). GFP expression was detected strongly in surface glial cells (filled triangle) and weakly in Kenyon cells (open triangle). (PDF) Total list of the 103 probes. Statistical analysis of the results shown in Table S1 (PDF) Table S2 Figures. (PDF) Acknowledgments We would like to thank S.-T. Hong for advice and comments, Y.K. Oh and J. Choe for help with locomotor test, and D. Kalderon, T. Zars, L.C. Griffith, G. Roman and the Bloomington stock center for providing fly stocks. Author Contributions Conceived and designed the experiments: J. Kang J. Kim. Performed the experiments: J. Kang. Analyzed the data: J. Kang KWC. Wrote the paper: J. Kang KWC. 1. Sayeed O, Benzer S (1996) Behavioral genetics of thermosensation and hygrosensation in Drosophila. Proc Natl Acad Sci USA 93: 6079–6084. 2. Zars T (2001) Two thermosensors in Drosophila have different behavioral functions. J Comp Phyiol A 187: 235–242. PLoS ONE | www.plosone.org 11 December 2011 | Volume 6 | Issue 12 | e29800