region upstream of the acr-16 ATG was PCR amplified with the following primers: ATTB1acr-16 and ATTB2acr-16. A 2.4 kb region upstream of the unc-63 ATG, plus the first three codons, was PCR amplified with the following primers: attb1unc-63 and unc-63attb2. A 4.3 kb genomic region that contained 1.2 kb upstream of the unc-38 ATG and all unc-38 introns and exons up to the stop codon was PCR amplified with the following primers: attb1unc-38fus and Attb2unc-38fusnew. A 2.2 kb genomic region that contained a 1.3 kb region upstream of the unc-29 ATG and part of the unc-29 genomic sequence up to the end of the second exon was PCR amplified with the following primers: ATTB1Punc- 29 and ATTB2Punc-29. A 2.3 kb genomic region that contained 1.2 kb upstream of the unc-29 ATG and all unc-29 introns and exons up to the stop codon was PCR amplified with the following primers: ATTB1Punc-29 and ATTB2U29lstex. A 6.2 kb region upstream of the acr-18 ATG was PCR amplified with the following primers: ATTB1acr-18 and ATTB2acr-18noatg. All these PCR primers contained Gateway ATTB sites, which allowed the acr-16, unc-63, unc-38, unc-29 and acr-18 PCR products to be recombined, using BP clonase (Invitrogen), into the low copy number Gateway entry vector pDG15, to generate pLR98, pLR149, pLR125, pYL21 (includes the partial unc-29 gene), pLR198 (includes the full-length unc-29 gene) and pLR162, respectively. In order to place the acetylcholine receptor sequences in front of YFP, pLR98, pLR149, pLR125, pYL21 and pLR198 were then individually recombined with the YFP destination vector pGW322YFP using LR clonase (Invitrogen) to make the plasmids pLR106, pLR152, pLR127, pYL22 and pLR199, respectively. pGW322YFP and pDG15 have been described in previous studies [6,44]. Cell-specific expression of the unc-29 cDNA. We inserted a SL2-accepting trans-splice site between the unc-29 cDNA (including the stop codon) and the GFP gene to use GFP flourescence as a proxy for unc-29 expression. The intergenic region between gpd-2 and gpd-3, containing a SL2-accepting trans- splice site, was PCR amplified with the following primers: igrgpd3 and gpd2igr. This region was then cloned into the SmaI site of pPD95.69 (Addgene plasmid 1491) to generate pJP1. pJP1 was cut with HindIII and ApaI and then cloned into the HindIII/AvaI sites of pBR322 to generate pDG4, which contains the SL2 site in front of the GFP gene. The unc-29 cDNA was then PCR amplified from a cDNA library using the primers Func-29 and unc-29R. The unc- 29 cDNA was cloned into the BamHI site of pDG4 to generate pDG5. pDG5 was then cut with XbaI, blunt-ended and then ligated with the Gateway Vector Conversion Reading frame Cassette C.1 (Invitrogen) to generate pYL16. The acr-8 and the lev- 11 promoter regions were recombined into the entry vector pDG15 to generate pLR92 and pLR22, respectively [6,34]. Then we recombined the promoter sequences in pLR92 and pLR22 into pYL16 using LR clonase (Invitrogen) to generate pYL18 and pYL20, respectively. Cell-specific expression of ChR2. To put a Gateway destination cassette in front of ChR2, a pmyo-3::ChR2(gf)::YFP plasmid (a gift from Dr. Gottschalk, Goethe University Frankfurt) [41] was cut with HindIII and BamHI, blunt-ended and then ligated with the Gateway Vector Conversion Reading frame Cassette C.1 (Invitrogen) to generate pZL5. A TGA stop codon 50 nucleotides downstream of the XbaI site was changed to GGA to generate pLR167. To express ChR2 from the promoter region of acr-18, unc- 103 (promoter E and F), lev-11 and gar-3 (promoter B), pLR162, pLR21, pLR28, pLR22 and pLR57 were subsequently recombined with pLR167 to make pLR165, pLR176, pYL41, pLR178 and pLR183, respectively. Construction of pLR21, pLR22, pLR28 and pLR57 have been described previously [5,26,34,44]. Schema of the Spicule Insertion Circuit Transgenics Plasmids were co-injected with pBX1 (50 ng/ml) into strains that contained the pha-1(e2123) allele. Animals that could survive beyond the L2 stage were then kept as transgenic lines. For strains that did not have the pha-1(e2123) allele, GFP or YFP encoded on one of the injected plasmids was used to identify transgenic animals. For all injections, pUC-18 was used to make the final concentration of DNA to 200 ng/ml. The expression constructs pLR106, pLR152, pLR127, pLR199 and pYL22 were injected at 50 ng/ml into the pha-1(e2123); him-5(e1490); lite-1(ce314) her- maphrodites. Multiple male-specific muscles are located within a small region of the male tail. Sometimes it was difficult to differentiate these muscles when the majority of them were expressing YFP. The expression of pLR165 (Pacr-18:ChR2::YFP) was located on the plasma membrane and the ER, making it easier to visualize muscle expression in the male tail (Figure S3Q-S3S). To rescue the locomotion defects of unc-29(lf) males, pYL18 (70 ng/ml) and pYL20 (20 ng/ml) were injected into unc-29(e193); him-5(e1490) or unc-29(e193); acr-16(ok789) him-5(e1490) hermaph- rodites. To express G-CaMP and DsRed simultaneously in the male-specific muscles and express ChR2 in the oblique-guberna- cular muscle group, pLR135 (20 ng/ml), pLR136 (50 ng/ml) and pLR165 (100ng/ml), were injected into the pha-1(e2123); him- 5(e1490); lite-1(ce314) hermaphrodites. pLR35(Punc103E::G-CaMP) and pLR36(Punc-103E::DsRed) are described elsewhere [5]. To express ChR2 in specific tissues, pLR176 (50 ng/ml), pLR178 (50 ng/ml), pLR183 (100 ng/ml) and pYL41 (150 ng/ml) were injected into the pha-1(e2123); him-5(e1490); lite-1(ce314) hermaph- rodites. To restore functional unc-103 to specific tissues of the unc- 103(0);Punc-103F:ChR2::YFP worms, the Punc-103F:unc-103F and Punc-103E:unc-103E constructs [44] were injected with pYL41 (Punc-103F:ChR2::YFP) (150 ng/ml)into the unc-103(0) hermaphro- dites at 20 ng/ml, respectively. Laser ablation Cell ablations were done using the standard protocol [86]. The operation was conducted using a Spectra-Physics VSL-337ND-S Nitrogen Laser (Mountain View, Ca) attached to an Olympus BX51 microscope. L2 worms were operated on 5% agar pads containing 0.5 mM NaN3, and L4 males were operated on 5% agar pads containing 2 mM NaN3. For each operated animal, a control animal was placed on the same agar pad for the same amount of time to rule out the possibility that behavioral changes are due to the anesthetic pads. Optical stimulation and detection of repetitive spicule thrusts lite-1(ce314) males that expressed Pacr-18:ChR2::YFP were used. At L4 stage, males were transferred onto NGM plates with OP50 supplemented with all-trans retinal. Males with the same transgenes were also placed onto NGM plates with regular OP50 as control. All-trans retinal-containing plates were prepared the day before by spreading OP50 culture that contained 50 mM all-trans retinal (A.G. Scientific). The next day, the males were immobilized on 10% agarose (in H2O) pad containing 0.5 ml of 0.1 mm diameter polystyrene microspheres, and covered with a coverglass [87]. Sequences of DIC images of the male tails were recorded under an Olympus BX51 microscope. A Dual View Simultaneous Imaging Systems by Photometrics (Surrey, BC) was used and adjusted to split the image signal, so that DIC image could be recorded in one field of view (field 1), and simultaneously, fluorescent signals could be PLoS Genetics | www.plosgenetics.org 15 March 2011 | Volume 7 | Issue 3 | e1001326