bioRxiv preprint doi: https://doi.org/10.1101/124305; this version posted January 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 A pleiotropic chemoreceptor facilitates the coupling of pheromonal signal perception 2 and production 3 4 Kathleen M. Zelle1,†, Cassondra Vernier1,†, Xitong Liang2, Sean Halloran3, Jocelyn G. 5 Millar3, Yehuda Ben-Shahar 1,4,* 6 7 1Department of Biology, Washington University in Saint Louis, 1 Brookings Drive, Saint 8 Louis, MO 63130 USA. 9 2Department of Neuroscience, Washington University School of Medicine, 660 South 10 Euclid Avenue, St. Louis, MO 63110, USA 11 3Department of Entomology, University of California, Riverside, 900 University Avenue, 12 Riverside, CA 92521, USA 13 4Lead Contact 14 *Correspondence: [email protected] (Y.B.) 15 †Denotes equal contribution 16 17 1 bioRxiv preprint doi: https://doi.org/10.1101/124305; this version posted January 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 18 SUMMARY 19 Optimal mating decisions depend on reliable and precise species-specific coupling of 20 the production and perception of mating communication signals because any functional 21 changes in either the signal or its perception should carry a fitness cost (Boake, 1991; 22 Brooks et al., 2005; Hoy et al., 1977b; Shaw et al., 2011; Shaw and Lesnick, 2009; 23 Steiger et al., 2010; Sweigart, 2010; Symonds and Elgar, 2008; Wyatt and Cambridge 24 University Press, 2014). Previously published theoretical models predict that genetic 25 linkage between sensory-driven mate choices and specific communication signals could 26 provide a robust mechanism for maintaining robust and stable signal production and 27 perception at the level of populations, while still retaining the capacity for future signal 28 diversification (Hoy et al., 1977b; Shaw et al., 2011; Shaw and Lesnick, 2009; Wiley et 29 al., 2011). Several experimental studies have provided empirical evidence that 30 pleiotropy is one possible genetic solution for maintaining functional coupling between 31 specific communication signals and the mate choice behaviors they elicit at the 32 population level (Hoy et al., 1977b; Shaw et al., 2011; Shaw and Lesnick, 2009; Wiley et 33 al., 2011). However, the complex characteristics of mating signals and behaviors 34 present a major barrier for identifying the actual pleiotropic genes and molecular 35 pathways that provide genetic linkages between the production and perception of 36 mating signals (Chenoweth and Blows, 2006; Singh and Shaw, 2012). Here we show 37 that in the fruit fly Drosophila melanogaster, some pheromone-driven mating choices 38 are coupled to the production of specific mating signals via pleiotropic chemoreceptors. 39 Specifically, we demonstrate that Gr8a, a member of the gustatory receptor family, 40 independently contributes to the perception of inhibitory mating signals in pheromone 41 sensing neurons, as well as in regulating the production of inhibitory mating signals in 42 oenocytes. Together, these data provide a relatively simple molecular explanation for 43 how coupling of pheromone perception and production, two independent physiological 44 processes, remains robust. 45 46 Keywords: Drosophila melanogaster; Vinegar fly; Fruit fly; Cuticular hydrocarbons; 47 Gr8a; Oenocyte. 2 bioRxiv preprint doi: https://doi.org/10.1101/124305; this version posted January 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 48 RESULTS AND DISCUSSION 49 Similar to other insect species, the fruit fly Drosophila melanogaster has evolved 50 cuticular hydrocarbons (CHCs), synthesized by the fat body and oenocytes (Billeter et 51 al., 2009; Gutierrez et al., 2006; Krupp et al., 2013; Makki et al., 2014), which primarily 52 provide a hydrophobic desiccation barrier, as well as play an important role as 53 pheromones that regulate diverse aspects of insect behavior (Blomquist and Bagnères, 54 2010; Chung and Carroll, 2015; Ferveur, 2005; Howard and Blomquist, 2005). 55 Specifically, complex blends of CHCs are often utilized by insect species to 56 communicate sex identity and female mating status, and well as defining the behavioral 57 reproductive boundaries between closely related species (Billeter et al., 2009; Chung 58 and Carroll, 2015; Chung et al., 2014; Coyne et al., 1994; Dweck et al., 2015; Ng et al., 59 2014; Shirangi et al., 2009; Yew and Chung, 2015). In Drosophila, the perception of 60 volatile CHCs is mediated by olfactory sensory neurons located in the antennae and 61 maxillary palps (Benton et al., 2007; Kurtovic et al., 2007; Lebreton et al., 2014; van der 62 Goes van Naters and Carlson, 2007), while less volatile CHCs, which require contact, 63 are sensed by specialized gustatory-like receptor neurons (GRNs) in the appendages 64 (legs and wings) and the proboscis (Koh et al., 2014; Lu et al., 2012; Lu et al., 2014; 65 Thistle et al., 2012; Toda et al., 2012). While some of the genes and pathways that 66 contribute to CHC synthesis in Drosophila are known, the molecular identities of the 67 majority of CHC receptors remain unknown. Previous work suggested that the gene 68 Desat1, which encodes a fatty acid desaturase enriched in oenocytes, might also 69 independently contribute to CHC perception (Bousquet et al., 2012). However, due to 70 the expression of Desat1 in central neurons (Billeter et al., 2009) and the broad impact 71 its mutant alleles have on the CHC profiles of both males and females (Labeur et al., 72 2002), whether Desat1 directly contributes to mating signal perception remains 73 unresolved. 74 Consequently, we chose to examine members of the Gustatory receptor (Gr) gene 75 family as candidates for pleiotropic factors that might contribute directly to both 76 perception and production of pheromonal mating signals in Drosophila. Because several 77 family members have already been implicated in the detection of specific excitatory and 78 inhibitory pheromones (Bray and Amrein, 2003; Hu et al., 2015; Miyamoto and Amrein, 3 bioRxiv preprint doi: https://doi.org/10.1101/124305; this version posted January 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 79 2008; Moon et al., 2009; Watanabe et al., 2011), and the majority of genes that encode 80 family members are already known to be enriched in gustatory receptor neurons 81 (GRNs) (Clyne et al., 2000; Dunipace et al., 2001; Scott et al., 2001; Wang et al., 2004), 82 we reasoned that any pleiotropic Gr genes should also be expressed in abdominal 83 oenocytes (Billeter et al., 2009). Subsequently, an RT-PCR screen revealed that 24 out 84 of the 60 genes that encode Gr family members in the Drosophila genome (Clyne et al., 85 2000; Dunipace et al., 2001; Robertson et al., 2003; Scott et al., 2001) exhibit enriched 86 expression in abdominal tissues (Table S1). 87 Although several members of the Gr receptor family, including Gr68a, Gr32a, Gr66a, 88 Gr39a, and Gr33a, have been previously linked to the sensory perception of mating 89 pheromones (Bray and Amrein, 2003; Lacaille et al., 2009; Miyamoto and Amrein, 2008; 90 Moon et al., 2009; Watanabe et al., 2011), none of these candidate pheromone 91 receptors were identified in our initial screen for Gr genes with enriched mRNA 92 expression in abdominal tissues of either males or females (Table S1). However, Gr8a, 93 which we have identified as a male-specific gene in our screen, is localized to a 94 chromosomal region that was previously implicated in chemoreception with enriched 95 expression in abdomens (Table S1) and was previously shown to specifically contribute 96 to the sensory detection of the non-proteinogenic amino acid L-Canavanine (Lee et al., 97 2012; Shim et al., 2015). Because our initial expression screen was based on whole- 98 abdomen RNAs, and it was previously reported that Gr8a is also expressed in some 99 abdominal neurons (Park and Kwon, 2011), we next used a GAL4 transgenic driver to 100 examine the spatial expression pattern of Gr8a in males and females. As was 101 previously reported, we found that Gr8a is expressed in 14-16 GRNs in the proboscis 102 (Figure 1A) (Lee et al., 2012) as well as in two paired GRNs in the foreleg pretarsus 103 (Figure 1B) of males and females. In addition, the Gr8a-GAL4 reporter was also highly 104 expressed in abdominal oenocyte-like cells in males but not females (Figure 1C). The 105 male-specific abdominal expression pattern was further supported by qRT-PCR (Figure 106 1D). Using immunohistochemistry, we then demonstrated that Gr8a is co-expressed 107 with the oenocyte marker Desat1 (Billeter et al., 2009), as well as in Desat1-negative 108 cells with fat-body-like morphology (Figure 1E-G). 4 bioRxiv preprint doi: https://doi.org/10.1101/124305; this version posted January 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 109 Together, the enriched expression of Gr8a in foreleg GRNs, a primary pheromone 110 chemosensory organ (Lu et al., 2012; Lu et al., 2014), and its sexually dimorphic 111 expression in oenocytes suggested that in addition to its role in L-Canavanine 112 perception, Gr8a also contributes to mating behaviors. Since mate choices of both male 113 and female flies are determined by a complex blend of excitatory and inhibitory 114 pheromones (Billeter et al., 2009; Krupp et al., 2013), we anticipated that if Gr8a is 115 indeed a pleiotropic factor that independently contributes to the perception and 116 production of specific components of the male mating pheromone bouquet, then 117 disruptions of the signal production in males or its perception in females should carry 118 similar impacts on female mate choice behavior.