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The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila Melanogaster

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The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila Melanogaster | INVESTIGATION The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster Jessica L. Sitnik,*,1 Dragan Gligorov,†,1 Robert K. Maeda,† François Karch,†,2 and Mariana F. Wolfner*,2 *Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, and †Department of Genetics and Evolution, University of Geneva, 1211 Geneva, Switzerland ORCID ID: 0000-0003-2701-9505 (M.F.W.) ABSTRACT Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a “sex peptide” (SP) and are maintained by SP’s binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these “main” and “secondary” cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6cocu males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of .53 in iab-6cocu males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles’ contents may be important for the machinery used to maintain the PMR. KEYWORDS reproduction; Drosophila; sex peptide; gene regulation; gene expression EMINAL fluid proteins (Sfps) play an essential role in cause the PMR (Takemori and Yamamoto 2009). Each lobe of Sreproduction. Sfps interact with the female, locally in the accessory gland consists of a monolayer of secretory cells the reproductive tract and more globally in the central nervous composed of two morphologically distinct cell types. Flat, system, resulting in changes in female gene expression, be- polygonal “main cells” make up 96% of each lobe. The re- havior (in insects), and physiology (reviewed in Sirot et al. maining 4% of the cells are large, spherical, vacuole-filled 2009). In Drosophila, these changes are collectively referred “secondary cells” (Bairati 1968; Bertram, et al. 1992) that to as the female post-mating response (PMR). The male ac- are located only at the distal tip of each lobe. The multi-cell cessory gland (AG) is the source of many important Sfps that state of the AG is not unique. Other secretory organs, such as the pancreas (Brissova et al. 2005) and the epididymis (re- Copyright © 2016 by the Genetics Society of America viewed in Cornwall 2009), are also composed of multiple cell doi: 10.1534/genetics.115.181644 types, the products of which work together to regulate com- Manuscript received August 4, 2015; accepted for publication December 23, 2015; published Early Online January 7, 2016. plex processes (Hermo 1995; Cheung et al. 2005; Pietrement Supporting information is available online at www.genetics.org/lookup/suppl/ et al. 2006; Kujala et al. 2007). doi:10.1534/genetics.115.181644/-/DC1. The two cell types of the Drosophila melanogaster male 1These authors contributed equally to this work. 2Corresponding authors: Department of Molecular Biology and Genetics, Cornell accessory gland make distinct products (Bertram et al. University, 423 Biotechnology Bldg., Ithaca, NY 14853-2703. 1992; Gligorov et al. 2013) that act in a network (Ravi E-mail: [email protected]; Department of Genetics and Evolution, University of Geneva, 30 Quai E. Ansermet, 1211 Geneva-4, Switzerland. Ram and Wolfner 2009; Findlay et al. 2014) that allows for E-mail: [email protected] the seminal sex peptide (SP) to bind to sperm, enter the Genetics, Vol. 202, 1029–1041 March 2016 1029 female sperm storage organs, and subsequently be cleaved Materials and Methods from the sperm in storage (Liu and Kubli 2003; Peng et al. Fly stocks and media 2005a). The binding of SP to sperm and its gradual release by proteolytic cleavage allows its effects to persist long after Flies were raised at room temperature (23° 6 1°) in bottles on mating has occurred (Chen and Buhler 1970; Chapman standard yeast–glucose media (8.2% w/w yeast, 8.2% w/w et al. 2003; Liu and Kubli 2003; Peng et al. 2005a). Through glucose, 1% w/w agar, 1.2% v/w acid mix). Virgin females this mechanism, SP has been implicated in regulating long- were aged 3–5 days from eclosion in groups of 5–12 in vials term changes in female egg laying, receptivity, and various with added yeast. Male flies were aged 3–5 days from eclo- other post-mating responses (Chapman et al. 2003; Liu and sion in groups of 10–20 in vials on standard yeast–glucose Kubli 2003; Peng et al. 2005a,b; Avila et al. 2010; Apger- media. Fly lines containing a UAS-hairpin construct specific McGlaughon and Wolfner 2013). Collectively, the prolonged to each gene of interest were obtained from the Vienna Dro- post-mating response that is mediated by SP is called the sophila RNAi Center (VDRC) (Dietzl et al. 2007); line num- long-term response (LTR) (Peng et al. 2005a; Ravi Ram bers for candidate genes discussed in this article are given in and Wolfner 2007). While main cells produce SP and other Supporting Information, Table S1 and Table S2. To generate proteins necessary to initiate and maintain the LTR (Styger knockdown males, each UAS-hairpin line was crossed to the 1992; Liu and Kubli 2003), secondary cell products are es- secondary cell driver iab-6D1-Gal4/CyO (Gligorov et al. 2013); sential to maintain the LTR (Leiblich et al. 2012; Minami et al. control flies were generated by crossing the driver to the 2012; Gligorov et al. 2013). A few of these secondary cell appropriate background line [w[1118];P{attP,y[+],w[39] products were identified by examining the cell type expres- (Attp60100)] for KK insertion lines and w1118 for GD insertion sion of known accessory gland proteins (Gligorov et al. lines. The level of knockdown of the targeted gene’s expres- 2013), but an unbiased search for other secondary cell prod- sion, relative to controls, was confirmed by RT-PCR as de- ucts that mediate the PMR has not been done. Previously, we scribed in Findlay et al. (2014) except that messenger RNA generated an enhancer deletion, iab-6cocu, that removes the (mRNA) samples were extracted from dissected accessory expression of the Hox gene, Abdominal-B, from the secondary glands rather than from whole flies (Table S1). In cases cells of the male accessory gland. iab-6cocu mutants display where knockdown was not detected with the iab-6D1-Gal4 abnormal secondary cell morphology and are unable to main- driver, which might occur for a gene that is normally tain the LTR in their mates (Gligorov et al. 2013). The three expressed in both main and secondary cell types, we also secondary cell proteins known to be important for the long- used tubulin-Gal4 to check for the function of the RNAi con- term storage of SP (CG1652, CG1656, and CG17575) were struct (Table S1). If both drivers suggested that knockdown still produced in iab-6cocu males (Ravi Ram and Wolfner did not occur, the line was excluded from analysis. To gener- 2009; Gligorov et al. 2013) and transferred to females during ate GFP expression in the secondary cells, the iab-6D1-Gal4 mating. This suggests that the inability of iab-6cocu mutants to driver was recombined with a UAS-GFP transgene on the maintain the LTR in their mates is caused by as-yet-unidentified second chromosome (generating w; iab-6D1-Gal4, UAS-GFP) protein(s) and that the mutant may provide a way to iden- and crossed to RNAi lines as previously described. tify new secondary cell products necessary for the regulation mRNA-seq of the LTR. To identify secondary cell genes whose function is needed Tocontrol forgenetic backgroundand for the PhiC31insertion for the LTR, we used RNA-seq to compare transcripts from that was used to make the iab-6cocu mutant (Gligorov et al. iab-6cocu and wild-type accessory glands. Using both unbiased 2013) the iab-5,6rescue line, which integrates a wild-type copy screening methods via secondary cell-specific RNA interfer- of the iab-6 cis-regulatory region instead of the iab-6cocu mu- ence (RNAi) and more targeted bioinformatics approaches to tation, was used as the source of control accessory glands. narrow down targets, we selected 20 transcripts that were Total RNA was isolated from 100 pairs of accessory glands downregulated in iab-6cocu for further study. Of these, 18 per genotype from iab5,6rescue and iab-6cocu (iab-5,6D5) males were efficiently knocked down by RNAi, and we character- using the miRNeasy Mini Kit (catalog no. 217004, Qiagen). ized their effects on the regulation of the PMR. Nine of these Approximately 10 mg of total RNA was obtained per genotype genes are necessary for the normal long-term increase in egg and was sent to Fasteris (Fasteris SA, Geneva) for sequencing laying and long-term reduction in female receptivity.
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