Capa GPCR and NMUR in Fluid Homeostasis Figure 6. capaR is tubule-specific and localized to principal cells; manipulation of capaR expression levels modulates [Ca2+]i and fluid transport rates. (A) Mean mRNA expression data 6 SEM were collated from Affymetrix tissue-specific array datasets described in flyatlas.org [14] for adult and larval tissues as indicated. Blue shading (dark-adult; light-larvae) indicates epithelial tissues; whereas green shading (dark-adult; light-larvae) indicates fat body or tissues containing fat body eg., adult head and carcass. ‘mRNA signal’ indicates how abundant capaR mRNA is; and for each tissue, capaR mRNA was detectably expressed in 4 out of 4 arrays (flyatlas.org). In order to assess the expression pattern of capaR in vivo, the capaR promoter-driven GAL4 line, capaR-GAL4, was generated and crossed with UAS-GFP, and fluorescence examined by GFP histochemistry in tissues from progeny of the cross (top left panel). For orientation, tubule regions are indicated by M (main segment); I (initial segment); L (lower tubule). Expression of capaR-driven GFP occurs in the principal cells in the tubule main segment, exclusion of a stellate cell (arrowed, top right panel). (B–D) Drosophila capa receptor is expressed in principal cells of the Malpighian tubule. (B) Tubules were processed with pre-immune serum and only low-level non-specific staining of intracellular vesicles was observed, confirming the specificity of the antibody. (C) Immunocytochemistry using anti- capaR rabbit polyclonal antibody and anti-Rabbit IgG-Texas Red conjugate reveal basolateral membrane localization of capaR in tubule principal cells. (D) Merge of z-stacks from (B) picture reveals exclusion of a stellate cell (arrowed). In panels A, B–D, nuclei are labelled blue with DAPI, scale bar represents 30 mM. (E) Manipulation of capaR affects cytosolic [Ca2+]i levels in intact tubules. Tubules were dissected from c42.UAS-apoaequorin flies (c42aeq), c42aeq.UAS-capaR RNAi flies and c42aeq.UAS-capaR. Resting cytoslic [Ca2+]i levels were measured, after which tubules were stimulated with 1027 M capa-1 to obtain stimulated cytosolic [Ca2+]i readings. Primary and secondary pooled data for cytosolic [Ca2+]i levels are shown as nM [Ca2+]i 6 SEM, N = 6, where * P,0.05, Student’s t-test. (F) Fluid transport by Drosophila c42-GAL4.capaR RNAi renal tubules is significantly decreased (as determined using a Student’s t-test (*P,0.05)) compared to the parental GAL4 line when the tubule is stimulated by application of capa-1 (1027 M). Secretion rates are expressed as nl/min 6 SEM (N = 6). doi:10.1371/journal.pone.0029897.g006 GAL4.UAS-apoaequorincyto transgenic tubules but Ca2+ re- sponses do not occur in response to stellate-cell specific drosokinin [21] (data not shown). Taken together, these data are consistent with the action of capa peptides on only principal cells of the tubule [3] and also confirm the exquisite principal-cell specificity of the capaR GAL4 driver. Manipulation of capaR expression levels in only the tubule principal cells results in direct modulation of capa-induced calcium and fluid-transport rates (Fig. 6E, 6F). Targeted over-expression of capaR to only tubule principal cells results in increased [Ca2+]i; whilst a cell-specific knockdown of capaR RNAi (Fig. S1) results in significantly decreased capa-1- stimulated [Ca2+] , and fluid transport. Thus, modulation of i expression of this single GPCR in vivo has physiological consequences for epithelial function. As capaR impacts so critically on fluid transport, we investigated the role of the capaR in starvation/desiccation stress. The tubule principal cell GAL4 driver (UO-GAL4) [17] was used to either over-express or knock-down capaR, with outcrossed (Canton-S) GAL4 and UAS lines. Perhaps unsurprisingly, over- expression of capaR (Fig. 7A) or of NMUR 2 (data not shown) did not affect survival under these stress conditions. The effect of over- expressing capaR in tubule principal cells does not impact critically on calcium signalling and fluid secretion, so may not impact on the physiological response to desiccation stress. Furthermore, we have found that lower levels of capa peptides are released during desiccation (data not shown) so increasing the levels of capaR will not necessarily make any difference to the desiccation stress response. By contrast, flies with reduced capaR levels exhibited significantly extended survival compared to controls under starvation/desiccation stress (Fig. 7B) (P,0.001 against both controls; Logrank test, Mantel-Cox), presumably due to reduced fluid loss by the tubule, which allows prolonged survival under desiccation. Thus, although capa/capaR signaling results in anti- diuresis in R. prolixus [42], in flies, the tubule-specific role of capaR in fluid homeostasis modulates desiccation stress responses of the whole organism by limiting fluid loss. Recent work has shown that production of Drosophila Short neuropeptide F and tachykinin by Figure 7. Knock-down of capaR expression in principal cells enhances organismal survival to desiccation stress. (A) Increased capaR levels in principal cells (UO-GAL4.UAS-capaR, black line) do not alter survival of desiccated flies. (B) Reduced capaR levels in principal cells (UO- GAL4.UAS-capaRi, red line) alter survival of desiccated flies. Desiccation resistance was significantly higher after knockdown of capa receptor in principal cells compared to controls (P,0.001 against both controls; Log rank test, Mantel-Cox). doi:10.1371/journal.pone.0029897.g007 PLoS ONE | www.plosone.org 11 January 2012 | Volume 7 | Issue 1 | e29897