Spatial analysis of AQP transcripts in different tsetse tissues/structures.

<p>(A) Relative expression levels of each AQP gene within specific tissues/structures based on qPCR analysis. (B) <i>gmmdripb</i> and (C) <i>gmmdripa in situ</i> hybridization, red, along with milk gland protein (MGP) immunohistochemistry, green, and DAPI staining of nuclei, blue, of a cross section of milk gland tubules. 1 = milk gland lumen; 2 = nuclei; 3 = secretory reservoir. Negative controls not treated with Digoxigenin-labeled sense RNA probes displayed no signal (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002517#pntd.0002517.s003" target="_blank">Figure S3</a>).</p

Expression of <i>gmmdripa</i>, <i>gmmdripb</i> and <i>gmmaqp5</i> transcripts during tsetse pregnancy.

<p>(A) <i>gmmdripa</i>, (B) <i>gmmdripb</i> and (C) <i>gmmaqp5</i>. Transcript levels were determined by qRT-PCR analysis. The data were analyzed with software version 3.1 (Bio-Rad). Data represent the mean ± SE for four samples and was normalized to <i>tubulin</i>. * indicates that the expression is significantly higher (P<0.05) than in newly emerged teneral flies (0 d).</p

Changes in tsetse physiology after suppression of aquaporins.

<p>(A) qPCR expression of <i>aquaporins</i> (<i>gmmdripa</i>, <i>gmmdripb</i> and <i>gmmaqp5</i>) after knockdown utilizing siRNA injection. Data represent the mean ± SE for three samples and was normalized to <i>tubulin</i>. (B) Rate of water loss (%/h, diuresis plus cuticular and respiratory water loss) after siRNA injection and a subsequent bloodmeal. The combined injection group received all three siRNAs for <i>dripa</i>, <i>dripb</i> and <i>aqp5</i>. Data represent the mean ± SE of three groups of 6 flies. (C) Images of bloodfed flies after injection of siGFP or combined siAQPs showing increased size due to delayed water loss (diuresis along with cuticular and respiratory water loss). (D) qPCR expression of transcripts for antioxidant enzymes (<i>Mn/Fe superoxide dismutase</i>, <i>Mn/Fe sod</i>; <i>Cu/Zn superoxide dismutase</i>, <i>Cu/Zn sod</i>; <i>catalase</i>, <i>cat</i>) 6 h after blood feeding after combined knockdown of AQPs. Data represent the mean ± SE for three samples and was normalized to <i>tubulin</i>. (E) Average duration of survival under dehydrating conditions following blood feeding after suppression by siRNA injection. Each point represents mean ± SE of five groups of 10 flies. Black point represents the mean. (F) Heat tolerance following knockdown of <i>aqps</i>. n Data are presented as mean ± SE of three groups of 6 flies. Black point represents the mean. * indicates that the value is significantly different (*P<0.05; **, P<0.01; ***, P<0.001) than control. siGFP, short-interfering green fluorescent protein that serves as a control.</p

Aquaporins Are Critical for Provision of Water during Lactation and Intrauterine Progeny Hydration to Maintain Tsetse Fly Reproductive Success

<div><p>Tsetse flies undergo drastic fluctuations in their water content throughout their adult life history due to events such as blood feeding, dehydration and lactation, an essential feature of the viviparous reproductive biology of tsetse. Aquaporins (AQPs) are transmembrane proteins that allow water and other solutes to permeate through cellular membranes. Here we identify tsetse aquaporin (AQP) genes, examine their expression patterns under different physiological conditions (blood feeding, lactation and stress response) and perform functional analysis of three specific genes utilizing RNA interference (RNAi) gene silencing. Ten putative aquaporins were identified in the <i>Glossina morsitans morsitans</i> (<i>Gmm</i>) genome, two more than has been previously documented in any other insect. All organs, tissues, and body parts examined had distinct AQP expression patterns. Two AQP genes, <i>gmmdripa</i> and <i>gmmdripb</i> ( = <i>gmmaqp1a</i> and <i>gmmaqp1b</i>) are highly expressed in the milk gland/fat body tissues. The whole-body transcript levels of these two genes vary over the course of pregnancy. A set of three AQPs (<i>gmmaqp5</i>, <i>gmmaqp2a</i>, and <i>gmmaqp4b</i>) are expressed highly in the Malpighian tubules. Knockdown of <i>gmmdripa</i> and <i>gmmdripb</i> reduced the efficiency of water loss following a blood meal, increased dehydration tolerance and reduced heat tolerance of adult females. Knockdown of <i>gmmdripa</i> extended pregnancy length, and <i>gmmdripb</i> knockdown resulted in extended pregnancy duration and reduced progeny production. We found that knockdown of AQPs increased tsetse milk osmolality and reduced the water content in developing larva. Combined knockdown of <i>gmmdripa</i>, <i>gmmdripb</i> and <i>gmmaqp5</i> extended pregnancy by 4–6 d, reduced pupal production by nearly 50%, increased milk osmolality by 20–25% and led to dehydration of feeding larvae. Based on these results, we conclude that gmmDripA and gmmDripB are critical for diuresis, stress tolerance and intrauterine lactation through the regulation of water and/or other uncharged solutes.</p></div

Sequence information for <i>Glossina</i> aquaporins.

<p>Sequence information for <i>Glossina</i> aquaporins.</p

Fecundity, gonotrophic cycle duration, milk osmolality and percent water content of intrauterine larva following knockdown of <i>aquaporins</i> in the mother.

<p>(A) Length of 1st gonotrophic cycle. Black point represents the mean. (B) Average pupae produced per female over 40 d after suppression of <i>gmmdripa</i>, <i>gmmdripb</i>, and <i>gmmaqp5</i> by siRNA injection. For “combined injection”, flies were simultaneously injected with <i>gmmdripa</i>, <i>gmmdripb</i> and <i>gmmaqp5</i> siRNA. Data are presented as mean ± SE of three groups of 20 flies. Black point represents the mean. (C) Percent water content in intrauterine larvae after suppression of gmm<i>dripa</i>, <i>gmmdripb</i>, <i>gmmaqp5</i>, <i>gfp</i> by siRNA injection. Data are presented as mean ± SE of three groups of 15 flies. Black point represents the mean. (D) Osmolality of tsetse milk recovered from larval gut contents after suppression of <i>gmmdripa</i>, <i>gmmdripb</i>, <i>gmmaqp5</i> and <i>gfp</i> by siRNA injection. Data are presented as mean ± SE of three groups of 6 flies. Black point represents the mean. * indicates that the value is significantly different (*, P<0.05; **, P<0.01; ***, P<0.001) than control. siGFP, short-interfering Green Fluorescent Protein was used for internal control. Black point represents the mean.</p

AQPs that were significantly higher than the temporal average at specific time points after blood feeding based on results from Table S3.

<p>Statistical differences were tested by ANOVA following Bonferroni correction (P<0.05).</p