Sexual transfer of the steroid hormone 20E induces thepostmating switch in Anopheles gambiaePaolo Gabrielia,1, Evdoxia G. Kakania,b,1, Sara N. Mitchellb,c,1, Enzo Mamelia, Elizabeth J. Wantd,Ainhoa Mariezcurrena Antonc, Aurelio Serraoa, Francesco Baldinia,b, and Flaminia Catterucciaa,b,c,2

aDipartimento di Medicina Sperimentale, Universit degli Studi di Perugia, Perugia 06100, Italy; bDepartment of Immunology and Infectious Diseases,Harvard School of Public Health, Boston, MA 02115; and Department of cLife Sciences and dSurgery and Cancer, Imperial College London, London SW7 2AZ,United Kingdom

Edited by Alexander S. Raikhel, University of California, Riverside, CA, and approved October 14, 2014 (received for review June 8, 2014)

Female insects generally mate multiple times during their lives. Anotable exception is the female malaria mosquito Anopheles gam-biae, which after sex loses her susceptibility to further copulation.Sex in this species also renders females competent to lay eggsdeveloped after blood feeding. Despite intense research efforts,the identity of the molecular triggers that cause the postmatingswitch in females, inducing a permanent refractoriness to furthermating and triggering egg-laying, remains elusive. Here we showthat the male-transferred steroid hormone 20-hydroxyecdysone(20E) is a key regulator of monandry and oviposition in An. gam-biae. When sexual transfer of 20E is impaired by partial inactiva-tion of the hormone and inhibition of its biosynthesis in males,oviposition and refractoriness to further mating in the female arestrongly reduced. Conversely, mimicking sexual delivery by inject-ing 20E into virgin females switches them to an artificial matedstatus, triggering egg-laying and reducing susceptibility to copu-lation. Sexual transfer of 20E appears to incapacitate femalesphysically from receiving seminal fluids by a second male. Com-parative analysis of microarray data from females after matingand after 20E treatment indicates that 20E-regulated molecularpathways likely are implicated in the postmating switch, includingcytoskeleton and musculature-associated genes that may renderthe atrium impenetrable to additional mates. By revealing signalsand pathways shaping key processes in the An. gambiae reproduc-tive biology, our data offer new opportunities for the control ofnatural populations of malaria vectors.

ecdysone | hormone | reproduction | malaria | mosquito

Sex profoundly affects the reproductive physiology of Anophelesgambiae mosquitoes, the major vector of human malaria.After copulation females of this species experience a permanentloss of receptivity to further copulation, so that they rely onsperm from a single male for a lifetime production of offspring(1). Sex also increases the number of eggs developed aftera blood meal and induces laying of mature eggs (2). Parallel tothese modifications in postcopulatory behavior, the female re-productive tract undergoes significant morphological changes,possibly reflecting a switch from an insemination to an egg-layingrequirement in these tissues (3). The evolutionary forces behindmonandry in An. gambiae are largely uncertain; however, thisreproductive strategy may have evolved because of the costsassociated with mating in swarms, including use of precious en-ergy resources and elevated risk of predation (4).What are the possible triggers of the postmating switch that

renders females refractory to copulation and allows them to layeggs? During mating, An. gambiae males package seminal fluidproduced in the male accessory glands (MAGs) into a co-agulated mating plug that is transferred to the female atrium(uterus), where it is digested in 12 d (57). Conversely, spermare permanently stored by the female in a storage organ namedthe spermatheca, where they must be maintained as viable duringmultiple reproductive cycles to fertilize eggs developed afterblood feeding. The relative roles of MAG secretions and sperm

transfer in inducing female postmating behavior in insects havebeen the subject of numerous studies. In Drosophila mela-nogaster, a species in which females experience a temporary lossof mating receptivity after copulation, a short 36-amino acid sexpeptide (SP) synthesized in the MAGs has a major role in theregulation of the postmating switch (8). Once transferred to thefemale, SP interacts with the sex peptide receptor, a G-proteincoupled receptor expressed in the reproductive tract and centralnervous system of females, to stimulate egg-laying and inducea temporary refractoriness to further mating (9). The refractoryperiod in fruit fly females is extended up to 1 wk beyond the firstday postcopulation by the slow release of SP bound to sperm tails(10). The An. gambiae postmating switch, on the other hand, doesnot seem to depend on sperm transfer, because females mated tospermless males are capable of laying eggs and do not mate again(11). A number of methodologies have been used to elucidate thetriggers of egg-laying and monandry in these mosquitoes (1215);although these studies yield contrasting results, they suggest thatthe MAG secretions that compose the mating plug have a prom-inent role in regulating these responses.Intriguing and rare among insects, the An. gambiaemating plug

contains the steroid hormone 20-hydroxyecdysone (20E) (16, 17)in addition to proteins and peptides (6, 7). 20E is the major

Significance

Anopheles gambiae females are the principal vectors ofmalaria, a disease that kills more than 600,000 people everyyear. Current control methods using insecticides to kill mos-quitoes are threatened by the spread of resistance in naturalpopulations. A promising alternative control strategy is basedon interfering with mosquito reproduction to reduce thenumber of malaria-transmitting females. Here we show thata male hormone transferred to the female during sex induceslarge changes in female behavior. These changes, defined asthe postmating switch, include a physical incapacity for fertil-ization by additional males and the ability to lay mature eggs.Tampering with the function of this hormone generates un-precedented opportunities to reduce the reproductive successof Anopheles mosquitoes and impact malaria transmission.

Author contributions: P.G., E.G.K., S.N.M., A.S., F.B., and F.C. designed research; P.G., E.G.K.,S.N.M., E.M., E.J.W., A.M.A., and F.B. performed research; P.G., E.G.K., S.N.M., E.J.W., and F.B.analyzed data; and P.G., E.G.K., S.N.M., and F.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The microarray data reported in this paper have been deposited in theArrayExpress database, www.ebi.ac.uk/arrayexpress (accession nos. E-MTAB-3022 andE-MTAB-3023).1P.G., E.G.K., and S.N.M. contributed equally to this work.2To whom correspondence should be addressed. Email: fcatter@hsph.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1410488111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1410488111 PNAS | November 18, 2014 | vol. 111 | no. 46 | 1635316358

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ecdysteroid in insects and regulates molting during juvenile stages(18). This hormone has been found to affect multiple aspects ofadult insect physiology: It controls lifespan (19), learning (20),stress-induced responses (21), sleep regulation, and social inter-actions (22). Moreover 20E acts as a sexual hormone, influencingmale sexual behavior in Drosophila (23). We recently have de-termined that in An. gambiae the interaction between sexuallytransferred 20E and the female atrial protein Mating-InducedStimulator of Oogenesis (MISO) increases female fecundity bystimulating a series of events that lead to a greater number ofeggs produced after blood feeding (17). In adult mosquitoes 20Eis also produced by the female after every blood meal to stimulatevitellogenesis and egg development (16, 17, 24). In An. gambiaemale-produced 20E is delivered specifically to the female atriumduring mating, whereas the synthesis of this hormone after bloodfeeding occurs in ovaries and, likely, in fat body and reaches peaklevels much lower than those transferred by males (16, 17).Here we demonstrate that sexually transferred 20E is a key

molecular trigger of the postmating switch in An. gambiae females.We show that this hormone induces a great transcriptional responsein two major female reproductive organs, the atrium and thespermatheca. The cascades of events triggered by 20E in these tis-sues have profound effects on female physiology, leading to a loss ofreceptivity to further insemination and triggering egg-laying afterblood feeding. Moreover, we identify the molecular pathways pos-sibly implicated in these postmating responses. Our findings unveilthe multiple roles of this steroid hormone in mosquito reproductivebiology and open possibilities for reducing the reproductive successof the most important malaria vector.

ResultsMating Induces a Large 20E-Mediated Transcriptional Response in theFemale Lower Reproductive Tract. To gain better insight into themolecular link between the postmating transcriptional andmorphological changes and the behavioral and physiological shiftinduced by copulation (3, 17, 25), we initially performed an in-depth transcriptional analysis of the female lower reproductivetract (LRT), comprising the atrium and the spermatheca. Toidentify the molecular pathways specifically regulated in these

female reproductive tissues, we analyzed three time points (3, 12,and 24 h post mating; hpm) that would capture transcriptionalchanges occurring within a broad time window after copulation.These analyses identified 708 genes induced and 412 genes re-pressed by mating across the different time points [P < 0.05;false-discovery rate (FDR) corrected] (Dataset S1), unveilinga greater induction of transcription than of repression and amuch larger transcriptional response than previously observed inthe whole-female analysis, in which only 141 mating-regulatedgenes were identified (3). Metabolic processes represented thegreat majority of assignable Gene Ontology (GO) terms in thedataset (Fig. 1A). Up-regulated metabolic genes included factorsassociated with proteolysis, amino acid biosynthesis, and energyproduction, suggestive of high-energy demand and protein cyclingpost mating. Down-regulated genes included cytochrome P450sand carboxy/metallopeptidases that may indicate a shift ofresources away from general metabolic activities toward morereproduction-associated processes. The response to mating in-creased with time, and the highest number of differentiallyexpressed genes (41% of all gene changes) was recorded at 24hpm. This finding suggests an accumulation of the mating signalduring the first day postcopulation, possibly extending into latertime points not analyzed here, in contrast to D. melanogaster, inwhich transcriptional changes were largest 16 hpm (26, 27).What factors could cause such a powerful and prolonged

transcriptional response in the female reproductive tract? Dur-ing mating males transfer large amounts of 20E, a strong regu-lator of gene expression (28), that is released gradually from themating plug into the female reproductive tract over the first dayafter copulation (17). We observed that a considerable numberof genes (vitellogenin, prophenoloxidases, proteasome subunits,V-type ATPases, cytochrome P450s,MISO, the heme peroxidaseHPX15) identified in our microarrays analysis generally areregulated by 20E in Anopheles and other insects (17, 25, 2932).Therefore we explored the full extent of a possible 20E-mediatedregulation of postmating transcriptional activity by comparingthe transcriptional profile of the female reproductive tract ob-served at 24 hpm and the transcriptional response induced in theatrium and spermatheca at the same time point after injection of

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