Mating induces developmental changes in the insect female reproductive tract

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ScienceDirect Mating induces developmental changes in the insect female reproductive tract I Carmel1, U Tram2 and Y Heifetz1 In response to mating, the Drosophila female undergoes a series of rapid molecular, morphological, behavioral and physiological changes. Studies in Drosophila and other organisms have shown that stimuli received during courtship and copulation, sperm, and seminal fluid are needed for the full mating response and thus reproductive success. Very little is known, however, about how females respond to these malederived stimuli/factors at the molecular level. More specifically, it is unclear what mechanisms regulate and mediate the mating response, how the signals received during mating are integrated and processed, and what network of molecules are essential for a successful mating response. Moreover, it is yet to be determined whether the rapid transition of the reproductive tract induced by mating is a general phenomenon in insects. This review highlights current knowledge and advances on the developmental switch that rapidly transitions the female from the ‘unmated’ to ‘mated’ state. Addresses 1 Department of Entomology, The Hebrew University, Rehovot, Israel 2 Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA Corresponding author: Heifetz, Y ([email protected])

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model organisms are compared [7,8]. Numerous studies in Drosophila and other organisms have shown that sperm, seminal fluid and other signals received during mating alter female behavior and physiology postmating, and that these changes increase the reproductive success of both the male and female [9–24]. Seminal fluid is composed of sperm and components secreted by the accessory glands, ejaculatory duct, and ejaculatory bulb of the male reproductive tract. Each of these seminal fluid components play an essential role in male and female reproductive success [10,23,25]. Despite this wealth of knowledge, little is known about how females respond to these male-derived stimuli/factors at the molecular level and more specifically what mechanisms regulate and mediate the mating response, how the signals received during mating are integrated and processed, and what network of molecules are essential for a successful mating response. In this review, we will focus on the female response in the first 24 hours postmating, highlighting the developmental switch that transitions the female from an ‘unmated’ to ‘mated’ state. We will discuss possible mechanisms through which external male-delivered signals modulate this developmental switch.

The mating response of D. melanogaster

Introduction

In response to mating, the Drosophila female (Figure 1) exhibits an increase in ovulation and oviposition rate [26–29] a decrease in female receptivity to courting males [30]; storage of sperm [31,32]; and a decrease in female life span [26,33,34]. Many of the stimuli that initiate the female response are in the seminal fluid, a major component of which is the male accessory gland proteins (accessory glands proteins, Acps). While it is well-established that Acps are essential for triggering several postmating changes in the female [10,19,26,35], it is only recently, with the advent of cellular and molecular analyses of the female postmating, that we have begun to understand how these signals transform the female from an ‘unmated’ to ‘mated’ state.

The female reproductive system plays several crucial roles in organisms with internal fertilization: it provides an environment the egg and sperm require for maturation prior to gamete fusion, facilitates fertilization, and supports embryo development [1–4]. Drosophila melanogaster, as a model system for studying how essential biological processes are regulated, has played a major role in unraveling the molecular mechanisms of development and physiology [5,6]. Drosophila remains the most important model system to study insect development and is the main reference species to which other emerging insect

At the morphological level, mating induces immediately after the start of copulation a series of conformational and size changes in the uterus and the oviduct which in part aid sperm to storage and allow the release and movement of oocytes through the tract [21,36,37]. Additionally, mating induces tissue-wide differentiation in epithelia, muscle and nerve tissues. In many epithelia, one of the last steps of differentiation is the development of a layer of extracellular matrix (ECM) that covers the apical and/ or basal membranes and the associated development of

This review comes from a themed issue on Development and regulation Edited by Leslie Pick and Cassandra Extavour For a complete overview see the Issue and the Editorial Available online 11th March 2016 http://dx.doi.org/10.1016/j.cois.2016.03.002 2214-5745/# 2016 Elsevier Inc. All rights reserved.

Current Opinion in Insect Science 2016, 13:106–113

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Mating induces developmental changes Carmel, Tram and Heifetz

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Phase I

MATING

Figure 1

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The female takes control

Changes in: • Transcription (miRNA, mRNA) • Translation • Morphology • Receptivity

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Sperm storage Increased ovulation rate Increased eggs production Increased oviposition

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Reproductive tract is poised for mating Current Opinion in Insect Science

Schematic model of the female Drosophila mating response. Phase I — the first few days post-eclosion, the reproductive system undergoes the first phase of differentiation, after which the reproductive tract is developmentally poised for a rapid response to an extrinsic cue (mating). Phase II — terminal differentiation is induced by stimuli (e.g. odorants, auditory, vision and tactile) perceived during courtship and largely by the transfer of seminal fluid proteins and sperm received during copulation. These stimuli and factors are necessary for the full female mating response [9–24]. In response to mating, the Drosophila female undergoes a series of rapid molecular, morphological, behavioral and physiological changes. During the initial phase, mating induces decrease or increase in the level of many miRNAs, mRNA transcripts and proteins. Other striking changes are also seen in reproductive tract morphology and female physiology. During this phase sperm reaches storage and ovulation begins. In addition, female receptivity to courting males decreases and persists for several days [9,17,19,21,37]. Phase III — a second switch point occurs at 6 hours postmating to sustain elevated fertilization and reproduction and is influenced by the female’s regulatory and physiological responses to seminal fluid components and newly stored sperm. At this time the female starts to produce and deposit eggs at high rate [20,23,37]; terminal differentiation continues along with the onset of increased egg production and deposition and terminates during this phase. Phase IV — Maintenance the developmental program of the reproductive tract has ended and the organ is ready to support high fertility. Different colors represent different phases of the mating response. Note that the schematic presents a clear difference between the different phases (donated by the dashed line), but the exact duration of each phase is unknown.

hemi-adherens junctions (HAJs). HAJs connect the cell cytoskeleton with the ECM and are formed in all cell surfaces that contact an ECM [38,39]. In the oviduct epithelia of mated females, the apical ECM (AECM) and the thin layer of cuticle above it have a ruffled appearance, unlike unmated females, suggesting that the AECM and cuticle have increased in surface area. It is possible that mating enhances or modulates apical secretion via the AECM. Increased apical secretion and surface of the AECM is likely to be essential for ovulation, activation and movement of eggs along the duct. Mating also increases secretion and/or deposition of the ECM in the basolateral membrane at the oviduct and brings the ECM to a threshold concentration that can support the development of HAJs [37]. Another striking postmating change observed in the oviduct epithelia is www.sciencedirect.com

the increase in the number of HAJs along the basolateral membrane in the upper oviduct. Because one function of the HAJ is to give shape and tension to cells and tissues [39], HAJs are particularly important in the upper oviduct, where the basolateral membrane is extensively infolded. Extensive infolding allows the expansion of epithelia during passage of an egg along the narrow duct. The infolded membrane also gives rise to a highly branched intercellular space that is filled with an ECM. The muscles of the upper oviduct appear more differentiated in mated than in unmated reproductive tracts; myofibrils and z-bodies are less dense, and there is little or no basal lamina. In addition, the number of nerve terminals or boutons innervating the lateral oviducts and the common oviduct increases 70% postmating. Interestingly, the increase is in type II boutons, which cause vesicle release Current Opinion in Insect Science 2016, 13:106–113

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Figure 2

OV

LO Upper RT CO

SR SP

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AG UT VA VU Current Opinion in Insect Science

The Drosohila female reproductive tract. Typical of the female insect reproductive system, the Drosophila female reproductive tract consists of a pair of ovaries (OV) connected to a median common oviduct (CO) by two lateral oviducts (LO), a pouch-like uterus (UT) that leads to the vagina (VA), which opens to the exterior through the vulva (VU). The reproductive tract also includes specialized organs such as the sperm storage organs (a pair of spermathecae (SP) and one seminal receptacle (SR)) and a pair of female accessory glands (parovaria) (AG) [25]. The upper RT (turquoise) includes the lateral and common oviducts; the lower RT (light brown) includes the sperm storage organs, female accessory glands, the posterior part of the common oviduct and the anterior part of the uterus (the fertilization site).

of neurohormones, such as octopamine, from nerve termini, in the common and lateral oviducts (Figure 2, [37]) [40–44]. Overall, these changes facilitate sperm storage and egg ovulation, activation, and deposition. At the molecular level, mating induces a change in the expression level of many mRNAs and proteins postmating. Microarray analyses comparing unmated vs mated females or their reproductive tracts have revealed how and to which male-derived component the female responds; in these studies, differentially expressed genes (1.5-fold) were labeled mating-responsive genes. Comparing whole female bodies, 38 mating-responsive transcripts were identified at 2 hours postmating [24], and 46 mating responsive transcripts at 3 hours postmating, Current Opinion in Insect Science 2016, 13:106–113

35 of which were mediated by sperm, 5 by male accessory glands proteins (Acps) and 6 by other mating signals [23]. Furthermore, 4 Acps have been shown to contribute to changes in the transcriptome between 1 and 3 hours after mating [45]. Prominently up-regulated transcripts included those related to immune response and proteolysis (proteases) functional groups [23,24,45]. Comparison of the expression profiles of the lower region of the female reproductive tract [20] (lower RT (LRT), Figure 2), sperm storage organs [46], or oviducts [9] of mated and unmated females revealed how the reproductive tract itself responds to mating. Immediately after the end of copulation, a small number of transcripts (71) is differential expressed in the LRT, most of which were down-regulated [20]. At 6 hours postmating, a large peak of 451 differentially expressed transcripts was observed, 305 of which were up-regulated. Interestingly, at 3 hour postmating the behavior of the vast majority of mating-responsive genes were not in accordance with the behavior of their protein products. The prominent functional groups of the LRT matingresponsive genes were nucleic acid, catalytic and transport activity as well as genes related to immune response and proteolysis (proteases), as observed in the whole body analyses. A comparison between the sperm storage organs (spermathecae (SP) and seminal receptacle (SR); Figure 2) of unmated and mated females, revealed a change in gene expression at 3-hour and 6-hour postmating. In the SP, the long-term sperm storage organs which appear to be specialized in maintenance of sperm viability and motility, 101 and 48 mating-responsive transcripts were identified at 3-hour and 6-hour postmating. In the SR, which provides a microenvironment that maintains active, motile sperm and prepares sperm for fertilization, 1168 and 865 mating-responsive transcripts were identified at 3-hour and 6-hour postmating. The proportion of up-regulated and down-regulated transcripts was 1:1 in the SP and SR at both timepoints postmating. Furthermore, the composition of differently expressed transcripts differed between the spermathecae and seminal receptacle reflective of their different functions. SP were enriched for genes involved in proteolysis and metabolism and the SR exhibited an over-representation of genes involved in localization, signaling, and ion transport [46]. Examination of unmated and mated oviducts revealed 155 differentially expressed transcripts, of which 122 increased and 33 decreased by 1.5-fold or more after mating [9]. As seen in whole body and LRT, transcripts up-regulated postmating were enriched for immune response genes. Molecular profiling of the oviduct proteome demonstrated that proteins associated with cellular junctions, such as a-Spectrin and b-Spectrin, Coracle, and Neuroglian, increased postmating [37]. Thus, mating promotes changes in actin-based cytoskeletal organization in the reproductive system and induces www.sciencedirect.com

Mating induces developmental changes Carmel, Tram and Heifetz

immune-related transcripts such as antimicrobial peptides in the reproductive system and whole body that likely contribute to creating the optimal environment for successful fertilization. Furthermore, the expression levels of many proteolysis-related transcripts are significantly altered postmating in whole body and the reproductive system. The products of proteolysis-related genes could be interacting with male proteins to influence the rate and duration of postmating responses by affecting the processing of seminal fluid proteins [18,47–49]. Modulating expression of proteolysis regulators in mated females could control the terminal differentiation of the reproductive tract and/or other physiological processes such as protecting sperm from degradation and induction of egg-laying.

Female ‘poised’ for mating Together, these studies support a model in which the female reproductive tract undergoes a two-phase maturation [9,20,23,37,40,45]. Initially, newly eclosed Drosophila females undergo a 3-day period of reproductive maturation during which the reproductive tract becomes ‘poised’ (Figure 1). Then mating triggers a second phase of maturation (tissue remodeling and modulation) which enables the reproductive system to produce, fertilize, and deposit numerous eggs rapidly over several days [21,36,37]. Kapelnikov et al. [37] further proposed that the second phase of maturation involves both mating-independent (differentiation continues irrespective of mating) and mating-dependent processes (differentiation is poised and continues upon mating) and that both of these pathways are essential to produce a functional oviduct. For example, muscles in the lower oviduct are highly differentiated prior to mating, while muscles in the upper oviduct are less differentiated. Although the onset of muscle differentiation in the upper and lower RT is mating-independent, further differentiation of muscle in the upper oviduct is mating-dependent and terminal of differentiation in the lower RT unrelated to mating. Furthermore, analysis of cytoskeleton mating-responsive protein abundance following different mating regimes (unmated, once-mated, twice-mated), demonstrated that the expression level of some of the proteins (e.g. Muscle LIM protein at 84B) does not change with female age and are only responsive to the first mating while others (e.g. Coracle, Neuroglian and Hu li tai shao) respond to the first and second mating [37]. It is possible that mating-responsive proteins which respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Hence, the response of the reproductive tract to an extrinsic cue (mating) is composed of rapid changes in the expression levels of mating responsive transcripts and proteins that are required for the final maturation of the reproductive system. www.sciencedirect.com

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What is the advantage of delaying the terminal differentiation until after mating? Unmated females are capable of depositing eggs, though at a reduced rate compared to mated females of the same age. One possible interpretation for delaying the terminal differentiation until postmating is that reproduction is energetically costly and delaying maturation until sperm is available is advantageous to the female.

How does mating induce the rapid transition to the ‘mated state’? The female response to mating on the molecular level occurs very rapidly. Three recent genomic screens demonstrate that this rapid response may be mediated posttranscriptionally via microRNA [50,51,52]. MicroRNAs (miRNA) are non-coding RNA sequences of 21–24 nucleotides that regulate the expression of their ‘target genes’ by binding to complementary sites on the 30 UTR of their mRNA transcripts and reducing or eliminating the level of protein products, even on the order of hours or less [53–56]. Fricke et al. [50] examined the effect of Acp70 (sex peptide) on the miRNA profile of the mated Drosophila female. For that, they compared the miRNA profile of the abdomen and that of the head and thorax of females mated to males lacking sex peptide or to control males. Out of miRNAs identified by Fricke et al. [50], 4 were functionally analyzed. Females in which expression of any of the 4 miRNAs (miR-279, miR-317, miR-278, and miR-184) was reduced or eliminated exhibited altered receptivity compared to control females. Interesting, egg-laying and lifespan were not affected. They concluded that the rapid behavioral response to sex-peptide is subjected to posttranscriptional regulation by miRNAs. Zhuo et al. [51], using deep-sequencing of RNA extracted from Drosophila female whole bodies, identified 27 miRNAs and 474 mRNA that were expressed significantly differently between mated and unmated females. Overexpression of several mating-responsive miRNAs in females affected egg laying behavior, and altered the expression of their putative candidate target genes suggesting that miRNAs could mediate changes in gene expression postmating. Carmel et al. [52] found an enrichment of mRNAs that are targets of miRNAs among the mating-responsive genes identified in the LRT, suggesting miRNA involvement. Using microarray for miRNAs, Carmel et al. [52] found that mating-responsive miRNAs are expressed throughout the LRT prior to mating and that mating changed their expression levels in one or more specific regions. When they further reduced the function of all miRNAs by silencing Dicer-1 (a key component in miRNA biogenesis) in female whole body pre-mating, they found that these females exhibited a significant decrease in fertility. During the initial maturation period, females have already synthesized most of the transcripts and proteins needed to rapidly respond to mating and for the early Current Opinion in Insect Science 2016, 13:106–113

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stages of postmating reproductive physiology. Mating rapidly relieves the arrest, shifting the female from a posteclosion developmental phase toward the postmating end of the developmental program during which they reach reproductive maturity and prepare for maximum reproduction. Mating-responsive miRNAs may sharpen the transition from the unmated to the mated state by eliminating many target genes simultaneously and suppressing residual transcripts that are specific to the unmated stage which interfere with the final stage of differentiation of epithelial, muscle and nerve tissues. Mating-responsive miRNAs may possibly also relieve inhibition of factors that promote differentiation that were kept in check to prevent precocious expression. It is therefore suggested that mating-responsive miRNAs reprogram the female expression profile to rapidly initiate the final differentiation of the reproductive tract tissues, allowing sperm storage and leading to a functional reproductive system.

Conclusions Regulation by miRNAs has been found to be critical for the functionality of reproductive tissues in a wide range of animals, from mammals to Caenorhabditis elegans [57– 60] and insects [61,62,63]. Studies reviewed here raise the possibility that the mating response in D. melanogaster also involves post-translational regulation via miRNA [20,37,45]. The transition from unmated to mated state in the D. melanogaster female is rapid, involving changes in the expression level of many mRNA transcripts. This transition would require molecular mechanisms that can rapidly end one program (unmated) and initiate the next program (mated) while preventing transcriptional and translational errors in the subsequent state. In silico simulations predict that regulation by miRNAs is faster and consumes less energy than conventional regulation via transcription factors [64]. In vivo studies of miRNA kinetics in mouse retina support this prediction [65]. Indeed, numerous miRNAs that are differentially expressed between unmated and mated females have been identified [50,51,52] and have been shown to affect the female mating response. Because D. melanogaster is short-lived, the duration of the mating response (the time from the start of copulation to oviposition) affects fitness. A fast mating response increases the chance a female has to establish the next generation of offspring before a possible catastrophe. Similar to Drosophila, mating induces changes in the female of many other insect species [66]. In Anopheles gambiae, for example, mating stimulates the female to start to feed on blood, which is required for the production of mature oocytes [67], and male seminal fluid stimulates oviposition, sperm storage and changes in the morphology and expression profile of the female reproductive tract [68–71]. At 24 hours postmating, the reproductive system Current Opinion in Insect Science 2016, 13:106–113

of the Anopheles female undergoes prominent transcriptional changes, including a dramatic down-regulation of 13 protease genes [69]. At the morphological level, postmating changes in the vagina include elimination of extensive smooth endoplasmatic reticulum from the apical side of the cells and induction of the formation of large electron dense vacuoles of uniform granule in some apical cells along the vagina [69]. In addition, rough endoplasmatic reticulum at the basal side of the vaginal cells was greatly reduced. These morphological changes suggest decreased capacity for protein synthesis in vaginal cells and a possible transfer of components between the vaginal lumen and epithelial cells. The rapid changes occurring in the secretory machinery and/or the epithelial cells of the Anopheles vagina may reflect a change in the communication mode between epithelial cells and/or epithelial cells and the lumen, reminiscent of the changes observed in the Drosophila oviduct postmating. Together, these studies suggest that the mosquito female reproductive tract undergoes a two-phase maturation, in which a rapid mating response leads to a functional reproductive system. In addition to Drosophila and Anopheles, seminal fluid induces physiological and behavioral changes (switch from unmated to mated state) in the females of many other insects, including moths, beetles, and honeybees [66]. However, not much is known about the mating response kinetics and/or developmental status of the system. We hypothesize that the model we describe for Drosophila can be also found in other insects (either dipteran or non-dipteran) which are short-lived and have similar reproductive strategy. We propose that delaying the final maturation allows the female to conserve the energy required to invest in the development of the reproductive tissues until conditions are optimal. It will be interesting to examine if mating-induced developmental changes take place in other insects. For this, further studies comparing the morphology and expression profile of reproductive tissues in virgin and mated female at different times postmating are required. These studies will determine if the rapid transition of the reproductive tract induced by mating is a general phenomenon in insects. Because the mating signal of organisms with internal reproduction is composed of stimuli perceived during courtship and the receipt of sperm and seminal fluid, it is likely that this rapid unmated to mated switch may reflect the evolution of a mechanism to optimize reproductive capacity in early adulthood in short-lived animals. As female reproductive molecules are identified in more insect species and their functions are better understood, we will get a deeper understanding of the dynamics of male-female reproductive molecule interactions and of how these interactions maximize reproductive success. www.sciencedirect.com

Mating induces developmental changes Carmel, Tram and Heifetz

Acknowledgements We thank Zohar Nir-Amitin for the graphics; support by US-Israel Binational Agricultural Research and Development Fund, research Grant 3492 (to YH), The Chief Scientist Ministry of Agriculture grant 872-0055-10 (to YH) and US-Israel Binational Science Foundation grant 2009270 (to YH and Mark Siegal).

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39. Harris TJ, Tepass U: Adherens junctions: from molecules to morphogenesis. Nat Rev Mol Cell Biol 2010, 11:502-514. 40. Heifetz Y, Wolfner MF: Mating, seminal fluid components, and sperm cause changes in vesicle release in the Drosophila female reproductive tract. Proc Natl Acad Sci U S A 2004, 101:6261-6266. 41. Avila FW, Bloch Qazi MC, Rubinstein CD, Wolfner MF: A requirement for the neuromodulators octopamine and tyramine in Drosophila melanogaster female sperm storage. Proc Natl Acad Sci U S A 2012, 109:4562-4567. 42. Rubinstein CD, Wolfner MF: Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling. Proc Natl Acad Sci U S A 2013, 110:17420-17425. 43. Heifetz Y, Lindner M, Garini Y, Wolfner MF: Mating regulates neuromodulator ensembles at nerve termini innervating the Drosophila reproductive tract. Curr Biol 2014, 24:731-737. 44. Rezaval C, Nojima T, Neville MC, Lin AC, Goodwin SF: Sexually dimorphic octopaminergic neurons modulate female postmating behaviors in Drosophila. Curr Biol 2014, 24:725-730. 45. McGraw LA, Clark AG, Wolfner MF: Post-mating gene expression profiles of female Drosophila melanogaster in response to time and to four male accessory gland proteins. Genetics 2008, 179:1395-1408. 46. Prokupek AM, Kachman SD, Ladunga I, Harshman LG: Transcriptional profiling of the sperm storage organs of Drosophila melanogaster. Insect Mol Biol 2009, 18:465-475. 47. Heifetz Y, Vandenberg LN, Cohn HI, Wolfner MF: Two cleavage products of the Drosophila accessory gland protein ovulin can independently induce ovulation. Proc Natl Acad Sci U S A 2005, 102:743-748. 48. Ram RK, Sirot LK, Wolfner MF: Predicted seminal astacin-like protease is required for processing of reproductive proteins in Drosophila melanogaster. Proc Natl Acad Sci U S A 2006, 103:18674-18679. 49. Laflamme BA, Avila FW, Michalski K, Wolfner MF: A Drosophila protease cascade member, seminal metalloprotease-1, is activated stepwise by male factors and requires female factors for full activity. Genetics 2014, 196:1117-1129. 50. Fricke C, Green D, Smith D, Dalmay T, Chapman T: microRNAs  influence reproductive responses by females to male sex peptide in Drosophila melanogaster. Genetics 2014, 198:16031619. This paper demonstrates that sex peptide induces post-transcriptional activity of miRNAs in the female. A comparison between miRNA profiles of females mated to males with and without sex peptide identified candidate miRNAs. Functional analysis of four selected miRNAs showed that mated females which lack these miRNAs had altered receptivity to courting males compared to control females. 51. Zhou S, Mackay T, Anholt RR: Transcriptional and epigenetic responses to mating and aging in Drosophila melanogaster.  BMC Genomics 2014, 15:927. The study of Zhou et al. supports two important themes in our review: (1) the phenotypic plasticity of mating and (2) the presence of functional miRNAs in the mating response of the Drosophila female. Zhou et al. preformed a comprehensive deep-Seq screen to examine the effect of mating and/or aging on the transcriptome, miRNome and histone modification (using Chip-seq) of the Drosophila female whole body. 52. Carmel I, Nave M, Zelinger E Apel I, Keidar T, Schnakenberg S, Widmayer P, Breer H, Siegal M, Heifetz Y: miRNAs regulate the mating response in the lower RT of the Drosophila melanogaster female. Unpublished results. 53. Giraldez AJ, Mishima Y, Rihel J, Grocock RJ, Van Dongen S, Inoue K, Enright AJ, Schier AF: Zebrafish miR-430 promotes deadenylation and clearance of maternal mRNAs. Science 2006, 312:75-79. 54. Hornstein E, Shomron N: Canalization of development by microRNAs. Nat Genet 2006, 38(Suppl.):S20-S24. Current Opinion in Insect Science 2016, 13:106–113

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Mating induces developmental changes Carmel, Tram and Heifetz

70. Gabrieli P, Kakani EG, Mitchell SN, Mameli E, Want EJ, Mariezcurrena Anton A, Serrao A, Baldini F, Catteruccia F: Sexual transfer of the steroid hormone 20E induces the postmating switch in Anopheles gambiae. Proc Natl Acad Sci U S A 2014, 111:16353-16358.

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71. Shaw WR, Teodori E, Mitchell SN, Baldini F, Gabrieli P, Rogers DW, Catteruccia F: Mating activates the heme peroxidase HPX15 in the sperm storage organ to ensure fertility in Anopheles gambiae. Proc Natl Acad Sci U S A 2014, 111:5854-5859.

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