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Pathogens and Global Health logoLink to Pathogens and Global Health
. 2012 May;106(2):82–93. doi: 10.1179/2047773212Y.0000000016

Function and composition of male accessory gland secretions in Anopheles gambiae: a comparison with other insect vectors of infectious diseases

Francesco Baldini 1, Paolo Gabrieli 1, David W Rogers 2, Flaminia Catteruccia 1; 3,3
PMCID: PMC4001493  PMID: 22943543

Abstract

Human malaria, a major public health burden in tropical and subtropical countries, is transmitted exclusively by the bite of a female Anopheles mosquito. Malaria control strategies aimed at inducing sexual sterility in natural vector populations are an attractive alternative to the use of insecticides. However, despite their importance as disease vectors, limited information is available on the molecular mechanisms regulating fertility in Anopheles mosquitoes. In the major malaria vector, An. gambiae, the full complement of sperm and seminal fluid required for a female’s lifelong egg production is obtained from a single mating event. This single mating has important consequences for the physiology and behavior of An. gambiae females: in particular, they become refractory to further insemination, and they start laying eggs. In other insects including Drosophila, similar post-copulatory changes are induced by seminal proteins secreted by the male accessory glands and transferred to the female during mating. In this review, we analyze the current state of knowledge on the function and characterization of male seminal proteins in An. gambiae, and provide a comparative assessment of the role of these male reproductive factors in other mosquito vectors of human disease in which female post-copulatory behavior has been studied. Knowledge of the factors and mechanisms regulating fertility in An. gambiae and other vectors can help the design of novel control strategies to fight the spread of disease.

Keywords: Anopheles, Fertility, Seminal fluid, Sperm, Post-mating response, Vector control, Malaria, Protease, Redox, Acps, Copulation, Reproduction, Sex, Sterile

Introduction

Mosquitoes transmit a variety of infectious diseases that severely affect human health. Malaria alone, transmitted by the bite of female Anopheles mosquitoes, annually infects more than 200 million people and causes nearly one million deaths. Infections by dengue and yellow fever virus, transmitted by Aedes mosquitoes, are a leading cause of illness and death in many tropical and subtropical countries. Current strategies aimed at targeting vector populations are mainly based on the use of insecticides; however, such efforts are hampered by the emergence of insecticide resistance in mosquitoes combined with the lack of novel chemicals. There is an urgent need for novel strategies to control mosquito disease-transmitting populations.

Among the hundreds of extant anopheline species, An. gambiae is the most important vector of human malaria. Plasmodium parasites, the causative agents of malaria, are transmitted when a female mosquito feeds on the blood of a host, releasing infective sporozoites into the blood stream.1 As blood feeding is necessary for egg production, the parasite exploits the mosquito’s reproductive needs to achieve its own transmission between vertebrate hosts. The high reproductive rate of An. gambiae mosquitoes is a major component of their capacity as malaria vectors. A female of this species can generate more than a hundred eggs from each blood meal, and can fertilize her lifetime egg production using sperm acquired from a single mating and stored in her sperm storage organ.

The acquisition of sperm by a female is a potential target for intervention aimed at vector control: An. gambiae females generally mate only once2 as mating with one male permanently switches off their receptivity to further insemination with other males and stimulates oviposition.3 This dependence of lifetime reproductive success on a single mating event offers an excellent target for intervention; interfering with insemination or oviposition would have a large impact on the size of natural mosquito populations. Fertility is a target in control strategies, such as the sterile insect technique (SIT),4 aimed at natural insect pests. SIT relies on the massive release of sterilized males into field populations. Females mated to sterile males lay infertile eggs, with a consequent decrease in population size. Despite the use of this technique for the control of many insect pests, to date SIT against Anopheles species has not been very successful.5 A deeper knowledge of mating and other processes underlying Anopheles fertility would definitely benefit the chances of SIT success, and would identify targets for the development of novel vector control strategies.

In this review, we describe the current understanding of reproductive biology in An. gambiae, with a particular focus on the mechanisms known to regulate female receptivity to mating and ability to lay eggs. In doing so, we provide a comparison of the events that influence female post-copulatory biology in other disease-transmitting mosquitoes, such as Aedes and Culex, and relate these factors to the primary model of insect reproductive biology, Drosophila melanogaster.

Mating Behavior and Physiology In Anopheles and Other Mosquitoes

Mosquitoes are members of a family of the nematocerid flies: the Culicidae. This family consists of three subgroubs: the toxorhynchitinae, the anophelinae, and the culicinae. Blood feeding mosquitoes, including vectors of human diseases, belong to the two latter groups. Anophelines mate predominantly in crepuscular station-keeping swarms formed by large aggregations of males above inanimate markers.612 Virgin females enter the swarm, are captured by a male, and leave the swarm while in copula. Most male culicines also aggregate in the proximity of visual markers, although members of the Aedes subgenus are known to swarm and mate in the vicinity of the host.1318 There is strong evidence that males and females recognize each other by the wing beat frequency specific to each species19,20 and interact acoustically by shifting their harmonic overtones to match.21,22 Furthermore, spatial segregation of the swarms may contribute to reproductive isolation of different species, as observed for the incipient M and S forms of An. gambiae species.23,24

Anopheline and culicine females are generally monandrous as after mating they become refractory to further insemination.2,2529 Field studies show that remating does not occur in anophelines,29 or is observed at very low rates.2,27,28 During mating, male mosquitoes transfer sperm, and seminal secretions from the male accessory glands (MAGs) (seeFig. 1 for a representation of the male and female reproductive tracts). Sperm are stored by the female in a dedicated sperm storage organ named the spermatheca. While Anopheles mosquitoes have a single spermatheca, Aedes and Culex have three, like Drosophila.30 Seminal secretions from the MAGs are transferred to the female reproductive tract, and in some anophelines including the major malaria vectors (An. gambiae, An. arabiensis, and An. funestus), these secretions are coagulated into a gelatinous mating plug that is deposited in the atrium (sometimes called the uterus).31

Figure 1.

Figure 1

Cartoon representing male and female reproductive tracts in An. gambiae. (A) The male reproductive tract, showing the testes (T) and male accessory glands (MAG). (B) The reproductive tract of a freshly mated female, showing the ovaries (Ov), the atrium (A) containing a mating plug, and the spermatheca (Sp) filled with sperm.

The Role of MAG Secretions in Modulating Female Behavior after Mating

In many insect species, the MAGs exert powerful control over female reproduction and behavior. In D. melanogaster, a wealth of studies has demonstrated that MAG secretions, composed of proteins, carbohydrates, and lipids,32 play a major role in inducing behavioral and physiological changes in the female. These changes include loss of mating receptivity, increased oogenesis and oviposition, increased feeding and sleeping activity, induction of immune responses, and decreased longevity (reviewed in Refs. 33 and 34). Seminal fluid proteins are also associated with the behavioral and physiological changes (namely, the loss of mating receptivity and increased oogenesis and oviposition) observed in females of some mosquito species after mating (reviewed in Refs. 32 and 35). The physical act of copulation is not always required to induce these post-mating responses; in Aedes and Culex species, simply transplanting the MAGs or injecting MAG extracts into the hemolymph of virgin females is sufficient to induce life-long mating refractoriness and to trigger oviposition.26,3640 Early studies showed some promise in the identification of MAG factors modulating oviposition and female remating.26,3840,41 However, in spite of recent advances in characterizing the components of the seminal fluid in Aedes mosquitoes,42,43 the nature of the molecule(s) responsible for inducing post-mating behavior remains elusive.

The mode of action of key seminal fluid proteins appears to be conserved across several species; heterologous transplant of MAGs between Ae. aegypti, C. pipiens, and D. melanogaster stimulates oviposition in virgins of all species.44 Similarly, oviposition can be triggered in virgin Ae. aegypti by the transplantation of MAGs from Ae. triseriatus.45 However, some functions of the seminal fluid may be species specific. Yeh and Klowden46 found that implanting MAGs or injecting MAG homogenates from a conspecific male stimulated pre-oviposition behavior (i.e. the attraction of gravid mosquitoes to mating sites) in Ae. aegypti. However, injection of MAG extracts from other mosquito species failed to induce pre-oviposition behavior.

The role of MAG secretions in shaping two of the major female post-mating responses in Anopheles, such as the acquisition of sexual refractoriness and the induction of oviposition, is controversial (see Table 1 for a summary of the experiments). Early studies, based on hybrid mating, suggested a role for the MAGs in triggering oviposition. Virgin An. gambiae, which rarely lay eggs,47 can be induced to oviposit unfertilized eggs if mated to hybrid An. gambiae/An. melas males with degenerate testes but normal accessory glands.48 However, attempts to replicate the MAG transplant and injection experiments that provide such clear results in other Diptera have produced mixed results in anophelines. Intraspecific MAG implant induced loss of mating receptivity in virgin An. quadrimaculatus26 but not in An. gambiae and An. albimanus.49 Furthermore, Klowden found that intra-abdominal injections of MAG extracts had no effect on mating receptivity or oviposition behavior of virgin An. gambiae.49 In contrast, recent experiments by Shutt et al.50 showed that intra-thoracic injections of MAG extracts into virgin An. gambiae females reduced their likelihood of subsequently becoming inseminated. To explain this discrepancy, Shutt et al. suggested that putative MAG protein receptors in An. gambiae might be located in the thorax, and that this would reduce the efficacy of abdominal injections.50 There might be several explanations for these conflicting results. In the field, Anopheles remating occurs at very low rates2,2729 while it is much higher under laboratory conditions.5153 A number of parameters can influence the frequency of mating (and remating) — as well as oviposition — in laboratory cages. These parameters include cage size, female age, female fitness after the injection (MAG homogenates contain many proteolytic enzymes that when injected may interfere with normal female physiology), and length of time that females are exposed to males.51 Further studies are needed to clarify the role of MAG secretions in modulating female post-mating changes. MAG products may need to be processed by atrial proteases in order to stimulate ovulation and oviposition, explaining why MAG implants or extract injections are not effective. In support of this hypothesis, proteases associated with processing of peptide hormones54 are highly expressed and synthesized in virgin atria.55

Table 1. Induction of post-mating response experiments in Anopheles.

Reference Species Mating status Operation Behavioral change
26 An. quadrimaculatus Virgin MAG implant Refractoriness
48 An. gambiae Mated Mated males with abnormal testes and normal MAGs Refractoriness,* oviposition
64 An. gambiae Mated Mated males with abnormal testes and abnormal MAGs No refractoriness, no oviposition†
49 An. gambiae/An. albimanus Virgin MAG implant No refractoriness, no oviposition
49 An. gambiae Virgin Injection of MAG homogenate (intra-abdominal) No refractoriness, no oviposition
49 An. gambiae Virgin Injection of MAG homogenate (into the genital tract) No refractoriness
49,51 An. gambiae Virgin Implant of spermatheca from a mated female No refractoriness, no oviposition
49,51 An. gambiae Mated Removal of spermatheca No oviposition
50 An. gambiae/An. stephensi Virgin Injection of MAG homogenate (intra-thoracic) Refractoriness
53 An. gambiae Mated Mated males with no sperm cells and functional MAGs Refractoriness, oviposition

Notes: MAG, male accessory gland.

Behavioral changes include the ability to lay eggs (oviposition) or the inhibition of remating (refractoriness) after treatment.

*Further mating was performed by forced copulation. No insemination was detected.

†Oviposition occurred at very low levels similar to those observed in virgins.

Sperm Effect in Female Post-Mating Responses

Injection of MAG secretions or purified components into female Drosophila induces post-mating changes for 1–2 days.56 The maintenance of the response for up to a week, as seen in natural matings, requires transfer of sperm,5759 a phenomenon known as the ‘sperm effect’. The sperm effect is mediated by the binding of MAG peptides to the sperm cell flagella; seminal proteins bound to sperm tails are carried into the female sperm storage organs, where their gradual release maintains the post-mating response.6062

Early studies suggested that sperm transfer could play an important role in influencing female behavior in mosquitoes. Gwadz found that refractoriness to mating was induced within 15 seconds of mating in female Ae. aegypti,63 in contrast to the 4 hour latency arising from MAG transplants described by Craig.26 This difference could be due to the time required by the injected females to recover from the operation or can be interpreted as an evidence for a sperm effect with the immediate post-mating response triggered by the filling of a female’s bursa by sperm.63 If a sperm effect is present in Ae. aegypti, it would only be effective over the short-term, unlike the long-term maintenance observed in D. melanogaster. Removal of the aedine spermathecae, where sperm migrate after their initial deposition in the bursa, prior to copulation did not interfere with the induction of oviposition upon mating.51

Due to the ambiguous results of MAG transplantation and injection experiments, sperm has often been viewed as a key trigger of post-mating changes in anophelines (Table 1). As with studies of the effects of MAG secretions, early studies of the sperm effect examined hybrid matings. Hybrid An. gambiae/An. melas males with degenerate testes but with well-developed MAGs were still capable of inducing post-mating behavior,48 whereas hybrid males lacking both MAGs and testes were not,64 suggesting that sperm are not required to trigger female behavioral changes. In a recent study, the role of sperm in modulating female behavior in An. gambiae has been unambiguously established by RNA interference-mediated silencing of zero population growth, a germ cell differentiation gene whose knockdown results in males lacking sperm cells but with functional accessory glands. These spermless males were capable of inducing oviposition and inhibiting remating in females,53 confirming the original findings by Bryan that in this species sperm have no role in triggering post-mating behavior. Furthermore, spermless males induced female transcriptional responses similar to those triggered by normal males.53

Although sperm do not play a major role in triggering behavioral changes in female An. gambiae, an intact (and possibly innervated) spermatheca may be needed for such responses to take place. Surgical removal of the spermatheca from mated An. gambiae results in inhibition of oviposition, while implantation of a mated spermatheca into virgin females does not stimulate egg laying or loss of sexual receptivity.49,51 In the grasshopper Gomphocerus rufus, microinjection of MAG secretion into the spermatheca induces female mating refractoriness and sperm have no role in the process.65 In this insect spermathecal gland cells digest and resorb seminal secretions, and the neural pathway between the spermatheca and the ventral nerve cord is required to maintain sexual refractoriness.66 Recently, it has been shown in Drosophila that spermathecal secretory cells attract sperm into the female sperm storage organ and participate in modulating sperm motility and stimulating oviposition.67 A possible role of spermathecal cells in Anopheles post-mating behavior needs to be fully established.

Mag Proteins in An. Gambiae and Other Insects

Despite the crucial role of MAG components in regulating many aspects of mosquito reproduction, elucidation of the seminal proteomes of mosquitoes has only been established in the last few years.42,43,68,69 The molecular composition of mosquito MAG secretions remained entirely unknown until Dottorini et al.’s69 bioinformatic comparison of D. melanogaster and An. gambiae identified 46 MAG-expressed anopheline genes. Since then, further components have been identified in An. gambiae68 and the Ae. aegypti seminal proteome has been well characterized.42,43 These analyses have identified 71 genes expressed in the MAGs of An. gambiae.6870 Moreover, for the purpose of this review, we have identified 50 additional An. gambiae MAG-specific genes by analyzing the data produced in a recent whole genome microarray study where gene expression was determined in multiple male and female tissues71 (Table 2).

Table 2. Genes identified in the MAGs of An. gambiae.

Functional class Anopheles Drosophila Aedes Expression data (%)
Acps
AGAP001510 nd AAEL017145 100
AGAP006362 CG13699 AAEL014767 100
AGAP006581 Acp62F* AAEL000356 13
AGAP006583 Acp63F* nd 100
AGAP006585 Acp63F* nd 100
AGAP006586 Acp62F* AAEL000356 17
AGAP006587 Acp62F* nd 100
AGAP006589 nd nd 100
AGAP008116 nd AAEL010264 100
AGAP008968 CG31704 nd nd
AGAP009352 (AGAP012681) Acp70A* nd 100
AGAP009353 (AGAP012680) Msopa* nd 100
AGAP009354 Mst57Da* nd 100
AGAP009355 Dro-PA nd 100
AGAP009356 Mst57Da* nd 100
AGAP009357 Dro-PA nd 100
AGAP009358 (AGAP012682/AGAP012830) Nplp4 nd 100
AGAP009359 Mst57Da* nd 100
AGAP009360 (AGAP012807) CG13230 nd 98
AGAP009361 Acp95EF* nd nd
AGAP009362 CG6409 nd 100
AGAP009367 Acp26Ab* nd 100
AGAP009368 CG14770 nd 100
AGAP009369 Acp53Ea* nd 100
AGAP009370 (AGAP012706) Acp53Ea* nd 100
AGAP009371 CG14302 nd 100
AGAP009372 CG32726 nd nd
AGAP009373 vsg Supp02310* 100
AGAP013714 nd nd 100
AGAP013731 CG13230 nd 98
AGAP013734 CG15065 nd 100
AGAP013776 Nplp4 nd 100
Redox
AGAP001039 spo AAEL009762 100
AGAP003067 Cyp304a1 AAEL014413 100
AGAP005784 PAM AAEL007732♮ 100
AGAP007420 PHM AAEL001394 0
AGAP007491 CG4670 AAEL012054 35
AGAP008019 Cyp12b2 AAEL002031♮ 99
AGAP008203 Cyp6a2 AAEL009120 100
AGAP009363 Cyp9f2 AAEL001312♮ 0
AGAP009584 Trx-2 AAEL010777 24
AGAP012855 Cyp6a2 AAEL009120 100
CYP302A1 dib AAEL015655 85
CYP306A1 phm AAEL004888 88
CYP314A1 shd AAEL011850 98
CYP315A1 sad AAEL010946 100
Proteases
AGAP005791 CG12951 nd 100
AGAP005792 CG12951 nd 100
AGAP008276 Try29F nd 100
AGAP008277 Try29F nd 100
AGAP008997 CG8172 AAEL000238 100
AGAP012315 CG34290 AAEL012447♮ 100
AGAP013150 CG9806* AAEL009108 100
ENSP017764 nd nd 100
ZCP1 nd nd 100
ZCP3 nd nd 100
ZCP4 nd nd 100
ZCP6 nd nd nd
ZCP7 nd nd 100
ZCP9 nd nd 100
Nucleic acid binding
AGAP000355 Mkrn1 AAEL007476 100
AGAP000754 CG15439 AAEL003032 100
AGAP000916 pAbp nd 100
AGAP000918 pAbp nd 100
AGAP000920 pAbp nd 100
AGAP003844 cwo AAEL010513 100
AGAP009339 CG6654 AAEL005029 100
AGAP009699 sens-2 AAEL001243 100
AGAP010358 gsb nd 100
AGAP010359 prd nd 100
Hydrolases
AGAP001649 CG31414 AAEL014321♮ 100
AGAP005255 Rab3 AAEL006267 100
AGAP006425 CG9701 nd 100
AGAP010720 pom1 AAEL013237 100
COEBE1D EST-6 nd nd
COEBE4D EST-7 nd nd
Protein folding
AGAP000831 CG7872 AAEL013114 100
AGAP001424 CG5520 AAEL012827 nd
AGAP001502 CG11267 AAEL001052 10
AGAP007088 CG2852 AAEL013279 3
AGAP008822 CG9847 AAEL004313 0
AGAP012407 PDI* AAEL000641* 4
Transferases
AGAP000843 CG4774 AAEL012719♮ 100
AGAP009099 CG7356 nd 100
AGAP009190 CG4688 AAEL007955 100
AGAP009191 CG16936 AAEL007954 100
AGAP009365 CG5973 AAEL001297 100
AGAP009377 CG5973 AAEL001297 93
Protein binding
AGAP000927 sqh AAEL008921 100
AGAP004761 mwh AAEL008269 100
AGAP005684 Syx17 AAEL000282♮ 100
AGAP007041 fibrinogen AAEL001713* 100
AGAP012986 spi AAEL011205 100
Transmembrane proteins
AGAP000107 pyx AAEL004179 98
AGAP002824 Takr86C AAEL017414 100
AGAP010637 Toll6 nd 100
AGAP010861 CG1698 AAEL003626 100
Immune peptides
AGAP007049 CG10433 AAEL009861 2
AGAP009429 Anp* nd 98
TEP15 CG10363 AAEL014755 nd
Serpins
AGAP005246 CG9334 AAEL007765 17
SRPN9 CG10956 AAEL008364 nd
Nucleic acid metabolism
AGAP000139 tam AAEL015671 100
AGAP009842 CG8194 AAEL001159 36
Protein metabolism
AGAP000926 l(1)G0196 AAEL008950♮ 100
AGAP009673 QPCT AAEL010727 100
Lipases
AGAP003083 CG17097 AAEL012343♮ 100
AGAP003749 CG6296 AAEL012790 100
CRISPs
AGAP006418 CG17575* AAEL009239*♮ 100
Kinases
AGAP002181 CG5644 AAEL010062 100
Others
AGAP004428 CG3359 AAEL014917 3
AGAP005239 CG8323 AAEL006262 93
AGAP005504 scramb1 AAEL010661 100
AGAP007339 TpnC47D AAEL000744 96
AGAP009001 Hdc AAEL014632 100
AGAP009189 Eps-15 AAEL007950 100
AGAP009364 CG5793 AAEL001308 0
CALRETICULIN Crc AAEL001005♮ nd
Unknown
AGAP003736 CG30053 nd 100
AGAP005859 nd nd 100
AGAP008439 CG31705 AAEL005017 100

Notes:</emph> Acps, accessory gland proteins; CRISPs, cysteine-rich secretory proteins.

The table contains all 121 genes identified to date in the accessory glands of An. gambiae (Anopheles) males, identified by a number of strategies (as described in the text).6871 Genes are organized based on their functional class. When known, the putative D. melanogaster (Drosophila) and Ae. aegypti (Aedes) orthologues are indicated. In the Anopheles column, identifiers in bold represent genes whose products have been detected in the male accessory glands (MAGs) by mass spectrometry68 and/or reverse transcription PCR (RT-PCR) data.6870 The ‘*’ symbol indicates D. melanogaster and Ae. aegypti genes that are expressed in the MAGs of these species. The ‘♮’ symbol in the Aedes column indicates the presence of multiple putative orthologues (only the most similar orthologue is reported). The expression data column refers to the degree of MAG specificity of the An. gambiae genes, as obtained from MozAtlas website (http://www.tissue-atlas.org/); the degree of specificity is reported as the percentage of expression observed in the MAGs relative to all male and female tissues where expression was present in a minimum of 3/4 calls.71 Numbers are rounded to the nearest integer. Numbers in bold represent expression data obtained by transcriptional and/or immuno blotting analyses.6870 Please note that An. gambiae genes in the following groups share a common probeset in mozatlas: (1) AGAP009354, AGAP009356, AGAP009359; (2) AGAP009355, AGAP009357; (3) AGAP009358, AGAP0013776; (4) AGAP009360, AGAP0013731; (5) AGAP009365, AGAP009377; and (6) AGAP009369, AGAP009370.

These studies allow comparative analysis of the MAG proteomes in An. gambiae, Ae. aegypti, and D. melanogaster. Many of the functional classes of MAG proteins are shared between the two mosquito species and D. melanogaster (Fig. 2 and Table 2). In all three species, accessory gland proteins (Acps) form the most abundant category. Acps are defined as MAG-specific proteins that are secreted and do not contain known functional domains. In the fruitfly, many of these proteins are known to be transferred to females and control a number of responses to mating. For instance, sex peptide (Acp70A) has been implicated in the inhibition of remating,56,61,62,72 increased egg production,73,74 decreased longevity,75 alteration of locomotion and feeding behaviors,76,77 and stimulation of the immune system.78,79 Sex peptide is detected by sensory neurons in the female reproductive tract80,81 where it binds to a G protein coupled receptor82 leading to the alteration of female physiology and behavior. Another category abundant in An. gambiae and D. melanogaster comprises peptides that have putative hormonal function. For instance, the hormone ovulin (Acp26Aa) is an important regulator of ovulation in D. melanogaster.83 Although functions have yet to be ascribed to MAG peptide hormones in An. gambiae, putative orthologues of Acp53Ea, another peptide hormone that in Drosophila is involved in sperm competition,84,85 were localized in close proximity to the spermatheca, suggesting a role in sperm function.86 In Ae. aegypti, a head peptide expressed primarily in the MAGs87 has been implicated in the short-term inhibition of host-seeking behavior in females.88

Figure 2.

Figure 2

Functional classes of MAG-specific genes in An. gambiae, Ae. aegypti, and D. melanogaster. The pie charts represent the functional classes of genes that are expressed in the male accessory glands of An. gambiae (A), Ae. aegypti (B), and D. melanogaster (C). Values indicate the percentage of genes that belongs to each class in the three species. These charts are based on published data derived from bioinformatics,34,42,69 transcriptional34,42,68,69,71 and proteomic34,43,68 analyses.

Proteases and peptidases are represented at high levels in all three species. These enzymes can be involved in the activating cleavage of many seminal fluid protein.83,89 In Drosophila, ovulin is transferred to females as a preprohormone where it is processed by a seminal astacin-like protease.89 Other proteases can play roles in controling the activity of these processing proteases.90,91 Similarly abundant are serpins and other protease inhibitors, which have been shown to have a role in male mammalian fertility,92 and chaperones, which can facilitate protein folding and sperm-egg interactions.93 Cysteine-rich secretory proteins (CRISPs) that in ascidians are involved in gamete interactions94 are instead more frequently observed in D. melanogaster than in mosquito species. Lipases are also more abundant in D. melanogaster than in mosquitoes; in Drosophila these enzymes are transferred to females during mating,95 influence egg-laying behavior and possibly receptivity to remating,96 and provide energy to sperm.97 Contrary to Drosophila and Aedes, in Anopheles, no lectins have been identified to date in the MAGs. Lectins are postulated to play a role in sperm-oocyte recognition.98

Finally, it is notable that proteins that participate in oxidation/reduction (redox) processes are more abundant in mosquitoes compared to D. melanogaster. In the fruitfly, many MAG-expressed redox proteins are prolyl 4-hydrohylases that are involved in the hydroxylation of collagen, whose function may be needed to ensure the integrity and functionality of the extracellular matrix, possibly necessary for the activity of the MAGs.99 In An. gambiae MAGs, the majority of identified redox proteins are oxidases. Among these, a number are involved in the synthesis of ecdysteroid hormones, which are transferred to females during mating70 and that control egg production after a blood meal.100 By contrast, in Ae. aegypti, MAG-specific redox proteins are mostly dehydrogenases involved in energetic metabolism, and subunits of the ATP synthase protein complex, which might supply the energetic requirement for protein synthesis in this secretory glands.42,43 A Rab3-like protein appears instead to be specific for An. gambiae MAGs. Rabs are proteins that regulate membrane trafficking and in particular Rab3 is associated with secretory vesicles.101

Although functional classes of seminal proteins are conserved across the species, the MAG-expression of individual genes rarely is. As shown in Table 2, among the 121 genes expressed in the An. gambiae MAGs, 109 and 71 have putative annotated D. melanogaster and Ae. aegypti orthologues, respectively. However, by comparing the tissue expression of orthologues in the three species, it is clear that only a small number of these are expressed in the MAGs of more than one species. Only 17% of the An. gambiae genes have orthologues in either D. melanogaster and/or Ae. aegypti that are expressed in the MAGs. Only two genes are expressed in the MAGs of all three species: they encode for a protein disulphide isomerase, which may promote protein folding, and a CRISP protein. A total of 16 genes are expressed in the MAGs of both An. gambiae and D. melanogaster but not Ae. aegypti, comprising predicted pro-hormonal peptides, antimicrobial peptides, protease inhibitors, and proteases. Putative orthologues of many Drosophila accessory gland peptides were identified in An. gambiae: among these is the putative An. gambiae orthologue of sex peptide. A further two genes are expressed in the MAGs of both An. gambiae and Ae. aegypti, but not D. melanogaster: a fibrinogen and a visgun-like peptide, whose functions in reproduction are unknown. The diversity of the factors synthetized in the MAGs may highlight different reproductive roles for these male reproductive tissues among insects, stressing the need for a detailed analysis of the reproductive molecular machinery in each species.

An. gambiae MAG Secretions are Coagulated to Form a Mating Plug

Unlike most mosquito species, An. gambiae (and its close relatives) transfers its seminal secretions as a gelatinous mating plug, which becomes coagulated during copulation. Many insects produce mating plugs with different functions. In the lepidopteran Cressida cressida, males transfer an external plug termed a sphragis that blocks the female copulatory opening, physically preventing remating by other males.102 In the hymenopteran Bombus terrestris, a linoleic fatty acid present in the mating plug renders females refractory to further insemination for life.103 In Diptera, there are examples of mating plugs that prevent remating, such as in the dung fly Coproica vagans,104 while in D. hibisci, the mating plug is associated with female loss of sexual receptivity and correct sperm storage.105,106 In D. melanogaster, mating plug function and composition have been well characterized. The plug is divided into anterior and posterior regions: the posterior part is composed of male ejaculatory bulb proteins (PEB-me, PEBII, and PEBIII) and is formed in the female reproductive tract 3 minutes after the start of mating but before sperm transfer.107 Acquisition of short-term refractoriness is associated with PEBII as shown in knockdown studies.108 The anterior region of the plug is formed after the transfer of sperm, and it is needed to prevent sperm backflow from the storage organs and is composed of MAG proteins such as Acp36DE.109 Acp36DE binds to sperm and enters the sperm storage organs,110,111 where it enhances the rate of sperm accumulation.112

Among mosquitoes mating plugs are only found in anophelines.113 The role of the plug in reproduction has been controversial for decades and various hypotheses ranged from a physical barrier against re-insemination or sperm loss to a vestigial trait with no function.31,114116 Recently, it has been shown that in An. gambiae, mating plug transfer is crucial for correct sperm storage by the female after mating.68 Through RNAi-mediated knockdown of a MAG-specific plug-forming transglutaminase (TGase), Rogers et al.74 were able to show that females mated to males who failed to form and transfer the plug could not store sperm in their spermatheca, uncovering a crucial role of this feature in the reproduction of An. gambiae. This MAG-specific TGase coagulates seminal secretions by cross-linking other MAG secreted proteins, primarily Plugin, a glutamine-rich protein that is highly abundant in the mating plug.68 This mechanism is remarkably similar to semen coagulation in mammals.117 While An. gambiae has three TGases, of which only one is active in the MAGs, the genomes of the culicines Ae. aegypti and C. quinquefasciatus contain only two TGase and none shows activity in the male glands, consistent with the inability of Aedes and Culex mosquitoes to produce a mating plug.

The An. gambiae plug is digested in the female atrium during the first 24–36 hours post-mating, possibly by female proteases,68,118 and this processing may produce factors that affect female post-copulatory behavior. This might explain the frequently observed inability of MAG transplantation or extract injections to induce oviposition and sexual refractoriness in An. gambiae, in contrast to what is observed in culicines.<</p>

Conclusions and Outlook

To date, the most effective strategies for the control of An. gambiae mosquitoes rely on the use of insecticides through indoor residual sprays and long-lasting insecticide treated bednets.119 In many regions where these tools are used, the size of vector populations is decreasing significantly, contributing to reducing malaria transmission and therefore placing these regions in the Malaria Elimination Group.120 However, in much of sub-Saharan Africa, the use of insecticides is not sufficient to stop the spread of disease. Furthermore, the origin and spread of insecticide resistance in vector populations is reducing the effectiveness of insecticide-based strategies.121 In this scenario, the study of the processes shaping the biology and physiology of An. gambiae mosquitoes and other disease vectors brings new promise to the generation of novel ideas and to the identification of targets for the manipulation of the mosquito vectorial capacity. Recent studies reviewed here have identified the factors produced and secreted by the MAGs in several species. However, the functions of the majority of these factors remain unknown.

Characterizing the effects of MAG proteins and of the female genes that they target can provide a gateway to understanding the genetic modulation of mosquito reproduction. This knowledge would benefit the development of novel control programs based on the genetic modification of the vector at two different but complementary levels. On the one hand, it may help generate males with increased mating competitiveness, crucial for a successful deployment of SIT and related strategies. Laboratory reared mosquitoes generally show extremely low mating success in competition with their wild counterparts (reviewed in Ref. 122). There is growing evidence that the MAGs are associated with male reproductive success across insects. In some species, including mosquitoes,123 depletion of the MAGs may result in infertility long before males exhaust their supplies of sperm. Moreover, seminal fluid availability can contribute to male motivation to mate in the first place,124 and a lack of accessory gland material as a result of sexual immaturity or exhaustion is often associated with low mating rates.125 In laboratory-reared anophelines, male mating rate reaches a maximum approximately 3–7 days after eclosion12,126128 which corresponds to the period of time required to synthesize MAG secretions (72–100 hours).129 Improving the diets of adult male mosquitoes may be a simple way to improve mating competitiveness by promoting the rapid maturation and final size of the MAGs. For instance, supplementing the pre-release diets of males with protein or juvenile hormone analogues has been shown to dramatically increase the mating competitiveness of sterile males in several fruit fly species (reviewed in Ref. 130).

On the other hand, the study of reproductive biology will identify male genes important for fertility that could be targeted to induce genetic sterility in males for release, while genes responsible for female fertility could be disrupted in homing endonuclease mediated population depletion.131 Understanding the mode of action of MAG proteins may also allow the development of novel chemosterilants. Synthetic compounds could be developed that mimic the behavior-modulating effects of MAG proteins, or prevent the function of factors essential for fertility. Inducing the post-mating response, particularly the inhibition of remating, in virgin females would provide an excellent addition to the vector control arsenal. Novel chemosterilants would provide a second line of defense when used in combination with traditional insecticides used as indoor residual sprays or insecticide treated bednets, as resistant mosquitoes that escape insecticide action would be rendered sterile, preventing the spread of resistance genes. Moreover, as many MAG genes evolve rapidly132 and are therefore highly divergent between closely related species, it may be possible to develop chemosterilants that would precisely target only the desired vector. One example might be the MAG-specific plug forming transglutaminase identified by Rogers et al.68 whose function is important for ensuring correct storage of sperm by the female and which has no direct orthologue in aedine or culicine mosquitoes. A molecule that specifically inhibits this TGase, but not other related enzymes, could provide a specific mechanism for reducing the fertility of anopheline mosquitoes. Although speculative at present, these strategies are potentially highly rewarding. The full feasibility of such measures will only become clear once we have an improved understanding of the multiple functions of the MAGs.

Acknowledgments

The authors wish to thank members of the Catteruccia laboratory for their critical reading of the manuscript and Daniela Di Lascio for drawing Fig. 1. FC has been sponsored on research related to this topic by an MRC Career Development Award (agreement ID: 78415, file no. G0600062), by the European Research Council FP7 ERC Starting Grant project ‘Anorep’ (grant ID: 260897) and by the EC FP7 Collaborative Project 223601 ‘Malvecblok’.

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