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Unlike vertebrates, insects are widely thought to lack male-biased sex steroid hormones.In Anopheles gambiae, the ecdysone steroid 20-hydroxyecdysone (20E) appears to have evolved to control egg development when synthesized by females2 and to induce a mating refractory period when sexually transferred by males3.Since egg development and mating are essential reproductive traits, understanding how female Anopheles mosquitoes integrate these hormonal signals could facilitate the design of new malaria control programs.Here, we reveal that these reproductive functions are regulated by distinct sex steroids through a complex network of ecdysteroid-activating/inactivating enzymes.We identified a male-specific oxidized ecdysone, 3-dehydro-20E (3D20E), that protects parentage by shutting down female sexual receptivity following sexual transfer and activation by dephosphorylation.Notably, 3D20E transfer also induced the expression of reproductive genes that maintain egg development during Plasmodium infection, ensuring the health of infected females.Female-derived 20E does not elicit a sexual response, but allows mating individuals to lay eggs after 20E-inhibiting kinases are inhibited.The identification of this male-specific insect steroid hormone and its role in regulating female sexual receptivity, fertility and interaction with Plasmodium suggests the potential to reduce the reproductive success of malaria-transmitting mosquitoes.
Malaria cases and deaths are on the rise again4 due to widespread insecticide resistance in Anopheles mosquitoes, the sole vector of human malaria parasites.The mating biology of these mosquitoes is a particularly attractive target for novel malaria control interventions because females only mate once5; making this single mating event sterile would have great potential to reduce mosquito populations in the field.
Women become sexually incapacitated after receiving high-titer steroid hormones from men.Studies have shown that the trigger for difficulty in further mating is 20-hydroxyecdysone (20E), a steroid hormone better known as a regulator of the molting cycle in the larval stage.The ability of males to synthesize and transfer 20E has evolved specifically in Anopheles species that are part of the subgenus Cellia7, which is distributed in Africa and includes the most dangerous vectors of malaria, including Anopheles gambiae.This is especially noteworthy because in these species females also produce 20E after each blood meal, and 20E drives the oogenesis cycle (see ref. 8).However, little is known about the way in which females integrate signals from two different sources of ecdysone (male transfer and blood feeding induction) without compromising their own ability to mate.In fact, if the 20E produced by the females triggers sexual intolerance, this will lead to infertility in virgin-feeding individuals, a very common behavior in these mosquitoes5.
A possible explanation is that A. gambiae males transfer a modified male-specific ecdysone, which activates a signaling cascade in the female reproductive tract, resulting in mating instability.However, although vertebrates have multiple steroid hormones, such as estrogen and androgen (reviewed in ref. 9), to our knowledge, androgenic-biased steroids have not been identified in insects.
We set out to determine the repertoire of steroid hormones in the male male accessory gland (MAG) of sexually mature A. gambiae in search of possible modifying steroids.Using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) rather than the less specific method previously used, we detected ecdysone (E) and 20E in this tissue, confirming previous result.However, the sample was dominated by oxidized phosphorylated steroids, consistent with the formula 3-dehydro-20E-22-phosphate (3D20E22P)12 (Figure 1).Other forms include 3-dehydro-20E (3D20E) and 20E-22-phosphate (20E22P).The HPLC-MS/MS signal intensity of 3D20E22P was two orders of magnitude higher than its dephosphorylated form, 3D20E, and three orders of magnitude higher than that of E and 20E (Figure 1).Although in other parts of the body and the lower reproductive tract (LRT; Extended Data Fig. 1a).We also analyzed ecdysteroids in newly closed (<1 day old) males and females and detected 3D20E and 3D20E22P only in MAG; E, 20E and 20E22P were present in both sexes (Extended Data Fig. 1b).These data suggest that A. gambiae adult males produce high titers of modifying hormones in their MAGs that are not synthesized by females.
MAG and female LRT (including atria, seminal vesicles, and parovarium) were dissected from 4-day-old (4-day-old) virgin males and virgin and mated females (0.5, 3, and 12 hpm).Ecdysone in these tissues was analyzed by HPLC-MS/MS (mean ± sem; unpaired t-test, two-sided, false discovery rate (FDR) corrected; NS, not significant; *P < 0.05, **P < 0.01 . 3D20E: 3 hours vs. 0.5 hours, P = 0.035; 12 hours vs. 3 hours, P = 0.0015; 12 hours vs. 0.5 hours, P = 0.030. 3D20E22P: 3 hours vs. 0.5 hours, P = 0.25; 12 hours vs. 3 hours, P = 0.0032; 12 hours vs. 0.5 hours, P = 0.015).Data are from three biological replicates.The peak area for each ecdysone of interest was calculated and normalized by the number of mosquitoes.Ecdysone is represented by color as follows: E, green; 20E, orange; 20E22P, purple; 3D20E, blue; 3D20E22P, pink.The inset increases the scale on the y-axis to show lower ecdysone levels.
To investigate whether 3D20E22P and 3D20E are transferred during mating, we dissected female LRTs at various time points after mating.Although ecdysone was not found in virgins, we observed substantial amounts of 3D20E22P in the LRT immediately after mating (0.5 h post-mating, hpm), decreasing over time, while 3D20E levels increased significantly (Fig. 1).Using chemically synthesized 3D20E as a standard, we determined that levels of this steroid hormone in mating LRTs were at least 100-fold higher than 20E (Extended Data Table 1).Thus, 3D20E22P is the major male ecdysone that is transferred to the female LRT during mating, and its dephosphorylated form, 3D20E, becomes highly abundant shortly after mating.This suggests an important role for the latter ecdysone in female post-mating biology.
After generating a new RNA sequencing (RNA-seq) dataset (Fig. 2a), using a custom-built bioinformatics pipeline, we searched for ecdysone kinase (EcK), ecdysone oxidase (EO), and ecdysone encoding 20E-modified phosphatase gene.EPP) is expressed in reproductive tissues.We identified one candidate EPP gene and two potential EcK genes (EcK1 and EcK2), but were unable to find a good candidate EO gene.Notably, individual EPP genes were expressed at high levels (98.9th percentile) in Gambian MAGs but not in female LRTs (Fig. 2b), contrary to our expectations since dephosphorylation of 3D20E22P occurred in this female tissue.Therefore, we believe that male EPP may be transferred during mating.Indeed, we used in vivo stable isotope labeling to mask the female protein after mating, an enzyme identified by MS in the female atrium (Fig. 2c and Supplementary Table 1).The presence of EPP in MAG and mated (but not virgin) female LRT was also confirmed using specific antibodies (Fig. 2d).
a, A custom-built bioinformatics pipeline to search the reproductive tissues of each sex for genes encoding EcKs, EOs, and EPPs.The numbers next to the arrows indicate the number of male and female candidates at each step.This analysis identified one EPP gene (EPP) and one EcK gene (EcK1) that are expressed in males, and one EcK gene (EcK2) that is expressed in both sexes but does not yield a candidate EO gene.b, Heatmap comparing candidate gene expression in virgin (V) and mating (M) Anopheles gambiae and Anopheles albicans tissues.Spca, fertilization; MAGs, accessory glands in males; other parts of the body, including breasts, wings, legs, fat bodies, and internal organs in both sexes, and ovaries in females.EcK2 is highly expressed in both MAG and atria of Gambia, whereas EPP is found only in MAG.c, Proteomic analysis of male ejaculate group translocation into female atria at 3, 12 and 24 hpm, showing the 67 most abundant proteins.Females were raised on a diet containing 15N to label (and mask) all proteins.Untagged males were mated with tagged females, and female LRTs were dissected at 3, 12 and 24 hpm for proteomic analysis (see Supplementary Table 1 for a complete list of ejaculatory proteins).Inset, EPP, Eck1 and EcK2 were detected in the MAG of virgin males by proteomic analysis of these tissues.d, EPP was detected by western blot in MAG and LRT of mated females, but not in virgin females or males or the rest of the female body.Membranes were simultaneously probed with anti-actin (loading control) and anti-EPP antibodies.All males are virgins.See Supplementary Figure 1 for gel source data.Western blots were performed twice with similar results.
The ecdysteroid phosphophosphatase activity of EPP was verified after incubation by HPLC-MS/MS with 3D20E22P isolated from MAG (Extended Data Fig. 2a).Furthermore, when we silenced EPP by RNA-mediated interference (RNAi), we detected a strong reduction in phosphatase activity in the reproductive tissues of these males (Fig. 3a), and females mated with EPP-silenced males showed significant A lower proportion of dephosphorylated 3D20E (Fig. 3b) despite partial gene silencing (Extended Data Fig. 2b,c).In contrast, we did not detect significant changes in the 20E22P/20E ratio in the same mosquitoes, which might suggest that the enzyme is specific for 3D20E22P (Fig. 3b).
a, Decreased phosphatase activity in MAG caused by EPP silencing using double-stranded EPP RNA (dsEPP) or double-stranded GFP RNA (dsGFP) controls.Twenty MAG pools were used in each replicate (P = 0.0046, paired t-test, two-sided), represented by separate dots.b, Females mated with EPP-silenced males had a significantly lower proportion of dephosphorylated 3D20E at 3 hpm (P = 0.0043, unpaired t-test, two-sided), whereas 20E levels were unaffected (P = 0.063, unpaired). t-test, two-sided).Data are presented as mean ± sem from three pools of 13, 16 and 19 females each.c, Females mated with EPP-silenced males had significantly higher rates of re-mating (P = 0.0002, Fisher’s exact test, two-sided).Females were first forced to mate to ensure their mating status; 2 days later, they were contacted with other males carrying transgenic sperm to assess re-mating rates by quantitative PCR detection of the transgene.d, Blood-fed females mated with EPP-silenced males had significantly reduced fertility (P < 0.0001; Mann-Whitney test, two-sided) and slightly reduced egg number (P = 0.088, Mann-Whitney test, two-sided) , while spawning rate was not affected (P = 0.94, Fisher’s exact test, two-sided).In all panels, n represents the number of biologically independent mosquito samples.NS, not significant.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Next, we assessed whether ecdysone dephosphorylation is important for inducing mating resistance in females.Notably, females mated with EPP-depleted males re-mated at a much higher frequency (44.9%) than control females (10.4%) when exposed to additional (transgenic) males (Fig. 3c).We also observed a significant decrease in fertility (Fig. 3d, left) and a slight decrease in the number of eggs laid by these females (Fig. 3d, middle), while the percentage of eggs laid by females (another response elicited in females by mating )) were not affected (Fig. 3d, right).Given the observed specificity of EPP for 3D20E22P, these results suggest that the activation of 3D20E by EPP transferred during mating may have an important role in turning off female receptivity to further mating, a behavior previously ascribed to the sexual transfer of 20E .Therefore, this male-specific hormone also strongly affects female fertility.
Next, we compared the activities of 20E and 3D20E in injection experiments in sexually mature virgins using chemically synthesized 3D20E (Fig. 4a–c) and commercially available 20E.We observed that 3D20E was significantly more effective than 20E in shutting off female sensitivity to mating at both concentrations (Fig. 4d).Notably, half the physiological level of 3D20E in the LRT (1,066 pg post-injection vs. 2,022 pg post-mating) induced a proportion of refractory females that was 20-fold higher than the physiological level of 20E (361 pg post-injection) 24 hours after injection at the highest concentration 18 pg post-mating; Extended Data Table 1).This result is consistent with the notion that sexual transfer of 20E does not cause mating refractory periods, and further points to 3D20E as a major factor in ensuring parent-child relationship.3D20E was also significantly more active than 20E in egg-laying assays in virgin females (Fig. 4e), suggesting that the normal egg-laying rate we observed after partial EPP silencing was due to the presence of residual 3D20E activity still produced by Mating-induced female factors.
(a,b) 3D20E chemically synthesized from 20E (a) with very high conversion/efficiency (data presented as mean ± s.e.m. from three independent synthesis reactions) (b).c, Mass spectrum (bottom half) exactly matches ecdysone found in mated female LRT (top half).d, Compared with 20E (0.63 µg, P = 0.02; 0.21 µg, P < 0.0001; Fisher’s exact test, two-sided) and 10% ethanol (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Fisher’s exact test, 2-sided), while 20E was significantly higher than control only at higher doses (0.63 µg, P = 0.0002; 0.21 µg, P = 0.54; Fisher’s exact test, 2-sided).e, 3D20E injection induced significantly higher spawning rates in virgin females than 10% ethanol controls (0.21 µg, P < 0.0001; 0.13 µg, P = 0.0003; Fisher’s exact test, two-sided), while 20E compared to controls Only at higher doses (0.21 µg, P = 0.022; 0.13 µg, P = 0.0823; Fisher’s exact test, two-sided).3D20E induced significantly higher spawning rates than 20E at higher doses (0.21 µg, P = 0.0019; 0.13 µg, P = 0.075; Fisher’s exact test, two-sided).In all panels, n represents the number of biologically independent mosquito samples.NS, not significant.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.Data are from three replicates.
In previous studies, we determined that sexual transfer of steroid hormones induces the expression of MISO (Mating-Induced Stimulator of Oogenesis 11), a female reproductive gene that protects A. gambiae females from P. falciparum infection Health costs caused by 13, the deadliest human malaria parasite.Given the importance of MISO to the reproductive fitness of Anopheles in malaria-endemic areas, we decided to determine which hormone 3D20E or 20E triggers the expression of this gene.We found that while 20E injection specifically or more potently induced some nuclear hormone receptors (HR), such as HR3 and HR4, and typical downstream steroid targets, such as the yolkogenic genes Vg14, 15, 16, MISO was more strongly was induced by 3D20E (Extended Data Fig. 3).Thus, the sexual transfer of this androgenic steroid hormone appears to induce mechanisms that protect females from the costs posed by parasitic infection.Furthermore, 3D20E differentially affects both isoforms of the E receptor EcR, inducing EcR-A and repressing EcR-B, and more strongly triggering other mating-inducing genes, including HPX15, which affects female fertility.This could explain the significant infertility observed in females mated with EPP-silenced males (Extended Data Fig. 3).These data suggest the existence of downstream pathways preferentially activated by two ecdysone hormones that may underlie sex-specific function.
Next, we tested the function of the two EcK genes identified in our bioinformatics pipeline.Silencing EcK1 or EcK2 resulted in significant mortality in males (Extended Data Fig. 4a), suggesting that ecdysone phosphorylation, and thus inactivation, is important for survival.Because EcK2 was expressed at higher levels than EcK1 and was detected in MAGs by proteomics (Fig. 2b,c and Supplementary Table 2), we validated its ecdysteroid kinase activity by incubating it with 20E, which resulted in phosphorylation 20E22P (Extended Data Figure 2).4b).When using 3D20E as a substrate, we were unable to detect the phosphorylated product 3D20E22P (Extended Data Fig. 4c), suggesting that 20E rather than 3D20E may be the preferred target of EcK2.
According to our RNA-seq analysis, EcK2 was also highly expressed in the LRT of virgin females, where it was turned off after mating (Fig. 2b).We confirmed these data and determined that EcK2 expression was not affected by blood feeding (Extended Data Fig. 5a).Extending our initial MS experiments, we determined that the peak of 20E22P was closely related to the peak of 20E (22-26 hours after blood meal; Extended Data Fig. 5b).Silencing of EcK2 in virgin females resulted in a 3-fold increase in the relative ratio of 20E to 20E22P at 26 h post blood meal (Extended Data Figures 2c and 5c), confirming that EcK2 also phosphorylates 20E in females.Notably, EcK2-depleted virgins maintained full sexual receptivity (Extended Data Fig. 5d,e), further suggesting that female production of 20E does not induce mating refractory periods.However, these females had significantly increased egg-laying rates compared to controls, with more than 30% of virgins laying eggs (Extended Data Fig. 5f).If double-stranded Eck2 RNA (dsEcK2) injections were performed after blood feeding, spawning did not occur, at which point the 20E peak due to blood ingestion had declined.Overall, these results support a model that 20E produced after blood sucking can induce spawning, but only when the spawning block (EcK2 and possibly other factors) is turned off by mating.Neither 20E nor 3D20E injections inhibited EcK2 expression in virgins (Extended Data Fig. 5g), suggesting that other factors mediate the inhibition of this kinase.However, 20E levels after blood feeding were not sufficient to induce mating discomfort, but were effectively triggered by high titers of sexually transferred 3D20E.
Our results provide important insights into the mechanisms regulating the reproductive success of A. gambiae.A model has emerged where males have evolved to synthesize high titers of 3D20E, a male-specific modified ecdysone that assures parentage by desensitizing females to further mating.At the same time, these malaria vectors have also evolved an efficient system to activate 3D20E in females in response to the sexual transfer of male-specific EPP.To our knowledge, this is the first example of a male- and female-dominated steroid hormone system performing a unique and critical function in insects.Male-specific ecdysone function has been postulated but not definitively demonstrated.For example, a largely disproved hypothesis 18 is that these functions may be performed by the 20E precursor E1.It is well known that in Drosophila, monandry is triggered by the sexual transfer of small sex peptides19,20 that interact with neurons innervating the female reproductive tract through specific sex peptide receptors21,22.Further work is required to determine the downstream signaling cascades controlled by 3D20E in A. gambiae females and to determine whether these cascades can be conserved between mosquitoes and Drosophila.
Given the important role of 3D20E on female fertility and behavior identified in our study, the pathways leading to 3D20E synthesis and activation offer new opportunities for future mosquito control strategies, such as the generation of competitive sterile males in sterile insect technology strategies Use for wild release or to imitate the 3D20E in virgin play.The male-specific function of 3D20E may have evolved when A. gambiae and other Cellia species acquired the ability to coagulate their semen into mating plugs, as this allows for the efficient transfer of large numbers of hormones and hormone-activating enzymes.In turn, 3D20E evolution implementing monandry provides a mechanism for females (through high expression of MISO) to favor their reproductive fitness in areas of high malaria prevalence, which indirectly contributes to Plasmodium transmission.Given that female 20E has been shown to have profound effects on the survival and growth of P. falciparum in female Anopheles mosquitoes,24 both male and female steroid hormone pathways are now key aspects of mosquito-parasite interactions.
A. gambiae G3 strains were reared under standard insect conditions (26-28 °C, 65-80% relative humidity, 12:12 h light/dark photoperiod).Larvae were fed with powdered fish food (TetraMin Tropical Flakes, Koi Pellets and Tetra Pond Sticks in a ratio of 7:7:2).Adult mosquitoes were fed ad libitum 10% dextrose solution and weekly human blood (study blood components).Virgin mosquitoes were obtained by segregating the sexes at the pupal stage after examining the ends by microscopy.Males carrying the DsRed transgene have been described previously.
Forced mating experiments were performed according to previously described protocols.For natural mating, 4-day-old virgin females were kept in a 1:3 ratio with sexually mature virgin males for two nights.For experiments in which males were injected with dsEPP, co-caging coincided with days 3-4 post-injection, when phosphatase activity was maximally silenced (Extended Data Fig. 2b).
Mosquito tissues, remaining cadavers (rest of the body), or whole body were dissected into 100% methanol and homogenized with a beader (2 mm glass beads, 2,400 rpm, 90 sec).Tissue amounts and methanol volumes were as follows: rest of body, 50 in 1,000 µl; MAG, 50–100 80 µl; female LRT, 25–50 80 µl.The precipitate was subjected to a second methanol extraction with the same volume of methanol.Cell debris was removed by centrifugation.The methanol from both extractions was combined and dried under nitrogen flow, then resuspended in the following volumes of 80% methanol in water: rest of the body, 50 µl; MAGs and female LRT, 30 µl.
Samples were analyzed on a mass spectrometer (ID-X, Thermo Fisher) coupled to an LC instrument (Vanquish, Thermo Fisher).5 µl of sample was injected onto a 3 µm, 100 × 4.6 mm column (Inspire C8, Dikma) maintained at 25 °C.The mobile phases for the LC were A (water, 0.1% formic acid) and B (acetonitrile, 0.1% formic acid).The LC gradient was as follows: 5% B for 1 minute, then increased to 100% B over 11 minutes.After 8 minutes at 100%, re-equilibrate the column at 5% B for 4 minutes.The flow rate was 0.3 ml min-1.Ionization in the MS source is accomplished by heated electrospray ionization in positive and negative modes.
The mass spectrometer measures data in the m/z range from 350 to 680 at 60,000 resolution in full MS mode.MS/MS data were acquired on [M + H]+ (all targets), [M - H2O + H]+ (all targets), and [M - H]- (phosphorylated targets).MS/MS data were used to confirm the ecdysone properties of targets for which no standard was available.To identify non-targeted ecdysteroids, MS/MS data for all HPLC peaks with >15% relative abundance were analyzed.Quantify using standard curves created from pure standards (20E, 3D20E) to calculate absolute amounts or dilutions of one specific sample (all other targets) to calculate their equivalence to the amounts found in one male.For 3D20E, quantification was performed using the sum of the following adducts: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-.Data were extracted and quantified using Tracefinder (version 4.1).MS/MS data were analyzed using Xcalibur (version 4.4).The MS spectra of E, 20E and 3D20E were compared to the respective standards.3D20E22P was analyzed by derivatization with Girard’s reagent.20E22P was analyzed by m/z ratio.
3D20E22P was purified from MAG.Purification was performed on an analytical scale using an ultra-performance liquid chromatograph (Acquity, Waters) with a quadrupole mass-based detector (QDa, Acquity, Waters) under the same LC conditions as the HPLC-MS/MS analysis.Fraction collection was triggered when the m/z corresponding to 3D20E22P was detected at the same retention time as previously determined.The purity of the extracted compounds was then checked by HPLC-MS/MS as described above.
Total RNA was extracted from 10-12 reproductive tissues or other parts of the body (headless) using TRI reagent (Thermo Fisher) following the manufacturer’s instructions.RNA was treated with TURBO DNase (Thermo Fisher).cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) following the manufacturer’s instructions.Primers for reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) were previously published24 or designed using Primer-BLAST26, with preference given to products of 70-150 bp in size and spanning exon-exon junctions or Primer pair primers separate exons.cDNA samples from three to four biological replicates were diluted four-fold in water for RT-qPCR.Quantification was performed in 15 µl replicate reactions containing 1× PowerUp SYBR Green Master Mix (Thermo Fisher), primers, and 5 µl of diluted cDNA.Reactions were run on a QuantStudio 6 Pro real-time PCR system (Thermo Fisher) and data were collected and analyzed using Design and Analysis (version 2.4.3).As demonstrated in this study, relative amounts were normalized to the ribosomal gene RpL19 (AGAP004422), whose expression did not change significantly with blood feeding 27 or mating 3 .
RNA quality was checked using an Agilent Bioanalyzer 2100 Bioanalyzer (Agilent).Illumina paired-end libraries were prepared and run at the Broad Institute of MIT and Harvard.Sequencing reads were aligned to the A. gambiae genome (PEST strain, version 4.12) using HISAT2 (version 2.0.5) with default parameters.Reads with mapping quality (MAPQ) scores <30 were removed using Samtools (version 1.3.1).The number of reads mapped to genes was counted using htseq-count (version 0.9.1) with default parameters.Normalized read counts were calculated and differential gene expression analyzed using the DESeq2 package (version 1.28.1) in R (version 4.0.3).
Ecdysone-modifying gene candidates were identified by first searching the A. gambiae genome using the PSI-BLAST algorithm (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), using default values Parameters with the following query protein sequences: from Bombyx mori (Accession No. NP_001038956.1), Musca domestica (Accession No. XP_005182020.1, XP_005175332.1 and XP_011294434.1) and Microplitis demolitor (Accession No. XP_008552646.1 and XP_008552645.1) EcK from B. mori (Accession No. NP_001036900), Drosophila melanogaster (Accession No. NP_651202), Apis mellifera (Accession No. XP_394838) and Acyrthosiphon pisum (Accession No. XP_001947166); and EPP from B. mori (Accession No. XP_001947166) NP_001177919.1 and NP_001243996.1) and EO of D. melanogaster (Accession No. NP_572986.1) (step 1).Next, filter hits based on high mRNA expression (>100 fragments/kilobase exons per million mapped reads (FPKM) or >85%) in reproductive tissue (female LRT or MAG) in Gambia (step 2 ). To improve specificity, we selected candidate enzymes that are also expressed in the reproductive tissue of A. albimanus, an anopheles species that does not synthesize or transfer ecdysone during mating.Candidate genes were filtered based on low expression (<100 FPKM or <85th percentile) in A. albimanus reproductive tissue (step 3).As a final filter (step 4), candidate genes need to satisfy at least one of the following: (1) significantly up-regulated after mating (P < 0.05) according to the analysis of differentially expressed genes and (2) in non-reproductive tissues (< 85 % or <100 FPKM).
We modified previously described methods 28,29,30 to achieve whole-organism isotopic labeling.Briefly, wild-type Saccharomyces cerevisiae type II (YSC2, Sigma) was tested in yeast nitrogen base (BD Difco, DF0335) containing (wt/vol) 2% glucose (G7528, Sigma), 1.7% amino acid-free and ammonium sulfate. culture medium) and 5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) as the sole source of nitrogen.Yeast was recovered by centrifugation and mosquito larvae were fed ad libitum until pupation.Supplement with fishmeal (0.5 mg per 300 larvae) to prevent fourth instar mortality.Only females were then used in mating experiments with unlabeled males to analyze the male proteome transferred during mating.
4-6 day old 15N-tagged virgin females were forced to mate with age-matched untagged virgin males.Successful mating was verified by detecting mating plugs under epifluorescence microscopy.At 3, 12, and 24 hpm, the atria of 45-55 mated females were dissected into 50 µl of ammonium bicarbonate buffer (pH 7.8) and homogenized with a pestle.The homogenate was centrifuged and the supernatant mixed with 50 µl of 0.1% RapiGest (186001860, Waters) in 50 mM ammonium bicarbonate.The supernatant and pellet from each sample were snap frozen on dry ice and shipped overnight to the MacCoss laboratory at the University of Washington, where sample preparation for LC-MS/MS was completed.Resuspend the pellet in 50 µl of 0.1% RapiGest in 50 mM ammonium bicarbonate and sonicate in a water bath.The protein concentration of the pellet and supernatant was measured by the BCA assay, samples were reduced with 5 mM dithiothreitol (DTT; Sigma), alkylated with 15 mM iodoacetamide (Sigma) and incubated at 37 °C (1:0 50) for 1 hour with trypsinization: trypsin:substrate ratio).RapiGest was lysed by the addition of 200 mM HCl, followed by incubation at 37 °C for 45 minutes and centrifugation at 14,000 rpm for 10 minutes at 4 °C to remove debris.Samples were washed by dual-mode solid-phase extraction (Oasis MCX cartridges, Waters) and resuspended in 0.1% formic acid for a final protein concentration of 0.33 µg µl-1.Unlabeled MAG proteomes were similarly analyzed from virgin males.Two analytical replicates were analyzed for each sample.Next, 1 µg of each was analyzed using a 25-cm fused silica 75-μm column with a 4-cm fused silica Kasil1 (PQ) frit trap packed with Jupiter C12 reversed-phase resin (Phenomenex) and 180-minute liquid chromatography. Sample digests – MS/MS was run on a Q-Exactive HF mass spectrometer (Thermo Fisher) with a nanoACQUITY UPLC System (Waters).Data-related acquisition data generated for each run were converted to mzML format using Proteowizard (version 3.0.20287) and using Comet31 (version 3.2) against the FASTA database containing protein sequences from Anopheles gambiae (VectorBase version 54), Anopheles coluzzi A search was performed on Mali-NIH (VectorBase version 54), Saccharomyces cerevisiae (Uniprot, March 2021), A. gambiae RNA-seq, and three-frame translations of known human contaminants.Peptide map-matched FDRs were determined using Percolator32 (version 3.05) with a threshold of 0.01, and peptides were assembled into protein identifications using protein parsimony in Limelight33 (version 2.2.0).Relative protein abundance was estimated using the normalized spectral abundance factor (NSAF) calculated for each protein in each run as previously described.NSAF relative to each protein was averaged across samples from two different biological replicates.15N labeling successfully masked the female proteome, although a small amount of unlabeled protein could be detected from the labeled virgins.We recorded detection of male protein reduction (1-5 spectra) in female raw samples only in technical runs, where raw samples were run after male/mating samples, as a result of HPLC “carry over”.Occasional proteins found as ‘contaminants’ from labeled virgins are listed in Supplementary Table 1.
Two antigenic peptides, QTTDRVAPAPDQQQ (within isotype PA) and MESDGTTPSGDSEQ (within isotype PA and PB) in Genscript.The two peptides were combined, then conjugated to the carrier protein KLH and injected into New Zealand rabbits.The rabbits were sacrificed after the fourth injection, and total IgG was isolated by affinity purification.IgG from the most EPP-specific rabbit was used for further western blotting.
For western blots, MAG (n = 10, where n represents the number of biologically independent mosquito samples) and female LRT (n = 30) from 4-day-old virgin males and virgin or force-mated females (<10 post-mating), Protein extraction buffer (50 mM Tris, pH 8.0; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1× protease inhibitor cocktail (Roche)) was added separately.Samples were homogenized immediately after dissection with a beader (2 mm glass beads, 2,400 rpm, 90 sec).Insoluble debris was removed by centrifugation at 20,000 g at 4 °C.Proteins were quantified by Bradford assay (Bio-Rad).Then, 20 µg of MAG protein, 40 µg of LRT protein, and 20 µg of residual bulk protein were denatured and separated by 10% Bis-Tris NuPAGE using MOPS buffer.Proteins were transferred to polyvinylidene fluoride membranes using the iBlot2 transfer system (Thermo Fisher).Membranes were washed twice in 1× PBS-T (0.1% Tween-20 in PBS) and then blocked in Odyssey blocking buffer (Li-Cor) for 1 hour at 22°C.Membranes were shaken overnight at 4 °C with custom rabbit anti-EPP polyclonal primary antibody (1:700 in blocking buffer) and rat anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000).Membranes were washed with PBS-T and then incubated with secondary antibodies (donkey anti-rabbit 800CW and goat anti-rat 680LT (Li-Cor), both 1:20,000) in blocking buffer containing 0.01% SDS and 0.2% Tween -20 for 1 hour at 22 °C.Membranes were washed with PBS-T and imaged with an Odyssey CLx scanner.Images were collected and processed in Image Studio (version 5.2).A specific band corresponding to the EPP-RA isoform (82 kDa) was not detected.
The coding regions of EPP (as isoform AGAP002463-RB containing histidine phosphatase domain, NCBI conserved domain search 34) and EcK2 (AGAP002181) were cloned into pET-21a(+) plasmid (Novagen Millipore Sigma) ; primers are listed in Extended Data Table 2.Eight GS4 linkers (in tandem) were inserted before the C-terminal 6xHis tag of the pET-21a(+)-EcK2 construct.Recombinant proteins were produced using the NEBExpress cell-free E. coli protein synthesis reaction (New England BioLabs).Recombinant proteins were purified using NEBExpress Ni spin columns (New England BioLabs).Dihydrofolate reductase (DHFR) control protein was produced using DNA template from the NEBExpress Cell-Free E. coli Protein Synthesis Kit.Proteins were stored in 50% glycerol in PBS at -20 °C for up to 3 months.
Phosphatase activity of EPP and tissue extracts was measured using 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich).The reaction buffer contained 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, and 1 mM DTT.Tissue was homogenized in reaction buffer and cell debris was removed by centrifugation.Initiate the reaction by adding enzyme or tissue extract to reaction buffer containing 2.5 mg ml-1 pNPP.The reaction mixture was incubated at room temperature in the dark, and the amount of pNP converted from pNPP was quantified by measuring the absorbance at 405 nm at various times.
For in vitro EcK activity, the protein was incubated with 0.2 mg 20E or 3D20E in 200 µl buffer (pH 7.5) containing 10 mM HEPES–NaOH, 0.1% BSA, 2 mM ATP and 10 mM MgCl2 for 2 h at 27 °C .The reaction was stopped by adding 800 µl methanol, then cooled at -20 °C for 1 hour, then centrifuged at 20,000 g for 10 minutes at 4 °C.The supernatant was then analyzed by HPLC-MS/MS.To heat-inactivate the proteins used in the control group, the proteins were incubated in 50% glycerol in PBS for 20 min at 95°C.
For in vitro EPP activity, the protein was incubated with 3D20E22P (equivalent to the amount found in 18 pairs of MAG, purified by HPLC-MS/MS) in 100 µl buffer (pH 7.5) containing 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, and 1 mM DTT for 3 hours at 27 °C.The reaction was stopped by adding 400 µl methanol and cooled at -20 °C for 1 hour, then centrifuged at 20,000 g for 10 minutes at 4 °C.The supernatant was analyzed by HPLC-MS/MS.
PCR fragments for EPP (362 bp), EcK1 (AGAP004574, 365 bp) and EcK2 (556 bp) were amplified from cDNA prepared from mixed-sex headless mosquito cadavers.The PCR fragment of the eGFP control (495 bp) was amplified from the previously described pCR2.1-eGFP; PCR primers are listed in Extended Data Table 2.The PCR fragment was inserted between the inverted T7 promoters on the pL4440 plasmid.Plasmid constructs were recovered from NEB 5-α competent E. coli (New England Biolabs) and verified by DNA sequencing before use (see Supplementary Data 1 for insert sequence).Primers matched to the T7 promoter (Extended Data Table 2) were used to amplify the insert from the pL4440-based plasmid.PCR product size was confirmed by agarose gel electrophoresis.dsRNA was transcribed from PCR templates using the Megascript T7 Transcription Kit (Thermo Fisher) and purified according to the manufacturer’s instructions with the modifications previously described.
For dsRNA injection, 1,380 ng of dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) was injected at a concentration of 10 ng nl-1 into the thorax of adult males or females (Nanoject III, Drummond) within 1 day after eclosion.Gene knockdown levels were determined in at least three biological replicates by RNA extraction, cDNA synthesis, and RT-qPCR.For ecdysone injection, 4-day-old virgin or 6-day-old virgin blood-fed females were injected with 0.13, 0.21, or 0.63 µg of 20E or 3D20E (Nanoject III, Drummond) at concentrations of 1.3, 2.1, respectively, depending on the experimental design or 6.3 ng nl-1.Inject 100 nl of 10% (vol/vol) ethanol in water; 100 nl of 3D20E22P in 10% ethanol (equivalent to 75% of the amount found in a pair of MAGs).Mosquitoes were randomly assigned to the injection group.
For spawning assays, 3-day-old females were fed ad libitum on human blood.Remove partially fed or unfed mosquitoes.Depending on the treatment, females were placed in separate spawning cups for four nights at least 48 hours after the blood meal.Eggs were counted under a stereoscope (Stemi 508, Zeiss); for mated females, eggs that hatched into larvae were considered fertile.
For mating trials, females were allowed at least 2 days depending on the treatment to develop resistance to mating, and wild-type age-matched males were subsequently introduced into the same cage.Two nights later, the female fertilized vesicles were dissected and genomic DNA was released by freeze-thaw and sonication in a buffer containing 10 mM Tris-HCl, 1 mM EDTA, and 25 mM NaCl (pH 8.2).Samples were incubated with Proteinase K (0.86 µg µl-1) for 15 minutes at 55 °C, followed by 10 minutes at 95 °C.Crude genomic DNA preparations were diluted 10-fold and subjected to qPCR detection of Y chromosome sequences; primers are listed in Extended Data Table 2.Absence of Y chromosome sequence indicates no mating.
For re-mating assays, force-mated females were examined for the presence of mating plugs to confirm mating status and allowed 2 days to develop refractoriness to mating in the absence of males, as previously described 36 .Males carrying DsRed transgenic sperm were then introduced into female cages.Two nights later, the fertilizing vesicles were dissected from the females, and genomic DNA was prepared as described above and subjected to qPCR detection of the DsRed transgene; primers are listed in Extended Data Table 2.The absence of the DsRed transgene indicated that no re-mating occurred.
3D20E was synthesized as previously described 37 .Briefly, 10 mg of 20E (Sigma-Aldrich) was dissolved in 10 ml of water, followed by the addition of 30 mg of platinum black (in powder form, Sigma-Aldrich).A gentle stream of O2 was continuously bubbled into the reaction mixture, which was stirred at room temperature.After 6 hours, 30 mL of methanol was added to stop the reaction.The mixture was centrifuged to remove catalyst particles.The supernatant was evaporated to dryness in vacuo at room temperature.The dried reaction product was dissolved in 10% ethanol and methanol for injection for HPLC-MS/MS analysis.The conversion rate (from 20E to 3D20E) was approximately 97% (Fig. 4b), and the MS spectrum of the synthesized 3D20E matched that found in mated females (Fig. 4c).
The legend contains specific details of the statistical tests performed.GraphPad (version 9.0) was used to perform Fisher’s exact test, Mantel-Cox test, and Student’s t-test.Cochran-Mantel-Haenszel tests were performed using a custom R script (available at https://github.com/duopeng/mantelhaen.test).The data distribution was tested for normality using the Shapiro-Wilk test with a significance threshold of 0.05.When the data failed the normality test, the Mann-Whitney test was performed.Survival data were analyzed using the Mantel-Cox test.The DESeq2 package (version 1.28.1) was used to perform RNA-seq gene-level differential expression analysis.The horizontal bar on the graph represents the median.A significance value of P = 0.05 was used as the threshold for all tests.
For more information on the study design, see the Nature Research Report abstract linked to this article.
MS proteomic data were deposited into the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) through the PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) with the dataset identifier PXD032157.
The RNA-seq dataset is deposited in the Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) under the serial record GSE198665.
Additional datasets generated and/or analyzed during the current study may be obtained from the corresponding authors upon reasonable request.This article provides the source data.
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