Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster

Abstract

Mating elicits two major changes in the reproductive behavior of many insect females. The egg-laying rate increases and the readiness to accept males (receptivity) is reduced. These postmating responses last approximately 1 week in Drosophila melanogaster. Males that do not transfer sperm but transfer seminal fluid during mating induce a short-term response of 1 day. The long-term response of 1 week requires the presence of sperm (sperm effect). Hence, sperm is essential for the long-term persistence of the postmating responses. Three seminal fluid peptides elicit postmating responses: ovulin, sex-peptide (SP), and DUP99B. Using the technique of targeted mutagenesis by homologous recombination, we have produced males with mutant SP genes. Here, we report that males lacking functional SP elicit only a weak short-term response. However, these males do transfer sperm. Thus, (i) SP is the major agent eliciting the short-term and the long-term postmating responses and (ii) sperm is merely the carrier for SP. The second conclusion is supported by the finding that SP binds to sperm. The 36-aa-encoding SP gene is the first small Drosophila gene knocked out with the method of homologous recombination.

Fig. 1.

(A–D) Strategy for targeted knockout of SP gene (not drawn to scale). Only the essential steps are shown. Arrowheads show ends of P elements. Half-arrowheads show FRT sequence. Star indicates site of the introduced stop codon. Translation stops in the signal peptide (see below, E). (A) Targeting construct. Because SP is a small gene of ≈200 bp, we used the flanking sequences as donors (ends-in technique). Fragment length is in kilobases. (B) On activation with FLP recombinase, the targeting construct jumps out as a circle. It is then cut at the I-SceI cutting site and binds to the homologous sequence on a host chromosome. (C) Targeting at the SP⁺ gene produces tandem repeats containing an SP⁺ and an SP⁰ gene. P1–P5, primers. (D) By homologous recombination, the SP⁺ gene is deleted to obtain an SP⁰ null mutant. (E) Introduced mutations. SP⁺ and SP⁰, partial DNA sequences of the WT and the mutant SP genes, respectively. The sequence starts 5′ with the AUG encoding the first amino acid of the SP signal sequence. Changed bases in the mutant are bold. The newly introduced AvrII site is underlined. SP⁺ and SP⁰, amino acid sequences of SP translated from the WT and the mutant SP genes, respectively.

Fig. 2.

Verification of homologous targeting. PCR was done with the primers P1 and P2 as indicated in Fig. 1C. M, λ DNA digested with EcoRI and HindIII. Lanes yw, TA, TB, and TC show the fragments amplified from DNA of the yw stock and the targeted lines TA, TB, and TC, respectively. Lanes overlined with AvrII, amplified DNA of the before-mentioned lines cut with AvrII. The AvrII site was introduced at the mutagenesis step. The arrow indicates a 729-bp fragment specific for targeted lines after cutting the DNA with AvrII enzyme.

Fig. 3.

Western blots showing the expression patterns of SP, ovulin (ACP26Aa), and DUP99B in different stocks. The nature of the polyclonal antibody used as probes is indicated. The expected phenotype for SP (WT or SP⁰) is indicated at the top of the corresponding lanes. Ovulin and DUP99B are expressed independent of the presence or absence of the SP gene. The numbers indicate the nature or source of the samples: 1, synthetic SP (sSP); 2–6, genotypes of the accessory gland extracts; 2, SP⁺/SP⁺; 3, SP⁰/SP⁰; 4, SP⁰, SP⁺/SP^Δ; 5, SP⁺/SP^Δ; and 6, SP⁰, SP⁺/SP⁰. Arrows indicate SP, ovulin, and DUP99B, respectively.

Fig. 4.

Effects of SP on oviposition and receptivity. In contrast to females mated to WT males, females mated to males lacking SP drastically reduce oviposition and regain receptivity quickly. (A) Time course of average number of eggs laid per day by virgin females and females mated once to Oregon R WT, SP⁰/SP^Δ, and SP⁰, SP⁺/SP^Δ males, respectively. (B) Time course of receptivity of virgin females and females mated once to Oregon R WT males, SP⁰/SP^Δ, and SP⁰, SP⁺/SP^Δ males, respectively. SP⁰,SP⁺/SP^Δ males produce the same amount of SP as Oregon R WT males. SP⁰/SP^Δ males do not synthesize functional SP. All females were collected from a virginizer stock (VC). Standard errors are indicated.

Fig. 5.

Sperm is transferred by males lacking functional SP and stored for many days. Sections through female seminal receptacles 9 days after matings with an SP⁰, SP⁺/SP^Δ control male (A) and an SP⁰/SP^Δ male lacking SP (B), respectively. Whereas no sperm can be found in the section of the female mated with the control male, many sperm can be identified in the female mated with the male lacking SP. The DNA of the sperm heads was stained with 4′,6-diamidino-2-phenylindole. (Magnification: ×1,000; bar = 10 μm.) Arrowheads point to sperm nuclei. Females were collected from a virginizer stock (VC).