Male accessory gland protein reduces egg laying in a simultaneous hermaphrodite

Abstract

Seminal fluid is an important part of the ejaculate of internally fertilizing animals. This fluid contains substances that nourish and activate sperm for successful fertilization. Additionally, it contains components that influence female physiology to further enhance fertilization success of the sperm donor, possibly beyond the recipient's optimum. Although evidence for such substances abounds, few studies have unraveled their identities, and focus has been exclusively on separate-sex species. We present the first detailed study into the seminal fluid composition of a hermaphrodite (Lymnaea stagnalis). Eight novel peptides and proteins were identified from the seminal-fluid-producing prostate gland and tested for effects on oviposition, hatching and consumption. The gene for the protein found to suppress egg mass production, Ovipostatin, was sequenced, thereby providing the first fully-characterized seminal fluid substance in a simultaneous hermaphrodite. Thus, seminal fluid peptides and proteins have evolved and can play a crucial role in sexual selection even when the sexes are combined.

Figure 1. Semen transfer in the hermaphroditic pond snail Lymnaea stagnalis.

A. The photo illustrates the typical mating position of this species. The top snail is performing the male role (sperm donor), its white preputium (penis-carrying organ, Pp) can be seen inserted under the shell of the sperm recipient, where the female opening is located. During insemination, sperm (from the seminal vesicles) and seminal fluids (from the prostate gland) are transferred. Since these are simultaneous hermaphrodites, sexual roles can be swapped immediately afterwards. B. Histological section of a prostate gland from L. stagnalis. The section illustrates the 5 different types of secretory cells present (numbered type 4 to 8; types 1 to 3 occur in the sperm duct) as well as the presence of secretions in the gland's central lumen through which the sperm pass on their way to the sperm recipient. This 7 µm section was stained with azan as well as hematoxylin and eosin.

Figure 2. Purification of Lymnaea stagnalis seminal fluid peptides and proteins.

A. C18 RP-HPLC profile of an extract of approximately 40 Lymnaea prostate glands that was purified on a Sep-Pak Vac cartridge and fractionated using a gradient of 0.1% HFBA and 100% ACN/0.1% HFBA. Subsequent microsequence analyses confirmed the presence of N-terminal sequences corresponding to the prominent peaks labeled with solid bars and numbers, while the white bars revealed no peptide presence. B. The inset shows the repurification of fraction 10 (Ovipostatin) using a gradient of 0.1% TFA and 100% ACN/0.1% TFA. Fractions containing Ovipostatin are indicated by the solid black bar.

Figure 3. Effect of Ovipostatin on egg mass production of Lymnaea stagnalis.

The graph shows the percentage of animals laying eggs after intravaginal injection of either the control substance (saline), Ovipostatin, or Ovipostatin accompanied with sperm. N = 15 for each treatment; see text for details and statistics.

Figure 4. Lymnaea stagnalis prostate gland Ovipostatin gene analysis.

A. Amino acid sequence of prostate gland Ovipostatin. The cDNA encodes a precursor protein of 167 amino acids. A putative glycosylation site is underlined and cysteines are in black shading. The predicted molecular mass of Ovipostatin is 18.9 kDa. B. Tissue expression of Ovipostatin mRNA in L. stagnalis. Transcript specific primers were used in RT-PCR to identify expression. Ovipostatin mRNA is present (631 bp) in the pooled central nervous system (CNS), foot tissue (Foot), penial complex (Pe), prostate gland (PG) and seminal vesicle (SV). No Ovipostatin mRNA was detected in the albumen gland (AG) or the control in which no cDNA template was used in PCR.