An Ectopic Expression Screen Reveals the Protective and Toxic Effects of Drosophila Seminal Fluid Proteins

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ABSTRACT

In Drosophila melanogaster, seminal fluid regulates the reproductive and immune responses of mated females. Some seminal fluid proteins may provide protective functions to mated females, such as antimicrobial activity and/or stimulation of antimicrobial gene expression levels, while others appear to have negative effects, contributing to a "cost of mating." To identify seminal proteins that could participate in these phenomena, we used a systemic ectopic expression screen to test the effects on unmated females of proteins normally produced by the male accessory gland (Acps). Of the 21 ectopically expressed Acps that we tested for ability to assist in the clearance of a bacterial infection with Serratia marcescens, 3 Acps significantly reduced the bacterial counts of infected females, suggesting a protective role. Of the 23 Acps that we tested for toxicity, 3 were toxic, including one that has been implicated in the cost of mating in another study. We also tested ectopic expression females for other Acp-induced effects, but found no additional Acps that affected egg laying or receptivity upon ectopic expression.

DURING mating in Drosophila melanogaster, seminal fluid proteins transferred to the female modulate her behavior, physiology, and reproduction. Approximately 70-106 seminal fluid accessory gland proteins (Acps) are produced in the male's accessory gland. Acps increase the egg-laying rate of mated females, decrease their receptivity to remating, induce them to store sperm, induce them to eat (CARVALHO et al. 2006), and decrease their life span (reviewed in KUBLI 2003; CHAPMAN and DAVIES 2004; WOLFNER et al. 2005). Acps also have the potential to provide immune-related functions to females: Acps upregulate antimicrobial gene transcript levels in mated females (McGRAW et al. 2004; PENG et al. 2005), and at least three Acps have antimicrobial activity in vitro (LUNG et al. 2001). Although these findings suggest that Acps might perform a protective role in improving a mated female's ability to clear an infection, the ability of Acps to affect a female's immune capacity has yet to be assessed directly in vivo.

The female reproductive tract is constantly in contact with the external environment, making it potentially susceptible to microbial infection. Transfer of protective Acps to females could lower a female's chance of infection, thus increasing her and her mate's reproductive success. Two extracellular protein classes play decisive roles in the innate immunity of mammals and insects: proteolysis regulators and antimicrobial peptides (reviewed in LECLERC and REICHHART 2004). For example, a D. melanogaster serine protease, spätzle-processing enzyme ( Jang et al. 2006), and a protease inhibitor, necrotic (LEVASHINA et al. 1999), regulate the proteolytic processing of a critical immunity protein, spätzle. Activation of spätzle, via its processing in the hemolymph, triggers the rapid upregulation of the expression of antifungal peptide genes (LEMAITRE et al. 1996). Of the 63 known D. melanogaster Acps (HOLLOWAY and BEGUN 2004; MUELLER et al. 2005; WALKER et al. 2006), 13 are predicted or known proteolysis regulators and 18 are peptides. Some have sequence/structural similarities with immunity proteolysis regulators and peptides (Swanson et al. 2001; LUNG et al. 2002; MUELLER et al. 2004).

Ectopic expression of antimicrobial peptides alone can combat a pathogenic infection (TZOU et al. 2002). We examined the immunity-promoting properties of 21 individual Acps (peptides and proteolysis regulators) for their ability to clear an infection of Serratia marcescens in females. We found that ectopic expression of three Acps with predicted proteolysis-regulating functions can assist in S. marcescens clearance.

Despite such potential beneficial effects of Acps on females, Acps have also been shown to have negative consequences, in particular by decreasing female viability postmating (CHAPMAN et al. …