Mating-increases trypsin in female Drosophila hemolymph

Area of Science:

• Reproductive Biology
• Insect Physiology
• Biochemistry

Background:

• Male accessory gland proteins (Acps) are transferred during mating, influencing female reproductive maturation and behavior.
• Some Acps enter the female hemolymph, suggesting humoral factors regulate their activity.
• Accessory gland proteins (Acps) are crucial for post-mating responses, but their regulation in the female body is not fully understood.

Purpose of the Study:

• To test the hypothesis that humoral proteases primarily mediate Acp bioactivity by processing or degrading them.
• To investigate the role of serine proteases in regulating the duration of Drosophila melanogaster Sex Peptide (DrmSP) activity in mated females.
• To elucidate the mechanism by which female hemolymph proteases affect male-derived protein function.

Main Methods:

• Kinetic analysis of synthetic DrmSP (T-SP) cleavage in hemolymph from unmated and mated female fruit flies.
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify DrmSP cleavage sites.
• In vitro verification using specific chromogenic serine-protease substrates and inhibitors.

Main Results:

• Synthetic DrmSP (T-SP) was cleaved more rapidly and completely in mated female hemolymph compared to unmated females.
• LC-MS/MS analysis identified trypsin as the major endopeptidase responsible for cleaving T-SP in female hemolymph.
• In vitro assays confirmed the role of serine proteases, specifically trypsin, in DrmSP degradation.

Conclusions:

• Post-mating cleavage of DrmSP in female hemolymph by serine proteases, particularly trypsin, regulates the duration of its bioactivity.
• This cleavage mechanism likely controls the persistence of post-mating responses in Drosophila melanogaster.
• Female hemolymph serine proteases represent a key regulatory system for male-derived accessory gland protein function.