Characterization of Melanoplus sanguinipes oviposition stimulating protein expression and re-examination of its potential role in stimulating oviposition

• MsOSP transcription is restricted to the long hyaline tubule of the male accessory gland.
• MsOSP is a major component of the spermatophore packet but little is transmitted to the female.
• MsOSP, either recombinant or LHT derived, increases oviposition when injected into females.

Melanoplus sanguinipes oviposition stimulating protein (MsOSP) was characterized and its role in stimulating oviposition in virgin females was examined. A 967 nt MsOSP mRNA sequence with homology to previously characterized N-terminal amino acid sequence data for MsOSP was identified in a RNAseq library generated from an mRNA pool from the long hyaline tubule (LHT) of the male accessory gland complex. This transcript contained a predicted 729 nt open reading frame encoding the 242 aa putative MsOSP protein and had the second highest read abundance in the library. The MsOSP transcript was detected exclusively in the LHT tissue of adult males and its abundance increased with time until 7 days post-eclosion. Western blot analysis using an anti-MsOSP antibody showed high levels of MsOSP protein in the LHT luminal secretions of virgin males and to a lesser degree was associated with the aedeagus and ejaculatory duct. MsOSP was shown to be a major protein component of the spermatophore packet transferred from the male to female during copulation. However, only minor amounts of MsOSP could be detected in the female bursa, spermatheca and oviduct. Intrahemocoelic injection of LHT luminal protein into mature virgin females stimulated oviposition in ∼65% of females. A similar but non-significant trend was observed upon injection of purified recombinant MsOSP protein, and immunoprecipitation of LHT protein with anti-MsOSP antibody led to abrogation of oviposition stimulation upon injection of mature virgin females. Despite the demonstration of stimulation of oviposition upon intrahemocoelic injection of LHT-derived-MsOSP into mature virgin females, the potential mode of action of MsOSP in this process remains to be determined. MsOSP cannot be detected in the tissues other than the bursa, spermatheca and oviduct of female grasshoppers and relatively large quantities of MsOSP are required to stimulate oviposition upon injection.

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