Upregulation of Transferrin and Major Royal Jelly Proteins in the Spermathecal Fluid of Mated Honeybee (Apis mellifera) Queens

Simple Summary To understand the mechanisms underlying long-term storage and survival of sperm in honeybee Apis mellifera queens, previous studies have elucidated the components of honeybee spermathecal fluid. However, the expression profiles of transferrin (Tf) and major royal jelly proteins 1-9 (MRJPs 1-9) in the spermatheca and spermathecal fluid of mated honeybee queens have still not been characterized. In this study, we confirmed upregulation of Tf and MRJPs in the spermatheca and spermathecal fluid of mated honeybee queens by using RNA sequencing, reverse transcription-polymerase chain reaction, and Western blot analyses. The levels of Tf and antioxidant enzymes were elevated in the spermathecal fluid of the mated queens, paralleling the levels of reactive oxygen species, H2O2, and iron. The increased levels of MRJPs, especially MRJP1, MRJP4, and MRJP6, in the spermathecal fluid of mated queens may be responsible for energy provision during sperm storage in honeybee queens. Overall, our findings indicate that Tf and MRJPs are upregulated in the spermatheca and spermathecal fluid of mated honeybee queens, providing a novel insight into antioxidant defense and energy metabolism for stored sperm in honeybee queens. Sperm storage in the spermathecae of honeybee (Apis mellifera) queens is vital for reproduction of honeybees. However, the molecular mechanisms whereby queens store sperm in a viable state over prolonged periods in the spermatheca are not fully understood. Here, we conducted RNA sequencing analysis of the spermathecae in both virgin and mated A. mellifera queens 24 h after mating and observed that the genes encoding transferrin (Tf) and major royal jelly proteins (MRJPs) were differentially expressed in the spermathecae of mated queens. The concentrations of Tf and antioxidant proteins such as superoxide dismutase 1, catalase, and glutathione peroxidase as well as the levels of reactive oxygen species, H2O2, and iron were higher in the spermathecal fluid of the mated queens than in virgin queens. Tf upregulation is likely to perform a protective role against the Fenton reaction occurring between iron and H2O2 in the antioxidant pathway in the mated queen's spermathecal fluid. Furthermore, MRJPs-especially MRJP1, MRJP4, and MRJP6-were upregulated in the mated queen's spermathecal fluid, indicating that they may serve as antimicrobial and antioxidant agents as well as an energy source for stored sperm in the spermathecal fluid of honeybee quee