D3039: Characterizing the effects of trypsin on eupyrene sperm cell bundles from Danaus plexippus

Despite decades of conservation efforts, populations of the North American monarch butterfly (Danaus plexippus) continue to decline. Artificial insemination could help increase genetic diversity if reliable sperm storage protocols and quality assessments are developed. Sperm motility is a common indicator of sperm quality in many animals. In most lepidopterans, males produce two types of sperm: nucleate eupyrene sperm and anucleate parasperm. Although parasperm are more abundant, only eupyrene sperm carry genetic material and fertilize eggs. Eupyrene sperm are stored in bundles within the male seminal duplex and are later dissociated in the female’s spermatheca with the aid of secretions from the male accessory gland.

Previous work has shown that animal-derived trypsin can induce sperm motility in several moth species. This study investigates the effects of trypsin on eupyrene bundle dissociation in monarch butterflies. Eupyrene bundles were isolated from the seminal duplex of adult males and incubated in Grace’s insect medium with either control (more Grace’s), low, medium, or high concentrations of trypsin. Bundle dissociation was assessed immediately after and 1.5-3 hours after treatment. Higher concentrations of trypsin led to faster and more complete dissociation. Live/dead assays with fluorescent stains, Helixyte-14^TM and propidium iodide, showed that sperm cells remained viable for at least 24 hours following treatment. 

These findings demonstrate that trypsin can effectively dissociate eupyrene sperm bundles without compromising cell viability, offering a potential method for assessing sperm quality in monarch butterflies and supporting future reproductive and conservation applications.