Both lipopolysaccharide and vehicle treatments upregulate complement-dependent and complement-independent innate immune responses in Diamond-backed Watersnakes (Squamata: Nerodia)

Introduction The innate immune response is complex and highly context dependent. In reptiles, relatively little information is available regarding how individual innate immune components interact and change over the course of an immune response. Methods We characterized innate immune responses of Diamond-backed Watersnakes ( Nerodia rhombifer ) over a 72-hour period after stimulation by a nonpathogenic antigen, lipopolysaccharide (LPS). Blood samples taken at predetermined time points were used to determine microbial killing ability of immune cells and proteins. Using two microbes that elicit unique responses (gram-negative Escherichia coli and gram-positive Staphylococcus aureus ), we explored the contribution of unique immune components through a series of microbial killing assays using fresh versus frozen-thawed serum and buffy layer (serum+BL). Results There was an effect of handling on the immune activity of the fresh serum+BL (leukocyte-dependent responses and proteins) as snakes across treatments showed increased response to E. coli and a decreased response to S. aureus . LPS-treated snakes had a stronger immune response overall to both E. coli and S. aureus when only proteins were measured. Discussion Our findings demonstrated the importance of including both vehicle saline injection and handling-only control groups in manipulative studies as the patterns for these two groups differed. This study describes the response of multiple metrics of innate immunity to different immune challenges over a 72-hour period, which provides novel insight into the immune response of a poorly understood taxon. Overall, this study provides evidence that non-antigenic saline vehicle injections stimulate an immune response and that leukocyte-dependent and complement responses are time-specific following an immune challenge.