Repeatability and sources of variation of the bacteria‐killing assay in the common snapping turtle

Abstract

Research on reptile ecoimmunology lags behind that on other vertebrates, despite the importance of such studies for conservation and evolution. Because the innate immune system is highly conserved across vertebrate lineages, assessments of its performance may be particularly useful in reptiles. The bacteria-killing assay requires a single, small blood sample and quantifies an individual's ability to kill microorganisms. The assay's construct validity and interpretability make it an attractive measure of innate immunity, but it requires proper optimization and sample storage. We optimized this assay for the common snapping turtle (Chelydra serpentina) to assess the repeatability of the assay and the effects of freezing and thawing on bactericidal capacity. We determined whether age (adult female and hatchlings) or incubation temperature influenced bactericidal capacity. We found that the assay was repeatable and that freezing plasma samples for 6 weeks at -80°C did not decrease bactericidal capacity nor did a single 30-min thaw and subsequent refreezing. However, we detected subtle interassay variation and results from one assay were 5-6% greater than those from the other two. Adult females had significantly greater bactericidal ability than hatchlings and we found no relationship between incubation temperature and bactericidal capacity. This assay is a useful tool in snapping turtles and may have applicability in other reptiles. However, species-specific optimization is required to ensure that variation among individuals exceeds interassay variation. Consideration should be given to optimization conditions that facilitate comparisons between or within groups, particularly groups that differ considerably in bactericidal capacity.

Plasma antibacterial activities in ornate (Terrapene ornata) and eastern box turtles (Terrapene carolina)

“…However, heparin is the preferred anticoagulant for most chelonians, including box turtles, due to the occurrence of hemolysis in whole blood samples anticoagulated with EDTA (Heatley & Russell, 2010; Muro, Cuenca, Pastor, Vinas, & Lavin, 1998). Furthermore, many existing chelonian innate immune function studies (including those focused on complement activity) use plasma samples containing heparin at approximately 20 USP/ml (Baker, Kessler, Darville‐Bowleg, et al, 2019; Baker, Kessler, & Merchant, 2019; Beck et al, 2017; Cochran et al, 2018; Ferronato et al, 2009; Goessling et al, 2016; Goessling, Guyer, & Mendonça, 2017; Goessling, Koler, et al, 2017; Goessling et al, 2019; Haskins, Hamilton, Jones, et al, 2017; Haskins, Hamilton, Stacy, et al, 2017; Refsnider et al, 2015; Sandmeier et al, 2016, 2018). While our findings may be biased by the use of heparinized samples, they are comparable to the existing chelonian innate immune function literature, which also relies heavily upon this anticoagulant.…”

“…Plasma was pooled from an even sex ratio of adult turtles for each species immediately before use. Multiple freeze‐thaw cycles were avoided to reduce variability in experimental results (Beck, Thompson, & Hopkins, 2017). All animal procedures were approved by the University of Illinois Institutional Animal Use and Care Committee (Protocol 18000).…”

Immune configuration in hatchling snakes is affected by incubation moisture, and is linked to subsequent growth and survival in the field

“…The resultant variation in WBC count may have fitness consequences, as evidenced by links to subsequent growth rates and probabilities of recapture (even after adjusting for sex and body size effects). Although incubation temperatures can affect the immune systems of hatchling reptiles (Freedberg et al., ; Les et al., ), the effect is not ubiquitous (Beck, Thompson, & Hopkins, ; Paitz, Clairardin, Griffin, Holgersson, & Bowden, ). Thus, the methods of the current study need to be replicated in a broader array of taxa, in order to assess the generality of our results.…”

Introduction to ecoimmunology: An integrative approach

“…Avian species include the house wren ( Troglodytes aedon ), the golden eagle ( Aquila chrysaetos ), the song sparrow ( Melospiza melodia ), the European starling ( Sturnus vulgaris ), and the zebra finch ( Taeniopygia guttata ) (Bowers, Sakaluk, & Thompson, ; Grindstaff & Merrill, ; Kelly et al., ; MacColl et al., ; Pryor & Casto, ). Reptilian species represented in the current issue are the green anole ( Anolis carolinensis ), the gopher tortoise ( Gopherus polyphemus ), and the snapping turtle ( Chelydra serpentina ) (Beck, Thompson, & Hopkins, ; Goessling et al., ; Tylan & Langkilde, ). Finally, one mammalian species is also represented, the polar bear ( Ursus maritimus ) (Neuman‐Lee et al., ).…”