Received: 22 March 2020 | Revised: 27 September 2020 | Accepted: 29 September 2020 DOI: 10.1002/jez.2423 RESEARCH PAPER Plasma complement activation mechanisms differ in ornate (Terrapene ornata ornata) and eastern box turtles (Terrapene carolina carolina) Laura Adamovicz 1 | Sarah J. Baker 1,2 | Mark Merchant 3 | Lancia Darville 4 | Matthew C. Allender 1 1 Wildlife Epidemiology Laboratory, University of Illinois College of Veterinary Medicine, Urbana, Illinois, USA 2 Arizona Game and Fish Department, Phoenix, Arizona, USA 3 Department of Chemistry, College of Science and Engineering, McNeese State University, Lake Charles, Louisiana, USA 4 Moffitt Cancer Center and Research Institute, Tampa, Florida, USA Correspondence Laura Adamovicz, Wildlife Epidemiology Laboratory, University of Illinois College of Veterinary Medicine, 2001 S Lincoln Ave, Urbana, IL 61802, USA. Email: adamovi2@illinois.edu Funding information Illinois Department of Natural Resources, Grant/Award Number: State Wildlife Grant T‐104‐R‐1 Abstract Eastern (Terrapene carolina carolina) and ornate (Terrapene ornata ornata) box turtles have robust plasma antibacterial activity, however, the mechanism behind this ac- tivity is unknown. We used sheep red blood cell (SRBC) hemolysis assays, mannan‐ affinity chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), and matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐ TOF) to explore the mechanisms of complement activity in box turtles. Plasma from both species demonstrated volume, time, and temperature‐dependent SRBC he- molysis, with significantly greater hemolytic activity in ornate box turtle plasma. Hemolytic activity was highly attenuated following treatment with heat, EDTA, and salicylaldoxime in both species, but was unchanged after treatment with methyla- mine and ammonium hydroxide. Two abundant mannan‐binding proteins (presumed C‐type lectins) were identified in eastern box turtle plasma using SDS‐PAGE and MALDI‐TOF, but ornate box turtles did not express either protein. Eastern box turtles appear to rely on the lectin pathway of complement activation while ornate box turtles utilize the alternative pathway. This study provides further evidence that mechanisms underlying immune function are not always conserved between closely related species. This finding may have important implications for explaining species differences in susceptibility to emerging threats such as disease, toxicants, and climate change. KEYWORDS box turtle, complement, innate immunity, Terrapene, reptile 1 | INTRODUCTION The vertebrate immune system is divided into innate and acquired branches which prevent and combat microbial infection. The innate immune system is composed of diverse molecules, proteins, and cells which provide rapid and nonspecific protection against invading pathogens (Janeway et al., 2001). Major components of the innate immune system include physical and chemical barriers to pathogen entry, molecular pattern recognition receptors, leukocytes, lysozyme, antimicrobial peptides, the complement cascade, and physiologic responses such as inflammation and fever (Janeway et al., 2001; Rios & Zimmerman, 2015; Zimmerman et al., 2010, 2016). The comple- ment system is one of the most important constituents of innate immunity, comprising dozens of blood proteins that become se- quentially activated following recognition of a pathogenic organism (Janeway et al., 2001). This enzyme cascade promotes inflammation J Exp Zool. 2020;1–12. wileyonlinelibrary.com/journal/jez © 2020 Wiley Periodicals LLC | 1