Effects of Sample Storage Conditions and Individual Characteristics on Innate Immune Assays in Box Turtles.

Area of Science:

• Wildlife immunology
• Conservation physiology
• Ecology and evolution

Background:

• Immune assays are vital for wildlife research but face challenges with non-model organisms and storage effects.
• Validating immune assays across diverse species and understanding storage impacts are crucial for reliable data.

Purpose of the Study:

• To assess the impact of plasma storage (refrigerated vs. frozen) on bacterial killing assay (BKA) and hemagglutination results in eastern box turtles.
• To investigate the influence of individual identity, sex, and environmental factors on immune assay outcomes.

Main Methods:

• Evaluated plasma samples from eastern box turtles stored under refrigeration and freezing conditions.
• Conducted bacterial killing assays (BKA) and hemagglutination assays to measure immune responses.
• Analyzed repeatability and differences in immune measures based on storage, sex, and individual variation.

Main Results:

• Freezing plasma for 3-17 days yielded more repeatable BKA results than refrigeration, without altering average performance.
• BKA performance decreased after 3-4 months of frozen storage; hemagglutination showed no significant storage effect.
• Males exhibited higher hemagglutination but lower BKA than females, with repeatable individual immune differences observed.

Conclusions:

• Freezing plasma samples for up to two weeks is appropriate for BKA and hemagglutination in eastern box turtles, potentially applicable to related species.
• Individual and sex differences significantly influence immune assay results, necessitating consideration in future wildlife immunology studies.
• Standardizing sample handling protocols is essential for robust immune assessments in wild populations.