Establishing Age-Based Color Changes for the American Burying Beetle, Nicrophorus americanus Olivier, with Implications for Conservation Efforts

SIMPLE SUMMARY: The American burying beetle, Nicrophorus americanus, is federally protected as a threatened species because of population decline in most of its historic range in North America. Recovery of the species requires observation and cataloging of individuals captured in the wild. Previous work has characterized adult beetles as teneral (young) or senescent (old) but improving age determination of adults that survive approximately 90 days is critical to future recovery outcomes. As an adult, the beetle has distinctive orange/red markings over a black background. Because a demonstrable color difference was observed under laboratory conditions, we determined that a gradual, age-based color gradient exists and can be visually assessed to estimate the age of an individual beetle within 15-day intervals. We photographed adult N. americanus at set time intervals over their lifespans and generated a pixel-based approach to age adult beetles in a nondestructive manner. The orange/red color near the head became predictably darker as the beetles aged. A color chart was created that should allow conservation practitioners to assign beetles to six distinct age categories in the field without requiring additional equipment. This technique will refine estimates of population structure and improve the selection of individuals for breeding, potentially improving reintroduction program success. ABSTRACT: The American burying beetle, Nicrophorus americanus Olivier, is a federally protected insect that once occupied most of eastern North America. Adult beetles feature distinct, recognizable markings on the pronotum and elytra, and color changes with age have been observed. Among the challenges faced by research scientists and conservation practitioners is the ability to determine beetle age in the field between and including teneral (young) and senescent (old) adult stages. Using 20 (10 male and 10 female) captive-bred beetles, we characterized the change in greyscale and red, green, and blue (RGB) color channels over the lifespan of each beetle for field-aging applications. Individual beetles were photographed at set intervals from eclosion to death, and color data were extracted using open-source ImageJ Version 1.54f software. A series of linear mixed-effects models determined that red color showed the steepest decrease among all color channels in the pronotum and elytral markings, with a more significant decrease in the pronotum. The change in greyscale between the pronotum and elytral markings was visibly different, with more rapid darkening in the pronotum. The resulting pronotum color chart was tested under field conditions in Oklahoma, aging 299 adult N. americanus, and six age categories (day range) were discernable by eye: teneral (0–15), late teneral (15–31), early mature (31–45), mature (45–59), early senescent (59–76), and senescent (76–90). The ability to more precisely estimate age will improve population structure estimates, laboratory breeding programs, and potential reintroduction efforts.