How to store a beetle larva? Comparing temporal effects of common fluid preservation methods on color, shape, and DNA quality.

Proper fixing and long-term preservation of entomological evidence are essential in collections and research and crucial in applied fields such as forensic entomology. Incorrectly stored samples may lose important morphological features over time, rendering molecular analyses exceedingly difficult. The most effective method for preserving soft samples such as larvae is fluid preservation. It uses a combination of a wide range of fixatives and storage fluids. However, very little comparative work has been done to determine the effects of long-term storage on sample quality in terms of color, shape, and DNA stability. Moreover, the current golden standard in forensic entomology has been tailored for age estimation of larvae of Diptera, which differ from larvae of Coleoptera in morphology and subsequently in applied methods. We compared the effects of combinations of 6 commonly used fixatives and 6 commonly used storage fluids on midsized larvae of the forensically important beetle, Necrodes littoralis (Linnaeus, 1758), in terms of color, shape, and suitability for DNA analyses over a 2-yr period. We were looking for combinations that can preserve specimens in a satisfactory state, can be used on a regular basis, do not require advanced protection or skills of the personnel, and are not toxic or too harmful to the environment. We found not only several methods that scored significantly better in the tested parameters compared with the golden standard but also several common methods that should be avoided. The effects of agents on each tested category are discussed in detail.