Evaluating the impact of hot water killing larvae on gene expression using the transformer gene in Cochliomyia macellaria (Diptera: Calliphoridae)

In forensic entomology, determining the age of a larva from a body to estimate time since death is commonly performed through the measurement of a physical trait. Gene expression has been studied as an alternative age estimation approach, but the storage conditions required for these studies are different than those used in forensic entomological casework. Studies analyzing gene expression prioritize the preservation of RNA, which requires fresh tissue and ultra-cold storage. Casework, in contrast, utilizes hot water killing specimens that may not be analyzed for a long period after collection. In the current study, the impact of hot water killing on gene expression was assessed for larval samples of the forensically important blow fly, Cochliomyia macellaria. Successful amplification of the sex-determining gene, transformer, was tested across larvae ranging in size from 3.22 to 16.85 mm in length after storage times of 1–2 weeks, 4–5 weeks, and 8–9 weeks at 4°C in RNAlater. Larvae hot water killed were processed in tandem with larvae stored live to allow for a direct assessment of the impact of boiling on gene expression. As expected, the transformer gene was successfully amplified in all larvae stored live. For the hot water-killed larvae, the success rate was only slightly lower, with 3 out of 75 larvae not generating a sex-specific band pattern. The results show gene expression can be used for hot water-killed samples, though future work across different genes, species, and extending to quantitative gene expression methods is needed.