Identification and Characterization of Small RNA Markers of Age in the Blow Fly Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae)

SIMPLE SUMMARY: Immature blow fly development information is critical for temporal estimations in death investigations. While there are phenotypic markers of development, they often do not provide adequate precision and resolution. In order to ameliorate these issues, we have investigated microRNAs as putative markers of immature development in the forensically relevant blow fly, Cochliomyia macellaria. Using RNA sequencing, we were able to identify miRNAs present in immature developmental stages, as well as identify miRNA which were significantly differentially expressed. Through these analyses, we found likely markers of development time, some of which were validated with qPCR methods. As a follow-up, we investigated factors which may influence expression of miRNA throughout development, such as selected genotypic variation and sex. It was very clear that genetic and abiotic factors can impact predictions of age, some of which may exhibit interactions. It is important that future work investigate the markers most robust to genetic effects on development. ABSTRACT: Blow fly development is important in decomposition ecology, agriculture, and forensics. Much of the impact of these species is from immature samples, thus knowledge of their development is important to enhance or ameliorate their effects. One application of this information is the estimation of immature insect age to provide temporal information for death investigations. While traditional markers of age such as stage and size are generally accurate, they lack precision in later developmental stages. We used miRNA sequencing to measure miRNA expression, throughout development, of the secondary screwworm, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae) and identified 217 miRNAs present across the samples. Ten were identified to be significantly differentially expressed in larval samples and seventeen were found to be significantly differentially expressed in intrapuparial samples. Twenty-eight miRNAs were identified to be differentially expressed between sexes. Expression patterns of two miRNAs, miR-92b and bantam, were qPCR-validated in intrapuparial samples; these and likely food-derived miRNAs appear to be stable markers of age in C. macellaria. Our results support the use of miRNAs for developmental markers of age and suggest further investigations across species and under a range of abiotic and biotic conditions.