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

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.

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

“…Differences in transcript size could lead to male and female forms experiencing different rates of degradation. Approaches using a single transcript size and smaller targets, such as small RNAs [24], may be more optimal choices for gene expression work in boiled larvae.…”

“…Now that a baseline of success has been shown using non‐quantitative gene expression, evaluating how quantitative gene expression is impacted by boiling should be assessed. Many articles evaluating gene expression in forensic entomology have focused on quantitative gene expression [6, 7, 24, 25], an approach that increases the number of suitable gene expression targets for insect aging beyond the presence/absence approach used here. For the current work, no attempt was made to evaluate RNA degradation levels, a factor that would impact quantitative gene expression methods more than the current presence/absence approach.…”