Abstract
Reliability of forensic entomology analyses to produce relevant information to a given case requires an understanding of the underlying arthropod population(s) of interest and the factors contributing to variability. Common traits for analyses are affected by a variety of genetic and environmental factors. One trait of interest in forensic investigations has been species-specific temperature-dependent growth rates. Recent work indicates sexual dimorphism may be important in the analysis of such traits and related genetic markers of age. However, studying sexual dimorphic patterns of gene expression throughout immature development in wild-type insects can be difficult due to a lack of genetic tools, and the limits of most sex-determination mechanisms. Chrysomya rufifacies, however, is a particularly tractable system to address these issues as it has a monogenic sex determination system, meaning females have only a single-sex of offspring throughout their life. Using modified breeding procedures (to ensure single-female egg clutches) and transcriptomics, we investigated sexual dimorphism in development rate and gene expression. Females develop slower than males (9 h difference from egg to eclosion respectively) even at 30°C, with an average egg-to-eclosion time of 225 h for males and 234 h for females. Given that many key genes rely on sex-specific splicing for the development and maintenance of sexually dimorphic traits, we used a transcriptomic approach to identify different expression of gene splice variants. We find that 98.4% of assembled nodes exhibited sex-specific, stage-specific, to sex-by-stage specific patterns of expression. However, the greatest signal in the expression data is differentiation by developmental stage, indicating that sexual dimorphism in gene expression during development may not be investigatively important and that markers of age may be relatively independent of sex. Subtle differences in these gene expression patterns can be detected as early as 4 h post-oviposition, and 12 of these nodes demonstrate homology with key Drosophila sex determination genes, providing clues regarding the distinct sex determination mechanism of C. rufifacies. Finally, we validated the transcriptome analyses through qPCR and have identified five genes that are developmentally informative within and between sexes.
Highlights
Accuracy and precision in forensic science often rely upon advanced statistical analyses requiring a solid understanding of both underlying statistical distributions, as well as the factors that affect variation of measures of interest (National Research Council, 2009)
Concerns about sexual dimorphism in forensically important species have generally focused on organismal phenotypes; the same consideration must be made for molecular markers of fly age, which are gaining traction as additional information that can improve precision in insect age estimates for forensic purposes (Picard et al, 2013; Jonika et al, 2020)
This study evaluated gene expression over immature C. rufifacies development, leveraging the distinct sex determination system to simultaneously evaluate sexual dimorphism in development and developmental gene expression
Summary
Accuracy and precision in forensic science often rely upon advanced statistical analyses requiring a solid understanding of both underlying statistical distributions, as well as the factors that affect variation of measures of interest (National Research Council, 2009) These concerns apply to insect growth rates in forensic entomology, the application of the scientific study of arthropods to legal investigations. Many fly species (the most important group of insects in forensic entomology) demonstrate dimorphism in both final adult size and/or development rate (Stillwell et al, 2010) Both of these factors may be important in the refinement and precision of insect age estimates (Blanckenhorn et al, 2007; Zuha and Omar, 2014; Blanckenhorn et al, 2015; FratczakŁagiewska and Matuszewski, 2018; Matuszewski and FratczakŁagiewska, 2018; Patrício Macedo et al, 2018). Some unknown proportion of genes used to predict fly age may be affected by sex, but they may be affected in ways that are relevant to fly age predictions or not
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