An Evaluation of Differentially Spliced Genes as Markers of Sex for Forensic Entomology,,

Abstract

Blow flies (Calliphoridae) are important medically and economically and are commonly used in forensics as temporal markers in death investigations. While phenotypic traits in adult flies can be sexually dimorphic, sex identification in immatures is difficult. Consequently, little is known about how sex may result in developmental disparities among sexes even though there are indications that they may be important in some instances. Since genetic mechanisms for sex are well studied in model flies and species of agricultural and medical importance, we exploit the sex‐specifically spliced genes transformer (tra) and doublesex (dsx) in the sex determination pathway to optimize a sex identification assay for immatures. Using known primer sets for tra and with a novel one for dsx, we develop PCR assays for identifying sex in four forensically relevant Calliphoridae species: Lucilia sericata (Meigen), Lucilia cuprina (Wiedemann), Cochliomyia macellaria (Fabricius), and Chrysomya rufifacies (Macquart) and evaluated their performance. Band detection rates were found to range from 71 to 100%, call rates ranged from 90 to 100%, and no error was found when bands could be called. Such information is informative for purposes of testimony and in preparation for development studies. The developed assays will assist in further differentiating sexually dimorphic differences in development of the Calliphoridae and aid in more accurately estimating insect age when age predictive markers (size, development time, molecular expression) are sexually dimorphic.

Sexual Dimorphism in Growth Rate and Gene Expression Throughout Immature Development in Wild Type Chrysomya rufifacies (Diptera: Calliphoridae) Macquart

“…The transcriptome was assembled with a program designed to preserve splicing information (Sze et al, 2017), as differential splicing of key sex-determination genes is responsible for many aspects of downstream sexual dimorphism. The error structure of sequences associated with this method have already been investigated, and this algorithm has been shown to be conservative and reliable (Sze et al, 2012(Sze et al, , 2017Pimsler et al, 2019); identifying dsx in C. rufifacies, which was confirmed by Sanger sequencing (Jonika et al, 2020), and recovering known sequences in several other model and nonmodel organisms. The assembly is fragmented, which may affect its usefulness for some purposes, but for the purpose of this project it was clearly sufficient for our needs.…”

“…Sex identification for each sample was performed as in Jonika et al (2020). Briefly, cDNA conversion was performed on original RNA extractions, as above, for PCR amplification of the sex specific DSX gene product.…”

“…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;Frątczak-Łagiewska and Matuszewski, 2018;Matuszewski and Fratczak-Łagiewska, 2018;Patrício Macedo et al, 2018). Concerns about sexual dimorphism in forensically important species have generally focused on organismal phenotypes; however, the same consideration must be made for molecular markers of fly age (e.g., gene expression), which are gaining traction as additional information that can (but may not always, see Smith and Cook, 2020) improve precision in insect age estimates for forensic purposes (Picard et al, 2013;Jonika et al, 2020). For any sexually dimorphic trait in forensic entomology, determining if the effect of sex is functionally important for predictions is important (see Smith and Cook, 2020).…”

“…Furthermore, as C. rufifacies has homomorphic sex chromosomes, maternally determined sex, and limited genetic data available, it is not currently possible to use karyotyping or genome-size for immature sex-identification (Ullerich, 1973(Ullerich, , 1975Picard et al, 2012;Andere et al, 2020). Given the atypical nature of the sex determination system, it is also unclear if expression of known sex determination factors will be useful (Scott et al, 2014;Andere et al, 2020), though preliminary work with doublesex has been promising (Sze et al, 2017;Jonika et al, 2020).…”

A review of Forensic Entomology literature in the southwestern United States

“…Genes that are expressed throughout development are associated with sex determination in C. rufifacies and the expression of genes biased toward females can be found in the eggs and adults, whereas male bias existed in pupal and larval stages of this fly (Pimsler, 2015). Specific markers for sex were identified in larvae of four species of blow flies ( L. sericata , L. cuprina , C. macellaria , and C. rufifacies ) by Jonika, Hjelmen, Faris, McGuane, and Tarone (2020), which could be useful for estimations of PMI. Pimsler et al (2019) identified genes associated with intraguild predator, C. rufifacies .…”