Evaluation of Reference Genes and Age Estimation of Forensically Useful Aldrichina grahami (Diptera: Calliphoridae) During Intrapuparial Period

Abstract

The minimum postmortem interval (PMImin) could be evaluated from the developmental stage of forensically important insects colonize a corpse, such as blow flies (Diptera: Calliphoridae). Unlike larvae, the developmental stage of which is well established according to their morphology, estimating the age of pupae is proven to be challenging. Recently, several studies reported the regulation of special genes during the development of blow fly pupae. However, gene regulation in Aldrichina grahami during the intrapuparial period remains to be studied. Therefore, we set out to investigate the mRNA levels of heat shock protein 23 (Hsp23), heat shock protein 24 (Hsp24), and 1_16 during the metamorphosis of A. grahami pupae. First, we examined seven candidate reference genes (ribosomal protein 49 (RP49), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), beta-tubulin at 56D (β-tubulin), Ribosomal protein L23 (RPL23), glutathione S-transferase (GST1), and Actin. Three widely used algorithms (NormFinder, BestKeeper, and geNorm) were applied to evaluate the mRNA levels of reference gene candidates in puparium at three stable temperatures (15, 22, and 27°C). Next, mRNA expression of Hsp23, Hsp24, and 1_16 during A. grahami metamorphosis was examined. We demonstrated that mRNA expression levels of Hsp23, Hsp24, and 1_16 showed time-specific regulation. In summary, our study identified three gene markers for the intrapuparial period of A. grahami and might provide a potential application in PMImin estimation.

Age-dependent gene expression of Calliphora vicina pupae (Diptera: Calliphoridae) at constant and fluctuating temperatures

“…The fold change values, ranging between approximately 0.0001 and 10,000, indicate high quantitative differences of gene expression. In contrast to previously described age markers, which only show fold change values of less than 15 [11][12][13][14][15][16][17], the analysed molecular age markers show a stronger change in gene expression during pupal development, which suggests a stronger predictive power.…”

“…Other studies investigated the age-dependent gene expression in each case 4 markers for Sarcophaga dux [12], Sarcophaga peregrina [11] and C. vicina [15][16][17]. Liu et al [14] analysed the gene expression of 3 markers for age estimation of Aldrichina grahami pupae. For age estimation of Lucilia sericata, 9 age-related markers were described [13].…”

“…But such landmarks are not easy to define and inhere a high degree of subjectivity [9,10]. To circumvent this, several molecular age markers for pupae of flesh flies [11,12] and blow flies [13][14][15][16][17] have been described. Molecular analysis provides quantitative and accurate gene expression data and can be standardised.…”

From Laboratory Studies to Court Evidence: Challenges in Forensic Entomology

“…This is demonstrated by the positive influence of mentors in stimulating the interest of medical students in a career in forensic pathology [79]. An article that discusses some of the challenges and rewards of a career as an expert witness in the forensic sciences concluded with a statement by Owen Jones, Professor of Law and Biological Sciences at the Vanderbilt Law School in Nashville, USA, saying, in effect, that legal systems will never be better informed than when those in science spend their time helping those systems advance along a more constructive and accurate path [80]. I agree strongly with that sentiment, but scientists, especially early-career researchers, need to be helped to do this in both their research and casework.…”

“…In addition to morphological observations, hemolymph soluble proteins [77], cuticular hydrocarbons [78], and differential gene expression [69,[79][80][81] have also been applied to estimate the age of immature stages. Cuticular hydrocarbons in fly puparium were found to significantly change with weathering time, indicating their potential use for PMI min estimation [82,83].…”