Sarcophaga dux (Diptera: Sarcophagidae) is a necrophagous flesh fly species with potential forensic value for estimating minimum postmortem interval (PMImin). The basic developmental data and precise age estimates of the pupae are significant for PMImin estimation in forensic investigations. In the present study, we investigated the development data of that species at seven constant temperatures varying from 16 °C to 34 °C, including body length changes of the larve, developmental duration and accumulated degree hours of the preadults. Several reference genes for relative quantification of the differentially expressed genes (DEGs) were firstly selected and evaluated in the pupae of different ages under different temperatures. The DEGs of the insects during the pupal period at different constant temperatures (34, 25 and 16 °C) were further analyzed for more precise age estimation. The results showed that the developmental durations of the preadults at 16, 19, 22, 25, 28, 31 and 34 °C were 1478.6 ± 18.3 h, 726.1 ± 15.8 h, 538.5 ± 0.9 h, 394.1 ± 9.5 h, 375.6 ± 10.8 h, 284.1 ± 7.3 h, and 252.5 ± 6.1 h, respectively. The developmental threshold temperature the flies was 12.27 ± 0.35 °C, and the thermal summation constant was 5341.71 ± 249.29° hours. The most reliable reference genes during the pupal period at different temperatures were found: GST1 and 18S rRNA for the 34 °C group, GST1 and RPL49 for 25 °C, and 18S rRNA and 28S rRNA for 16 °C. The four differential expression genes (Hsp60, A-alpha, ARP, and RPL8) have the potential to be used for more precise age estimation of pupal S. dux. This work provides important basic developmental data and a more precise age estimation method for pupal S. dux, and improves the value of this species for PMImin estimation in forensic investigations.

中文翻译：

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Sarcophaga dux（双翅目：Sarcophagidae）在恒温条件下的发育和用于蛹年龄估计的差异基因表达

Sarcophaga dux（双翅目：Sarcophagidae）是一种食肉性肉蝇物种，具有估计最小死后间隔 (PMImin) 的潜在法医价值。蛹的基本发育数据和精确的年龄估计对于法医调查中的 PMImin 估计具有重要意义。在本研究中，我们调查了该物种在 16°C 至 34°C 的七个恒定温度下的发育数据，包括幼虫的体长变化、发育持续时间和前成体的累积度数。首先在不同温度下不同年龄的蛹中选择和评估差异表达基因（DEGs）的相对定量的几个参考基因。不同恒温条件下蛹期昆虫的DEGs(34, 25 和 16 °C) 进行了进一步分析，以进行更精确的年龄估计。结果表明，前成体在16、19、22、25、28、31和34℃的发育持续时间分别为1478.6±18.3h、726.1±15.8h、538.5±0.9h、394.1.6±10±9小时、284.1±7.3 小时和 252.5±6.1 小时。果蝇的发育阈温度为 12.27 ± 0.35 °C，热总和常数为 5341.71 ± 249.29° 小时。发现不同温度下蛹期最可靠的参考基因：34℃组为GST1和​​18S rRNA，25℃组为GST1和​​RPL49，16℃组为18S rRNA和28S rRNA。四种差异表达基因（Hsp60、A-alpha、ARP 和 RPL8）有可能用于更精确地估计蛹 S. dux 的年龄。