Shao Shipeng, Hu Gengwang, Li Liangliang, Sheng Yi, Wang Yinghui, Zhang Yanan, Guo Yi, Kang Chengtao, Xu Wang, Chen Jie, Wang Jiangfeng, Wang Yu
Department of Forensic Medicine, Soochow University, Ganjiang East Road, Suzhou, China.
Clinical Experimental Center, Xi'an International Medical Center Hospital, Xi'an, Shaanxi 710100, China.
Acta Trop. 2023 Jun;242:106910. doi: 10.1016/j.actatropica.2023.106910. Epub 2023 Mar 23.
Hydrotaea spinigera (Stein, 1910) (Diptera: Muscidae) is a forensically important sarcosaprophagous species widely distributed throughout the Oriental and Australasian regions. At the advanced decomposition stage or the skeletonize stage, the immature stages of H. spinigera, especially the pupae, can still be found in large quantities and could be used as important indicators to estimate the minimum postmortem interval (PMI). However, there have been no studies on the intra-puparial period of this species. Herein, we studied morphological and differential gene expression changes during the intra-puparial development of H. spinigera, aiming to accurately estimate the intra-puparial age of H. spinigera. The intra-puparial morphological changes of H. spinigera were observed at seven constant temperatures ranging from 16 °C to 34 °C and divided into 12 sub-stages. Structures that could be used to estimate the intra-puparial age, such as compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, were observed in detail, and the developmental process of each structure was divided into 5 to 10 stages. The time range of each sub-stage, or when a structure appeared, was recorded. For the gene expression section, the most suitable reference genes were screened by geNorm, NormFinder, BestKeeper, and ΔCt methods. Based on the selected reference genes, real-time quantitative PCR (RT qPCR) was used to detect the expression changes of the white and hsp90 genes with developmental time at 19 °C, 25 °C, and 31 °C. Results showed that the trend of hsp90 gene expression under different temperatures was not consistent, while white genes exhibited regular changes during development, and could thus be used for age estimation of H. spinigera. This study provides an important basis for forensic entomology to use morphological and differential gene expression for estimating the age of H. spinigera during the intra-puparial period. Moreover, the combination of the two methods can produce a more accurate minimum postmortem interval (PMI) estimate compared to when each method is used separately.
刺鬃黑蝇（Hydrotaea spinigera (Stein, 1910)）（双翅目：蝇科）是一种在法医学上具有重要意义的尸食性物种，广泛分布于东洋区和澳大拉西亚地区。在尸体分解的后期或白骨化阶段，仍能大量发现刺鬃黑蝇的未成熟阶段，尤其是蛹，它们可作为估计最短死后间隔时间（PMI）的重要指标。然而，目前尚未有关于该物种蛹期的研究。在此，我们研究了刺鬃黑蝇蛹期发育过程中的形态变化和差异基因表达，旨在准确估计其蛹期年龄。在16℃至34℃的七个恒定温度下观察了刺鬃黑蝇蛹期的形态变化，并将其分为12个亚阶段。详细观察了可用于估计蛹期年龄的结构，如复眼、口器、触角、胸部、腿部、翅膀和腹部，并将每个结构的发育过程分为5至10个阶段。记录了每个亚阶段的时间范围或结构出现的时间。对于基因表达部分，通过geNorm、NormFinder、BestKeeper和ΔCt方法筛选出最合适的内参基因。基于选定的内参基因，采用实时定量PCR（RT qPCR）检测19℃、25℃和31℃下白色基因和热休克蛋白90（hsp90）基因随发育时间的表达变化。结果表明，不同温度下hsp90基因的表达趋势不一致，而白色基因在发育过程中呈现出规律变化，因此可用于刺鬃黑蝇的年龄估计。本研究为法医昆虫学利用形态学和差异基因表达来估计刺鬃黑蝇蛹期年龄提供了重要依据。此外，与单独使用每种方法相比，两种方法结合使用可以更准确地估计最短死后间隔时间（PMI）。
