- Conclusions: This study identified functional genes and showed differences in the expression pattern of diverse temperatures. - Forensic entomology, which explores the succession pat- tern and developmental stages of insects found on the decomposed cadavers, has been increasingly recognized as an important tool in the medico-legal discipline [1].. - time of the immature stages of necrophagous flies (eggs, larvae, pupae) has been a good indicator in determining the minimum PMI (PMI min ) [2, 3]. - makes it active in the cold winter when other necropha- gous flies almost inactive [11–13]. - In the present study, we used RNA-seq technique to identify the cold tolerance related genes by building tran- scriptomes profile of eggs, and larvae (1st instar to 3rd in- star) under three different temperatures (4 °C, 12 °C and 20 °C). - DEGs involved in the three different temperatures In a set condition (p-value <. - In the egg stage, there were DEGs in the comparison of L0 vs. - In first-instar genes were found to be DEGs in the compari- son of L1 vs M1, M1 vs H1. - In the second-instar and third-instar larvae stage, thousands of DEGs were also obtained in a similar comparison (Fig. - In egg stage, plenty of DEGs were screened in the two of the three comparisons of L0 vs M0, M0 vs H0, L0 vs H0. - In first-instar, 440 DEGs were found to be an intersection in the two of the three comparisons of L1 vs M1, M1 vs H1. - In the second-instar and third-instar larvae stage, 927 and 530 DEGs were also obtained in a similar com- parison (Fig. - 3e) were the intersection in the two of these four comparisons, suggesting to be the most import- ant for cold tolerance. - Hence, DEGs of intersection in the two of these three comparisons were also worth studying.. - Series-cluster analysis and functional annotation of the clusters. - In the egg libraries (0 libraries), a total of 13,356 genes were found to be DEGs. - There were 1272 DEGs in the cluster and among them was novel genes.. - Moreover, in the first- instar larvae libraries (1 libraries) (Additional file 5: Figure S3), the second-instar larvae libraries (2 libraries) (Add- itional file 6: Figure S4), and the third-instar larvae librar- ies (3 libraries) (Additional file 7: Figure S5), there were and 11,242 DEGs had been analyzed as series-cluster, respectively. - Series-cluster analysis and functional annotation of the clusters provided. - GO and KEGG pathway analysis of DEGs in 0 libraries A total of 7250 DEGs were annotated into GO terms in- volved in the egg stage. - “Cellular process” and “metabolic process” were the most enriched in the biological. - In cellular component category, “mem- brane” and “membrane part” were the highest enriched, while “binding” and “catalytic activity” were the most enriched in the molecular function category. - 0.05) in- volved in the egg stage were determined from the DEGs of L0 vs M0, M0 vs H0, L0 vs H0 (See for a full list of GO terms in Table 2). - 4 The series-clusters for DEGs in the egg stage. - GO and KEGG pathway analysis of DEGs in 1 and 2 library A total of 5121 DEGs and 6354 DEGs were annotated into GO terms involved in the first-instar and the second-instar, respectively. - “Cellular process” and “biological regulation” were the most enriched terms in the biological process domain. - “membrane part” were the highest enriched, while “bind- ing” and “catalytic activity” were mostly enriched terms in the molecular function category. - 0.05) involved in the first-instar larvae stage were determined from the DEGs of L1 vs M1, M1 vs H1. - process, structural constituent of cuticle and amino sugar metabolic process were all significantly enriched in the DEGs of L1 vs M1, M1 vs H1. - GO and KEGG pathway analysis of DEGs in 3 libraries A total of 5593 DEGs were annotated into GO terms in- volved in the third-instar. - “biological regulation” were the most enriched in the biological process domain. - And “binding” and “catalytic activity” were the most enriched in the molecular function category. - 0.05) involved in the third-instar larvae stage were determined Table 2 GO functional enrichment analysis related with temperature of the DEGs of H0 vs L0, H0 vs M0 and M0 vs L0. - The gene co-expression net-work of DEGs in the cold temperatures was analyzed (Fig. - There are several core genes with the highest degrees connect with most adjacent genes in the network, which are frequently identified as key indicators. - A comparative analysis of all the selected genes showed a similar ex- pression pattern in the qRT-PCR analysis as observed in RNAseq data (Fig. - In the results of gene-co-expression, it also showed the importance of Chitin metabolic process.. - Espe- cially, the cAMP and cGMP-PKG signaling pathway were all significantly enriched in the DEGs of H0 vs L0, H1 vs L1, and H2 vs L2, indicating the involvement of energy metabolism [43, 44] and cell growth and differentiation [45, 46]. - In the third-instar larvae, response to starvation, energy reserve metabolic process, glucosamine-containing compound metabolic process and amino sugar metabolic process were all significantly enriched. - Unfortunately, none showed consistent trend in the four developmental. - 7 The gene co-expression net-work of DEGs in the cold temperatures (4 °C) was analyzed. - OBPs are the key step in the insect olfaction [57], suggesting that A. - Then we selected the key DEGs related to development time, which could be applied in PMI min deduction in the future.. - makes it more important among necrophagous flies, es- pecially in the cold environment. - Hence, it would be mean- ingful to explore a low temperature between 4 °C and 12 °C in the future, at which the A. - Expression level of the 9 DEGs in Aldrichina grahami.. - The series-clusters for DEGs in the first- instar larvae stage. - The series-clusters for DEGs in the second- instar larvae stage. - The gene co-expression net-work of DEGs in the middle temperatures (12 °C) was analyzed. - The gene co-expression net-work of DEGs in the relatively high temperatures (20 °C) was analyzed. - The funding body played no role in the design of the study, collection, analysis of data and in writing the manuscript.. - Klong-Klaew T, et al. - Amendt J, et al. - Boehme P, et al. - Sukontason K, et al. - Chen LS, et al. - Xu H, et al. - Guo YD, et al. - Li X, et al. - Gruner SV, et al. - Effect of temperature on development of the blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae). - Wang Y, et al. - Chen W, et al. - Kikuo M, et al. - Kostal V, et al. - Ma RY, et al. - Khani A, et al. - Spranghers T, et al. - Cold hardiness of the black soldier Fly (Diptera:. - Teets NM, et al. - Telonis-Scott M, et al. - 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