| Background: Forensic entomology plays a major role in the investigation of decomposed corpses,the age determination of necrophagous flies is one of the key tasks in providing evidence for postmortem interval(PMI)estimation.Sarcophaga peregrina(RobineauDesvoidy 1830)can be commonly found in insect succession patterns of human and animal corpses,and is regarded as an important necrophagous flesh fly in forensic entomology.Generally,the developmental patterns of immature insects take place in a predictable manner under a controlled temperature.So far,the age estimation has mostly relied on morphological characteristics of larvae and pupae.Temperature is the most significant abiotic factor affecting behavior,life history,physiology,abundance,and distribution of organisms.However,as the global climate continues to warm and the extreme heat increases significantly,heat stress obviously affects the accuracy and scientificity of entomology evidence,which becomes a vital issue limiting the validity of entomology evidence.Object:(1)To obtain the high-quality genome data and functional annotation of S.peregrina and provide a basis for further research on heat stress.(2)To explore the effects of heat stress on its growth and development and its regulatory mechanism from the aspects of physiology,transcription and metabolism.(3)The influence of temperature on its growth and development was explored by actual cases to provide a reference for correcting the accuracy of its application in PMI estimation.Method:(1)Mating pairs of adult S.peregrina trapped were inbred for six generations.Newly hatched adult females were used for further studies.Genome size was assessed by survey and flow cytometry.The whole genome was sequenced by illumina Xten+Pac Bio Seque,and also including pseudomolecules construction by Hi-C,de novo assembly and polishing of the genome,gene prediction and functional annotation,gene family analysis.(2)40 °C was selected as the optimal temperature of heat stress based on preliminary experiments.We exposed S.peregrina to heat stress at 40 °C for 3h every day throughout developmental stages from newly hatched larva to adult,and that as a control at constant temperature of 25°C.Samples were selected for transcriptome sequencing,differential gene expression and GO/KEGG functional enrichment analysis,s HSP family gene identification and analysis,and q RT-PCR was used to verify the expression pattern.(3)On the basis of the above studies,LC-MS was used to further analyze their metabolic level.After quality control and data pretreatment,multivariate statistical analysis such as PCA and OPLS-DA,T test and multiple variation analysis,etc.,the differential changes of metabolites after heat stress during growth and development were screened and functional enrichment analysis was conducted.(4)Larvae of necrophagous flies were collected from human corpses in indoor and outdoor actual cases in summer.The PMI estimation was blind tested.Results:(1)We present a de novo-assembled genome at chromosome scale for S.peregrina.The final assembled genome was 560.31 Mb with contig N50 of 3.84 Mb.Hi-C scaffolding reliably anchored six pseudochromosomes,accounting for 97.76% of the assembled genome.A total of 14,476 protein-coding genes were functionally annotated,accounting for 92.14% of all predicted genes.The six pseudochromosomes in the assembled genome can be aligned against the D.melanogaster genome.9,636 gene families were identified in the assembled genome,covering 13,039 genes.Among these,13 gene families containing 106 genes were unique to S.peregrina.This approach revealed 1,191 contracted gene families,and only 568 gene families had a greater degree of expansion.The heat shock protein(HSP)family members were identified,including12 s HSP,29 HSP40,12 HSP70 and 2 HSP90.(2)Combined with the actual cases and experimental data,the development cycle of S.peregrina was shortened after heat stress,and the body length at the heat stress group was significantly higher than that of control group,and the growth rate was also faster.(3)We performed comparative transcriptome analysis during the developmental stages of S.peregrina to investigate the effect of heat stress,changes in expression level of HSP22,HSP23,HSP27,HSP68,HSP70,and HSP90 were significantly up-regulated,also including these genes involved in serine-type endopeptidase activity,iron ion binding,and digestion and metabolism pathways.Twelve s HSP family genes were identified.RT-q PCR analysis showed that the expression pattern of s HSP family genes was consistent with the expression trend of transcriptomic sequencing,and they were significantly upregulated after heat stress.(4)Moreover,proline,alanine,tyrosine,glutamine,and phosphocholine metabolism were closely associated with thermal acclimation,as these pathways were significantly enriched in both the transcriptome and metabolome,suggesting that these candidate genes,metabolites,and metabolic pathways in response to heat tolerance are particularly robust.Conclusion:(1)This study provides a valuable genomic resource for S.peregrina,and sheds insight into further revealing the underlying molecular mechanisms of adaptive evolution.HSP family genes were also identified,which laid a foundation for further study on the regulation mechanism of S.peregrina in response to heat stress.(2)In the actual cases of outdoor natural environment in summer,the influence of heat stress on the growth and development of necrophagous flies should be considered when PMI is estimated based on the development data of necrophagous flies in order to avoid high deviation of PMI estimation.(3)Based on multiomics analysis,from the aspects of physiology,transcriptome and metabolism to explore the effect of heat stress on the growth and development of S.peregrina.The transcriptional regulatory factors that play a key role in response to heat stress were verified and their expression patterns were analyzed under heat stress,providing theoretical support for PMI estimation using necrophagous flies in high-temperature environment. |
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