| Objective:The rapid identification of necrophagous insect species and the estimation of pupal age are the key problems to be solved in the promotion of forensic entomology.This study verified the effectiveness of 658-bp COI gene fragment in the identification of common necrophagous flies in the Yangtze River Delta,and provided the first mitochondrial genome sequence of short horn exposed tail beetle,which provided a molecular basis for the rapid identification of necrophagous insect species.This study also explored the temporal changes of RNA and bacteria in the pupal stage of necrophagous flies and the changes of Fourier transform infrared spectrum of pupal shell after eclosion,which provided ideas for inferring the age of pupal stage and late pupal stage.Method:(1)The seven fly species used in the experiment were collected from Suzhou City,Jiangsu Province and raised for several generations under laboratory conditions at 25℃.After extracting the genomic DNA of each fly species,PCR amplification was carried out with universal primer LCO-1490/HCO-2198.The amplified products were first-generation sequenced,and the sequencing results were uploaded to NCBI and blast to determine the identification efficacy of 658-bp COI gene fragment for common necrophagous flies in the Yangtze River Delta.(2)Genomic DNA was extracted from Omosita colon.After the library was established,the second-generation sequencing was carried out on Illumina platform.After obtaining the original sequencing data,data evaluation and quality control,mitochondrial genome splicing and mitochondrial gene prediction were carried out in turn.After sequencing,the nucleotide composition,protein coding gene and codon usage frequency,tRNA secondary structure were analyzed,and the phylogenetic tree was constructed.(3)Hydrotaea spinigera larvae were raised at 31℃,25℃ and 19℃ respectively.After pupation,pupal tissue was taken every day and total RNA was extracted.After screening the most suitable reference genes at various temperatures,the changes of White and Hsp90 genes with pupal development at different temperatures were studied by RT-qPCR,and the change models of pupal age and gene expression were established.(4)Chrysomya megacephala larvae were raised at 25℃.After pupation,pupal tissue was taken every day and bacterial samples were extracted.PCR amplification was carried out with 16S rDNA full-length primer 27F/1492R.The amplified products were sequenced for three generations,the microbial composition and its changes were analyzed.(5)C.megacephala larvae were raised at 25℃.After eclosion,the pupae were placed in a constant temperature incubator at 25℃,and several pupae were taken out at 0d,5d,10d,15d,20d,25d and 30d respectively for infrared spectrum collection.Result:(1)The average base composition of different fly species was A(30.14%),T(38.23%),C(15.98%)and G(15.65%).The rate of interspecific evolutionary divergence ranged from 2.2%to 15.3%,the lowest was between C.megacephala and Chrysomya pinguis,and the highest was between Boettcherisca peregrina and Muscina stabulans.(2)The total length of mitochondrial genome of short horn exposed tail beetle is 16544bp,including 13 protein coding genes,22 tRNA genes,two rRNA genes and a non-coding region.The nucleotide composition was A(41.1%),T(38.7%),C(8.3%),G(11.9%),with obvious AT-skew(0.029).(3)The first two internal reference genes of pupal stability of black fly with thick ring at all temperatures were 18S and 28s.The White gene first increased and then decreased at 19℃ and 25℃,while it gradually increased at 31℃.At 19℃,Hsp90 gene increased first,then decreased,and finally increased.The expression increased first and then decreased at 25℃,while the expression did not change significantly at 31℃.(4)The results of cluster analysis showed that when the similarity was≥97%,154 operational taxonomic units were obtained,which were divided into 15 classes,28 orders,50 families,88 genera and 130 species.In the pupal stage,Proteobacteria,Firmicutes,and Bacteroidetes are the dominant phyla of bacteria.At the genus level,the relative abundance of Wolbachia decreased significantly at the end of pupal development.(5)The prediction results of PLS regression model show that the R2CV of calibration data set is 0.864,RMSECV=3.711 D,the R2P of prediction data set is 0.909,RMSEP=3.045 D,RMSEP/RMSECV=0.821.Conclusion:(1)658-bp COI gene fragment can be used to better identify common necrophagous flies in the Yangtze River Delta..(2)It provides the first complete mitochondrial genome sequence of O.colon,enriches the mitochondrial genome data of the Nitidulidae family,and provides a molecular basis for using O.colon to infer the time of death in forensic entomology.(3)White gene and Hsp90 gene can be used to infer the pupal age of H.spinigera.(4)The bacterial population and its changes at the C.megacephala pupal stage provide a basis for explaining the development,but the significance of indicating pupal age is not obvious.(5)The infrared spectrum of pupariums of C.megacephala changed regularly after eclosion,which can be used to infer the time of late death. |
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