| Objective:To investigate the breeding conditions for Carpoglyphus lactis to raise the mites,and conduct sequencing and analysis of mitochondrial genome of the Carpoglyphus lactis and the comparative analysis of the other mites belonging to Astigmata.Methods:(1)The stored dried fruits were collected from stores,from which the mites were isolated;(2)Three different formulations of the mite feedstuffs were prepared,and 200 capita of the isolated C.lactis were artificially fed in the box with identicalenvironmental conditions for consecutive 4 weeks,followed by measuring multiplication of the mites.The one of the feedstuff was selected,and 33 diverse levels of temperature and humudity conditions were set up to observe the survival rate of the mites on the 6~thh day and the average number of eggs laid in six days under each humiture conditions;(3)The genomic DNA was extracted from the mites bred in our laboratory using phenol-chloroform or commercial DNA extraction kit.Some sequences of mitochondrial cox1,cob,rrnS and nad4-nad5 genes were amplified with PCR using common primers for mites or arthropods.The amplified genes were cloned and purified,and undergone two-way sequencing by the contracted biological company.Specific primers for the target gene sequences were designed for long-PCR amplification and step-by-step sequencing until the complete mitochondrial genome was detected.Some bioinformatics softwares like Seqman,SEQUIN 9.0 and tRNAscan were used to predict and analyze the genetic structure,order,base composition,PCGs and condon usage,RNA gene and non-coding region structure of complete mitgenome of the C.lactis.Finally,the complete mitgenome sequences of the four other mites belonging to Astigmata were downloaded from GenBank were downloaded for comparative analysis.Results:(1)Nineteen species of acaroid mites,belonging to 16 genera under 6families,were identified from 61 samples of the dried fruits in which Carpoglyphus lactis,Lepidoglyphusdestructor,GlycyphagusdomesticusandTyrophagus putrescentiae were predominant;(2)After 4 weeks of feeding,the breeding density of the mites in aforementioned three different feedstuffs was 5598.62 captia/g,2013.15captia/g and 411.23 captia/g,respectively,and the multiplication rate was approximately1400,500 and 100 times,respectively.However,survival rate of female adults was 0 at all temperature gradients as the relative humidity maintained lower than 60%-66%,and suitable humidity range was significantly correlated with the survival rate and average number of eggs laid per day for female adult mites;(3)The complete length of mitochondrial genome was 14 060 bp for C.lactis,and the DNA was presented in typically colosed-circular double-stranded molecule fanshion,consisting of 37 coding genes including 13 protein-coding genes,2 ribosomal RNA genes,22 transfer RNA genes and 1 largest non-coding region.Comparison with Aleuroglyphus ovatus and Caloglyphus berlesei of Acaridae and Dermatophagoides pteronyssinus and Dermatophagoides farinae of Pyroglyphidae demonstrated no gene deletion or rearrangement in the mitochondrial genome of the C.lactis,and that the gene sequence was identical with the four mites mentioned above.Conclutions:(1)The suitable formula for rapid breeding of the C.lactis was screened out in this study,and the laboratiry conditions for pure breeding of the C.lactis were found out:the growth of C.lactis requires higher humidity environment within certain temperature of 15-35?,relative humidity over 75.5%is recommended,and optimal relatively humidity should be maitained approximately between 85%and 90%,which provided reference and experimental basis for the breeding of acarid mites;(2)We sequenced and inteperated the complete mitochondrial genome of the C.lactis,and this is the first attempt to sequence the complete mitochondrial genome for one species in the Acaridae(Acari:Carpoglyphidae).Data have been accumulated to promoto the sequencing and inteperating of the Astigmata species and the development of molecular systematics of the Astigmata. |
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