Molecular Mechanism Of Fenpropathrin Resistance In Neoseiulus Barkeri

Neoseiuhus barkeri is considered to be one of effective biocontrol agents in citrus red mite controlling in the orange orchard, functioned as an alternative stratage to solve the conflict between chemical control and biological control in integrated pest management. In order to obtain the resistance strain of N.barkeri to fenpropathrin, resistance selection was carried out for a long time. Besides, high-throughput sequencing technology was applied to analyze the transcriptome information of N.barkeri. Moreover, clone and quantitative PCR （qPCR） were performed to detect and compare the gene mutations and expression profils of some target genes in pesticide resistance between these two strains. In conclusion, this study provided a clear vision of resistance mechanism of N.barkeri to fenpropathrin at molecular level, and provided a theoretical basis for molecular transformation of natural enemy, particularly shed new light on resistance obtaining. The main results were stated as follows:1. The resistance selection of N.barkeri to fenpropathrin and evaluation of ecological fitness on N.barkeri.The toxicity of different pesticides to N.barkeri were tested in laboratory, pyrethroid had the most toxicity against N.barkeri. Therefore, we chosed the fenpropathrin as the reagment for resistance selection of N.barkeri. Fenpropathrin was sprayed for a long time with50%lethal concentration in the laboratory to obtain the resistance strain of N.barkeri in resistance selection. The LC50finally increased from35.79mg/L to22190.91mg/L, with the resistance ratio of619.96in resistant strain （R-strain）. Moderate cross-resistance to beta-cyfluthrin （RR=45.89）, chlorpyrifos （RR=19.10）, etoxazole （RR=11.45） and thiamethoxam （RR=10.32） were observed in the R-strain. Developmental duration, funcundity and predacious ability of R-strain and susceptible strain （S-strain） of N.barkeri. Interestingly, the oviposition period, the ovisiposition peak, the average spawning per female, total ovisiposition number and predacious ability showed no significant difference between R-strain and S-strain, but the developmental duration were significantly different in all stages （P<0.05）. The effects of different pesticides on hatchability between R-strain and S-strain were studied, majority treatments had little influence on hatchability. And after pyridaben, cyflumetofen, beta-cyfluthrin treatment, there was significant differences between R-strain and S-strain （P<0.05）.2. Transcriptome analysis of N.barkeri and analysis of resistance-related genes.Next-generation sequencing technology was perform to obtain the database of N.barkeri in this experiment. A total of50,462transcripts and34,211Unigenes of this species were obtained by sequencing the transcriptome. To annotate Unigenes, sequences were searched in the nonredundant （Nr） NCBI protein database using BLASTP with a cut off E-value of10-5. A total of15,987distinct sequences returned a blast result, which meant that these Unigenes were successfully mapped to known function genes. Besides the Nr database, there were10,486Unigenes successfully annotated in Swiss-Prot,5,445in KEGG,8,707in GO and5,673in COG. Totally,15,987Unigenes were annotated across these databases.3. Cloning and difference analysis of voltage-gated sodium channel gene cDNA in N.barkeri.Three unigenes comp28931_c2, comp28931_c3and comp28696_c0were identified in the transcriptome database, which related with the sodium channel. The cDNA sequences was obtained via PCR and compared the nucleic acid and putative amino acid sequences between R-strain and S-strain. The full-length of the cDNA sequence was6175bp, encoding2058amino acids. And the protein sequence had all of the typical motifs of VGSC. The amino acid shared91.04%,80.48%,55.09%and41.98%identity with sodium channel genes of Metaseiulus occidentalis（GeaB&nk Accession No. XP₀03741737）, Varroa destructor（GenBank Accession No. AAP13992）, Drosophila melanogaster（GenBank Accession No. AAB59195） and Homo sapiens（GenBank Accession No. NP₀66287）, respectively. There were5SNPs （single nucleotide polymorphisms） after comparing the R-strain and S-strain. They were mainly used interchangeably A and G. The mutations from G to A located in3228and3243, and the mutation from A to G located in3844,4730and4874. There were three amino acids found in the sodium channel gene mutations through translation, that were S1282G, H1577R and E1665G, that means the mutations in the sodium channel gene may be the main causes of resistance to fenpropathrin.4. Selection of valid reference genes and the expression model of detoxification between susceptible and resistant strains of N.barkeri.Ten candidate reference genes, including beta actin （ACTB）, alpha tublin （TUBA）, elongation factor-1alpha （ELF1A）, RNA polymerase Ⅱ largest subunit （RNAP Ⅱ）,28s ribosomal RNA （28S rRNA）, heat shock protein90（Hsp90）, heat shock protein70（Hsp70）, heat shock protein40（Hsp40）, glyceraldehydes3phosphate dehydrogenase （GAPDH）, ubiquitin conjugating enzyme （UBC） were chosen as the potential canidate reference genes. The stability of these genes was estimate by three softwares （GeNorm, NormFinder and BestKeeper） between R-strain and S-strain. The ranking after GeNorm analysis of candidate reference genes between R-strain and S-strain with mean M values from the lowest to highest was:ACTB=UBC> Hsp40> Hsp70> GAPDH> ELF1A> Hsp90> TUBA>28s RNA> RNAP Ⅱ. By NormFinder software to calculate that the ranking of candidate reference genes across R-strain and S-strain with mean M values from the lowest to highest was:ACTB> UBC> Hsp70> Hsp40> GAPDH> ELF1A> TUBA> Hsp90>28S rRNA> RNAP Ⅱ. BestKeeper software evaluation results are basically consistent with the results of GeNorm and NormFinder, the ranking of candidate reference genes across R-strain and S-strain with mean M values from the lowest to highest was:ACTB> UBC> Hsp70> GAPDH> ELF1A> Hsp40> Hsp90> TUBA>28S rRNA> RNAP Ⅱ. Consolidated results of three kinds of software, The most suitable reference genes is ACTB between R-strain and S-strain of N.barkeri. After determined the reference gene, the expression profils of P450s, CarEs and GSTs were analyzed by using the corresponding reference gene in different treatemnts.By manually searching the database, three categories of metabolic resistance genes （P450, CarE and GST） were selected to conducted the following experiment. Thirty-two P450unigenes were found in the aforementioned database, of which9unigenes were cataloged into Clan2,19unigenes into Clan3,2unigenes into Clan2and Clan M. qPCR demenstrated that comp24004_c0and comp32672_c0from Clan4, comp29873_c0, comp24471_c0from Clan2were up-regulated the four top up-regulated46.69,2.90,3.03and1.96folds in R-strain, respectively. In addition, the expression profil of comp24004_c0and comp32672_c0were significantly increased. Fourteen CarE unigenes were found in the database, which belonged to Clade J, Clade K and Clade L. The results showed that7genes were up-regulated and7genes were down-regulated in the selected samples. Among of which, comp21521_c0was highly up-regulated, with the log2ratio（RS/SS） of3.52, followed by comp30704_c0with the log2ratio（RS/SS） of2.69. In addition,11GST unigenes were identified, which distributed in Delta, Kappa, Mu and Omega, with the number of3,2,4and2in their clade, respectively. GSTd01, GSTd02and GSTd03from Delta were significantly up-regulated, with log2ratio（RS/SS） of11.19,5.43and4.04, respectively.In a word, We got a resistances train of N. bakeri by bioassay in this study, and the cross-resistance of R-strain to any pesticides was also considered in the same time. Application of high-throughput sequencing technology to N. bakeri to complete transcriptome sequencing and annoting. Further more, using RT-PCR technology and molecular biology software, we cloned and bioinformatics analysis of voltage-gated sodium channel gene in N. bakeri, and found the sequence mutations. While we evaluated the stability of housekeeping genes between R-strain and S-strain by qPCR technology. On this basis, the analysis of differential expression of P450, CarE and GST genes between different strains. The results of this study not only enriches the predatory mite-resistant materials, and compares the difference between R-strain and S-strain at the molecular level, laid the foundation for the study of N. bakeri molecular mechanism of resistance.