Comparison Of Bacteria Community And NAChR α3 Subunit In Insecticide-susceptible And-resistant Strains Of Bemisia Tabaci

The sweetpotato whitefly,Bemisia tabaci Gennadius(Homoptera: Aleyrodidae),is an important worldwide agricultural pest.There are 26 cryptic species(previously 36 biotypes),but the invasive B [also known as Middle East Asia Minor 1(MEAM1)species] and Q [Mediterranean(MED)species] biotypes are the two most destructive cryptic species in China and elsewhere.Management of B.tabaci mainly depends on chemical insecticides.Field evolved resistance to many commercially available conventional pesticides,especially neonicotinoids,has been documented.To reveal if bacteria community and nAChR α3 subunit contribute to whitefly resistance,I studied the bacteria community and nAChR α3 subunit(only Yuma-04-B-S and Composite 11-B-R)of the following insecticide-susceptible and-resistant strains of B.tabaci: Yuma-04-B-S(susceptible to imidacloprid),Composite 11-B-R(resistance ratio=15600),12-10-Gadsen-B-S(susceptible to pyriproxyfen),QC-02-B-R(resistance ratio=25.43),08-52-Q-R(resistance ratio=144500),11-50-WFP-Q-R(resistance ratio=133800).I compared the bacteria community among different strains of B and Q biotypes that are susceptible or resistant to pypriproxyfen or neonicotinoids by 16 S rRNA amplicon sequencing technology.We also RT-PCR-cloned the c DNAs of acetylcholine receptor(AchR)α3 subunit of the the neonicotinoids-resistant Composite 11-B-R and the neonicotinoids-susceptible Yuma-04-B-S,the target of neonicotinoids,and compared their sequence differences.The results are summarized as follows.I determined the species and amount compostions of the microbes among the above 6 strains using Illuminon sequencing of 16 s rRNA amplicons.The results show that Candidatus Portiera,Rickettsia and Candidatus Hamiltonella were the most abundant microbes shared by all the six strains.The resistant Q biotype strain 08-52-Q-R contained 668 Candidatus Nitrososphaera,489 Arthrobacter,262 Bacillus,15 Wolbachia,113 Pseudomonas,73 Sphingobium,72 Rubrobacter,53 JanThinobacterium,53 Sphingobium,whereas none of the aformentioned endosymbionts was detected in the resistant B biotype strain.Comparison of Composite 11-B-R with another resistant Q biotype strain 11-50-WFP-Q-R,the latter contained 325 Arthrobacte,281 Candidatus Nitrososphaera,189 Bacillus,50 Sphingobium,but the former did not harbor these bacteria.These differences suggest that the above microbes may contribute to the B/Q-biotype differentiation of B.tabaci.Comparison of the bacteria community of the neonicotinoid-susceptible Yuma-04-B-S and the neonicotinoid-resistant Composite 11-B-R shows that their major bacteria species were the same,but the relative amount of these bacteria differed significantly.For example,the sequence reads of Rickettsia-OTU10,Candidatus Portiera-OTU85,Candidatus Hamiltonella-OTU324,S24-7-OTU297,[Prevotella]-OTU70,Candidatus Portiera-OTU190 were 17.36,15.0,3.51,3.25,3.18,and 3.0 times more in Composite 11-B-R than in Yuma-04-B –S,respectively.Likewise,the pyriproxyfen-resistant QC-02-B-R harbored 1215,86.67,28,20.8,10.95,and 10.03 times more Candidatus Hamiltonella-OTU7,Candidatus Portiera-OTU85,Candidatus Portiera-OTU5,Candidatus Portiera-OTU190,Rickettsia-OTU4,and Rickettsia-OTU2,respectively,than did 12-10-Gadsen-B-S.These quantatative differnces suggest that the above microbes may confer at least partially to B.tabaci’s resistance to neonicotinoids and the juvenile hormone analogue pyriproxyfen.I also RTPCR-cloned the cDNA sequences of B.tabaci n AChR α3 subunit gene from three 100-ppm imidacloprid treatment male survivors of the neonicotionids-resistant Composite 11-B-R and three neonicotionids-susceptible Yuma-04-B-S male adults.Nucleotide sequence alignment of the obtained three resistant and three susceptible alleles of B.tabaci nAChR α3 subunit cDNA reveled 4 necleotide polymorphisms,including A52 G,G91A,G152 A and T1463 C.Among the four point muations,A52 G,G91A and G152 A are synonymous substitutions and also occurred in two of the three susceptible alleles,indicating they are not involved in neonicotinoid resistance.By contrast,T1463 C was a resistance allele-unique subsititution and causes an amino acid replacement of V488 A.It is likely that T1463 C mutation contributes to B.tabaci’s resistance to neonicotionids,but further functional and/or linkage experiments are needed to verify this speculation.