Studies On ATP-binding Cassette(ABC) Transporters In Small Brown Planthopper, Laodelphax Striatellus(Fallén) And Their Involvement In Insecticide Resistance

The gene superfamily of ATP-binding cassette(ABC)transporters encodes ubiquitous membrane-bound proteins and is present in all living organism from bacteria to human.The first eukaryotic ABC transporter was found in human cancer cells associated with multidrug resistance(MDR)phenotype and was named as P-(gp(ABCB1).Further studies revealed that ABC transporters can mediate the transport of a broad spectrum of insecticides in vivo and vitro studies,and an increasing number of studies focused on their involvement in insecticide resistance.ABC transporters have been considered as an important detoxification component in resistance mechanism of endogenous and exogenous compounds after Cytochrome P450 manooxygenases,esterases and Glutathione-S-transferase,and play an important role in transport or resistant to different insecticides.The small brown planthopper,Laodelphax stiriatellus(Fallen)(Hemiptera:Delphacidae),is a major agricultural pest in China.The abused application of chemical insecticides leads to insecticide resistance,which has become a crucial problem for pest control.Previous studies in our laboratory established three strain of L.slriatellus resistant to chlorpyrifos,deltamethrin and imidacloprid,respectively,and a susceptible strain resistant and focused on tradition detoxification enzymes such as P450s,ESTs and GSTs.With the availability of transcriptome of L.striatellus,ABC transporters were identified and used for screening the potential involvements in resistance mechanism of different insecticides.The aim of this study was to reveal the resistant mechanism associated with ABC transporters using q-PCR technique to analysis the constitutive or induced expression levels of ABC transporters in different strains and in combination with verapamil synergism studies.Our studies indicated the involvement of a novel gene family in the complicated resistance mechanism of L.striatellus,and provided a new explanation for mechanism of cross resistance.In addition,this work provided a theoretical basis on establishing reasonable standard for chemical application and developing more efficient resistance management strategies.1.Molecular cloning and analysis of ABC transporters in L.striatellusSearching from the transcriptome of L.strialellus,99 of putative ABC transporter sequences(>200 bp)were obtained after removing the redundancy by GeneDOC alignments.The NCBI BlastX analysis indicated that all these sequences showed high levels of identity with the ABC transporters in other species and belonged to ABC transporter superfamily.RT-PCR and RACE techniques were used for identifying the ABC transporters.With 40 of full length ABC transporter genes were obtained after removing the redundant sequences using ContigExpress software.The open reading fragments(ORFs)of the 40 ABC transporters ranged from 603 aa to 2221 aa.The phylogenetic tree was constructed based on the NBDs identified by Pfam.Similar to other insect species,the 40 ABC transporters of L.striatellus were divided into ABCA-ABCH eight subfamilies,including 2 ABCAs,6 ABCBs,5 ABCCs,2 ABCDs,1 ABCE,2 ABCFs,14 ABCGs and 8 ABCHs.There were 9 full transporters and they belonged to ABCA,ABCB and ABCC subfamilies.With 70%of ABC genes were half transporters and they were present in ABCB,ABCD,ABCG and ABCH subfamilies.Based on the comparisons among other insect species,the number of ABC transporters annotated at genome or transcript wide level ranged from 41 in Apis mellifera to 73 in Tribilium cataneum.The ABC transporters of L.striatellus have a full coverage of ABC transporter subfamilies in insect species and similar amount of ABC genes in A.melliera identified at genome wide level.In addition,the same amout of genes in well conserved subfamilies ABCD,ABCE and ABCF among all insect species were identified,which indicated that the majority of ABC transporters have been discovered in L.striatellus.This provides a foundation for characterization the involvement of ABC genes in insecticide resistance.2.Analysis of ABC transporter genes associated with chlorpyrifos resistance in L.striatellusIncreased detoxification of metabolism enzymes has been considered as an important part in resistance mechanism of chlorpyrifos,however,ABC transporters as a significant component of detoxification process may contribute to resistance of chlorpyrifos in L.striatellus.In this study verapamil was used as the ABC transporter inhibitor to evaluate the toxicity of chlorpyrifos in the chlorpyrifos resistant strain(JH-chl)and the susceptible strain(JH-sus)in vivo.The synergism studies showed that verapamil increased the toxicity of chlorpyrifos by a factor of 3.02 in JH-chl strain,but no significant synergism was observed in JH-sus strain.Then the q-PCR was used to quantify the expression levels of 40 ABC transporters at the transcript level.Compared to the susceptible strain,9 and 25 ABC transporters were found significantly(p<0.5)overexpressed(>2-fold)in JH-chl strain at the adult and 4th instar larvae stages,respectively,and no genes were found significantly down-regulated under the same standard.The results indicate that overexpressed ABC transporters are involved in chlorpyrifos resistance in JH-chl strain.With the treatment of 3-4-day-old female adults in JH-chl strain using the LD25 of chlorpyrifos,13 ABC transporters were found significantly overexpressed compared to the control(treated with acetone only)and 12 of them were found consititutively overexpressed in resistant strain,which indicate that they may play an important role in transport of chlorpyrifos.In summary,there were 28 ABC transporter genes belong to ABCA/B/C/D/F/G/H seven subfamilies,which have significant constitutive overexpression or induced overexpression associated with chlorpyrifos.In addition,seven genes were found significantly overexpressed at three different conditions,which are LsABCB2,LsABCC4,LsABCD1,LsABCF1,LsABCG2,LsABCG6 and LsABCG11.These results indicated that multiple ABC transporter genes were associated with chlorpyrifos in transport and resistance mechanism in resistant strain of L.striatellus.3.Analysis of ABC transporter genes associated with deltamethrin resistance in L.striatellusIncreased detoxification of metabolism enzymes has also been considered as an important step in resistance mechanism of deltamethrin,however,ABC transporters have been found to be involved in pyrethroid resistance in several insect species.In this study verapamil was used as a synergist to evaluate the toxicity of β-cypermethrin in the deltamethrin resistant strain(JH-del)and the susceptible strain(JH-sus)in vivo.The synergism studies showed that verapamil increased the toxicity of β-cypermethrin by a factor of 2.24 in JH-del strain,but no synergism was observed in JH-sus strain.Then the q-PCR was used to quantify the expression levels of 40 ABC transporters at the transcript level.Compared to the susceptible strain,13 and 26 ABC transporters were found significantly(p<0.5)overexpressed(>2-fold)in JH-del strain at the adult and 4th instar larvae stages,respectively,and no genes were found significantly down-regulated under the same standard.The results indicate that overexpressed ABC transporters are involved in deltamethrin resistance in JH-del strain.With the treatment of 3-4-day-old female adults in JH-del strain using the LD25 of β-cypermethrin,6 ABC transporters were found significantly overexpressed compared to the control(treated with acetone only)and all of them were found consititutively overexpressed in resistant strain,which indicate that they may play an important role in transport of pyrethroids.In summary,there were 28 ABC transporter genes belong to ABCA-ABCH eight subfamilies,which have significant constitutive overexpression or induced overexpression associated with deltamethrin or(3-cypermethrin.In addition,five genes were significantly overexpressed at three different conditions,including LsABCB2,LsABCC4,LsABCG2,LsABCG6 and LsABCG7.These results indicated that multiple ABC transporter genes were associated with pyrethroids(deltamethrin and β-cypermethrin)in transport and resistance mechanism in resistant strain of L.striatellus.4.Analysis of ABC transporter genes associated with imidacloprid resistance in L.striatellusAs an effective neonicotinoid insecticide,imidacloprid has been widely used for chemical control of plant hoppers in rice field.The resistant levels of imidacloprid in the field populations of L.striatellus were monitored and highly resistant populations were found since then.Same as the other type of insecticides,ABC transporters may play an important role in resistance mechanism of imidacloprid in L.striatellus.In this study verapamil was used as a synergist to evaluate the toxicity of imidacloprid in the imidacloprid resistant strain(JH-imi)and the susceptible strain(JH-sus)in vivo.The synergism studies showed that verapamil increased the toxicity of imidacloprid by a factor of 1.52 in JH-imi strain,but no synergism was observed in JH-sus strain.Then the q-PCR was used to quantify the expression levels of 40 ABC transporters at the transcript level.Compared to the susceptible strain,11 and 8 ABC transporters were found significantly(p<0.5)overexpressed(>2-fold)in JH-imi strain at the adult and 4th instar larvae stages,respectively,and no genes were found significantly down-regulated under the same standard.The results indicate that overexpressed ABC transporters are involved in imidacloprid resistance in JH-imi strain.With the treatment of 3-4-day-old female adults in JH-imi strain using the LD25 of imidacloprid,no ABC transporter was found significantly overexpressed compared to the control(treated with acetone only).In summary,there were 17 ABC transporter genes belong to ABCB/C/D/G/H five subfamilies,which have significant constitutive overexpression associated with imidacloprid.In addition,only two genes LsABCG10 and LsABCH4 were significantly overexpressed at the adult and 4th instar larvae stages.These results indicated that multiple ABC transporter genes were associated with imidacloprid in resistance mechanism in resistant strain of L.striatellus.5.Analysis of multidrug resistance candidate genes in L.striatellusAs one of the most important steps for insecticide detoxification,the ABC transporter genes have been involved in insecticide transport and resistance against different types of insecticides,such as chlorpyrifos.deltamethrin(β-cypermethrin)and imidacloprid.Most of the ABC transporters were found overexpressed at different stages or insecticide treatments in different resistant strains.However,there are eight ABC transporter genes were overexpressed at the female adults among all three resistant strains,and it is reasonable to assume that they are candidates as multidrug resistance genes which could play an important role in transport or resistant to multiple insecticides.The q-PCR was used to compare gene copy number between the three resistant and the susceptible L.striatellus strains.The copy number of LsABCG10 gene was about 3-,2-and 2-fold higher in the resistant JH-chl,JH-del and JH-imi strains respectively compared with the JH-sus strain,and no changes were observed for the other 7 genes.The q-PCR analysis was used to quantify the expression level of all eight genes at different tissues and stages of L.striatellus.The results showed that all the genes were expressed at all tissues and stages checked and most of the genes were found relative highly expressed at head and 1-2th instar larvae.These results indicated that the constitutive overexpression were mostly due to expression regulation,and the multidrug candidate genes were mostly expressed in brain of L.striatellus that could be involved in resistance to different types of insecticides.6.Gene analysis and heterogenous expression in Sf9 for full transporter LsABCC4Among the eight multidrug candidate genes,LsABCC4 gene was the only full transporter,and it is also from ABCC subfamily which contains multidrug resistance associated proteins(MRPs).LsABCC4 was assumed to have a similar function as the human MRP1.The complete ORF of LsABCC4 was amplified from the JH-sus strain,and three different isoforms of LsABCC4 were found and named as LsABCC4-1,LsABC4-2 and LsABCC4-3,respectively.Based on the amino acid alignment and conserved domains searches,the three isoforms of LsABCC4 contain all four conserved domains(2 NBDs and 2 TMDs),and the differences were observed at the sixth a helix near to the N end.The phylogenetic analysis revealed that the amino acid sequences of MRPs from other species were highly conserved with LsABCC1 and LsABCC4 and the insects belonging to the same order tend to cluster together.The transcript frequencies of the three different isoformis were determined.and no significant differences were observed for transcriptal preferences among resistant and susceptible strains.To futher validate the function of LsABCC4,heterogyenous expression system was used to express LsABCC4 gene in vitro.The Bacmid-LsABCC4-1 and Bacmid-LsABCC4-2 were established successfully.