Resistance Mechanisms To Fipronil In Laodelphax Striatelius (Fallen)

The small brown planthopper (SBPH), Laodelphax striatellus (Hemiptera:Delph acidae), has a wide distribution from south-east Asia to Siberia and Europe. In recent years, population density and its damage intensity increased dramatically in Jiangsu, Zhejiang, Shanghai, Anhui, and Shandong, which posed a serious threat to the trait of high and stable yield of the crop. Currently, control of Laodelphax striatellus relies mainly on chemical Insecticides. To control the pest, neonicotinoid and phenyl-pyrazole insectcides, such as imidacloprid and fipronil, have been widely used. Extensive use of fipronil against rice planthopper has led to resistance problems in East and South-east Asia.Fipronil is the first phenyl pyrazole insecticide, and the mechanism of fipronil resistance in Laodelphax Striatellus is not clear. Therefore, it was important to target insensiti-vity, biochemical mechanism, cross resistance of fipronil. The results could provide theory basis for using insecticides scientifically, and enrich the theory of insecticide toxicology.1. Cloning and sequence analysis of Glutamate-gated chloride channel gene from Laodelphax striatellusClone Glutamate-gated chloride channel gene from L.striatellus by using degenerate primers and nested PCR technology. A cDNA fragment of L.striatellus Glutamate-gated chloride channel gene was cloned. According to this fragment, a full-length cDNA was amplified by using specificity primers. The sequence includes2112nucleotides (GenBank ID:JF430868), and the open reading frame range from208to1566bp. It encoded452amino acid residues. The protein molecular weight is52.12560KDa, and the isoelectric point is8.3. Alignment amino acid sequences of Glutamate-gated chloride channel between L.striatellus and other insects, the identity of sequences up to79%-86%. 2. Polymorphism analysis and predict the mutation of Glutamate-gated chloride channel gene from Laodelphax striatellusIn order to search for the target mutation of fipronil resistance in L.Striatellus, the open reading frame of Glutamate-gated chloride channel gene were cloned from the primary strain and resistant strain. It included twenty individuals from the primary strain and ten individuals from the resistant strain respectively. Alignment the sequence of Glutamate-gated chloride channel gene, there were seventy-five site take place the replace-ment of amino acids because of the very of bases. It contained nine locuses (L13P、 D63A、 P65H、 A66T、V67I、 V74L、 S76P、 V83T、T84K) from part of the primary strain and resistant strain simultaneously. Thirty-one locuses (T7A、119T、 N23D、 T33H、 P104S、 P128L、E132G、H136R, I149V、L176P. D194V、 P197S、E201G、G201R、 V205E、 C237R、 L242E、 Y250H、 V263M、S275G、P278S、 M291T、 N299D、 L302P、 Y309R、 E328G、 Q344R、 E353G、 N373S、K389R、 K394R) Only were found in the part of the resistant strain. And other thirty-five locuses (S27P、 Q43R、 G46D、 S48C、172L、 I77T、 K79T、 D81S、 D82N、V83I、 W97R、 L102P、 T146C、 I161V、 C167R、D194G、 Y250C、 Y256C、 S282P、 V285A、 L288P、 S306P、 T315A、 M345V、 M345T、 Q351R、C352Y、 V395A、 C406R、 T415P、 R417H、 I426T、P428Y、 L437P、 R446Q) Only were found in the part of the primary strain. From the above case, there were two locuses (V83T and V83I, M345and M345T) occured different replacement of amino acids in the same place in the primary strain. And two locuse (D194V and D194G, Y250H and Y250C) occured different replacement of amino acids in the same place between the primary strain and the resistant strain. The P278S mutation was found in the M2domain of Glutamate-gated chlo-ride channel gene in the L.Striatellus. The P299S mutation is a useful tool in mapping sites on ligand-gated ion channels that play a role in channel activation, and P278S and P299S were in the same amino acid mutation but in the different insect. Therefore, the P278S mutation may relate to the fipronil resistance.3. Clone and predict the mutation of Rdl GABA receptor gene from Laodelphax striatellusIn order to search for the target mutation of fipronil resistance in L.Striatellus, at least20individuals from each strain were cloned by RT-PCR. As expected, we found that all individuals tested from the primary strain were genotypes for the LsRdl-Ala allele. However, the mutant LsRdl-Asn allele frequency in the fipronil resistant strain was up to80%. Considering high frequency of the LsRdl-Asn allele remaining in the selected resistant strain, it is suggested that the A2’N mutation in the LsRdl gene is associated with fipronil resistance. In addition to the A2’N mutation, a novel R305Q or R305W mutation was found in the cytoplasmic loop between M3and M4. The R305Q or R305W was only found in cDNA clones carrying the A2’N mutation.4. Biochemical mechanism for fipronil resistance in L.StriatellusDEF or TPP, PBO and DEM inhibit esterases, P450monooxygenases and glutathione S-transferases, respectively. The inhibition of these metabolic enzymes can enhance insect-cide toxicity. Synergistic effects of DEF, TPP, PBO and DEM on fipronil were tested on the primary strain and fipronil-resistance strain of L.striatellus. The results showed thatsynergistic ratios of DEF, TPP, PBO and DEM were0.93,0.89,0.91and0.77-fold in the primary strain, and1.00,1.18,1.18and1.27-fold in the fipronil-resistance strain, respective-ly. To confirm whether the metabolic enzymes are involved in fipronil resistance, metabo-lic enzyme activities, including P450monooxygenases (against the substrates p-NA), esterases (against the substrate a-NA), and GSTs (against the substrates CDNB), were tested between the primary strain and fipronil resistance strain. The results showed no significant difference in detoxification enzyme activities between these two strains, indicating that metabolic mechanisms are not the main factor in the high fipronil resistance strain.5. Cross-resistance of fipronil in L.StriatellusTo further confirm whether the binding site or mechanisms of action of fipronil and cyclodiene insecticides was identical, in this study, cross-resistance of fipronil to cyclodiene was determined by rice seeding dipping method in laboratory. The fipronil resistant strain of Laodelphax striatellus (Hemiptera:Delphacidae) was derived from a field-collected strain (primary strain) by77generations of continuous selection with fipronil. The selection resulted in86.6-fold resistance to fipronil, and it showed no cross-resistance to dieldrin (1.8-fold) and low level cross-resistance to endosulfan (3.7-fold), respectively, in the fipronil resistant strain, compared with the primary strain.