Establishment Of A Spinosad Resistant Near-isogenic Line And The Resistance Mechanism In Frankliniella Occidentalis (Pergande)

Western flower thrips,Frankliniella occidentalis?Pergande??Thysanoptera:Thripidae?is an economically important agricultural pest worldwide,as it could damage host plants by feeding and transmitting viruses.It is very difficult to control F.occidentalis in the fields because of its unique characteristics like small body size,affinity for enclosed space,high fecundity,haplo-diploid reproduction system and short life cycle.These characteristics facilitate F.occidentalis to develop resistance to a wide range of pesticides.Spinosad is an eco-friendly macrocyclic lactone insecticide.The primary target of spinosad is nicotinic acetylcholine receptors?nAChRs?and the secondary target is GABA receptor.Even though spinosad is one of the most effective pesticides in controlling thrips,many field resistant strains have been detected all around the world.Illustration of spinosad resistance mechanism has significant importance in field control and resistance management of F.occidentalis.In the study,we investigated the control index of F.occidentalis in lettuce,and evaluated resistance levels of six field strains to seven insecticides.Beside these,we established a spinosad resistant near-isogenic line in F.occidentalis,which is also the first NILs established in parthenogenetical animals.We also screened ISSR primers and optimized the reaction system of ISSR-PCR for F.occidentalis.Moreover,a linear model between the proportion of truncated nAChR a6 transcripts and resistance level of spinosad resistance was constructed,and verified using field strains.This finding not only enriched the molecular mechanism study of F.occidentalis spinosad resistance,but also provide a potential molecular diagnostic for field moitoring of spinosad resistance.1.Resistance monitoring of field populations of F.occidentalis to insecticidesThe resistance levels of six field populations to seven insecticides were evaluated.The results showed that chlorfenapyr had the highest toxicity(LC₅₀:0.035-1.668 mg·L^-1).The resistance to?-cypermethrin and thiamethoxam were low-to-middle level,with 0.039-6.534 fold and 0.427-11.633fold resistance ratio individually.The resistance levels to abamectin and emamectin benzoate varied greatly in different strains,and the highest resistance level was detected in SS strain.The resistance to spinosad and spinetoram had reached very high level,with 8.4-191.8 fold and 190-2395 fold resistance ratio individually.2.The control index of F.occidentalis on lettuceThe relationship between population density of F.occidentalis and lettuce?Lactuca sativa L.?yield loss ratio was studied by artificial inoculating method.Two field trials were conducted in spring and autumn,respectively.The results of linear-regression analysis indicated that the correlation between lettuce yield loss ratio and thrips population density was significant positive.Control measures should be taken,when the density of F.occidentalis reached 5-6 per plant,otherwise significant yield loss will be caused3.Establishment of a spinosad resistant near-isogenic line in F.occidentalisA spinosad resistant near-isogenic line NIL-R was established in F.occidentalis through eight rounds of backcrossing.The genetic similarity between susceptible parental strain Ivf03 and resistant parental strain Spin-R was 0.6395.After eight rounds of backcrossing,the genetic similarity between NIL-R and Ivf03 reached 0.9890.And the resistance ratio of NIL-R was 36,000-fold,which is comparable to the resistance level of Spin-R.Moreover,we screened eight ISSR primers that has stable polymorphic amplification in F.occidentalis,and designed orthogonal experiment to optimize ISSR-PCR reaction system.Optimization results showed that the ranking of factors from the most to least effective was dNTPs>primer>Taq polymerase>DNA>Mg²⁺,and the optimal concentration for each factor was 1.0 mmol·L^-1 Mg²⁺,0.25 mmol·L^-1 dNTPs,2?mol·L^-1 primer,0.5 U Taq polymerase,and30 ng DNA template.4.Investigation of resistance mechanism in spinosad resistant F.occidentalisFive laboratory strains with different spinosad resistance levels were established.nAChR a6transcripts in the five laboratory strains with different spinosad resistance level were obtained using RT-PCR and mass sequencing.Then the proportions of truncated nAChR a6 transcripts were compared with corresponding resistance ratios.A linear correlation was found between these two datasets,indicating that loss of function mutations in nAChR a6 subunit may lead to spinosad resistance in F.Occidentalis?y=5.4392x-1.2038,R²=0.9765?,which means the higher level of spinosad resistance,the greater proportion of truncated nAChR a6 transcripts exists..Therefore the loss-of-function mutation of nAChR a6 subunit might be the molecular mechanism of spinosad resistance in F.occidentalis.Five field strains were collected and used for model verification.The Chi-square value was 0.2914(?²=0.2914<?²_3,0.05=7.815),demonstrating no significant difference existed between field data and model prediction.This model also works in the fields,and could be developed as a molecular diagnostic for field monitoring.