| The oriental fruit fly B.dorsalis was a worldwide pest that had caused dramatic financial losses to crops.The long-term applications of insecticide had led to different degrees of insecticide resistance even though chemical control has been regarded as an important management measure.Therefore,the management of B.dorsalis is becoming more and more challenging with the emerging insecticide resistance and it is an urgent task to find novel and effective methods to control B.dorsalis.Studies in this thesis were conducted on insecticide resistance of 5 trains B.dorsalis with different pupa colorations,transcriptome of 5 strains pupae,specific activities of Acetylcholinesterase(ACh E),glutathione S-transferases(GST)and cytochrome P450 in pupae and adults of 5 strains,quantities of nine candidate genes,quantities of three candidate genes in adults and midgut of B.dorsalis treated in 3 kinds of insecticides,specific activities of ACh E and GST in adults and midgut of B.dorsalis treated in 3 kinds of insecticides,and proved that there were relationship between pigmentations of pupae and adults insecticide resistance of B.dorsalis.Three genes were proved to be associated with pigmentation of B.dorsalis pupae and contributed to insecticide resistance of B.dorsalis adults and as follow: yellow gene MK529913 and MK529915 of dopachrome conversion enzyme(DDC),gene MK515144 of arylalkylamine-N-acetyltransferase(AANAT).These results could lead to better understanding on mechanism of B.dorsalis resistance against insecticide and provide new clues for pest control strategy.The brief of results were as follow:1 Comparison of the resistance of five strains to abamectin,beta-cypermethrin and trichlorfon.Insecticide resistance of 5 strains to abamectin,beta-cypermethrin and trichlorfon were meastrued using pesticide-membrane method.Results showed that regular pattern of resistances of 5 strains to abamectin was same to that of 5 strains to beta-cypermethrin as CZ > CB > CK > LC ≈ LX(high to low),while regular pattern of resistances of 5 strains to trichlorfon were roughly as CZ > LC > CK > LX ≈ CB(high to low).Strain CZ was a beta-cypermethrin-resistant strain with brown pupae coloration while Strain CK was a wild strain with brown pupae coloration.Strain CB was a pigmentation mutation strain with white pupae coloration.Strain LC was the female of pigmentation sex genetic strain whose pupae coloration was white.Strain LX was the male of pigmentation sex genetic strain whose pupae coloration was brown.2 Transcriptome of B.dorsalis pupae.Basing on the transcriptome of pupae of 5 strains,studies were conducted on the relationships of 5 strains genes,functions distributions of genes associated with pigmentation,functions distributions of genes associated with detoxification and functions distributions of genes up-regulated or down-regulated,showed that the gene expression model of strain CK was similar to gene expression model of strain CB(spearman correlation coefficients 0.85)while strain LC was similar to strain LX(spearman correlation coefficients 0.86),and revealed that genes associated with pigmentation and detoxification were involved in some biological process,such as “Amino acid metabolism”and “Xenobiotics biodegradation and metabolism”.Results in this part revealed that there were relationship between pigmentations of pupae and adult insecticide resistance of B.dorsalis preliminarily,and proved that the differences of insecticide resistance of 5 trains were mostly distributed in “Amino acid metabolism” and “Xenobiotics biodegradation and metabolism”.Besides,nine candidate genes were indentified for subsequent studies and were as follow: four yellow genes of dopachrome conversion enzyme family(MK529912,MK529913,MK529914,MK529915),AANAT gene MK515144 of arylalkylamine-N-acetyltransferase,gene MK515141 of dopa decarboxylase(DDC),gene MK515140 of Phenoloxidase(PO),laccase2 gene MK515143 of Phenoloxidase and P450 gene MK515142.3 Specific activities of ACh E,GST and P450 in pupae and adults of 5 strains.Studies were conducted on specific activities of ACh E,GST and P450 in pupae and adults of 5 strains.Results and conclusions of these studies were includes three parts and were as following:(1)Results showed that specific activities of ACh E in adults of strain LC and strain LX were 55.44 μg/mgprot and 309.75 μg/mgprot respectively,lower than specific activities of ACh E in other three strains respectively,specific activities of ACh E in pupae with white coloration were higher than that of ACh E in pupae with brown coloration,such as specific activity of ACh E in strain CB was highest as 335.84 μg/mgprot,proved that ACh E could contribute to insecticide resistance of B.dorsalis,and indicated that white pigmentation might contribute to insecticide resistance of B.dorsalis.(2)Results showed that specific activities of GST in pupae of strain CB and strain LC were 167.94μmol/min/mg protein and 290.86 μmol/min/mg protein respectively,higher than that in pupae of strain CK and strain LC,specific activities of GST in adults of strain CB and strain LC were 145.98 μmol/min/mg protein and 185.35 μmol/min/mg protein respectively,higher than that in adults of strain CK and LX,specific activity of GST in adults of strain CZ was 224.01 μmol/min/mg protein,higher than that in adults of other four strains,proved that GST could contribute to insecticide resistance of B.dorsalis,and indicated that white pigmentation might contribute to insecticide resistance of B.dorsalis.(3)Results showed that relative activities of P450 in adults of strain LC were 3.14 pmol/min/mg,significantly higher than that in adults of other four strains,but insecticide resistance of strain LC to abamectin and beta-cypermethrin had been proved to be lower than strain CK,strain CZ and strain CB,indicated that there were other factors besides of P450 that could promote the insecticide resistance of B.dorsalis.4 Quantifications of nine candidate genes in pupae and adults of 5 strains.According to the results of analysis of transcriptome of B.dorsalis,the functions of nine candidate genes had been understood preliminarily.In purpose of speculating the functions of these nine candidate genes,studies were conducted on the quantifications of nine candidate genes in pupae and adults of 5 strains,showed that eight candidate genes could contribute to insecticide resistance of B.dorsalis to abamectin and beta-cypermethri and were as follow: four yellow genes of dopachrome conversion enzyme family(MK529912,MK529913,MK529914,MK529915),AANAT gene MK515144 of arylalkylamine-N-acetyltransferase,gene MK515141 of dopa decarboxylase,gene MK515140 of Phenoloxidase and P450 gene MK515142.Studies showed that laccase2 gene MK515143 of Phenoloxidase could contribute to insecticide resistance of B.dorsalis to trichlorfon.5 Quantifications of yellow gene MK529913,yellow gene MK529915 and AANAT gene MK515144 in midgut of 5 strains.Studies were conducted on quantifications of two yellow genes of dopachrome conversion enzyme family(MK529913,MK529915)and AANAT gene MK515144 of arylalkylamine-N-acetyltransferase in the midgut of 5 strains,showed that relative elative quantity of yellow gene MK529913 in the midgut of strain CZ was 1.89 times of that in the midgut of strain CK,relative quantity of AANAT gene MK515144 in the midgut of strain CZ was 2.64 times of that in the midgut of strain CK,significantly higher than that in the midgut of strain CK,relative quantity of yellow gene MK529915 in the midgut of strain LX was 16.28 times of that in the midgut of strain CK,significantly higher than that in the midgut of other 4 strains,relative quantities of AANAT gene MK515144 in the midgut of 2strains(strain CB,strain LC)whose pupae coloration were white and strain LX were 2.93,2.24 and 6.13 times of strain CK respectively,significantly higher than relative quantities of this gene in the midgut of strain CK,proved that these three genes could contribute to insecticide resistance of B.dorsalis,and could play important roles in midgut of B.dorsalis either.6 Specific activities of ACh E and GST in adults and midgut of B.dorsalis which were treated with abamectin,beta-cypermethrin and trichlorfon.Studies were conducted on specific activities of ACh E and GST in the strain CK adults which were treated with abamectin,beta-cypermethrin and trichlorfon.Results and conclusions of these studies were includes two parts and were as following:(1)Results showed that specific activities of ACh E in adults of B.dorsalis which were treated in abamectin for 30 minutes and 24 hours were promoted to 143.50 μg/mgprot and 237.79μg/mgprot respectively,significantly higher than that in adults of B.dorsalis which were treated without any insecticide,proved that ACh E participated in the metabolic process of B.dorsalis to insecticides.(2)Results showed that the specific activity of GST in B.dorsalis adults which were treated in trichlorfon for a short time(30 minutes)was 113.53μmol/min/mg protein,significantly higher than that in B.dorsalis adults which were treated without any insecticide,specific activities of GST could be promoted in midgut of B.dorsalis which were treated in abamectin,beta-cypermethrin and trichlorfon for a long time(12 hours or more than 12 hours),proved that GST could contribute to the metabolism of B.dorsalis to trichlorfon.7 Quantifications of AANAT gene MK515144,yellow gene MK529913 and yellow gene MK529915 in adults and midgut of 5 strains which were treated with 3 kinds of insecticides.Studies were conducted on quantifications of AANAT gene MK515144,yellow gene MK529913 and yellow gene MK529915 in adults and midgut of 5 strains which were treated with abamectin,beta-cypermethrin and trichlorfon.Results and conclusions of these studies were includes three parts and were as following:(1)Results showed that relative quantities of AANAT gene MK515144 in adults which were treated with abamectin and trichlorfon for 30 minutes were 1.12 and 1.27 times to that in adults which were treated without any insecticide,relative quantities of this gene in adults which were treated with abamectin and trichlorfon for 12 hours were 1.47 and1.13 times to that in adults which were treated without any insecticide,relative quantities of this gene in the midgut of adults which were treated with abamectin,beta-cypermethrin and trichlorfon for 6 hours were 1.11,1.15 and 1.29 times to that in adults which were treated without any insecticide,proved that AANAT gene MK515144 participated in the reaction and metabolism of B.dorsalis to abamectin,beta-cypermethrin and trichlorfon.(2)Results showed that relative quantities of gene MK529913 in adults and midgut of B.dorsalis which were treated in trichlorfon for 30 minutes were significantly up-regulated to be 1.58 and 2.42 times of that in adults and midgut of B.dorsalis which were treated without any insecticide respectively,compared to relative quantities of yellow gene MK529913 in adults,relative quantities of this gene in midgut of B.dorsalis which were treated with abamectin,beta-cypermethrin and trichlorfon showed larger regulations,such as relative quantities of this gene in midgut were significantly up-regulated to be 2.42 times of that in midgut of B.dorsalis which were treated without any insecticide while relative quantities of this gene in adults were significantly up-regulated to be 1.58 times of that in adults of B.dorsalis which were treated without any insecticide,proved that yellow gene MK529913 participated in the reaction and metabolism of B.dorsalis to abamectin,beta-cypermethrin and trichlorfon,and proved that yellow gene MK529913 played important roles in midgut of B.dorsalis either.(3)Result showed that the relative quantity of gene MK529915 in adults of B.dorsalis which were treated with trichlorfon for 30 minutes were significantly up-regulated to be 1.15 of that in adults which were treated without any insecticide,relative quantity of gene MK529915 in midgut of B.dorsalis which were treated in trichlorfon for 6 hours and12 hours were significantly up-regulated to be 1.46 and 1.81 times of that in B.dorsalis adults which were treated without any insecticide,proved that yellow gene MK529915 participated in the reaction and metabolism of B.dorsalis to trichlorfon,and proved that yellow gene MK529915 played important roles in midgut of B.dorsalis either. |
PDF Full Text Request