복숭아혹진딧물(Myzus persicae)은 다식성으로 담배, 감자, 고추, 배추, 복숭아 등에 심각한 피해를 입히는 대표적인 농업해충이다. 본 연구에 서는 국내 복숭아혹진딧물 야외개체군의 λ-cyhalothrin, imidacloprid 및 flupyradifurone에 대한 약제 저항성 발달 수준과 점 돌연변이(R81T, L1014F, M918L)의 발생 여부를 확인하였다. 또한, qRT-PCR을 통해 사이토크롬 P450 유전자인 CYP6CY3 발현량을 확인하였다. 그 결과, λ -cyhalothrin은 저항성비(Resistance Ratio, RR)가 12개 모든 지역이 > 200으로 높은 저항성을 보였다. Imidacloprid와 flupyradifurone은 YS, UR, HY, 그리고 WJ 개체군에서 > 200의 저항성비로 높은 저항성을 나타냈다. R81T는 12개 집단 중 약 50%, L1014F는 약 33.3%, M918L은 100%에서 발현하였다. 또한 qRT-PCR을 통해 imidacloprid 저항성 개체에서 subunit CYP6CY3의 발현량이 높게 나타난 것을 확인하였다. 이러 한 결과는 M918L 점 돌연변이는 λ-cyhalothrin 저항성 진단마커로, R81T와 CYP6CY3의 높은 발현은 imidacloprid 저항성 진단마커로 활용이 가능하다는 것을 시사한다.

The cotton aphid, Aphis gossypii (Glover), is one of the main pests in various vegetable crops due to insecticide resistance in Korea. Some insect pests noticed neonicotinoid insecticide resistance such as Nilaparvata lugens, Bemisia tabaci and Myzus persicae etc. and the major player which contributed for developing the resistance was over-expression of P450, particularly CPY6 family. However, A. gossypii was a unique case that they developed non-P450 dependent resistant mechanism. Previously we reported that two point mutations (RtoT in nicotinic acetylcholine receptor, nAChR, beta 1 subunit and RtoT with LtoS in a transcript variant) contribute to develop the imidacolprid resistance in A. gossypii. Moreover we surveyed the mutation(s) in various local field populations. Based on the 3D modeling, we hypothesize that RtoT mutation can reduce the imidacloprid sensitivity. A stretch of 33 amino acid was deleted in the N-terminal region of original transcript of nAChR beta 1 subunit that contained RtoT with LtoS mutations in resistant strain. Among the two transcripts, only original transcript differently expressed between imidacloprid susceptible and resistant strain (resistant ratio = 3,800). Six alpha subunit (1∼5, 7) transcript levels were not significantly different between two strains. Therefore mutation and down-regulation of nAChR beta 1 subunit is also associated with imidacloprid resistance in the A. gossypii.

The effect of electron beam irradiation on development and reproduction of susceptible strain (S) and imidacloprid-resistance strain (IMI-R) of Aphis gossypii were compared. Nymphs and adults of S and IMI-R strain were irradiated at target doses of 50, 100, 150, and 200 Gy. When nymphs were irradiated, emergence rate was not affected at all target dose, but number of F1 nymphs was perfectly inhibited at 150 Gy in both strains when irradiated to the adults, longevity slightly decrease at 150 Gy and above. Fecundity was strongly decreased at 100 Gy, but was not completely inhibited even at 200 Gy. Emergence rate of F1 nymph was decreased at 100 Gy and completely inhibited at 200 Gy. However, there was no significant differences on development and reproduction of S and IMI-R strain. We also conducted the comet assay immediately after irradiation and over the following 7 day periods. In addition, we performed quantitative real-time PCR on several genes.

The melon and cotton aphid Aphis gossypii Glover (Hemiptera; Aphididae) is one of the most serious pests worldwide. We surveyed insecticide susceptibility in A. gossypii field populations to 12 insecticides (6 neonicotinoids, 3 pyrethroids and 3 others) to examine resistance ratios. The levels of insecticide resistance were extremely high, especially to neonicotinoids, such as acetamiprid, clothianidin, thiacloprid and imidacloprid. To identify the neonicotinoid resistance mechanisms, we used an imidacloprid-resistant (IMI-R) strain as a model strain. IMI-R showed an extremely high resistance ratio and also cross-resistance to all the test neonicotinoids. However, there was little or no cross-resistance to the other insecticides, including sulfoxaflor. Synergist tests and enzyme activity assays suggested the absence of resistance mechanisms based on enhanced detoxification enzymes, such as cytochrome P450, esterase and glutathione S-transferase. One point mutation was found in the beta1 subunit loop D region of the nicotinic acetylcholine receptor (nAChR) of the IMI-R strain. This R81T point mutation was also found in field populations collected from 5 regions. Therefore, the R81T point mutation was identified as an important mechanism of imidacloprid resistance in A. gossypii.

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pests in various vegetable crops. In Korea, some field populations of A. gossypii especially in greenhouse showed high resistance against neonicotinoids. The imidaclopridresistant strain (IR) selected from one of the greenhouse strains was found to be about 3,800 folds more resistant to imidacloprid, compared to the susceptible strain (S), as judged by LC50 values. To identify differentially expressed genes in IR, an isogenic strain, reverse susceptible strain (IRS) was generated from IR and comparative transcriptome analyses based on GS-FLX were conducted using total RNAs extracted from both IR and IRS. Also we confirmed protein expression patterns by 2DE and detoxification enzyme over-expression by synergist test. However there was no significant variation among IR, IRS and S. Comparison of the nucleotide sequence of seven nicotinic acetylcholine receptor (nAChR) subunit (alpha 1-5,7 and beta 1) genes from S and IR strain revealed a point mutation causing an arginine to threonine substitution (R81T) in the loop D region of the nAChR beta 1 subunit of the IR. These mechanisms were also reported in M. persicae and this amino acid change confers a vertebrate-like character to the insect nAChR and results in reduced sensitivity to neonicotinoids. Moreover an extra point mutation, L80S (leucine to serine substitution) was also detected nearby R81T mutation in nAChR beta 1 subunit variant. These mutations can be an additive factor in imidacloprid resistance in A. gossypii. This is the first report of imidacloprid resistance mechanism in A.gossypii. Further, this would be helpful in managing A. gossypii resistant populations in field.

The cotton aphid, Aphis gossypii (Glover) (Hemiptera:Aphididae), is a highly polyphagous pest that directly or indirectly damages cultivated plants. Six field-collected populations of cotton aphid, A. gossypii (BY-A, BY-B, YJ-A, YJ-B, CJ-A, and CJ-B) were tested for susceptibility to 14 different insecticides. Most population exhibited high to very high levels of resistance to neonicotinoid. Among them, a strain showing resistance to imidacloprid were selected and showed 1,543-fold in resistance as compared with susceptible strain. Piperonyl butoxide (PBO), diethyl maleate (DEM), and S,S,S tributyl-phosphorothiolate (DEF) failed to synergize imidacloprid in this resistant population. In addition, the activity of detoxification enzymes (P450, EST, GST) were no differences between susceptibility and imidalcoprid resistance strain. However, by analyzing the nicotinic acetylcholine receptor β1 subunit loop D, R81T point mutation was detected in BY-A, BY-B, YJ-A, and YJ-B strain.

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pest in seed potato and various vegetable cultivation. The imidacloprid-resistant strain (IR) was over 300-fold more resistant to imidacloprid compared to a susceptible strain (S) as judged by LC50 values. A highly imidacloprid-resistant local field population (L) was collected from cucumber at Gangwha island in 4th August 2011. Even though neonicotinoid insecticides especially imidacloprid were sprayed six times during June and July, aphid density was too high to be counted. To identify differentially expressed genes in IR or L, comparative transcriptome analyses based on GS-FLX were conducted using total RNAs extracted from IR, L and S strains. Futhermore, to search the resistance associated proteins in IR or L, comparative proteome analyses based on 2DE were conducted using total proteins extracted from IR, L and S strains. Few common candidate genes detected among IR and L such as ABC genes. Comparison of the nucleotide sequence of six nicotinic acetylcholine receptor (nAChR) subunit (alpha 1-5, beta 1) genes from IR, L and S strain revealed a point mutation in the loop D region of the nAChR beta 1 subunit of the IR, causing an arginine to threonine substitution (R81T). These mechanisms also reported in Myzus persicae and this amino acid change confers a vertebrate-like character to the insect nAChR and results in reduced sensitivity to neonicotinoids.

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pest in the cultivation of various vegetables. A highly imidacloprid-resistant field population (CA-L) was collected from cucumber at Gangwha island in 4th August 2011. Even though neonicotinoid insecticides especially imidacloprid were sprayed six times during June and July, aphid density was too high to be counted. IEF and 2DE analyses revealed that general esterase isozyme (pI. 5) in CA-L were dramatically overexpressed and more isozyme spots identified in CA-L compared to susceptible (CA-S) strain. To identify differentially expressed genes in CA-L, comparative transcriptome analyses based on GS-FLX were conducted with total RNA extracted from CA-L, which generated ca. 143 Mb reads. Previously reported, comparative transcriptome analyses performed in imidacloprid resistant (CA-IR) and CA-S. The comparative transcriptome analyses re-investigated after all data sets were combined together. As previously reported, seven ATP-binding cassette (ABC) transporter genes were newly identified in A. gossypii, among which only ABCC9 gene was highly expressed in CA-IR and L. These results suggested that ABCC subfamily associated with imidacloprid resistance in A. gossypii.

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pest in seed potato and various vegetable cultivation. The imidacloprid-resistant strain (IR) was over 200-fold more resistant to imidacloprid compared to a susceptible strain (S) as judged by LC50 values. The IR showed cross resistances to other neonicotinoid insecticides. IEF and 2DE analyses revealed that general esterase isozyme patterns in IR were almost identical to those of S. Nevertheless, a significantly overexpressed protein spot was detected in IR. To identify differentially expressed genes in IR, comparative transcriptome analyses based on GS-FLX were conducted using total RNAs extracted from both IR and S strains, which generated ca. 290 Mb reads for each strain. Generally, most nicotinic acetylcholine receptor subunit genes, such as alpha 2 and beta 1, were more transcribed in S than in IR. In contrast, only alpha 5 subunit gene was 1.8 fold more expressed in IR. Seven ATP-binding cassette (ABC) transporter genes were newly identified in A. gossypii, among which only ABCC9 gene was highly expressed in IR. Therefore, this ABCC subfamily, a member of the MRP subfamily which is involved in multi-drug resistance, could be one of the main factors associated with imidacloprid resistance in A. gossypii.

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pest in seed potato and various vegetable cultivation. The imidacloprid resistant strain (IR) was over 200 fold more resistant to imidacloprid compared to the susceptible strain (S) as judged by the LC50 values and IR showed cross resistant to acetamiprid, thiamethoxam, thiacloprid, clothianidin. By using the suppression subtractive hybridization method, a imidacloprid resistant associated cDNA library was constructed in adult cotton aphid. In total 115 differentially expressed cDNA clones were obtained. Any point mutation detected in nicotinic acetylcholine receptor alpha 1~5 and beta 1 subunits in the IR. Based on IEF, the IR general esterase isozyme banding patterns were identical with that of S.

Imidacloprid에 저항성을 보이는 복숭아혹진딧물에 대해 몇가지 저항성 기작을 조사하였다. 복숭아혹진딧물에 약제를 처리한 후의 체벽잔류량은 처리 후 시간이 지남에 따라 서서히 감소되었으나 감수성계통과 저항성계통 간에 체벽침투력의 유의성은 없었다. 체내잔류량은 양 계통에서 시간이 지남에 따라 점차 증가되었으며 감수성에서 많았다. 배설량은 저항성계통이 감수성계통보다 많아 약제 대시가 빠르게 나타났다. Imidacloprid 저항성계통의 acetylcholine-sterase (AChE) 활성은 감수성계통 보다 약 1.4배 높았으며, imidacloprid는 AChE를 저해하지 않았다. 저항성계통에 대해 산화효소 저해제인 PBO(piperonyl butoxide)와 esterase 저해제인 IBP (iprobenfos)를 혼합하여 사용한 결과 Imidacloprid : PBO의 비율은 1 : 1과 1 : 5에서 각각 69.4, 250배의 독성을 보였으며, IBP와 혼합사용(1 : 1과 1 : 5)에서는 각각 227, 80.6배의 독성을 보였다. 감수성계통에 PBO와 IBP를 imidacloprid와 같은 비율로 혼합처리 하였을 경우 단독처리와 독성차이가 보이지 않았다. -naphtyl butyrate와 -naphtyl acetate 기질을 사용하여 비특이적 esterase의 활성을 측정한 결과 저항성계통의 감수성 계통보다 esteraseghkf성이 높게 나타났다. 따라서 imidacloprid 저항성 복숭아혹진딧물의 저항성 기작에는 산화효소와 esterase가 관여되고 있음을 알 수 있었다.