棉蚜2个乙酰胆碱酯酶cDNA片段的基因克隆与序列分析

采用RT－PCR技术,利用简并引物从棉蚜（Aphis gossypii Glover)中克隆出2个乙酰胆碱酯酶基因的cDNA片段,Ag,ace 1l和Ag.ace2.Ag.ace1基因的cDNA片段为282bp,编码94个氨基酸;Ag.ace2基因的cDNA片段为264bp,编码88个氨基酸。扩增获得的2个乙酰胆碱酯酶基因cDNA片段所编码的氨基酸序列均与其他昆虫的乙酰胆碱酯酶基因有很高的同源性。首次从一种昆虫中克隆出2个乙酰胆碱酯酶基因片段,为同一种昆虫中存在多个乙酰胆碱酯酶基因的假设提供了直接的分子生物学证据。     

Oral Delivery Mediated RNA Interference of a Carboxylesterase Gene Results in Reduced Resistance to Organophosphorus Insecticides in the Cotton Aphid,Aphis gossypii Glover

Background

RNA interference (RNAi) is an effective tool to examine the function of individual genes. Carboxylesterases (CarE, EC 3.1.1.1) are known to play significant roles in the metabolism of xenobiotic compounds in many insect species. Previous studies in our laboratory found that CarE expression was up-regulated in Aphis gossypii (Glover) (Hemiptera: Aphididae) adults of both omethoate and malathion resistant strains, indicating the potential involvement of CarE in organophosphorus (OP) insecticide resistance. Functional analysis (RNAi) is therefore warranted to investigate the role of CarE in A. gossypii to OPs resistance.

Result

CarE expression in omethoate resistant individuals of Aphis gossypii was dramatically suppressed following ingestion of dsRNA-CarE. The highest knockdown efficiency (33%) was observed at 72 h after feeding when dsRNA-CarE concentration was 100 ng/µL. The CarE activities from the CarE knockdown aphids were consistent with the correspondingly significant reduction in CarE expression. The CarE activity in the individuals of control aphids was concentrated in the range of 650–900 mOD/per/min, while in the individuals of dsRNA-CarE-fed aphids, the CarE activity was concentrated in the range of 500–800 mOD/per/min. In vitro inhibition experiments also demonstrated that total CarE activity in the CarE knockdown aphids decreased significantly as compared to control aphids. Bioassay results of aphids fed dsRNA-CarE indicated that suppression of CarE expression increased susceptibility to omethoate in individuals of the resistant aphid strains.

Conclusion

The results of this study not only suggest that ingestion of dsRNA through artificial diet could be exploited for functional genomic studies in cotton aphids, but also indicate that CarE can be considered as a major target of organophosphorus insecticide (OPs) resistance in A. gossypii. Further, our results suggest that the CarE would be a propitious target for OPs resistant aphid control, and insect-resistant transgenic plants may be obtained through plant RNAi-mediated silencing of insect CarE expression.     

Ecological specialization of the aphid Aphis gossypii Glover on cultivated host plants

Many plant-feeding insect species considered to be polyphagous are in fact composed of genetically differentiated sympatric populations that use different hosts and between which gene flow still exists. We studied the population genetic structure of the cotton-melon aphid Aphis gossypii that is considered as one of the most polyphagous aphid species. We used eight microsatellites to analyse the genetic diversity of numerous samples of A. gossypii collected over several years at a large geographical scale on annual crops from different plant families. The number of multilocus genotypes detected was extremely low and the genotypes were found to be associated with host plants. Five host races were unambiguously identified (Cucurbitaceae, cotton, eggplant, potato and chili- or sweet pepper). These host races were dominated by asexual clones. Plant transfer experiments using several specialized clones further confirmed the existence of host-associated trade-offs. Finally, both genetic and experimental data suggested that plants of the genus Hibiscus may be used as refuge for the specialized clones. Resource abundance is discussed as a key factor involved in the process of ecological specialization in A. gossypii.     

Interspecific Associations between Cycloneda sanguinea and Two Aphid Species (Aphis gossypii and Hyadaphis foeniculi) in Sole-Crop and Fennel-Cotton Intercropping Systems

Aphids cause significant damage to crop plants. Studies regarding predator-prey relationships in fennel (Foeniculum vulgare Mill.) and cotton (Gossypium hirsutum L.) crops are important for understanding essential ecological interactions in the context of intercropping and for establishing pest management programs for aphids. This study evaluated the association among Hyadaphis foeniculi (Passerini) (Hemiptera: Aphididae), Aphis gossypii Glover (Hemiptera: Aphididae) and Cycloneda sanguinea (L.) (Coleoptera: Coccinellidae) in cotton with coloured fibres, fennel and cotton intercropped with fennel. Association analysis was used to investigate whether the presence or absence of prey and predator species can indicate possible interactions between aphids and ladybugs. Significant associations among both apterous and alate H. foeniculi and C. sanguinea were observed in both the fennel and fennel-cotton intercropping systems. The similarity analysis showed that the presence of aphids and ladybugs in the same system is significantly dependent on the type of crop. A substantial amount of evidence indicates that the presence of the ladybug C. sanguinea, is associated with apterous or alate A. gossypii and H. foeniculi in fennel-cotton intercropping system. We recommend that future research vising integrated aphid management taking into account these associations for take decisions.     

Identification of Aphis gossypii Glover (Hemiptera: Aphididae) Biotypes from Different Host Plants in North China

Background

The cotton-melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a polyphagous species with a worldwide distribution and a variety of biotypes. North China is a traditional agricultural area with abundant winter and summer hosts of A. gossypii. While the life cycles of A. gossypii on different plants have been well studied, those of the biotypes of North China are still unclear.

Results

Host transfer experiments showed that A. gossypii from North China has two host-specialized biotypes: cotton and cucumber. Based on complete mitochondrial sequences, we identified a molecular marker with five single-nucleotide polymorphisms to distinguish the biotypes. Using this marker, a large-scale study of biotypes on primary winter and summer hosts was conducted. All A. gossypii collected from three primary hosts—hibiscus, pomegranate, and Chinese prickly ash—were cotton biotypes, with more cotton-melon aphids found on hibiscus than the other two species. In May, alate cotton and cucumber biotypes coexisted on cotton and cucumber seedlings, but each preferred its natal host. Both biotypes existed on zucchini, although the cucumber biotype was more numerous. Aphids on muskmelon were all cucumber biotypes, whereas most aphids on kidney bean were cotton biotypes. Aphids on seedlings of potato and cowpea belong to other species. In August, aphids on cotton and cucumber were the respective biotypes, with zucchini still hosting both biotypes as before. Thus, the biotypes had different fitnesses on different host plants.

Conclusions

Two host-specialized biotypes (cotton and cucumber) are present in North China. Hibiscus, pomegranate, and Chinese prickly ash can serve as winter hosts for the cotton biotype but not the cucumber biotype in North China. The fitnesses of the two host-specialized biotypes differ on various summer hosts. When alate aphids migrate to summer hosts, they cannot accurately land on the corresponding plant.     

缩节胺对棉蚜体内能源物质的影响

为探究缩节胺对棉蚜体内能源物质的影响,以室内繁殖棉蚜为试材,采用生化酶学方法,测定了缩节胺对棉蚜代谢过程中主要能源物质脂类、蛋白质、总糖、游离氨基酸含量。结果表明:15 mg/L、25 mg/L、35 mg/L、50 mg/L缩节胺处理下的棉蚜体内三油酸甘油酯含量均比对照组低,且处理初期随缩节胺浓度增大,甘油酯含量逐渐下降;总糖、游离氨基酸及蛋白质含量均比对照组高,游离氨基酸含量变化趋势与蛋白质含量变化趋势相似。表明棉蚜受到缩节胺胁迫初期时,棉蚜以消耗体内脂肪能源物质来应对缩节胺对自身的胁迫作用,与此同时,棉蚜体内糖类、游离氨基酸、蛋白质能源物质开始积累,以维持棉蚜正常的生命活动。     

Genotypic diversity of the cotton-melon aphid Aphis gossypii (Glover) in Tunisia is structured by host plants

The study of intraspecific variation with respect to host plant utilization in polyphagous insects is crucial for understanding evolutionary patterns of insect-plant interactions. Aphis gossypii (Glover) is a cosmopolitan and extremely polyphagous aphid species. If host plant species or families constitute selective regimes to these aphids, genetic differentiation and host associated adaptation may occur. In this study, we describe the genetic structure of A. gossypii collected in six localities in Tunisia on different vegetable crops, on citrus trees and on Hibiscus. The aim was to determine if the aphid populations are structured in relation to the host plants and if such differentiation is consistent among localities. The genetic variability of A. gossypii samples was examined at eight microsatellite loci. We identified only 11 multilocus genotypes among 559 individuals. Significant deviations from Hardy-Weinberg equilibrium, linkage disequilibria and absence of recombinant genotypes, confirmed that A. gossypii reproduces by continuous apomictic parthenogenesis. Genetic differentiation between localities was not significant, whereas a strong differentiation was observed between host plant families (0.175相似文献   

Organophosphorus resistance in Aphis gossypii (Hemiptera: Aphididae) on chrysanthemum in the UK

The FAO standard dip-test was used to test resistance of glasshouse populations of Aphis gossypii from chrysanthemums to the organophosphorus insecticides heptenophos and diazinon. Compared with a susceptible population collected in 1981, resistance factors to diazinon of up to 538 were found in populations collected after 1984. Resistance to heptenophos was less than that to diazinon but some populations collected after 1984 were 34 times as resistant as the susceptible strain. Possible reasons for the sudden increase in resistance in A. gossypii and current prospects for control of resistant populations are discussed.     

Vertical and Temporal Distribution of Aphis gossypii Glover and Coccinellid Populations on Different Chilli (Capsicum annuum) Varieties

The vertical and temporal distribution of an aphid, Aphis gossypii Glover, and the coccinellid populations on six chilli varieties were studied. The total number of apterous aphid per plant stratum was significantly different among plant strata of a particular variety (treatment) as well as among the treatments. Generally, the total number of aphids was significantly greater in the lower stratum than in the middle and upper strata. However, the varieties with erect and open plant architecture (Kulai and MC 11) had significantly less number of apterous aphids at all strata as compared to varieties with compact or prostrate plant architectures. There was a significant difference in the total number of coccinellids per plant strata among the treatments but not within a treatment. The distribution of apterous aphid populations varied significantly among sampling periods and treatments. The temporal distribution of coccinellids showed a similar trend as that of apterous aphids. The total number of alate aphids caught per week was significantly different among the sampling periods. However, its population was significantly greater during the early season and gradually declined as the season progressed except during June 18 to 24. The importance of recording the most observed coccinellids species, which limit the aphid populations at each particular plant stratum per variety, and the conditions that favor natural enemies are also discussed.     

Purification and characterization of acetylcholinesterase from cotton aphid (Aphis gossypii Glover)

A simple and effective method was set up to purify acetylcholinesterase (AChE, EC3.1.1.7) from the cotton aphid, Aphis gossypii Glover. The procedure involved filtration on a sephadex G-25 column, separation with sephadex G-200 and procainamide affinity column. AChE from both susceptible and resistant strains were purified to a single band as resolved on denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity increased by 35,100- and 33,680-fold with a yield of 30.3 and 29.8%, respectively. The molecular mass of the purified AChE was about 63,500 Dalton as determined by SDS-PAGE. However, three bands resolved on PAGE gel electrophoresis, leading to the inference that native AChE exists in three forms. The optimum conditions for measuring the activity of purified AChE with kinetic method were 0.02M phosphate buffer, pH7.2, 0.02 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and 25 degrees C. Investigation also revealed that crude extract and purified AChE had different kinetic characteristics and inhibitory properties. They responded differently to varied DTNB, ATChI, and phosphate buffer ion concentrations, as well as pH, temperature, and inhibitors. The purified AChE was more sensitive to eserine, methamidophos, and pirimicarb. Especially for resistant aphids, the sensitivity of purified AChE to methamidophos and pirimicarb was enhanced 6.43 and 11.73 times, respectively. We infer that one or more factors in the crude extract from the resistance strain have more influence on AChE sensitivity. Further study is needed to investigate the basis of these observations.     

Antibiosis and antixenosis to Aphis gossypii (Homoptera: Aphididac) in Colocasia esculenta

Fifty cultivars of taro, Colocasia esculenta (L.) Schott (Araceae), collected from islands in Micronesia and Polynesia, eight cultivars from the University of Hawaii's taro germplasm collection, and a closely related aroid, Xanthosorna sagittifolium (L.) (Araceae), were screened for antibiosis and antixenosis to Aphis gossypii Clover. Life history data for A. gossypii were collected by assessing survivorship and fecundity of aphids caged on taro leaves in the field. Significant differences in aphid reproductive rate and longevity were observed among the taro cultivars, and cultivars were ranked from most resistant to most susceptible. Antixenosis was assayed in the laboratory in a multiround choice test where A. gossypii were offered four leaf discs excised from different taro cultivars. Additionally, field observations of aphid abundance on taro cultivars were made to corroborate clip cage studies and laboratory experiments. 'Iliuaua','Rumung Mary','Maria', 'Ketan 36', and'Agaga' were the most resistant in terms of reducing aphid fecundity and survivorship, whereas the Iliuana,'Purple', 'TC-83001', and 'Putih 24' were least preferred in aphid choice tests. X. sagittifolium consistently exhibited strong aphid resistance. Resistant cultivars identified in this study may form the basis of breeding programs seeking to combine aphid resistance with other desirable agronomic traits in taro.     

Different exposure methods to neonicotinoids influenced biochemical characteristics in cotton aphid,Aphis gossypii Glover (Hemiptera:Aphididae)

We measured lipid, carbohydrate and protein content in three strains of cotton aphid, Aphis gossypii (Hemiptera: Aphididae) from very resistant to neonicotinoids up to semi sensitive and sensitive strains in different exposure method of spraying to neonicotinoids. We observed changes in energy source rates at which each substrate was metabolised under starvation, selection and residue stress method spraying that assist in metabolisation of their biochemical parameters. These particular exposure methods influenced some of biochemical parameters in cotton aphid. Results indicated that Sugar, glycogen, total protein and lipid in aphids have significant changes. Studies showed that among three strains called Ag-R, Ag-M and Ag-D which have different susceptibilities to neonicotinoids, strain Ag-R was the most tolerated aphids in counter of imidacloprid and thiametoxam, strain Ag-D was somewhat more tolerated to these insecticides and strain Ag-M was the most sensitive strain which has the resistance factor of nearly 890 in starvation method and was different depending on the method of exposure. Among energy sources, the total lipid in susceptible strain were decreasing more than resistant strain, whereas total proteins were increasing in resistant strain compared with sensitive strain. Total Glycogen was affected significantly in the stress condition which caused an increase and was the most resistant strain.     

Relationship between Aphis gossypii (Homoptera: Aphididae) and sticky lint in cotton

The study was conducted in the northern Texas Rolling Plains in 1999 to define the relationship between number of cotton aphids, Aphis gossypii Glover, and resulting contamination of cotton lint by honeydew. Whole-plot treatments were three furrow irrigation management treatments: cotton grown without supplemental irrigation (dryland), irrigated cotton with last irrigation in mid August, and irrigated cotton with last irrigation in late August. Subplots within each irrigation treatment included an untreated check, a plot treated with lambda-cyhalothrin to stimulate aphid population increase, a plot treated with lambda-cyhalothrin followed by pymetrozine after aphids began to increase, and a plot treated with lambda-cyhalothrin followed by thiamethoxam after aphids began to increase. Cotton aphids were counted on leaves picked from the top and bottom half of the plant. Cotton lint was analyzed for contamination by glucose, fructose, sucrose, and melezitose secreted by cotton aphids, and percentage leaf moisture and nitrogen and leaf sucrose concentrations were determined. The manual sticky cotton thermodetector was used to determine degree of lint stickinesss. There was a significant relationship between thermodetector counts and melezitose contamination on lint, and a melezitose concentration of 90.9 microg/g of lint was associated with a thermodetector count of 10, the threshold for sticky lint problems in textile mills. An equation was developed to estimate melezitose concentration on lint as a function of average numbers of aphids per leaf and the interaction between percentage leaf moisture and nitrogen. The number of aphids per leaf associated with a melezitose concentration of 90.9 microg/g of lint ranged from 11.1 to 50.1, depending on percentage leaf moisture and nitrogen. The threshold for sticky lint problems occurred when aphid numbers ranged between 11.1 and 50.1 per leaf after bolls open.     

Development of neonicotinoid resistance in the cotton aphid Aphis gossypii (Hemiptera: Aphididae) in Japan

A neonicotinoid-resistant Aphis gossypii Glover population was first detected from cucumber and sweet pepper crops in Miyazaki Prefecture, Japan, in April 2012. In this study, we determined the insecticide susceptibility of five field-collected populations of A. gossypii to seven neonicotinoids. Insecticide susceptibility was determined by performing bioassays and using the seedling-treatment method. Insecticide susceptibility of a susceptible population of A. gossypii, originally collected from a cucumber field in 2008, was also determined. High mortality (96.4–100 %) was observed in the susceptible population for the seven insecticides. In contrast, the mortality rate of the five field-collected populations collected in 2012 was low for five of the seven neonicotinoid insecticides tested: imidacloprid (26.7–65.5 %), dinotefuran (0–27.3 %), clothianidin (20.0–35.7 %), thiamethoxam (7.1–42.3 %), and nitenpyram (6.7–32.1 %). Mortality was 86.2–100 % for acetamiprid and 90.2–100 % for thiacloprid. The resistance ratio in comparison with the susceptible population was the highest for clothianidin (687-fold), lowest for thiacloprid (17-fold), and ranged from 43- to 253-fold for the other five neonicotinoids. Thus, the frequency of insecticide use needs to be reduced and other insecticides need to be employed in order to prevent the development of resistance to these two insecticides in the future.     

Morphological discrimination of Aphis gossypii (Hemiptera: Aphididae) populations feeding on Compositae

Aphis gossypii Glover is a polyphagous aphid pest with a worldwide distribution. However, there is evidence that on a global scale the name A. gossypii is being applied to a number of forms with different life cycles and/or host-plant associations. Morphometric variation of A. gossypii samples from crops and non-cultivated plants in many parts of the world was examined, to determine whether this variation is correlated with the hosts from which the aphids originated. Samples of A. gossypii were collected from Cucurbitaceae and Malvaceae in Europe, and from Compositae in various parts of the world. Morphometric data for 13 parameters measured from 97 clonal lineages (728 specimens) and 27 field-collected samples (313 specimens) were analysed by a series of canonical variates analyses, using the field sample/clonal lineage as grouping factor. Clonal lineages were reared on a common host in controlled conditions to standardize the effect of host and environment on morphology. The analyses provided a clear morphometric separation of the aphids originating from Compositae and those collected on Cucurbitaceae and Malvaceae, regardless of the geographical origin of the aphids and the host plant on which they were reared. This indicates that within A. gossypii there are two widely distributed host races or subspecies with different plant family associations. The taxonomic implications are discussed.     

Fitness cost associated with carbosulfan resistance in Aphis gossypii Glover (Hemiptera: Aphididae)

In order to collect basic information for a resistance management program of Aphis gossypii Glover to carbosulfan, studies to evaluate fitness costs associated to this resistance were conducted under laboratory conditions. For this purpose, we studied the stability of carbosulfan resistance in A. gossypii populations with differing initial frequencies of resistance and compared life-history traits of carbosulfan-susceptible and resistant strains. The stability of resistance was followed in three populations with initial frequencies of 20, 50 and 80% of resistant individuals. The susceptibility to carbosulfan was monthly estimated by using the diagnostic concentration of 32 ppm. The biology of carbosulfan-susceptible and resistant strains was evaluated in cotton plants kept under controlled climatic conditions. The resistance to carbosulfan was unstable, that is, there was a significant increase in the susceptibility of different populations of A. gossypii to this insecticide in the absence of selection pressure. Comparison of biological traits between carbosulfan-susceptible and resistant strains showed that there is a fitness cost associated with resistance. The resistant strain showed lower fecundity, shorter reproductive period and lower longevity than the susceptible strain. The net reproductive rate (Ro) was 14.04 and 43.18 and the finite rate of increase (lambda) was 1.25 and 1.30 females/female/day for the resistant and susceptible strains respectively. Therefore, the instability of carbosulfan resistance in A. gossypii can be exploited in resistance management programs.