Microsatellites isolated from diamondback moth,Plutella xylostella (L.), for studies of dispersal in Australian populations

Diamondback moth, Plutella xylostella (L.), is a worldwide agricultural pest that has developed resistance to many insecticides used for its control. Population structure and gene flow are yet to be determined for P. xylostella in Australia, but are important factors for the design of effective control strategies. We have isolated six polymorphic microsatellite markers: three from a partial genomic library, two from an Expressed Sequence Tagged library and one from an aminopeptidase intron of P. xylostella. These microsatellites will be used to determine population structure and gene flow in Australian populations of P. xylostella to improve insecticide resistance management.     

小菜蛾对几种常用药剂的敏感性测定

采用叶片浸溃法,在2001年10月和2002年10月分别测定了福州上街蔬菜基地的小菜蛾,对10种杀虫剂的敏感性。结果表明,上街菜区小菜蛾对灭多威、杀虫双、辛硫磷、毒死蜱、杀灭菊酯和功夫菊酯等农药极不敏感,已不能有效控制小菜蛾的危害。而小菜蛾对氟啶脲、阿维菌素、氟虫腈和虫螨腈比较敏感。通过两次毒力测定比较(2002年10月的LC50;2001年10月的LC50),结果表明,大多数杀虫剂敏感性变化较小。但小菜蛾对阿维菌素敏感性明显下降,毒力倍数达6．65倍,对氟虫腈毒力倍数相差1．48倍。此外,2002年10月小菜蛾对功夫菊酯的LC50为2001年10月的2．02倍。用浸叶法和浸溃法测定多杀菌素、氟虫腈与虫螨腈推荐浓度对小菜蛾幼虫的毒杀作用。结果表明3种药剂浸叶法处理效果均较理想。     

小菜蛾酪氨酸酶生物化学性质研究

采用时间动力学法对小菜蛾Plutella xylostella酪氨酸酶的最适反应条件、对抑制剂的敏感度以及发育期变化规律进行了研究。结果表明：小菜蛾酪氨酸酶反应的最适pH值为6.4,反应线性时间为0～2 min,在22℃和4℃下其活性分别可保持12 h 和72 h,在-20℃下至少可保持7 天。不同发育阶段小菜蛾酪氨酸酶活性的大小依次为预蛹＞4龄幼虫＞蛹＞1龄、2龄、3龄幼虫＞成虫。苯基硫脲（phenyl thiourea,PTU）对小菜蛾阿维菌素抗性品系和敏感品系酪氨酸酶活性的抑制中浓度（I₅₀）值分别为1.1796 μmol/L和1.2795 μmol/L,二者无明显差异。     

小菜蛾的耐热性

小菜蛾是世界范围内十字花科蔬菜上的重要害虫.临界高温(critical thermal maxi-mum,CTMax)是昆虫耐热性的常用指标.采用动态加热方法,利用自行组装的装置测定了小菜蛾的临界高温,以此作为其耐热性指标,研究发育阶段、饲养温度、世代、性别和热激对小菜蛾耐热性的影响.结果表明:25℃下饲养的小菜蛾4龄幼虫的CTMax均值为50.31℃,显著高于1龄幼虫(43.03℃)、2龄幼虫(46.39℃)、3龄幼虫(49.67℃)以及雌性成虫(45.76℃)和雄性成虫(47.73℃);不同饲养温度(20、25和30℃)下成虫耐热性无显著差异;30℃下饲养1代、3代及6代的不同世代成虫CTMax也无显著变化;所有处理雌雄成虫的CTMax无显著差异;40℃下45 min热激可使5日龄雄成虫的CTMax值从45.51℃增加到46.49℃.     

Evidence for behavioural resistance by the diamondback moth, Plutella xylostella (L.)

Abstract:  The diamondback moth (DBM), Plutella xylostella (L.) (Lep., Plutellidae), is an important pest of brassicaceous crops worldwide. It has developed resistance to almost every synthetic insecticide applied in the field and consequently is often difficult to control in crucifer-growing areas. DBM oviposition behaviour was investigated in greenhouse and growth chamber conditions with seven cruciferous and one non-cruciferous plant varieties. In free choice tests, females deposited more eggs on the stem near the soil-stem interface than on leaves. Our findings suggest that DBM is capable of developing behavioural resistance through oviposition site selection to avoid lethal doses of foliar-applied insecticides in the field.     

小菜蛾对苦皮藤素抗性选育及交互抗性测定

敏感小菜蛾Plutella xylostella (L.)经苦皮藤素20代的抗性选育,其抗性增长21.57倍。选育的小菜蛾抗性品系对杀虫双、杀螟丹和叶蝉散分别有4.63、4.11和3.71倍的交互抗性;对溴氰菊酯、氯菊酯、氯氰菊酯分别有0.22、0.01和0.26倍的负交互抗性。高抗杀螟丹、杀虫双小菜蛾品系对苦皮藤素无明显交互抗性,而高抗溴氰菊酯小菜蛾品系对苦皮藤素有3.61倍的交互抗性。     

小菜蛾饲养技术与规范

小菜蛾Plutella xylostella(L.)是一种世界性危害的十字花科蔬菜重要害虫,其抗药性问题尤为突出。目前,小菜蛾已成为害虫抗药性等多个研究领域的重要研究对象,而小菜蛾室内大规模人工饲养是开展相关研究的基础。针对目前小菜蛾室内大规模人工饲养技术存在不规范、不科学、不标准等问题,本文重点介绍了本实验室成熟系统的小菜蛾室内大规模人工饲养方法——甘蓝萝卜苗法。该方法简单、易操作、实用性强,因此,可以作为一种小菜蛾室内大规模人工饲养的技术规范在全国范围内推广。     

印楝素乳油x对-小菜蛾种群的控制作用

通过室内外系统试验探讨了印楝素乳油对小菜蛾Plutella xylostella种群的控制作用.结果表明,其控制作用主要表现在对成虫产卵的显著忌避作用、对幼虫的拒食作用以及对生长发育的影响和减少幼虫危害力等方面.印楝素乳油0.005 mL/L处理对成虫的忌避率为78.3%,对幼虫的选择性和非选择性拒食率分别为71.8%和62.9%,幼虫总取食量是对照的59.0%,使小菜蛾种群趋势指数(I值)降为2.4,干扰作用控制指数(1lpc)为0.1079.采用状态空间分析法就印楝素乳油施用后小菜蛾种群数量动态的模拟分析亦表明,其显著的控制作用表现在种群发展初期对成虫的忌避作用.     

印楝素乳油对小菜蛾种群的控制作用

通过室内外系统试验探讨了印楝素乳油对小菜蛾Plutella xylostella种群的控制作用。结果表明,其控制作用主要表现在对成虫产卵的显忌避作用。对幼虫的拒食作用以及对生长发育的影响和减少幼虫危害为等方面,印楝素乳油0.005mL/59.0%,使小菜蛾种群趋势指数（I值）降为2.4干扰作用控制指数（IIPC）为0.1079,采用状态空间分析法就印楝素乳油施用后小菜蛾种群数量动态的模拟分析亦表明,其显的控制作用表现在种群发展初期对成虫的忌避作用。     

云南弥渡小菜蛾发生及防治

通过田间调查 ,查明弥渡县小菜蛾年发生 1 0～ 1 2代 ,发生危害期有 9个月 ,一般有两个发生高峰期 ,即 4～ 6月和 9～ 1 0月 ,春季危害比秋季重 ;其发生影响因素主要有温度、降雨、十字花科蔬菜种植面积以及寄生蜂的种类和数量。针对小菜蛾发生规律及影响因素 ,提出了相应的防治措施。     

小菜蛾在温带地区越冬研究进展

小菜蛾是世界性重要害虫。在热带和亚热带地区冬季的十字花科植物上能正常发育繁殖,可见到各种虫态。但在温带冬季十字花科植物不能生长的地区,小菜蛾的越冬成为一个重要生态学问题。综述了亚洲、北美洲和欧洲小菜蛾越冬的研究进展。小菜蛾在日本的北海道、本州岛的北陆和东北大部分地区不能越冬,越冬北限相当于冬季积雪覆盖时间为60d的区域;在中国,小菜蛾不能在寒冷的东北地区越冬,在长江中下游以南地区冬季可见各虫态,但越冬北限尚不清楚;小菜蛾在冬天气候温和的韩国以及澳大利亚东南部继续发生;北美洲的加拿大西部和安大略地区大量的试验证明小菜蛾不能成功越冬,在美国南部小菜蛾冬季可正常发生,北部小菜蛾的越冬尚未见系统研究报道,但确认春季从南部运输的受小菜蛾感染的甘蓝等种苗是美国北部的重要虫源。小菜蛾在欧洲各地越冬的系统研究未见报道,没有证据表明在英国小菜蛾会发生有显著意义的越冬。目前小菜蛾越冬研究主要采用冬季直接试验观察和基于耐寒性试验的越冬预测两种方法。冬季直接试验观察法包括:(1)利用人工饲养的小菜蛾在田间各种潜在的越冬场所的越冬试验;(2)在秋播、野生或残留的十字花科植物上进行冬季种群的系统抽样调查;(3)越冬前后在前茬为十字花科植物的田块广泛搜寻普查小菜蛾的存活个体。基于耐寒性试验的越冬预测法:在获取小菜蛾越冬场所温度的基础上设计低温处理模式,试验低温处理后小菜蛾的存活率及后续发育和生殖。将试验数据和各地气温或小气候相结合,对小菜蛾在的越冬可能性进行推断。     

Molecular characterization of pheromone biosynthesis activating neuropeptide from the diamondback moth, Plutella xylostella (L.)

Pheromone biosynthesis activating neuropeptide (PBAN) produced in the subesophageal ganglion stimulates pheromone production in the pheromone gland. A cDNA isolated from female adult heads of the diamondback moth (Plutella xylostella (L.)) encodes 193 amino acids including PBAN, designated as Plx-PBAN, and four other neuropeptides (NPs): diapause hormone (DH) homologue, -NP, β-NP and γ-NP. All of the peptides are amidated in their C-termini and shared a conserved motif, FXPR(or K)L structure, as reported from other PBAN cDNAs. Plx-PBAN consists of 30 amino acids, the shortest PBAN so far reported. Plx-PBAN exhibited below 50% homology, compared with other known PBANs. The Plx-DH homologue is structurally different from DH of Bombyx mori. The length of Plx-β-NP (16 amino acids) was the shortest and showed relatively low similarity, whereas γ-NP (10 amino acids in length) was the longest among examined γ-NPs. When female adults were injected with synthetic Plx-PBAN, pheromone production showed a maximal increase 1 h post-injection. RT-PCR screening revealed that Plx-PBAN cDNA was expressed in all examined body parts, with the highest expression level in the head of female adults. Analysis of RT-PCR products indicated the Plx-PBAN sequence was identical in all examined body parts of both sexes. Phylogenetic analysis revealed that the Plx-PBAN gene is distantly related to other PBANs, demonstrated by the relatively low similarity.     

Implication for DNA methylation involved in the host transfer of diamondback moth,Plutella xylostella (L.)

DNA methylation exerts extensive impacts on gene expression of various living organisms exposed to environmental variation. However, little is known whether DNA methylation is involved in the host transfer of diamondback moth, Plutella xylostella (L.), a worldwide destructive pest of crucifers. In this study, we found that P. xylostella genome exhibited a relatively low level of DNA methylation on the basis of the CpG O/E prediction and experimental validation. A significant positive linear correlation was observed between the stage‐specific expressions of PxDNMT1 and DNA methylation levels (5mC content). Particularly, high levels of DNA methylation and gene expression of PxDNMT1 were observed in eggs and mature females of P. xylostella. After host transfer of P. xylostella from Raphanus sativus to Arabidopsis thaliana, we identified some potential genomic loci that might have changed methylation levels. Using the method of fluorescence‐labeled methylation‐sensitive amplified polymorphism (F‐MSAP), we also found the corresponding genes primarily involved in neural system and signaling. The expressions of six candidate genes were verified by qRT‐PCR. One of the genes, Px009600, might be regulated by a DNA methylation‐mediated mechanism in response to host transfer. Our study provides evidence for a functional system of DNA methylation in P. xylostella and its possible role in adaptation during host transfer. Further studies should examine methylation as responsive factors to different host plants and environmental cues in insect pests.     

Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella

Braconid wasps, Cotesia plutellae (Kurdjumov), were collected from parasitized host larvae of diamondback moth, Plutella xylostella (L.) in Korea. Virus particles were found in the oviduct lumen of C. plutellae females. Multiple nucleocapsids with approximately 30-nm diameter and variable length (30-80 nm) were surrounded with a single unit membrane envelope. The parasitization of C. plutellae completely inhibited pupal metamorphosis. The parasitized larvae showed significant decrease in feeding activity and total hemolymph proteins, especially as larval storage proteins. They also showed a significant decrease in immune capacity as evidenced by reduced ability to form hemocyte nodules and reduced phenoloxidase and lysozyme activity. Here, we show that C. plutellae has an endosymbiotic virus like other reported species in Microgastrinae, and suggest that it causes host developmental arrest and immune-depression at parasitization.     

Inheritance of resistance and cross resistance pattern in indoxacarb-resistant diamondback moth Plutella xylostella L.

Leaf-dip assay of Plutella xylostella against indoxacarb showed that the concentration that produced 50% mortality (LC₅₀) of indoxacarb ranged from 20.1 to 11.9 ppm, with highest in Nasik and lowest levels in Coimbatore strains. In selection studies, the LC₅₀ of indoxacarb was 18.5 ppm at generation 1 (G1), which increased to 31.3-fold (167.8 ppm) resistance after ten exposed generations (G10) as compared to unexposed. The LC₅₀ of quinalphos was 74.4 ppm, which increased to 10.0-fold (631.5 ppm) resistance after G10. The LC₅₀ of cypermethrin resistant strain resulted in an 11.5-fold increase in resistance after G10. In P. xylostella , heritability (h²) after ten generations of selection was estimated at 0.4. The number of generations required for tenfold increase in LC₅₀ (1/R) were 6.7. The response to indoxacarb selection in P. xylostella was 0.2 and the selection differential was estimated as 0.4. The phenotypic standard deviation was 0.2. Reciprocal crosses between indoxacarb-resistant and susceptible strains showed that the inheritance of indoxacarb resistance was autosomal. The degree of heritability (D_LC) (0.4, 0.4) indicated incomplete recessive inheritance of indoxacarb resistance.     

Widespread pyrethroid resistance in Australian diamondback moth, Plutella xylostella (L.), is related to multiple mutations in the para sodium channel gene

Populations of Plutella xylostella, extending over 3800 km in southern Australia, show no genetic structure as assessed by microsatellite markers; yet outbreaks of pyrethroid resistance occur sporadically in cropping areas. Since mutations in the para voltage-gated sodium channel gene have been implicated in pyrethroid resistance, we looked for DNA sequence variation at this target among Australian moths. We found two resistance mutations previously reported for this species (L1014F and T929I), as well as a novel substitution (F1020S). Of the eight possible haplotypes formed by combinations of these three biallelic polymorphisms, only four were found in Australian populations: the wild-type allele (w), the kdr mutation allele (kdr) with only L1014F, the super-kdr-like combination of L1014F and T929I (skdrl), and the crashdown allele with only F1020S (cdr). Comparison of genotype frequencies among survivors of permethrin assays with those from untreated controls identified three resistant genotypes: skdrl homozygotes, cdr homozygotes and the corresponding heterozygote, cdr/skrdl - the heterozygote being at least as resistant as either homozygote. Spatial heterogeneity of allele frequencies was conspicuous, both across the continent and among local collections, consistent with reported spatial heterogeneity of pyrethroid resistance. Further, high resistance samples were sometimes associated with high frequency of cdr, sometimes high frequency of skdrl, or sometimes with a high combined cdr+skdrl frequency. The skdrl and cdr alleles explain a high proportion of the Australia-wide resistance variation. These data add to evidence that nerve insensitivity by mutations in the para-sodium channel gene is a common pyrethroid resistance mechanism in P. xylostella.     

Disentangling factors limiting diamondback moth,Plutella xylostella (L.), spatio‐temporal population abundance: A tool for pest forecasting

Data‐mining techniques play an important role in hyperparameter optimization of heterogeneous environmental factors and their relative contribution as determinants of incidences in insect pest ecological studies. A multidimensional field‐based surveillance was conducted in two seasons (24 months), July–June of each season (2015/2016 ‐ season 1 and 2016/2017 ‐ season 2) using sex‐pheromone‐baited traps and Thermocron i‐Buttons to identify key determinants of population abundance of diamondback moth, Plutella xylostella L., across spatial horticultural hotspots of Botswana. The moth is a notorious global brassica pest. Pearson's product moment correlation matrix showed month of the year (M), mean temperature (T_mean) and maximum temperature (T_max) as positively correlated (p < 0.001) to number of moths (N), while minimum temperature (T_min), minimum relative humidity (RH_min), mean relative humidity (RH_mean), maximum relative humidity (RH_max) and host plant (h) were negatively correlated (p < 0.001) to N. Using Waikato Environment for Knowledge Analysis (WEKA) data‐mining techniques, two models were developed: (a) M5P decision‐tree algorithm associated with nine linear models (LMs) and (b) principal component analysis (PCA) based on four principal components. Both approaches identified M as the major predictor of moth abundance, followed by h and farming region (R). However, R was a function of T_max (positive auto‐correlation) and RH_max (negative auto‐correlation). These results provide simplified relative contribution of heterogeneous factors in influencing P. xylostella spatio‐temporal abundance, essential for early warning systems in pest management. This is an important component of sustainable pest management aimed at managing insect pests and minimizing pesticides abuse in brassica production systems.     

Interactions between the diamondback moth, Plutella xylostella L. and glasshouse and outdoor-grown cabbage cultivars

Interactions between Brassica cultivars (cvs) and adult and larval stages of Plutella xylostella L. were examined. In six-way choice oviposition experiments, glasshouse-grown Chinese cabbage cv. Tip Top was preferred to five outdoor-grown temperate cabbage cultivars but was similar to four out of five tropical cultivars. Bionomic studies with neonate larvae on temperate cabbage cultivars showed that mean larval duration was longer and percentage survival less on older compared with younger plants but that plant age or cultivar had little effect on pupal weight. The fecundity of P. xylostella reared on outdoor-grown cabbage cultivars varied ten-fold between the poorest and the best cultivar but plant age had little effect on P. xylostella reared on Chinese cabbage. In a glasshouse experiment, survival of P. xylostella on Chinese cabbage was significantly greater compared with field-grown glossy-leaved, normal bloom green cabbage and red cabbage. In bioassays with neonate larvae, when the leaves were placed vertically instead of horizontally a significant level of intrinsic resistance was revealed for glasshouse and field-grown glossy-leaved cabbage but not for Chinese cabbage. Observed differences in host status to P. xylostella are discussed in relation to the potential for partial plant resistance in control programmes.     

Oviposition site selection by the diamondback moth,Plutella xylostella (L.) (Lepidoptera: Plutellidae)

AlthoughPlutella xylostella (L.) is a worldwide pest of cruciferous crops, relatively little is known about its oviposition behaviour. This study was undertaken to provide necessary information about mechanisms involved inP. xylostella host selection. Four oviposition behaviours were described. Moths were given artificial substrates treated with water, sinigrin, orBrassica napus (cv. Westar) squashes, combined withB. napus volatiles and/or grooves in the substrate. No eggs were deposited in the absence of olfactory and gustatory stimuli. Moths given gustatory but not olfactory stimuli deposited similar numbers of eggs but spent significantly more time performing olfactory-related behaviours. Conversely, moths given olfactory but not gustatory stimuli did not oviposit.SSubstrate grooves did not influence egg numbers but appeared to influence egg location. The order in which oviposition behaviours occurs and the relative importance of stimuli type are discussed.