Evaluating trap crops for diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

Potential trap crops for the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), were evaluated through a series of ovipositional preference and larval survival experiments in outdoor screenhouses in 2002 and 2003. Hosts examined as trap crops were glossy and waxy collards, Brassica oleracea L. variety acephala; Indian mustard, Brassica juncea (L.) Czern; and yellow rocket, Barbarea vulgaris (R. Br.) variety arcuata. More eggs were laid on the potential trap crops, with the exception of waxy collards, than on cabbage. When P. xylostella was offered multiple hosts at the same time, numbers of eggs laid on glossy collards, Indian mustard, and yellow rocket were 3, 18, and 12 times greater than on cabbage, respectively. Similarly, when P. xylostella was offered a single trap crop host and cabbage, numbers of eggs laid on glossy collards, Indian mustard, and yellow rocket were 300, 19, and 110 times greater than on cabbage, respectively. Our studies suggest differences in oviposition between the potential trap crops and cabbage were likely due to host volatiles, leaf morphology and color, or a combination of these factors, rather than to total leaf areas, leaf shape, or plant architecture. Two-choice tests with a Y-tube olfactometer indicated that plant volatiles were major factors in P. xylostella host preference. The percentage larval survival from egg to pupation was 22.2% on cabbage, 18.9% on waxy collards, and 24.4% on Indian mustard, whereas survival was significantly lower on glossy collards (6.7%) and yellow rocket (0%). Based on our tests, it seems that yellow rocket may be the best candidate for use as a trap crop for P. xylostella because it is highly attractive for oviposition, but larvae do not survive on it.     

小菜蛾化学感受蛋白基因PxylCSP1的克隆和表达

利用RT-PCR和RACE技术克隆到小菜蛾Plutella xylostella 化学感受蛋白（CSP）基因PxylCSP1（GenBank登录号: FJ361903）,其核苷酸序列全长405 bp,编码134个氨基酸残基,预测N-末端包含19个氨基酸组成的信号肽序列,估测其成熟蛋白分子量为13.56 kD,等电点为6.12。该基因编码氨基酸序列和其他鳞翅目昆虫CSP的氨基酸序列比对同源性较高（70%~80%）。RT-PCR结果表明PxylCSP1不仅存在于小菜蛾的触角中,还存在于头、足、腹和翅中。Real-time PCR结果表明PxylCSP1的表达水平因被测小菜蛾的性别、日龄、组织不同和交配与否而异。     

Functional and genetic characteristics of Chlorantraniliprole resistance in the diamondback moth,Plutella xylostella (Lepidoptera: Plutellidae)

The diamondback moth (Plutella xylostella) is a globally distributed and important economic pest, and it has developed resistance to all conventional insecticide classes used in the field. Chlorantraniliprole is a new chemical class of insecticide that acts as a conformation‐sensitive activator of the insect ryanodine receptor (RyR). In the present study, a field strain (16.3‐fold resistance to chlorantraniliprole) was collected in Korea and lab‐selected with chlorantraniliprole for more than one year. The resulting strain presented 2,157‐fold resistance to chlorantraniliprole. A point mutation (G4946E) in the RyR gene was observed at a high frequency in the resistant strain. Enzyme assays indicated that glutathione S‐transferase (GST) and P450 activity in the resistant strain were 2.4‐ and 1.96‐times higher than that of the susceptible strain, respectively. The expression of the RyR, GST (sigma, omega, and zeta) and CYP321E1 gene was higher in the resistant strain than in the susceptible strain. The F₁ progeny resulting from reciprocal crosses did not reveal maternal effects or a diamide‐susceptible phenotype, which suggests an autosomal nearly recessive mode of inheritance. In addition, we surveyed the susceptibility to 13 insecticides (3 diamides, 2 synthetic pyrethroids, 2 spinosyns, 1 organophosphate, 1 oxadiazine, 1 avermectin, and 3 others) in the chlorantraniliprole‐resistant strain. The resistant strain exhibited high cross‐resistance to flubendiamide (5,910 fold) and showed no cross‐resistance to spinetoram, spinosad, indoxacarb, and metaflumizone. These results can serve as an important basis for guiding the use of insecticides in the field.     

羧酸酯酶介导的小菜蛾对氟虫腈的抗性

【目的】羧酸酯酶（carboxylesterases, CarEs）是昆虫重要的解毒代谢酶之一,可以介导靶标昆虫对多种杀虫剂的代谢抗性。本研究检测了羧酸酯酶对小菜蛾Plutella xylostella 抗药性的介导功能,旨在阐明羧酸酯酶在小菜蛾代谢解毒中的生理生化和分子机理。【方法】采用点滴法测定氟虫腈对小菜蛾敏感种群和抗氟虫腈种群的毒力,以及羧酸酯酶抑制剂磷酸三苯酯（triphenyl phosphate, TPP）对氟虫腈的增效作用;以LC₃₀和LC₅₀浓度的氟虫腈处理抗性小菜蛾,测定药剂处理后CarEs酶活性的变化;利用qRT-PCR技术分析Pxae22和Pxae31两个基因在小菜蛾不同发育阶段、组织和种群的表达模式;利用dsRNA干扰Pxae22和Pxae31后观察基因的表达变化和小菜蛾3龄幼虫对药剂敏感性的变化。【结果】TPP可以削弱小菜蛾3龄幼虫对氟虫腈的抗性,增效倍数约为6倍;使用较低剂量（LC₃₀和LC₅₀）氟虫腈处理小菜蛾3龄幼虫后,处理组CarEs比活力明显高于对照,提示氟虫腈对小菜蛾CarEs活性具有诱导作用。对羧酸酯酶基因Pxae22和Pxae31在小菜蛾不同发育阶段、4龄幼虫不同组织和不同种群3龄幼虫中的表达模式分析发现,这两个基因在小菜蛾4龄幼虫中的表达量最高;在4龄幼虫中以中肠组织中的表达量较高,头、表皮、脂肪体中的表达量很低; 抗性种群中的表达量显著高于敏感种群。通过干扰 Pxae22和 Pxae31后的qRT-PCR验证,两个基因的表达量均显著降低,进一步的氟虫腈毒力测定发现,干扰P xae22和 Pxae31后的小菜蛾3龄幼虫对氟虫腈的敏感性分别增加了1.63倍和1.73倍。【结论】羧酸酯酶在小菜蛾对氟虫腈解毒代谢中具有重要作用;Pxae22和Pxae31是小菜蛾的两个抗性相关基因,其表达水平的变化直接影响小菜蛾对氟虫腈的敏感性。     

Management of diamondback moth,Plutella xylostella (Lepidoptera: Plutellidae) by mating disruption

Abstract Field trials were conducted in China in 2008 and 2009 to evaluate the efficacy of mating disruption (MD) on diamondback moth, Plutella xylostella, in cabbage, Brassica oleracea var. capitata. Effectiveness was positively correlated with the MD dispenser density in the field. A density of 167 MD dispensers per ha produced an average population decrease of about 50% compared to the conventional‐practice field. Significant fewer males were captured in pheromone‐treated and conventional‐practice fields than in the blank control field, but the difference was not significant between the pheromone‐treated and conventional‐practice fields. In addition, fewer eggs and larvae were observed in pheromone‐treated fields. Our results suggest mating disruption coupled with minimal insecticidal supplements is a promising solution for resistance management and control of diamondback moth infestation.     

Monitoring and characterization of diamondback moth (Lepidoptera: Plutellidae) resistance to spinosad

Fourteen populations of the diamondback moth, Plutella xylostella (L.), were collected from fields of crucifer vegetables in the United States, Mexico, and Thailand in 1999 and 2000 for susceptibility tests with spinosad. Most populations were susceptible to spinosad and similar to earlier baseline values, but populations from Thailand and Hawaii showed high levels of tolerance. A statewide survey in Hawaii in 2000 and 2001 indicated resistance problems on several islands. One colony collected in October 2000 from Pearl City, HI, was subjected to further selection pressure, using spinosad in the laboratory, and then was used as the resistant strain (Pearl-Sel) for other tests. Spray tests using the recommended field rates of spinosad on potted broccoli plants in the greenhouse confirmed that field control failures due to resistance were possible in the areas of these collections. Analysis of probit lines from F1 reciprocal crosses between the Pearl-Sel and S strain indicated that resistance to spinosad was inherited autosomally and was incompletely recessive. A direct test of monogenic inheritance based on the F1 x Pearl-Sel backcrosses suggested that resistance to spinosad was probably controlled by one locus. The synergists S,S,S-tributyl phosphorotrithioate and piperonyl butoxide did not enhance the toxicity of spinosad to the resistant colony, indicating metabolic mediated detoxification was probably not responsible for the spinosad resistance. Two field colonies in Hawaii that were resistant to spinosad were not cross-resistant to emamectin benzoate or indoxacarb. Resistance developed in Hawaii due to the continuous cultivation of crucifers in which as many as 50 applications of spinosad per year may have been made to a common population of P. xylostella in sequential plantings, although each grower might have used the labeled restrictions for resistance management. Resistance management strategies will need to address such cropping and pest management practices.     

Genetic differentiation among various populations of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae)

Genetic variation among 14 populations of Plutella xylostella (Linnaeus) from USA (Geneva, New York), Brazil (Brasilia), Japan (Okayama), The Philippines (Caragan de Oyo), Uzbekistan (Tashkent), France (Montpellier), Benin (Cotonou), South Africa (Johannesburg), Réunion Island (Montvert), and five localities in Australia (Adelaide, Brisbane, Mareeba, Melbourne, Sydney) were assessed by analysis of allozyme frequencies at seven polymorphic loci. Most of the populations were not in Hardy-Weinberg equilibrium and had a deficit in heterozygotes. The global differentiation among populations was estimated by the fixation index (Fst) at 0.103 for the 14 populations and at 0.047 when populations from Australia and Japan, which differed most and had a strong genetic structure, were excluded from the analysis. By contrast, the populations from Benin (West Africa) and Brazil (South America) were very similar to each other. Genetic differentiation among the populations was not correlated with geographical distance.     

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.     

Monitoring of diamondback moth (Lepidoptera: Plutellidae) resistance to spinosad, indoxacarb, and emamectin benzoate

Six to nine populations of the diamondback moth, Plutella xylostella (L.), were collected annually from fields of crucifer vegetables in the United States and Mexico from 2001 to 2004 for baseline susceptibility tests and resistance monitoring to spinosad, indoxacarb, and emamectin benzoate. A discriminating concentration for resistance monitoring to indoxacarb and emamectin benzoate was determined based on baseline data in 2001 and was used in the diagnostic assay for each population in 2002-2004 together with a discriminating concentration for spinosad determined previously. Most populations were susceptible to all three insecticides, but a population from Hawaii in 2003 showed high levels of resistance to indoxacarb. Instances of resistance to spinosad occurred in Hawaii (2000), Georgia (2001), and California (2002) as a consequence of a few years of extensive applications in each region. The collaborative monitoring program between university and industry scientists we discuss in this article has provided useful information to both parties as well as growers who use the products. These studies provide a baseline for developing a more effective resistance management program for diamondback moth.     

小菜蛾鞣化激素基因克隆及其功能分析

鞣化激素是调节昆虫表皮骨化和翅膀发育的一种神经激素, 尽管已经在许多不同种昆虫上克隆了鞣化激素基因, 但是关于小菜蛾 Plutella xylostella鞣化激素及其基因的研究至今未见报道。本研究克隆了两个小菜蛾鞣化激素基因Pxbursα和Pxbursβ （GenBank 登录号分别为KF498645和KF498646）全长cDNA, 其序列长度分别为537 bp和360 bp, 与已报道的其他昆虫的鞣化激素氨基酸序列一致性分别为51%~68% 和37%~57%。实时定量PCR分析发现Pxbursα和Pxbursβ均在蛹期表达量高, 而在幼虫期和成虫期的表达量低。以Pxbursα部分序列的双链RNA（dsRNA）饲喂小菜蛾4龄末期幼虫, 发现蛹期Pxbursα的表达受到了显著抑制, 小菜蛾的发育停滞在蛹期而无法正常羽化, 并最终死亡。由此推测, 小菜蛾鞣化激素基因在蛹期的大量表达对其生长发育和羽化具有重要的作用。     

光周期对小菜蛾羽化节律的影响

【目的】本研究旨在探究光周期对小菜蛾Plutella xylostella(Linn.)羽化节律的调控作用,为小菜蛾的季节性种群动态预测提供理论基础。【方法】在自然光周期和逆转光周期条件下,观察小菜蛾每日的羽化情况。【结果】在自然光周期和逆转光周期条件下,小菜蛾蛹在24 h均可以羽化。正常光周期下,羽化行为表现为近似抛物线形昼夜节律,雌成虫通常在黑暗阶段羽化（18:00-6:00）,而雄成虫大多在光照阶段羽化（6:00-18:00）。在逆转光周期下,小菜蛾蛹的羽化无明显规律。在正常光周期下,雌成虫的羽化频率在光期和暗期没有显著差异,但是,相比于暗期,雄成虫在光期羽化率较高;在逆转光周期后,雌成虫在暗期（6:00-18:00）的羽化数量比光期（18:00-6:00）高,雄成虫暗期的羽化数量比光期低。【结论】光周期逆转后小菜蛾雌成虫和雄成虫的羽化节律明显改变,这将有助于阐明害虫光周期变化与生物钟之间的关系。     

Hexameric storage proteins during metamorphosis and egg production in the diamondback moth, Plutella xylostella (Lepidoptera)

As in many Lepidoptera, Plutella xylostella adults do not feed on protein and females must use accumulated reserves to supply vitellogenin synthesis. Storage proteins were quantified in females and males from the late larval stage through day 4 of adult life. The level of storage protein peaked in the early pupal stage, with females having about twice as much as males. In males, the level fell through pupal development and dropped to a trace by one day after eclosion. In females, level of storage proteins fell until eclosion, and then rose dramatically within four hours after the molt to about 2/3 of the original peak level. This post-eclosion increase, which has not been reported previously in insects, suggests that adult females synthesize hexamerins to resequester amino acids. Subsequently, the level of storage proteins fell as vitellogenin appeared and eggs were laid. The ability to synthesize and sequester amino acids as storage proteins during the adult stage has wide-ranging implication for protein management in insects, particularly those that are long-lived and have flexible schedules of reproduction.     

Genetic, biochemical, and physiological characterization of spinosad resistance in Plutella xylostella (Lepidoptera: Plutellidae)

Bioassays (at generation G2) with a newly collected field population (designated MN) of Plutella xylostella (L.) (Lepidoptera: Plutellidae) from Multan, Pakistan, indicated resistance to spinosad, indoxacarb, deltamethrin, abamectin, and acetamiprid. At G2 the field-derived population was divided into two subpopulations, one was selected (G2 to G11) with spinosad (Spino-SEL), whereas the second was left unselected (UNSEL). A significant reduction in the resistance ratio for each compound was observed in UNSEL at G12, indicating that the observed resistance to each insecticide was unstable. For Spino-SEL, bioassays at G12 found that selection with spinosad gave a resistance ratio of 283 compared with MN at G2. The resistance to indoxacarb and acetamiprid in the Spino-SEL population increased to 13- and 67-fold, respectively, compared with MN at G2. The toxicity of deltamethrin to Spino-SEL was similar to its toxicity to the MN population at G2. This suggests that spinosad selection maintained the otherwise unstable resistance to the compound. In contrast, resistance to abamectin decreased significantly from G2 to G12 in Spino-SEL. Logit regression analysis of F1 reciprocal crosses between Spino-SEL and the susceptible Lab-UK indicated that resistance to spinosad was inherited as an autosomal, incompletely recessive trait. The spinosad resistance allele significantly delays the developmental time, reduced pupal weight, number of eggs laid, and number of eggs hatched compared with Lab-UK. Further analysis suggests Spino-SEL exhibited a significantly lower intrinsic rate of population increase (r(m)) to all other populations tested.     

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.     

小菜蛾对杀虫双和杀螟丹抗性的现实遗传力

利用室内选育119代的小菜蛾Plutella xylostella抗性品系,分析了小菜蛾对杀虫双和杀螟丹抗药性的现实遗传力,并对遗传力和杀虫剂的杀死率对抗性发展速率的影响进行了预测,结果表明,室内继代药剂选择119代后,小菜蛾对杀虫双和杀螟丹抗性的遗传力很低（h^2=0.03)。因此,小菜蛾对这两种药剂产生更高水平抗性的潜力不大,在选择的前半段（F30）,杀虫双和杀螟丹的抗性遗传力（分别为0.14t 0.11)高于选择后半段（F80)的遗传力（均为0.05),选择前、后半段的选择差数没有明显差别。即使在室内选择条件下,假如斜率=2.0(δp=0.5),h^2=0.05,群体中70%个体致死时要获得10倍的抗性,约需34代,田间条件下,由于等位基因频率的变化、环境变异等因素的影响,抗性增长10倍则需要更长的时间,因此田间条件下小菜蛾在短期内不易对杀虫双和杀螟丹产生高水平抗性。     

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℃.     

Sublethal effects of hexaflumuron on development and reproduction of the diamondback moth,Plutella xylostella (Lepidoptera: Yponomeutidae)

Abstract Effects of hexaflumuron at 10% lethal concentration (LC₁₀) and LC₂₅ on development and reproduction parameters of the diamondback moth, Plutella xylostella (Linnaeus, 1753) (Lep.: Yponomeutidae) were investigated. Estimated LC₅₀, LC₁₀ and LC₂₅ values of leaf dip bioassay of hexaflumuron on the third instar larvae of the P. xylostella were 1.48, 0.59 and 0.91 mg/L, respectively. Hexaflumuron decreased pupal weight in the parent generation at sublethal concentrations but in the offspring generation, this effect was not observed. Sublethal concentrations increased egg, first and second larval instar and pupa developmental time and shortened life span of adults, but did not change the third and fourth larval instars and pre‐pupa developmental period. Also fecundity of females reduced significantly but hatchability of treatments and control were similar. Survival rate of pre‐adult stages declined significantly at LC₂₅ concentration. Reproduction parameters such as reproductive rate (R₀) and intrinsic rate of increase in sublethal concentrations were significantly lower compared with control, but gross reproduction rate (GRR) at the LC₁₀ concentration was increased and it could be hormoligosis. Also hexaflumuron significantly increased doubling time (Dt). We conclude that the sublethal effects of hexaflumuron might exhibit significant effects on the population dynamics of P. xylostella.     

Cloning of CYP9G2 from the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae).

Cytochrome P450s constitute a superfamily of hemoproteins, important in the metabolism of endogenous and xenobiotic compounds. The full-length cDNA of a novel cytochrome P450, CYP9G2, was isolated from a cDNA library. The cDNA is 2143 bp in length and contains an open reading frame from 50 to 1615 bp, encoding a protein of 521 amino acid residues. The putative P450 protein contains a highly hydrophobic N terminus and a P450 protein signature motif, FG/S*G*R*C*G***A/G, known as the important ligand for heme binding, analysis of the NH2-terminal sequence indicated that CYP9G2 is a microsomal P450. Using polymerase chain reaction with primers specific to CYP9G2, the genomic structure of CYP9G2 was analyzed, and it was found that the gene contains seven introns and eight exons within the coding region, all the sequences of the exon-intron junctions are consistent with the AG-GT rule. Multiple alignment indicated that CYP9G2 is most similar to CYP9E2 from the Blattella germanica (42.7% identity), it is also similar to the insect P450s in family 9, including CYP9L1 from Anopheles gambiae (38.7%) and CYP9A1 from Heliothis virescens (39.5%).