Tritrophic Choice Experiments with Bt Plants,the Diamondback Moth (Plutella xylostella) and the parasitoid Cotesia plutellae

Parasitoids are important natural enemies of many pest species and are used extensively in biological and integrated control programmes. Crop plants transformed to express toxin genes derived from Bacillus thuringiensis (Bt) provide high levels of resistance to certain pest species, which is likely to have consequent effects on parasitoids specialising on such pests. A better understanding of the interaction between transgenic plants, pests and parasitoids is important to limit disruption of biological control and to provide background knowledge essential for implementing measures for the conservation of parasitoid populations. It is also essential for investigations into the potential role of parasitoids in delaying the build-up of Bt-resistant pest populations. The diamondback moth (Plutella xylostella), a major pest of brassica crops, is normally highly susceptible to a range of Bt toxins. However, extensive use of microbial Bt sprays has led to the selection of resistance to Bt toxins in P. xylostella. Cotesia plutellae is an important endoparasitoid of P. xylostella larvae. Although unable to survive in Bt-susceptible P. xylostella larvae on highly resistant Bt oilseed rape plants due to premature host mortality, C. plutellae is able to complete its larval development in Bt-resistant P. xylostella larvae. Experiments of parasitoid flight and foraging behaviour presented in this paper showed that adult C. plutellae females do not distinguish between Bt and wildtype oilseed rape plants, and are more attracted to Bt plants damaged by Bt-resistant hosts than by susceptible hosts. This stronger attraction to Bt plants damaged by resistant hosts was due to more extensive feeding damage. Population scale experiments with mixtures of Bt and wildtype plants demonstrated that the parasitoid is as effective in controlling Bt-resistant P. xylostella larvae on Bt plants as on wildtype plants. In these experiments equal or higher numbers of parasitoid adults emerged per transgenic as per wildtype plant. The implications for integrated pest management and the evolution of resistance to Bt in P. xylostella are discussed.     

菜蛾盘绒茧蜂多分DNA病毒的特性及其对小菜蛾幼虫的生理效应

对菜蛾盘绒茧蜂Cotesia plutellae多分DNA病毒的特性及其对寄主小菜蛾Plutella xylostella幼虫的生理效应进行了研究。结果表明：菜蛾盘绒茧蜂雌蜂输卵管萼中含有大量的多分DNA病毒（polydnavirus, PDV）;一个PDV内含多个核衣壳,最多可达16个;核衣壳长40～168 nm,直径39～40 nm;PDV仅在输卵管萼细胞内复制;雌蜂产卵时,随蜂卵将PDV注入寄主血腔,并扩散到寄主的许多组织中;PDV可能先通过脱膜再侵染寄主组织。雌蜂经Co⁶⁰辐射处理后再寄生（即假寄生）小菜蛾2龄、3龄和4龄初期的幼虫,被寄生后的寄主幼虫几乎全部不能化蛹,但末龄（即4龄）幼虫期显著延长,并在寄生后期,幼虫胸部有褐色的短翅芽出现;即将化蛹的4龄末小菜蛾幼虫被假寄生后,即使每头寄主被过寄生9次,依然能正常化蛹,但不能羽化。假寄生与正常寄生后寄主的脂肪体数量和形态结构有明显的不同,推测在正常寄生的情况下蜂卵孵化时释放的畸形细胞及随后的幼蜂可能对脂肪体的结构产生了作用。     

Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella

Cotesia plutellae, a solitary endoparasitoid wasp, parasitizes the diamondback moth, Plutella xylostella, and induces host immunosuppression and lethality in the late larval stage. This study focused on changes of cellular immunity in the parasitized P. xylostella in terms of hemocyte composition and cellular functions. In third and fourth instar larvae of nonparasitized P. xylostella, granular cells represented the main hemocyte type (60-70%) and plasmatocytes were also present at around 15% among the total hemocytes. Following parasitization by C. plutellae, the relative proportions of these two major hemocytes changed very little, but the total hemocyte counts exhibited a significant reduction. Functionally, the granular cells played a significant role in phagocytosis based on a fluorescence assay using fluorecein isothiocyanate-labeled bacteria. The phagocytic activity of the granular cells occurred as early as 5 min after incubation with the bacteria, and increased during the first 40 min of incubation. The parasitism by C. plutellae significantly inhibited phagocytosis of the granular cells. Plasmatocytes also exhibited minor phagocytic activity. Moreover, plasmatocyte phagocytosis was not inhibited by parasitism. On the other hand, hemocyte-spreading behavior in response to pathogen infection was significant only for plasmatocytes, which exhibited a characteristic spindle shape upon infection. A significant spreading of the plasmatocytes was found as early as 5 min after pathogen incubation and their ratio increased during the first 40 min.An insect cytokine, plasmatocyte-spreading peptide 1 (PSP1) from Pseudoplusia includens, was highly active in inducing plasmatocyte-spreading behavior of P. xylostella in a dose-dependent manner. P. xylostella parasitized by C. plutella was significantly inhibited in plasmatocyte-spreading in response to an active dose of PSP1. An in vivo encapsulation assay showed that the parasitized P. xylostella could not effectively form the hemocyte capsules around injected agarose beads. This research demonstrates that the parasitism of C. plutellae adversely affects the total hemocyte populations in number and function, which would contribute to host immunosuppression.     

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。     

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。     

Altered actin polymerization of Plutella xylostella (L.) in response to ovarian calyx components of an endoparasitoid Cotesia plutellae (Kurdjumov)

Abstract Cotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae), a solitary braconid endoparasitoid wasp, parasitizes the diamondback moth Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) by suppressing the host defense response, thereby resulting in successful parasitization. During parasitization, ovarian calyx fluid is also delivered into the haemocoel of the host along with the wasp egg. The effect of calyx fluid constituents on haemocyte‐spreading behaviour of P. xylostella is analysed by measuring F‐actin development in the haemocytes. For this purpose, the calyx fluid of C. plutellae is separated into ovarian protein and C. plutellae bracovirus (CpBV). The ovarian protein consists of a wide range of molecular weight proteins, which are apparently different from those of CpBV. When nonparasitized P. xylostella haemocytes are incubated with either ovarian protein or CpBV for 1 or 2 h, haemocytes lose their responsiveness to a cytokine, plasmatocyte‐spreading peptide, in a dose‐dependent manner for each calyx component and fail to exhibit haemocyte‐spreading behaviour. Some CpBV genes are expressed within 1 h of parasitization. The inhibition of haemocyte‐spreading could be explained by measuring F‐actin contents, in which parasitization by C. plutellae inhibits F‐actin development in the haemocytes of P. xylostella. Either ovarian protein or CpBV could inhibit F‐actin development in the nonparasitized haemocytes. In addition, co‐incubation of ovarian protein and CpBV results in significant additive inhibition of both haemocyte‐spreading and F‐actin development in the haemocytes in response to cytokine. These results suggest that both components of C. plutellae calyx fluid function in a synergistic manner, leading to immunosuppression during the early stage of parasitization.     

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。     

Effects of Bacillus thuringiensis on Adults of Cotesia plutellae (Hymenoptera: Braconidae), a Parasitoid of the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae)

To complement existing information on the mortality of larvae of the wasp Cotesia plutellae attacking moth caterpillars infected with Bacillus thuringiensis (Bt) we tested the direct and indirect effects of the bacterium on adult wasp longevity and oviposition behaviour. In one experiment with female parasitoids, mean longevity (SEM) was not significantly different between females exposed to Bt (1.98 +/- 0.08 days) and those not exposed (2.18 +/- 0.13 days). In a second experiment with both males and females, Bt treatment did not significantly effect either male or female parasitoids exposed to Bt. To observe the possible effects of Bt on oviposition behaviour of C. plutellae each of 10 females were given five larvae that had been treated with Bt and five untreated larvae at the same time. All parasitoids were observed to make oviposition attempts in both untreated and treated larvae. Upon dissection of the host larvae, one or more C. plutellae eggs were found in each of the larvae in which a parasitoid attempted oviposition. There was no effect of Bt treatment on parasitoid oviposition. The mean number of ovipositions in treated larvae (4.3 +/- 0.3) was not significantly different from untreated larvae (4.7 +/- 0.2).     

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.     

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.     

Bottom-up and top-down effects in a tritrophic system: the population dynamics of Plutella xylostella (L.)–Cotesia plutellae (Kurdjumov) on different host plants

Abstract.  1. The effects of host-plant resistance on the population dynamics of the Diamondback moth, Plutella xylostella L., and its solitary parasitoid, Cotesia plutellae (Kurdjumov), were studied in replicated time-series experiments.
2. Host-plant resistance did not affect the equilibrial abundance of the Diamondback moth, but it affected the dynamics of Diamondback moth populations.
3. The mean population size of Diamondback moth showed no significant difference between Brassica rapa (a susceptible host plant) and Brassica napus (a partially resistant host plant) either in the presence or absence of the parasitoid.
4. Time-series analysis suggests that the dynamics of Diamondback moth on B. rapa were underpinned by delayed density-dependent processes. In contrast, the dynamics of the moth on B. napus were influenced by a direct density-dependent process.
5. Although measures of short-term parasitism showed a significantly higher rate of parasitism by C. plutellae on Diamondback moth feeding on B. napus compared with B. rapa , this individual performance does not translate into differences in the population dynamics. Analysis shows no significant difference in the persistence time of the population-level interaction between the host and parasitoid on the two different host plants.     

Differences between attractive diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), sex pheromone lures are not determinable through analysis of emissions

1 Conjoint comparisons were made between trap captures of male diamondback moths, Plutella xylostella (L.), and gas chromatographic measures of sex pheromone emissions from grey and red rubber septa. The objective was to correlate trap captures with emissions. 2 Irrespective of their composition, all mixtures that were released from grey septa effected the capture of more males than the same mixtures released from red septa. There was no difference between captures by any of the mixtures when emitted from grey septa. However, when emitted from red septa, a nominal 50 : 50 ratio of (Z)-11-hexadecenal (Z11–16:Al) and (Z)-11-hexadecenyl acetate (Z11–16:Ac) effected the capture of more males than either nominal 70 : 30 or 67 : 23 ratios. Generally, the emitted ratios of Z11–16:Al and Z11–16:Ac from both types of septa were variable, ranging between 6 : 10 to about 40 : 10. 3 No statistical correlation was found between the emissions and trap captures by any single component, group or ratio of components. These results suggest that the environment substantially affects the emission of sex pheromone components from rubber septa. As a result, the demonstration of geographical biotypes of diamondback moths and other insects may be difficult.     

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.     

印楝素乳油对小菜蛾种群的控制作用模拟

应用害虫种群系统控制控制的状态空间方程和作用因子添加分析法,模拟1％印楝素乳油对小菜蛾种群的控制作用,结果表明,印楝素浮油对小菜蛾种群的控制机理,主要是对小菜蛾成虫的显忌避作用,尤其是在种群发展的初期,由于其显的忌避作用,降低田间卵量,使其种群数量明显降低下来,有效控制其为害。     

Microsatellites reveal a lack of structure in Australian populations of the diamondback moth, Plutella xylostella (L.)

The diamondback moth, Plutella xylostella, is renowned for developing resistance to insecticides and causing significant economic damage to Brassica vegetable crops throughout the world. Yet despite its economic importance, little is known about the population structure and movement patterns of this pest both at local and regional scales. In Australia, the movement patterns and insecticide resistance status of P. xylostella infesting canola, vegetables, forage brassicas and weeds have fundamental implications for the management of this pest. Here we use six polymorphic microsatellite loci to investigate population structure and gene flow in Australian populations of P. xylostella. Samples of P. xylostella from New Zealand, Malaysia, Indonesia and Kenya were also scored at these loci. We found no evidence of population structure within Australia, with most populations having low inbreeding coefficients and in Hardy-Weinberg equilibrium. In addition, a sample from the North Island of New Zealand was indistinguishable from the Australian samples. However, large genetic differences were found between the Australia/New Zealand samples and samples from Kenya, Malaysia and Indonesia. There was no relationship between genetic distance and geographic distance among Australian and New Zealand samples. Two of the loci were found to have null alleles, the frequency of which was increased in the populations outside the Australia/New Zealand region. We discuss these results with reference to insecticide resistance management strategies for P. xylostella in Australia.     

小菜蛾乙酰胆碱酯酶cDNA片段的克隆和序列分析

利用反转录-多聚酶链式反应(RT-PCR)的方法对小菜蛾Plutella xylostella的乙酰胆碱酯酶基因cDNA片段进行了克隆和序列分析。通过简并性上游引物和下游引物扩增出了小菜蛾乙酰胆碱酯酶基因281bp的cDNA片段。同源性分析表明, 该cDNA片段与其它昆虫乙酰胆碱酯酶基因序列具有较高的同源性。     

Efficacy of entomopathogenic nematode,Steinernema carpocapsae against the diamondback moth,Plutella xylostella (L.) in laboratory condition

In vitro studies were carried out on the diamondback moth, Plutella xylostella larvae using an insect entomopathogenic nematode isolate, Steinernema carpocapsae obtained from the Koppert company, the Netherlands. Larvae of P. xylostella were collected from cabbage farms around Mashhad city of Iran. During the study, the responses of larvae at 25?°C for three periods of 24, 48 and 72?h with different concentrations of 0, 5, 10, 20, 40, 80, 160 and 320 third instar larvae of nematode (infective stage?=?IJs) per insect into 10?cm Petri dishes containing filter paper soaked with 1?ml of nematodes suspension were compared. Maximum mortality caused by S. carpocapsae nematode was 88% at 24?h, and it was 100% at 48 and 72 h. With increasing nematode population level and exposure time (ET in hour), mortality of P. xylostella larvae was increased. Based on probit analysis, LC₅₀ values of S. carpocapsae nematode in three test periods were 45.61, 12.02 and 40.80 IJs per insect, respectively. Initial ANOVA was performed for S. carpocapsae nematode. The effect of both nematode population levels (IJ) and ET on third instar larvae of the diamondback moth, P. xylostella and interaction between IJ and ET were significant. In general, it is recommended to apply this nematode in suitable condition for controlling diamondback moth.