昆虫病原线虫共生细菌共生性的分子生物学研究进展

嗜线虫致病杆菌属Xenorhabdus和发光杆菌属Photorhabdus细菌隶属肠杆菌科Enterobacteriaceae,对多种害虫致病能力强,分别与斯氏属Steinernema和异小杆属Heterorhabditis昆虫病原线虫互惠共生。该两属共生细菌既存在对昆虫寄主的病原性,又存在与线虫寄主的共生性。共生细菌与其线虫寄主的共生性主要表现以下4方面：（1）细菌产生食物信号诱导滞育不取食的感染期线虫恢复;（2）细菌为线虫生长与繁殖提供营养;（3）细菌能于感染期线虫的肠道定殖与生长;（4）细菌产生杀线虫毒素杀死非共生线虫。本文综述了共生菌以上4方面的共生性及其相关的分子机制。     

昆虫病原线虫感染寄主行为研究进展

昆虫病原线虫斯氏属Steinernema和异小杆属Heterorhabditids线虫是新型的生物杀虫剂。其感染期幼虫是惟一能够侵染寄主昆虫的虫态。这类线虫感染寄主的行为分为寻找寄主、识别寄主和侵染寄主。文章综述昆虫病原线虫感染寄主昆虫的行为以及在感染寄主过程中的影响因素。     

根结线虫对斜纹夜蛾幼虫生长及营养利用随龄期变化的研究

【目的】为明确南方根结线虫Meloidogyne incognita侵染对斜纹夜蛾Spodoptera litura幼虫的影响是否因幼虫的龄期而异。【方法】采用被南方根结线虫侵染和未被侵染的乌桕叶片饲喂斜纹夜蛾幼虫,并测定不同龄期幼虫的生长（幼虫体重、发育历期、相对生长率）和营养利用（取食量、近似消化率、食物转化率）情况。【结果】随着幼虫龄期的增加,斜纹夜蛾幼虫的取食量及发育历期呈上升趋势,幼虫体重、相对生长率和食物转化率先上升后下降,而近似消化率则先下降后上升。与饲喂未侵染南方根结线虫的乌桕叶片相比,取食线虫侵染的乌桕叶片的斜纹夜蛾2龄幼虫近似消化率（0.78±0.07）%显著增加了约30%,但斜纹夜蛾的4龄幼虫体重（0.12±0.04）g、相对生长率（0.20±0.06）g·g^(-1)·d(-1)·d^(-1)和食物转化率（0.54±0.18）%分别降低了61%、36%和73%。线虫侵染处理与龄期互作对斜纹夜蛾的发育历期和取食量无显著影响。【结论】根结线虫侵染对斜纹夜蛾幼虫生长及其营养利用的影响因龄期而异,线虫侵染处理虽显著增加了斜纹夜蛾2龄幼虫的近似消化率,但却抑制了其4龄幼虫的生长及其营养利用。     

一种从小卷蛾斯氏线虫怀卵成虫制备感染期幼虫的简易方法

昆虫病原线虫是新型的生物杀虫剂,其感染期幼虫是昆虫病原线虫产业化生产和应用的唯一虫态,对昆虫病原线虫基因功能的研究及转基因改造有助于推进昆虫病原线虫的产业化。本研究基于昆虫病原线虫"噬母现象"的原理,以不同的孵育液孵育小卷蛾斯氏线虫Steinernema carpocapsae的怀卵成虫,找到可以简单快速从怀卵成虫直接获得整齐龄期的感染期幼虫的方法,为该线虫卵或性腺的RNA干扰后感染期幼虫的收集及生物测定提供基础,为昆虫病原线虫的转基因改造以提高其环境耐受力提供技术支持。     

营养改变对潜蝇姬小蜂寄生行为和寄主取食行为的影响

为了研究营养状态对卵育型寄生蜂潜蝇姬小蜂Diglyphus isaea (Walker)雌蜂的寄主取食行为和产卵寄生行为及其二者行为权衡的影响, 在培养皿条件下, 比较了饥饿、加蜂蜜水和不加蜂蜜水3种营养状态的潜蝇姬小蜂雌蜂对美洲斑潜蝇Liriomyza sativae Blanchard各龄幼虫（低、中、高）的寄生、取食及致死能力。结果表明: 非选择条件下, 3种营养状态寄生蜂对高龄幼虫均具有较高的寄生率, 对中龄幼虫具有较高的取食率, 致死率和致死量之间存在显著性差异。3种状态的寄生蜂对低龄幼虫均没有表现出致死能力。有选择条件下, 饥饿状态的寄生蜂对寄主的寄生率最低（5.0%±1.6%）, 取食率最高（16.0%±2.9%）, 特别是对高龄幼虫的取食率占到了整个寄主食物取食率的91.9%; 加蜂蜜水状态下, 寄生蜂对寄主有最低的取食率（8.3%±0.9%）和致死率（17.7%±1.1%）; 不加蜂蜜水状态下, 寄生蜂对寄主有最高的寄生率（13.3%±1.1%）和致死率（28.4%±1.8%）。综合分析发现, 取食寄主的雌蜂比取食蜂蜜水的雌蜂具有更强的致死能力和活动能力。     

新烟碱类杀虫剂拌种对马铃薯甲虫幼虫食物利用和生长发育的影响

为研究马铃薯甲虫Leptinotarsa decemlineata（Say）幼虫取食新烟碱杀虫剂噻虫嗪和吡虫啉处理（有效成分18 g/100 kg种薯拌种）播种90 d后马铃薯叶片的消化吸收和生长发育情况,在室内采用重量法测定了幼虫取食量、体重增加量、食物利用率,近似消化率以及蛹重等营养指标。 结果表明：70%噻虫嗪种子处理可分散粉剂拌种后,马铃薯甲虫幼虫的取食量、体重增加量、食物利用率、食物转化率、近似消化率和相对生长率以及蛹重显著低于10%吡虫啉可湿性粉剂拌种和空白对照（P＜0.05）,发育历期显著长于10%吡虫啉可湿性粉剂拌种和空白对照（P＜0.05）;而10%吡虫啉可湿性粉剂拌种后,马铃薯甲虫幼虫的取食量和3～4龄期的体重增加量与空白对照差异显著（P＜0.05）,蛹重、1～2龄幼虫的体重增长量、食物利用率、食物转化率、近似消化率、相对生长率和生长发育历期与空白对照差异不显著（P＞0.05）。 说明70%噻虫嗪种子处理可分散粉剂拌种对马铃薯甲虫幼虫的取食有显著抑制作用,不能很好地满足其生长发育营养物质的需要,幼虫取食其叶片后,近似消化率和相对生长速率显著降低,生长发育历期显著延长。 这有利于减少当季世代数,降低危害。     

外源水杨酸甲酯(MeSA)处理茶树对茶园主要害虫及其天敌的影响

研究了4种豆科寄主植物及5个不同温度处理对西花蓟马生长发育的影响,并对各虫态的发育起点温度和有效积温进行了分析。结果表明:同一温度条件下,西花蓟马取食不同豆科寄主植物时,各虫态的发育历期差异显著,但不同温度下不同寄主处理间的差异性不同;西花蓟马的未成熟期取食四季豆叶片时发育最快,取食蚕豆叶片时发育最慢,两寄主间存在显著差异;取食同一寄主植物时,不同温度处理下,西花蓟马各虫态的发育历期差异显著,且随温度升高历期缩短,发育速率和温度之间为直线关系;西花蓟马各虫态在不同寄主植物上的发育起点温度和有效积温各不相同,未成熟期取食蚕豆叶片时发育起点温度最低为6.35℃,但需要的有效积温最高为331.36日度。     

黑水虻幼虫的发育速率及食物转化率研究

本文研究了黑水虻Hermetia illucens幼虫取食花生麸的发育速率及其食物转化率。仔细考察了黑水虻卵孵化后,其幼虫平均体重的增长情况,结果显示,黑水虻在实验条件下（T=30℃,RH=60%）,幼虫发育的参考周期为20 d,其中最初5 d体重增长9433倍,从第6 d至第10 d体重增长7倍,最后10 d体重增长095倍,从初孵幼虫到老熟幼虫体重增长148667倍,发育期内的体重增长趋势类似于逻辑斯谛曲线,而第5 天（W=143 g/百头虫）与第10 天（W=1145g/百头虫）则为逻辑斯谛曲线的两个拐点。食物转化率测定结果表明,黑水虻幼虫取食花生麸的转化率为2888%。     

我国结膜吸吮线虫在中间宿主——变色纵眼果蝇体内发育过程的研究

以结膜吸吮线虫产在家兔眼分泌物内的初产蚴和解剖雌虫子宫内的幼虫,喂饲实验室繁殖的变色纵眼果蝇进行感染,证明了变色纵眼果蝇可作为我国结膜吸吮线虫的中间宿主。以终宿主眼分泌物中的初产蚴喂饲感染果蝇,结果在感染后第16天,于存活的11只果蝇中,在2只雄蝇的口器和头部检出感染期幼虫9条。用取自雌虫体内的幼虫,平均每蝇5条幼虫的比例喂饲感染608只果蝇,结果检出阳性蝇106只,其感染率为17.4％。并发现幼虫在果蝇体内发育主要侵入雄蝇睾丸外层组织和雌蝇的血腔壁形成“虫泡囊”,幼虫在囊内发育至腊肠蚴蜕1次皮,进入感染前期发育阶段,再行第2次蜕皮,而发育成感染期幼虫。发育速度与外界温度密切相关,当外界温度为26.1—31.8℃,发育到感染期幼虫最早天数需要17天。     

外源茉莉酸诱导的青杨叶片保护性酶活性变化及其对舞毒蛾幼虫生长发育的影响

为探讨外源茉莉酸对青杨Populus cathayana的诱导抗性及其对舞毒蛾Lymantria dispar的影响, 室内对青杨扦插苗喷施不同浓度的茉莉酸（对照为0.17%丙酮）, 分别在喷施后1, 5和10 d采集叶片分析其保护性酶活性的变化, 并接舞毒蛾幼虫于青杨苗木上观测其长发育情况。结果表明： 0.1 和0.001 mmol/L两种浓度的茉莉酸（JA）处理均使青杨叶过氧化物酶（POD）、 多酚氧化酶（PPO）、 苯丙氨酸解氨酶（PAL）、 胰凝乳蛋白酶抑制剂（CI）和胰蛋白酶抑制剂（TI）活性较对照增加(P<0.05)。取食茉莉酸诱导的青杨苗木后, 舞毒蛾幼虫的发育历期延长, 体重降低。0.1 mmol/L茉莉酸诱导的青杨苗木5 d后接虫, 使舞毒蛾幼虫的发育历期显著延长, 较对照长8 d; 接虫21 d后称重时, 取食茉莉酸诱导的青杨叶片的幼虫体重较对照组降低了50%~100%, 该结果说明外源茉莉酸诱导青杨产生了对舞毒蛾明显的抗虫性。     

Nematodes, bacteria, and flies: a tripartite model for nematode parasitism

More than a quarter of the world's population is infected with nematode parasites, and more than a hundred species of nematodes are parasites of humans [1-3]. Despite extensive morbidity and mortality caused by nematode parasites, the biological mechanisms of host-parasite interactions are poorly understood, largely because of the lack of genetically tractable model systems. We have demonstrated that the insect parasitic nematode Heterorhabditis bacteriophora, its bacterial symbiont Photorhabdus luminescens, and the fruit fly Drosophila melanogaster constitute a tripartite model for nematode parasitism and parasitic infection. We find that infective juveniles (IJs) of Heterorhabditis, which contain Photorhabdus in their gut, can infect and kill Drosophila larvae. We show that infection activates an immune response in Drosophila that results in the temporally dynamic expression of a subset of antimicrobial peptide (AMP) genes, and that this immune response is induced specifically by Photorhabdus. We also investigated the cellular and molecular mechanisms underlying IJ recovery, the developmental process that occurs in parasitic nematodes upon host invasion and that is necessary for successful parasitism. We find that the chemosensory neurons and signaling pathways that control dauer recovery in Caenorhabditis elegans also control IJ recovery in Heterorhabditis, suggesting conservation of these developmental processes across free-living and parasitic nematodes.     

Identification of genes involved in the mutualistic colonization of the nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> by the bacterium <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis>

Background  

Photorhabdus are Gram negative entomopathogenic bacteria that also have a mutualistic association with nematodes from the family Heterorhabditis. An essential part of this symbiosis is the ability of the bacterium to colonize the gut of the freeliving form of the nematode called the infective juvenile (IJ). Although the colonization process (also called transmission) has been described phenomonologically very little is known about the underlying molecular mechanisms. Therefore, in this study, we were interested in identifying genes in Photorhabdus that are important for IJ colonization.     

Heritability of the Liquid Culture Mass Production Potential of the Entomopathogenic Nematode Heterorhabditis bacteriophora

The infective stage of entomopathogenic nematodes ( Heterorhabditis spp.) is the mobile, but developmentally arrested dauer juvenile (DJ). For commercial application, nematodes are produced in liquid culture. Prior to the inoculation of the DJ, their symbiotic bacterium Photorhabdus luminescens is cultured. The DJ exit from the arrested stage (recovery) and develop to reproductive adults. Recovery is a response to bacterial food signals. In liquid culture the percentage of DJs recovering from the DJ stage is highly variable, which significantly influences the number of reproducing hermaphrodites and the final DJ yields. The liquid culture yield is defined by the number of DJ mL -1 harvested from the medium. The heritability of the disposition to recover from the DJ stage and of the final DJ yield in liquid culture has been evaluated. From a hybrid strain of H. bacteriophora 30 homozygous inbred lines were established by inbreeding over seven generations. These inbred lines were propagated in liquid culture and DJ recovery and yields were recorded. The calculated heritability for the DJ recovery was low ( h 2 = 0.38). No significant genetic variability could be detected for this trait. In contrast, a high heritability ( h 2 = 0.90) was found for the total number of DJs produced in the liquid medium.     

Bionomics of a Phoretic Association Between Paenibacillus sp. and the Entomopathogenic Nematode Steinernema diaprepesi

Spores of an unidentified bacterium were discovered adhering to cuticles of third-stage infective juvenile (IJ) Steinernema diaprepesi endemic in a central Florida citrus orchard. The spores were cup-shaped, 5 to 6 mm in length, and contained a central endospore. Based on 16S rDNA gene sequencing, the bacterium is closely related to the insect pathogens Paenibacillus popilliae and P. lentimorbus. However, unlike the latter bacteria, the Paenibacillus sp. is non-fastidious and grew readily on several standard media. The bacterium did not attach to cuticles of several entomopathogenic or plant-parasitic nematodes tested, suggesting host specificity to S. diaprepesi. Attachment of Paenibacillus sp. to the third-stage cuticle of S. diaprepesi differed from Paenibacillus spp. associated with heterorhabditid entomopathogenic nematodes, which attach to the IJ sheath (second-stage cuticle). The inability to detect endospores within the body of S. diaprepesi indicates that the bacterial association with the nematode is phoretic. The Paenibacillus sp. showed limited virulence to Diaprepes abbreviatus, requiring inoculation of larvae with 108 spores to achieve death of the insect and reproduction of the bacterium. The effect of the bacterium on the nematode population biology was studied in 25-cm-long vertical sand columns. A single D. abbreviatus larva was confined below 15-cm depth, and the soil surface was inoculated with either spore-free or spore-encumbered IJ nematodes. After 7 days, the proportion of IJ below 5-cm depth was seven-fold greater for spore-free IJ than for spore-encumbered nematodes. Mortality of D. abbreviatus larvae was 72% greater (P <= 0.01) for spore-free compared to spore-encumbered S. diaprepesi. More than 5 times as many progeny IJs (P <= 0.01) were produced by spore-free compared to spore-encumbered nematodes. These data suggest that the bacterium is a component of the D. abbreviatus food web with some potential to regulate a natural enemy of the insect.     

Liquid culture of the entomopathogenic nematode-bacterium-complex Heterorhabditis megidis/Photorhabdus luminescens

A process technology for production of the entomopathogenic biocontrol nematode-bacterium complex Heterorhabditis megidis/Photorhabdus luminescens in monoxenic liquid culture in laboratory scale bioreactors is described. Dauer juvenile yields varied between 21 and 68 million dauer juveniles/l medium. The maximum density was reached at 13 to 25 days after inoculation of P. luminescens. The reason for the high variability in yield was identified. After the 24-h bacterial preculture the bioreactor is inoculated with nematode dauer juveniles which develop to self-fertilizing hermaphrodites. The exit from enduring dauer stages (recovery) was between 18 and 90% of the inoculum density. Low dauer juvenile recovery resulted in the development of two-generations within 20 to 25 days. In contrast, high dauer juvenile recovery led to a one-generation process terminated within 15 days. Factors influencing dauer recovery are still unknown.     

Mass Production Potential of the Bacto-Helminthic Biocontrol Complex Heterorhabditis indica - Photorhabdus luminescens

Heterorhabditis indica is a potential agent for the biological control of grubs in sugarcane fields in India. The type strain LN 2 was transferred to monoxenic cultures on its symbiont Photorhabdus luminescens and successfully produced on solid media. In liquid cultures, a mean dauer juvenile yield of 457 000 was obtained with a maximum of 648 000 per ml. Comparatively high yields have not been reported before. Therefore, costs related to the liquid culture production of H. indica will be lower than for other entomopathogenic nematodes currently used in biocontrol. Different bacterial clones had no significant influence on the dauer juvenile yields in liquid media. The exit from the dauer juvenile stage (recovery) after inoculation and the number of hermaphrodites significantly decreased when culture temperature was increased from 25-30 ° C; the dauer juvenile yields were not affected. The cell density of P. luminescens in batch cultures was higher at 25 and 30 ° C than at growth temperatures of 35 and 37 ° C. In continuous culture, the bacterial growth was inhibited when the growth temperature reached 38 ° C. After approximately 60 h, the bacteria adapted to higher temperature and the growth rate increased again. When the temperature was further increased to 40 ° C, the bacterial growth was inhibited.     

Role of Mrx fimbriae of Xenorhabdus nematophila in competitive colonization of the nematode host

Xenorhabdus nematophila engages in mutualistic associations with the infective juvenile (IJ) stage of specific entomopathogenic nematodes. Mannose-resistant (Mrx) chaperone-usher-type fimbriae are produced when the bacteria are grown on nutrient broth agar (NB agar). The role of Mrx fimbriae in the colonization of the nematode host has remained unresolved. We show that X. nematophila grown on LB agar produced flagella rather than fimbriae. IJs propagated on X. nematophila grown on LB agar were colonized to the same extent as those propagated on NB agar. Further, progeny IJs were normally colonized by mrx mutant strains that lacked fimbriae both when bacteria were grown on NB agar and when coinjected into the insect host with aposymbiotic nematodes. The mrx strains were not competitively defective for colonization when grown in the presence of wild-type cells on NB agar. In addition, a phenotypic variant strain that lacked fimbriae colonized as well as the wild-type strain. In contrast, the mrx strains displayed a competitive colonization defect in vivo. IJ progeny obtained from insects injected with comixtures of nematodes carrying either the wild-type or the mrx strain were colonized almost exclusively with the wild-type strain. Likewise, when insects were coinjected with aposymbiotic IJs together with a comixture of the wild-type and mrx strains, the resulting IJ progeny were predominantly colonized with the wild-type strain. These results revealed that Mrx fimbriae confer a competitive advantage during colonization in vivo and provide new insights into the role of chaperone-usher fimbriae in the life cycle of X. nematophila.     

Life Cycle and Reproductive Potential of the Nematode Heterorhabditis bacteriophora Strain HP88

Development of the entomopathogenic nematode Heterorhabditis bacteriophora strain HP88 was studied in vivo with larvae of the greater wax moth, Galleria mellonella, as host and in vitro. At 25 C in vivo, the duration of the life cycle from egg hatch to egg hatch was 96 hours. Juvenile development took 48 hours, with the duration of each juvenile stage ranging from 8 to 12 hours. Under crowded conditions, development proceeded to the infective juvenile (IJ) stage instead of the third juvenile stage (J3). Life-cycle duration and proportion of the various developmental stages in the population were similar in in vitro and in vivo cultures. When in vivo or in vitro development was initiated from the IJ stage, only hermaphrodites developed in the first generation and males appeared only in the second generation. The average (±SD) number of progeny per hermaphrodite was 243 ± 98. The ratio of males to hermaphrodites in the second generation was 1:9.4 ± 6.8.     

Host cadavers protect entomopathogenic nematodes during freezing

The entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema glaseri, and Steinernema feltiae were exposed to freezing while inside their hosts. Survival was assessed by observing live and dead nematodes inside cadavers and by counting the infective juveniles (IJs) that emerged after freezing. We (1) measured the effects of 24h of freezing at different times throughout the course of an infection, (2) determined the duration of freezing entomopathogenic nematodes could survive, (3) determined species differences in freezing survival. Highest stage-specific survival was IJs for S. carpocapsae, and adults for H. bacteriophora. When cadavers were frozen two or three days after infection, few IJs emerged from them. Freezing between five and seven days after infection had no negative effect on IJ production. No decrease in IJ production was measured for H. bacteriophora after freezing. H. bacteriophora also showed improved survival inside versus outside their host when exposed to freezing.