Three microsporidian pathogens infecting Lymantria dispar larvae do not differ in their success in horizontal transmission

We quantified horizontal transmission of three microsporidian pathogens, Endoreticulatus schubergi, Nosema lymantriae and Vairimorpha disparis that infect Lymantria dispar larvae in an experiment using caged, potted oak plants. Despite marked differences in the modes of spore release from infectious hosts, no significant differences in the transmission success to uninfected, susceptible test hosts were ascertained between the tested microsporidian species. The density of initially inoculated larvae and the exposure period, on the other hand, did influence the number of infected test larvae. Depending on the density of inoculated larvae (10%, 30% or 50%), between 0% and 26% of the test larvae became infected with one of the three tested microsporidian pathogens after an exposure period of 6 days. When the exposure period was 12 days, between 11% and 76% of the test larvae became infected.     

Food utilization values of gypsy moth Lymantria dispar (Lepidoptera: Lymantriidae) larvae infected with the microsporidium Vairimorpha sp. (Microsporidia: Burenellidae)

Infection of the gypsy moth, Lymantria dispar, with the microsporidium Vairimorpha sp. strongly influences the development of the host in ways typical of many species of terrestrial entomopathogenic Microsporidia; growth is reduced while development time is extended in infected insects. The appearance of the different stages of the parasite in the host relative to the elapsed time after oral infection, as well as the influence of the parasite proliferation on food utilization of the host, were examined. At 3 days postinfection, midgut muscle cells were infected with primary spores, and the fat body tissues contained meronts, sporonts, and primary spores. Many more fat body cells contained vegetative stages and primary spores at 4 and 5 days postinfection, and diplokaryotic spores and immature octospores were also present. Approximate digestibility of infected larvae increased during this time period, whereas the conversion of ingested and digested food to body substance decreased. The relative growth rate of infected and uninfected groups did not differ significantly between 4 and 5 days postinfection, although the relative consumption rate in infected L. dispar larvae was higher. Between 8 and 10 days postinfection, the relative growth rate of uninfected larvae increased. The infected group did not demonstrate this increase at a time period characterized by maturation of diplokaryotic spores and octospores in larval fat body tissues. Total body weight of uninfected larvae remained higher than that of infected larvae after 8 days postinfection.     

Parasitoid complex of the Asian gypsy moth (Lymantria dispar) (Lepidoptera: Lymantriidae) in Primorye Territory,Russian Far East

A study of the moth parasitoid complex attacking gypsy moth, Lymantria dispar (L.) was carried out in Primorye territory, Russia Far East. Season-long collections at three sites in the Vladivostok area and collections at seven sites of central region of Primorye revealed the 18 primary parasites: one egg parasitoid, 11 larval parasitoids, one larval-pupal parasitoid, one parasitic nematode, one ectoparasitoid, and three diseases including NPV (nuclear polyhedrosis virus) and Entomophaga maimaiga. Phobocampe species (Ichneumonidae) dominated the parasitoid complex, parasitizing 5.5% of the larvae in the Vladivostok area and 9.3% in central Primorye, rates which are much higher than those detected from other Asiatic regions of Russia and Northeastern Asia. The insect parasitoid complex was found to be somewhat depauperate. The 11.8% average total parasitism in eastern Russian is similar to the 12% recorded in the US. Both regions have large gypsy moth outbreaks, but other factors including diseases have compensated for the rather low mortality exerted by the parasitoid complex in the Russian Far East.     

Larviposition of Compsilura concinnata (meigen) (diptera: Tachinidae), a parasitoid of the gypsy moth,Lymantria dispar (L.) (lepidoptera: Lymantriidae)

Larviposition of Compsilura concinnata, a polyphagous tachinid parasitoid of Lepidoptera, is described. The conventional assessment, long‐established in the literature, places the site of larval deposition by C. concinnata as directly into the host's gut. New evidence, reported here, contradicts the earlier view. Dissection of 38 freshly parasitized gypsy moth (Lymantria dispar) larvae revealed as many maggots (n = 26) situated in the lumen of the midgut as were free in the hemocoel. Observations on maggot behavior and morphology in dissected hosts are briefly presented.     

Horizontal and vertical transmission of a Nosema sp. (Microsporidia) from Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

The gypsy moth, Lymantria dispar L. (Lepidoptera, Lymantriidae), a serious defoliator of deciduous trees, is an economically important pest when population densities are high. Outbreaking populations are, however, subject to some moderating influences in the form of entomopathogens, including several species of microsporidia. In this study, we conducted laboratory experiments to investigate the transmission of an unusual Nosema sp. isolated from L. dispar in Schweinfurt, Germany; this isolate infects only the silk glands and, to a lesser extent, Malpighian tubules of the larval host. The latent period ended between 8 and 15 days after oral inoculation and spores were continuously released in the feces of infected larvae until pupation. Exclusion of feces from the rearing cages resulted in a 58% decrease in horizontal transmission. The silk of only 2 of 25 infected larvae contained microsporidian spores. When larvae were exposed to silk that was artificially contaminated with Nosema sp., 5% became infected. No evidence was found for venereal or transovum (including transovarial) transmission of this parasite.     

四种植物酚类物质对舞毒蛾生长发育及繁殖的影响

【目的】为明确植物酚类次生物质对舞毒蛾Lymantria dispar (L.) 的影响,并为应用其防治害虫奠定基础。【方法】本研究选用4种酚酸（水杨酸、丁香酸、绿原酸和水解单宁酸）分别加入人工饲料中饲养舞毒蛾2龄幼虫,调查其对幼虫生长发育及繁殖的影响。【结果】不同酚酸对舞毒蛾的影响差异显著。单宁酸处理第12天时幼虫死亡率达到22.2%,显著高于对照（取食不添加任何酚酸的人工饲料）死亡率（3.3%）（P＜0.01）及其他酚酸处理时的死亡率。到第22天单宁酸及绿原酸使幼虫死亡率迅速上升,至第34天时死亡率达到100%;二者处理的幼虫虫体瘦小,发育历期显著延长,不能正常蜕皮,到4龄期时全部死亡。取食含丁香酸或水杨酸饲料的幼虫可幸存至蛹和成虫,雌性蛹重较对照显著增加,但产卵量和卵受精率均显著降低。其中,取食含丁香酸饲料的成虫产卵量和卵受精率分别比对照减少近90粒和降低约35%,雌性成虫比例下降。【结论】结果说明不同酚酸在害虫体内的积累会对其生长发育及繁殖产生不同程度的影响。     

Starvation resistance of gypsy moth,Lymantria dispar (L.) (Lepidoptera: Lymantriidae): tradeoffs among growth,body size,and survival

Summary Survival and body composition of starving gypsy moth larvae initially reared on aspen foliage or artificial diet differeing in nitrogen (N) and carbohydrate concentration were examined under laboratory conditions. Diet nitrogen concentration strongly affected starvation resistance and body composition, but diet carbohydrate content had no effects on these. Within any single diet treatment, greater body mass afforded greater resistance to starvation. However, starving larvae reared on 1.5% N diet survived nearly three days longer than larvae reared on 3.5% N diet. Larvae reared on artificial diet survived longer than larvae reared on aspen. Differences in survival of larvae reared on artificial diet with low and high nitrogen concentrations could not be attributed to variation in respiration rates, but were associated with differences in body composition. Although percentage lipid in larvae was unaffected by diet nitrogen concentration, larvae reared on 1.5% N diet had a higher percentage carbohydrate and lower percentage protein in their bodies prior to starvation than larvae reared on 3.5% N diet. Hence, larger energy reserves of larvae reared on low nitrogen diet may have contributed to their greater starvation resistance. Whereas survival under food stress was lower for larvae reared on high N diets, growth rates and pupal weights were higher, suggesting a tradeoff between rapid growth and survival. Larger body size does not necessarily reflect larger energy reserves, and, in fact, larger body size accured via greater protein accumulation may be at the expense of energy reserves. Large, fast-growing larvae may be more fit when food is abundant, but this advantage may be severely diminished under food stress. The potential ecological and evolutionary implications of a growth/survival tradeoff are discussed.     

中国舞毒蛾六个地理种群的RAPD分析及SCAR标记构建

舞毒蛾Lymantria dispar L.是世界性农林害虫, 包含不同的亚种, 其中亚洲舞毒蛾的雌蛾具有较强的飞行能力, 已成为国际性的重要检疫性有害生物。然而, 不同舞毒蛾亚种及种群间形态难辨, 因此采用传统的手段鉴别舞毒蛾亚种种群是很困难的。本研究首先采用RAPD标记分析了中国舞毒蛾6个地理种群的遗传多态性。结果表明, 所检测的舞毒蛾种群的遗传分化系数G_st为0.7571, 由此推算出的平均有效迁移数（基因流参数）N_em为0.1604, 说明不同舞毒蛾种群间的遗传分化程度较高, 缺乏广泛的基因流动。本研究在RAPD遗传分析基础之上, 筛选出了4个舞毒蛾种群的特异性遗传位点, 然后对这些特异性位点进行了克隆测序、 序列分析和位点特异性引物设计。结果表明, 其中2个舞毒蛾种群的位点特异性引物可产生序列特征性扩增区域（SCAR）标记。经验证, 这些标记可被用来鉴别特定的舞毒蛾地理种群, 因此有助于对这些舞毒蛾地理种群的分布与扩散进行监测。     

Quantifying horizontal transmission of Nosema lymantriae, a microsporidian pathogen of the gypsy moth, Lymantria dispar (Lep., Lymantriidae) in field cage studies

Nosema lymantriae is a microsporidian pathogen of the gypsy moth, Lymantria dispar that has been documented to be at least partially responsible for the collapse of L. dispar outbreak populations in Europe. To quantify horizontal transmission of this pathogen under field conditions we performed caged-tree experiments that varied (1) the density of the pathogen through the introduction of laboratory-infected larvae, and (2) the total time that susceptible (test) larvae were exposed to these infected larvae. The time frame of the experiments extended from the early phase of colonization of the target tissues by the microsporidium to the onset of pathogen-induced mortality or pupation of test larvae. Upon termination of each experiment, the prevalence of infection in test larvae was evaluated. In the experiments performed over a range of pathogen densities, infection of test larvae increased with increasing density of inoculated larvae, from 14.2 ± 3.5% at density of 10 inoculated per 100 larvae to 36.7 ± 5.7% at 30 inoculated per 100 larvae. At higher densities, percent infection in test larvae appeared to level off (35.7 ± 5.5% at 50 inoculated per 100 larvae). When larval exposure to the pathogen was varied, transmission of N. lymantriae did not occur within the first 15 d post-inoculation (dpi) (11 d post-exposure of test larvae to inoculated larvae). We found the first infected test larvae in samples taken 20 dpi (16 d post-exposure). Transmission increased over time; in the cages sampled 25 dpi (21 d post-exposure), Nosema prevalence in test larvae ranged from 20.6% to 39.2%.     

TAT-Cloan-DH-EGFP融合蛋白的原核表达及其在舞毒蛾幼虫体内的跨膜转导

【目的】为了探究滞育激素经昆虫取食的生物效应, 需设计一种能够加速跨膜运输的融合蛋白, 克服经昆虫消化道的降解作用。【方法】本研究以分月扇舟蛾Clostera anastomosis滞育激素基因Cloan-DH和TAT穿膜肽编码序列为基础, 构建TAT-Cloan-DH融合基因; 然后以增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）为标签, 构建在pET-22b载体中。在0.2 mmol/L IPTG诱导下, 在大肠杆菌Escherichia coli BL21 Rosetta(DE3)中表达出TAT-Cloan-DH-EGFP融合蛋白。 再分别将表达有TAT-Cloan-DH-EGFP和EGFP融合蛋白的大肠杆菌BL21菌体拌入人工饲料,经舞毒蛾Lymantria dispar幼虫持续取食0, 8, 24, 48, 72和90 h后, 随机挑选试虫进行组织固定并制作石蜡切片, 选取中肠所在体段切片进行组织荧光分析。【结果】37℃ 0.2 mmol/L IPTG诱导条件下, 表达的TAT-Cloan-DH-EGFP融合蛋白的分子量为61 kD, 主要以包涵体形式表达, 融合蛋白约占细胞总蛋白含量的18.1%; 随着试虫取食含TAT-Cloan-DH-EGFP融合蛋白饲料的增加, 虫体组织的绿色荧光强度逐渐增加, 取食72 h后, 组织荧光强度达到最高。【结论】实验结果说明融合蛋白TAT-Cloan-DH-EGFP可以通过消化道横向跨膜运输, 到达虫体其他组织, 且该过程与蛋白的摄入量成比例。     

The effect of varying alkaloid concentrations on the feeding behavior of gypsy moth larvae, Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Nine alkaloids (acridine, aristolochic acid, atropine, berberine, caffeine, nicotine, scopolamine, sparteine, and strychnine) were evaluated as feeding deterrents for gypsy moth larvae (Lymantria dispar (L.); Lepidoptera: Lymantriidae). Our aim was to determine and compare the taste threshold concentrations, as well as the ED₅₀ values, of the nine alkaloids to determine their potency as feeding deterrents. The alkaloids were applied to disks cut from red oak leaves (Quercus rubra) (L.), a plant species highly favored by larvae of this polyphagous insect species. We used two-choice feeding bioassays to test a broad range of biologically relevant alkaloid concentrations spanning five logarithmic steps. We observed increasing feeding deterrent responses for all the alkaloids tested and found that the alkaloids tested exhibited different deterrency threshold concentrations ranging from 0.1 to 10 mM. In conclusion, it appears that this generalist insect species bears a relatively high sensitivity to these alkaloids, which confirms behavioral observations that it avoids foliage containing alkaloids. Berberine and aristolochic acid were found to have the lowest ED₅₀ values and were the most potent antifeedants. Handling Editor: Joseph Dickens.     

Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.     

Increased mortality of gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) exposed to gypsy moth nuclear polyhedrosis virus in combination with the phenolic gycoside salicin

Second instar gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), larvae suffered significantly greater mortality from aerially applied gypsy moth nuclear polyhedrosis virus (Gypchek) when the virus was consumed on quaking aspen, Populus tremuloides Michx., versus red oak, Quercus spp. L., foliage. Laboratory assays in which various doses of Gypchek and salicin (a phenolic glycoside present in aspen foliage) were tested in combination demonstrated that salicin significantly increased total larval mortality and lowered the LD50 estimates (dose of Gypchek that resulted in 50% population mortality) for the virus, although not significantly. While salicin did not impact larval survival in the absence of Gypcek, it did act to significantly deter feeding when it was present in high concentrations (up to 5.0%) within the treatment formulations. The enhanced activity of Gypchek in the presence of salicin is similar to prior reports of enhanced activity of the bacterial pathogen Bacillus thuringiensis when consumed concurrently with phenolic glycosides commonly present in aspen foliage. The enhancement of viral activity is in contrast to the inhibitory effects on the virus reported for another common group of phenolic compounds, tannins.     

The impact of mixed infection of three species of microsporidia isolated from the gypsy moth,Lymantria dispar L. (Lepidoptera: Lymantriidae)

The outcome of mixed infection by three species of microsporidia in the genera Endoreticulatus, Nosema, and Vairimorpha, isolated from different populations of Lymantria dispar in Bulgaria, was evaluated in the laboratory. All possible combinations of two species were administered either simultaneously or sequentially to larvae, and mortality, duration of development, and larval weight at 20 days post-infection (simultaneous inoculation) or 23 days post-infection (sequential inoculation) were chosen as the outcome variables. Larvae were also dissected and the presence of each species of microsporidia and the tissues infected were recorded for each treatment. Effects of infection were dependent on both host sex and the type of exposure. Infected larvae were more likely to die than uninfected larvae, but there were no differences in mortality between single and mixed infections. Addition of Endoreticulatus to infections of Nosema or Vairimorpha significantly increased duration of development to the fourth ecdysis; this effect was additive. Addition of Nosema or Vairimorpha to an existing infection had no such effect. When Nosema was administered simultaneously with Endoreticulatus or Vairimorpha, infected larvae weighed more than larvae that had single infections with either pathogen. Nosema was displaced from the silk glands by Vairimorpha and Nosema suppressed octospore formation by Vairimorpha in fat body. The histological evidence combined with the data on larval weight supports the hypothesis that competition occurred in mixed infections.     

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。     

Nosema chrysorrhoeae n. sp. (Microsporidia), isolated from browntail moth (Euproctis chrysorrhoea L.) (Lepidoptera, Lymantriidae) in Bulgaria: characterization and phylogenetic relationships

A new microsporidian parasite Nosema chrysorrhoeae n. sp., isolated in Bulgaria from the browntail moth (Euproctis chrysorrhoea L.), is described. Its life cycle includes two sequential developmental cycles that are similar to the general developmental cycles of the Nosema-like microsporidia and are indistinguishable from those of two Nosema spp. from Lymantria dispar. The primary cycle takes place in the midgut tissues and produces binucleate primary spores. The secondary developmental cycle takes place exclusively in the silk glands and produces binucleate environmental spores. N. chrysorrhoeae is specific to the browntail moth. Phylogenetic analysis based on the ssu rRNA gene sequence places N. chrysorrhoeae in the Nosema/Vairimorpha clade, with the microsporidia from lymantriid and hymenopteran hosts. Partial sequences of the lsu rRNA gene and ITS of related species Nosema kovacevici (Purrini K., Weiser J., 1975. Natürliche Feinde des Goldafters, Euproctis chrysorrhoea L., im Gebiet von Kosovo, FSR Jugoslawien. Anzeiger fuer Sch?dlingskunde, Pflanzen-Umweltschutz, 48, 11-12), Nosema serbica Weiser, 1963 and Nosema sp. from Lymantria monacha was obtained and compared with N. chrysorrhoeae. The molecular data indicate the necessity of future taxonomic reevaluation of the genera Nosema and Vairimorpha.     

Changes in range boundaries of the gypsy moth and the nun moth (Lymantria dispar,L. monacha,Lymantriidae, Lepidoptera) due to the global warming: a model approach

Entomological Review - Modeling of the gypsy moth and nun moth ranges using climatic predictors and the updated climate database suggested potential consequences of the observed climate changes for...     

Occurrence of Cystosporogenes sp. (Protozoa, Microsporidia) in a multi-species insect production facility and its elimination from a colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae)

We have isolated a microsporidium from a laboratory colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). Light and electron microscopic investigations showed that gross pathology and ultrastructure of our isolate are similar to those described for Cystosporogenes legeri from the European grape vine moth, Lobesia botrana. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods revealed perfect homology with the C. legeri sequence. The microsporidian was infectious to other Choristoneura species, as well as Malacosoma disstria, Lymantria dispar, and Lambdina fiscellaria. Incubation of infected egg masses at 41 degrees C for 20 min followed by 30 min in 33% formaldehyde did not reduce disease incidence in larval offspring. Exposure of one or two generations to fumagillin at 6000 ppm or higher eliminated infection in adult moths, but also reduced colony fitness. A clean colony was established by conducting individual matings and selecting disease-free offspring.