Utilization of the polymerase chain reaction in the diagnosis of nuclear polyhedrosis virus infections of gypsy moth (Lymantria dispar, Lep., Lymantriidae) populations

Abstract:  Three specific DNA probes were used for the detection of the nuclear polyhedrosis (NPV) virus of Lymantria dispar ( Ld NPV) genome. Two of these probes, H2 and H3 were obtained by classical cloning method and one (TR6) by polymerase chain reaction (PCR). These probes, used individually or in a pool in the standard slot–blot hybridizations, were able to detect 10⁹ genome copies. By performing 35 cycles of PCR amplification before hybridization with primers specific to Ld NPV genome on DNA extracted from infected larvae, the sensitivity of the hybridization technique was increased, so that as little as 10 copies of the Ld NPV genome could be detected. Using these methods, L. dispar naturally infected by Ld NPV were identified among field populations in Canada and in the United States near the eastern Canadian border. Using a combination of PCR and hybridization, Ld NPV contamination of egg masses were also detected. By disinfecting the eggs with sodium hypochlorite prior to PCR amplification and hybridization, it was also demonstrated that transmission of viral infection in the natural populations is mainly caused by external contamination of the egg and is unlikely to occur through the transovarial route.     

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%.     

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.     

Impact of predators on artificially augmented populations of Lymantria dispar L. pupae (Lep., Lymantriidae)

The impact of predators on artificially augmented populations of Lymantria dispar pupae (Lep., Lymantriidae) and the species composition of the predator community were investigated in two oak stands in eastern Austria. The population density of L. dispar has been at innocuous levels for several years at both sites. From mid-June to mid-July, we created artificial prey populations by mounting gypsy moth pupae with beeswax on burlap bands placed at the base, 25, 50 and 100 cm height on 25 trees at each site. A total of 6600 pupae were exposed at each site. A total of 92% of exposed pupae were destroyed by predators, at site I (with dense understorey vegetation) whereas 67% were destroyed on site II (with sparse understorey vegetation). Initially, pupal mortality was highest at the base of trees, but differences in mortality among the four locations where pupae were exposed decreased during the course of the study period. Spatial differences in predation were ascertained, suggesting that the aggregation of small mammals, for example, is related to patches of dense shrub vegetation. Pupal mortality was primarily caused by mice (46.3% at site I and 36.4% at site II). Many exposed pupae simply disappeared (40.4% at site I and 22.8% at site II). Calosoma spp. and other invertebrate predators caused very little mortality among L. dispar pupae (5.5% at site I and 7.8% at site II). Trap catches on separate study plots at both sites revealed that Apodemus flavicollis (Rodentia, Muridae) was the most important predator species present, whereas Apodemus sylvaticus (Rodentia, Muridae) and Clethrionomys glareolus (Rodentia, Arvicolidae) were captured in low numbers.     

Towards pheromone-based monitoring of nun moth, Lymantria monacha (L.) (Lep., Lymantriidae) populations

The research objective was to develop pheromone-based monitoring of the nun moth, Lymantria monacha (L.), an important defoliator of spruce and pine forests in central Europe. In 38 spruce or pine forests in central Europe, captures of male L. monacha in nonsaturating Unitraps and saturating Delta sticky traps baited with 0.2, 2, 20, or 200 μg of the L. monacha (pheromone) volatile blend [(±)-disparlure, (±)-monachalure, and 2-methyl- Z 7-octadecene at a 20 : 20 : 1 ratio] were compared with estimates of population densities obtained by counts of larval faecal pellets, pupal cases, and adult moths resting on tree trunks. Total captures of male L. monacha throughout the flight season in both types of trap were correlated with numbers of larval faecal pellets, irrespective of pheromone dose. Nonsaturating Unitraps baited with 2 μg of the L. monacha volatile blend seem to provide a cost-effective tool for monitoring densities of L. monacha populations. Long-term testing of this monitoring system has been initiated to substantiate the quantitative relationship between larval populations and trap captures of male L. monacha and to determine the threshold number of captured male moths that indicates an incipient outbreak.     

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.     

Natural occurrence of the nucleopolyhedrosis virus of the gypsy moth,Lymantria dispar [Lep.: Lymantriidae] in wild birds and mammals

Three species of birds and 5 species of mammals were captured in the wild from 2 plots in which mortality from naturally occurring nucleopolyhedrosis virus (NPV) among gypsy moth,Lymantria dispar (L.), larvae was 15% and 70%. Bioassays of intestinal contents showed that blue jays,Cyanocitta cristata (L.), towhees,Pipilo erythrophthalmus (L.), white-footed mice,Peromyscus leucopus (Rafinesque), redback voles,Clethrionomys gapperi (Vigers), raccoons,Procyon lotor (L.), and a chipmunk,Tamias striatus (L.), contained infectious (L.), and masked shrews,Sorex cinereus (Kerr), did not. Comparisons among mice and voles indicated that those collected from the plot in which the NPV mortality was greatest (70%) contained the most virus. We concluded that birds and mammals can passively transport infectious gypsy moth NPV in the wild.

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Résumé Trois espèces d'oiseaux et 5 espèces de mammifères ont été capturées en 2 endroits où la mortalité due à la présence naturelle du virus de la nucléopolyhédrose (VNP) chez les larves deLymantria dispar (L.), était de 15% et de 70%. Le système digestif des geais bleux,Cyanocitta cristata (L.), guiraca à poitrine rose,Pipilo erythrophthalmus (L.), souris-au-pied-blanc,Peromyscus leucopus (Rafinesque), campagnols-au-dos-rouge,Clethrionomys gapperi (Vigers), ratons laveurs,Procyon lotor (L.) et d'un tamia,Tamias striatus (L.), était infecté par le virus alors que celui des rouges-gorges,Turdus migratorius (L.) et des musaraignes,Sorex cinereus (Kerr), ne l'état pas. Une comparaison entre les campagnols et les souris a indiqué que les animaux provenant de l'endroit où la mortalité par virose était la plus forte (70%) contenaient le plus de virus. Nous concluons que les oiseaux et les mammifères peuvent transporter passivement dans la nature les polyèdres nucléaires deL. dispar.

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The work reported was funded in part by a U.S. Department of Agriculture sponsored program entitled, “The Expanded Gypsy Moth Research, Development and Applications Program”.     

Response of beech (Fagus sylvatica) to elevated CO2 and N: Influence on larval performance of the gypsy moth Lymantria dispar (Lep., Lymantriidae)

Two-year-old beech seedlings were kept from germination to bioassays with Lymantria dispar under the following conditions: ambient CO₂/low N, elevated CO₂/low N, ambient CO₂/elevated N, and elevated CO₂/elevated N. The effect of these growing conditions of the trees on the performance of the defoliator L. dispar was studied 2 years after initiating the tree cultivation. The developmental success of third-instar larvae of L. dispar was characterized by the weight gained, percentage of weight gain, relative growth rate (RGR), relative consumption rate (RCR), and efficiency of conversion of ingested food into body substance (ECI). Contrary to our expectations, additional N-fertilization did not increase and elevated CO₂ did not delay larval growth rate. However, the environmental treatments of the beech seedlings were found to affect the larval performance. Larvae consumed significantly higher amounts of foliage (RCR) on beech trees under controlled conditions (ambient CO₂ and low N) compared to those under elevated CO₂ and enhanced N. The opposite was true for ECI. The lowest efficacy to convert consumed food to body substance was observed under control conditions and the highest when the larvae were kept on beech trees grown under elevated CO₂ and additional N-fertilization. These opposite effects resulted in the weight gain-based parameters (absolute growth, percentage of growth, and RGR) of the gypsy moth larvae remaining unaffected. The results indicate that the gypsy moth larvae are able to change their ECI and RCR to obtain a specific growth rate. This is discussed as an adaptation to specific food qualities.     

Phenology of Parasetigena silvestris (Diptera: Tachinidae), gypsy moth (Lymantria dispar) (Lepidoptera: Lymantriidae) larval parasitoid and its efficiency for parasitisation

Parasetigena silvestris is a univoltine, solitary, larval endoparasitoid which lays its eggs on the surface of gypsy moth larvae. Field collection of the host larvae (2nd through 5th instar) from an artificially established gypsy moth population were made to compare stage specific parasitism between larvae without and with P. silvestris tachinid eggs. The tachinid oviposition rate detected was highest in second instar larvae, and then decreased as larvae developed toward full maturity. The opposite was true for tachinid parasitoid emergence which had no emergence from second through third host instar larvae. Fourth instar gypsy moth larvae, however, experienced significantly higher parasitism by P. silvestris in the larvae with eggs than those without the eggs. The braconid wasp Cotesia melanoscelus caused significantly higher parasitism in early instar larvae with P. silvestris eggs than in those without the eggs. The tachinid prefers to lay more eggs on parasitised larvae by the braconid even though the braconid is a superior competitor to the fly during multiparasitism. Factors influencing parasitism rates by P. silvestris such as host-parasitoid synchronisation and the multiparasitism interaction with C. melanoscelus are discussed.     

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。     

Effects of a kaolin-based particle film on oviposition and feeding of gypsy moth (Lep., Lymantriidae) and forest tent caterpillar (Lep., Lasiocampidae) in the laboratory

Abstract:  In the laboratory we investigated a kaolin-based crop protectant, Surround^® WP, that was mixed in a spray carrier (94% water, 2% methanol, 4% Triton X100) and applied as particle film against gypsy moth Lymantria dispar (L.) and forest tent caterpillar Malacosoma disstria Hubner. Paired-choice feeding tests showed that gypsy moth larvae were significantly (P < 0.05) less disposed to feed on red oak Quercus rubrus L. treated with high concentrations (60 and 120 g/l carrier) of kaolin than on untreated foliage. There was no significant difference (P > 0.05) between gypsy moth's consumption of kaolin-treated and untreated red oak when given a single food choice, but the adults were less inclined to oviposit on the kaolin-treated surfaces than on controls. However, no kaolin treatment completely deterred gypsy moth feeding or oviposition. Kaolin particle film did not influence forest tent caterpillar feeding and there were insufficient data to evaluate the effect of kaolin on the species' oviposition. The spray carrier did not increase the efficacy of the kaolin, but as an independent, albeit impractical treatment it constrained feeding by both insect species but did not affect gypsy moth oviposition. These initial laboratory findings suggest that applying kaolin-based particle film to forests would present significant challenges; but if these could be surmounted, the technique might assist in the management of gypsy moth but not likely in that of forest tent caterpillar.     

Biomodal patterns of mortality from nuclear polyhedrosis virus in gypsy moth (Lymantria dispar) populations

A bimodal temporal pattern of mortality caused by the nuclear polyhedrosis virus (NPV) was observed in nine gypsy moth (Lymantria dispar) populations of varying densities. In all cases, peak mortality from NPV occurred during the second wave (late larval instars) and the highest mortality occurred in high density populations. Patterns of NPV mortality were established several weeks before being expressed. There was no discernible correlation between weekly mortality rates and temperature, rainfall, or total solar radiation. The bimodality was also apparent in NPV contamination on foliage which was measured by bioassay. A similar pattern was observed in the laboratory among larvae reared in groups from field-collected egg masses and from eggs artificially contaminated with NPV from a laboratory population. As in field populations, the period of low mortality from NPV between the two waves occurred when most larvae were late third and fourth instars. Larvae reared individually did not exhibit the second wave of mortality.     

Intestinal modifications of oak leaf tannins by Lymantria dispar L. (Lep., Lymantriidae) and possible effect on larval development

The mode of action of polyphenols within an insect is not well understood and it would be useful to investigate the behaviour pattern of single phenolic compounds within the gut of an insect in this context. In the present study, the phenolic composition of the food of the gypsy moth, Lymantria dispar , was compared with that of its frass by using high-performance liquid chromatography. The main difference was a shift in the concentration of phenolics in the frass with short retention times to those with longer ones, which, additionally, resulted in the appearance of new phenolic compounds. These new phenolics were hydrolysable tannins because after a cleavage via hydrolysation the hydrolysate contained high amounts of gallic acid. The appearance of these new phenolics in the frass of the larvae influenced growth negatively, as there was a negative correlation (P < 0.05) between their concentrations and the growth percentages of the larvae.     

Response of the gypsy moth Lymantria dispar L. (Lepidoptera, Lymantriidae) to the content of allelochemicals in birch leaves (Betula pendula Roth)

Structural and functional characteristics of the gypsy moth population in a birch forest were found to be correlated with the level of defoliation and the content of allelochemicals in the foliage. One year after defoliation, the content of flavonoids, total lipid fractions, fatty alcohols, and alcohols increased in the foliage of the heavily (by 75%) damaged trees, whereas the content of free sterols and triterpenes decreased. These changes were associated with low vitality of the insects and a sharp drop in the population density.     

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。     

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.     

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.     

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.     

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.