Characterization and polyhedrin gene cloning of Lymantria xylina multiple nucleopolyhedrovirus

A baculovirus has been isolated from infected larvae of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae) in Taiwan. Ultrastructural observation revealed that this virus is a multiple nucleopolyhedrovirus (MNPV), and the name L. xylina MNPV (LyxyMNPV) was proposed. Restriction endonuclease (BamHI, EcoRI, and EcoRV) profiles of LyxyMNPV genome differed from those of other known NPVs. The size of the LyxyMNPV genome was estimated to be 154+/-1.26 kbp (mean+/-SE). The polyhedrin gene is located in the BamHI-D, EcoRI-C, and EcoRV-K fragments of LyxyMNPV genome. The gene organization of the LyxyMNPV EcoRV-K fragment and the phylogenetic analysis based on the polyhedrin gene sequences showed that LyxyMNPV is closely related to the L. dispar MNPV (LdMNPV). Furthermore, a rapid assay method was developed to distinguish LyxyMNPV from LdMNPV based on PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis of polyhedrin genes.     

New cell lines from larval fat bodies of Spodoptera exigua: characterization and susceptibility to baculoviruses (Lepidoptera: Noctuidae)

Two new cell lines, designated IOZCAS-Spex-II and IOZCAS-Spex-III, were initiated from the fat bodies of larvae of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. The spherical cells were predominant among the various cell types and measures approximately 15 microm in diameter. The cell lines were mainly composed of tetraploid cells with chromosome numbers ranging from 116 to 131 (n=31). The cell lines were confirmed to have originated from the S. exigua by DAF-PCR technique. They were susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses from S. exigua.     

A cell line (NTU-MV) established from Maruca vitrata (Lepidoptera: Pyralidae): Characterization, viral susceptibility, and polyhedra production

Here we describe the establishment of a new cell line, NTU-MV, derived from pupal tissues of an economically important pest, the legume pod borer Maruca vitrata. This cell line contained four major cell types: polymorphic cells, round cells, spindle-shaped cells, and comma cells. The doubling time of MV cells in TNM-FH medium supplemented with 8% FBS at 28 degrees C was 27h. The chromosome numbers of MV cells varied widely from 16 to 268. Compared to other insect cell lines, the MV cell line produced distinct isozyme patterns with esterase, malate dehydrogenase (MDH), and lactate dehydrogenase (LDH). Confirmation that NTU-MV was derived from M. vitrata was demonstrated by showing that the sequence of the internal transcribed spacer regions (ITS) of the MV cells was 98% identical to that of M. vitrata larvae. Two NTU-MV cell strains, NTU-MV1 and NTU-MV56, were selected based on susceptibility to MaviMNPV (M. vitrata multiple nucleopolyhedrovirus). NTU-MV, MV1, and MV56 cells showed a high susceptibility to MaviMNPV and produced high yields of polyhedra (47-50OBs/cell, 4x10(7)-5.96x10(7)OBs/ml) after 2 weeks of MaviMNPV infection. We conclude that the NTU-MV cell line will be a useful tool for studying MaviMNPV as well as for the mass production of MaviMNPV polyhedra for the biocontrol of M. vitrata.     

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.     

Larvicidal and antifeedant activity of some plant-derived compounds to Lymantria dispar L. (Lepidoptera: Limantriidae)

Ethanol solutions of essential oil of Ocimum basilicum and its main component, linalool (both isomer forms), all in three concentrations, as well as botanical standard Bioneem (0.5%), were tested for their toxicity and antifeedant activity against the second instar gypsy moth larvae in the laboratory bioassay. The essential oil of O. basilicum was subjected to gas chromatography analysis, and totally 37 compounds were detected, of which linalool was predominantly present. All tested solutions showed low to moderate larvicidal effect in both residual toxicity test and in chronic larval mortality bioassay. Chronic mortality tests showed that obtained mortality was a consequence of starving rather than ingestion of treated leaves. However, antifeedant index achieved by application of tested solutions in feeding choice assay was remarkable. Foliar application of all tested compounds deterred feeding by L2 in the same percent as Bioneem. Antifeedant index was relatively high at all tested treatments (85-94%); moreover, the larval desensitization to repelling volatiles has not occurred after five days of observation. Low toxic and high antifeedant properties make these plant-derived compounds suitable for incorporation in integrated pest management programs, especially in urban environments.     

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.     

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.     

Response of Lymantria dispar L. (Lepidoptera: Lymantriidae) to Bacillus thuringiensis subsp. kurstaki at different ingested doses and temperatures

We examined mortality and feeding inhibition response of Lymantria dispar L. (Lepidoptera: Lymantriidae) larvae to ingested doses of Bacillus thuringiensis subsp. kurstaki as a function of dose, instar and temperature. We developed generalized (logistic) linear mixed models and a mixture survival model, commonly used in medical statistics, to analyze the complex data set. We conducted bioassays of Foray 48B with larvae from the NJSS laboratory stock, using droplet imbibing or force-feeding to ensure dose ingestion. The dose causing mortality in 50% of the test population (LD₅₀) under standard test conditions (22 °C) ranged from 0.019 International Units (IU)/larva for first instar larvae (L₁) to 1.6 IU/larva for L₄. Temperature affected larval mortality in two ways. Mortality occurred sooner and progressed more rapidly with increasing temperature (13-25 °C) at each dose level and instar, while the maximum level of mortality attained by each instar decreased with increasing rearing temperature. The mechanisms underlying this effect are being investigated. Larvae that survived exposure to B. thuringiensis resumed feeding after a period that was dependent on instar, dose, and temperature. The equations describing observed mortality and feeding recovery responses were used to construct a simulation model, which was able to predict both processes, and which forms the basis for a process-oriented model that can be used as a decision support tool in aerial sprays.     

Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae)

Glyptapanteles liparidis is a gregarious, polydnavirus (PDV)-carrying braconid wasp that parasitizes larval stages of Lymantria dispar. In previous studies we showed that parasitized hosts dramatically increase juvenile hormone (JH) titers, whereas JH degradation is significantly inhibited in the hemolymph. Here we (i) quantified the effects of parasitism on JH esterase (JHE) activity in hemolymph and fat body of penultimate and final instars of L. dispar hosts and (ii) assessed the relative contribution of individual and combined wasp factors (PDV/venom, teratocytes, and wasp larvae) to the inhibition of host JHE activity. The effects of PDV/venom was investigated through the use of gamma-irradiated wasps, which lay non-viable eggs (leading to pseudoparasitization), while the effects of teratocytes and wasp larvae were examined by injection or insertion of these two components in either control or pseudoparasitized L. dispar larvae. Parasitism strongly suppressed host JHE activity in both hemolymph and fat body irrespective of whether the host was parasitized early (premolt-third instar) or late (mid-fourth instar). Down-regulation of JHE activity is primarily due to the injection of PDV/venom at the time of oviposition, with only very small additive effects of teratocytes and wasp larvae under certain experimental conditions. We compare the results with those reported earlier for L. dispar larvae parasitized by G. liparidis and discuss the possible role of JH alterations in host development disruption.     

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.     

Characterization of a new insect cell line that is derived from the neonate larvae of Papilio xuthus (Lepidoptera: Papilionidae) and its susceptibility to AcNPV

The cell line RIRI-PX1 was established from neonate larval tissues of Papilio xuthus by performing primary cultures in the modified Grace medium that was supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of spindle-shaped and spherical cells which attached themselves to the flask. The population-doubling times (PDTs) at the 50th and 60th passage were 42.5 h and 42.1 h respectively. The average chromosome numbers of RIRI-PX1 cell line from passage 5 to passage 50 ranged from 103 to 199. It was confirmed that RIRI-PX1 cell line was derived from P. xuthus by comparing the mitochondrial cytochrome c oxidase subunit I gene (COI) of RIRI-PX1 cells and P. xuthus eggs. This cell line was susceptible to the Autographa californica nucleopolyhedrovirus (AcNPV) and produced high yield of polyhedral occlusion bodies (43.9 OBs/cell) after 10 days of infection by AcNPV. The virus titer of AcNPV infected RIRI-PX1 cells was 3.25 × 10⁷ TCID₅₀/ml. We concluded that the RIRI-PX1 cell line is established from the neonate larvae tissues successfully and the cells of the cell line are sensitive to AcNPV.     

Relative activity of baculoviruses of the diamondback moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis> (L.) (Lepidoptera: Plutellidae)

The relative activities of the granulovirus (PxGV) and the nucleopolyhedrovirus (PxMNPV) of the diamondback moth, Plutella xylostella (L.), against this insect were evaluated in the laboratory. Similar numbers of occlusion bodies (OBs) of PxGV and PxMNPV were required to kill P. xylostella. However, cadavers of insects infected with PxGV contained more OBs per unit weight than did cadavers infected with PxMNPV. Less cadaver material was thus required to kill insects with PxGV. Larvae that were killed by PxGV survived approximately 2 d longer than did those killed by PxMNPV. When P. xylostella larvae were fed both viruses together, mortality was higher than that caused by either virus alone. The virulence of PxMNPV produced in P.␣xylostella did not differ from that of PxMNPV produced in the beet armyworm, Spodoptera exigua (Hübner). S. exigua was less susceptible to PxMNPV than to its homologous nucleopolyhedrovirus, SeMNPV. Use of trade names does not imply endorsements by USDA-ARS of products named nor criticism of similar ones not mentioned.     

Diel pattern in the structure and function of the gut microbial community in Lymantria dispar asiatica (Lepidoptera: Lymantriidae) larvae

In the present study, diel pattern in gut microbial communities in insects were evaluated. Lymantria dispar asiatica fourth instar larvae (72 ± 2 hr after molting) at noon (LdD) and midnight (LdN) were used for a comparative analysis of the gut microbial community. Ten bacterial operational taxonomic units (OTUs) were shared between LdD and LdN samples. One bacterial OTU was specific to LdD. The dominant gut microbes were OTU72 in LdD and OTU75 in LdN. A linear discriminant analysis effect size cladogram suggested that ten bacterial OTUs maintain significant differences in relative abundances between LdD and LdN. These results agreed with the discrete ellipses between LdD and LdN in principal coordinates analysis plots. Additionally, using phylogenetic investigation of communities by reconstruction of unobserved states, the gut microbial community was assigned to 23 functional terms, among which 22 exhibited significant differences between LdD and LdN. To conclude, the present study documented a diel pattern in the gut microbial community of L. dispar asiatica larvae.     

Development of cell lines from the cactophagous insect: Cactoblastis cactorum (Lepidoptera: Pyralidae) and their susceptibility to three baculoviruses

The unintentional introduction of the cactus moth, Cactoblastis cactorum, a successful biological control agent formerly employed in the control of invasive prickly pear cactus species (Opuntia spp.) in Australia, Hawaii, South Africa, and various Caribbean islands, has posed great concern as to the possible threat to native, endangered species of cactus in the southeastern USA as well as with the potential to cause a major infestation of commercial and agricultural cactus crops in Mexico. A number of control measures have been investigated with varying degrees of success including, field exploration for cactus moth-specific parasitoids, insecticides, fungal, bacterial, and nematode agents. Current tactics used by the USA-Mexico binational program to eradicate cactus moth from Mexico and mitigate its westward movement in the USA include host plant removal, the manual removal and destruction of egg sticks and infected cacti stems, and the Sterile Insect Technique. One other approach not taken until now is the development of a cactus moth cell line as a tool to facilitate the investigation of baculoviruses as an alternative biocontrol method for the cactus moth. Consequently, we established C. cactorum cell lines derived from adult ovarian tissue designated as BCIRL-Cc-AM and BCIRL-Cc-JG. The mean cell population doubling time was 204.3 and 112 h for BCIRL-Cc-AM and BCIRL-Cc-JG, respectively, with weekly medium change, while the doubling time was 176.6 and 192.6 h for BCIRL-Cc-AM and BCIRL-Cc-JG, respectively, with a daily change of medium. In addition, the daily versus weekly change in medium was reflected in the percentage viability with both cell lines showing higher levels with a daily medium change. Of the three baculoviruses tested, only the recombinant AcMNPV-hsp70Red and GmMNPV at a multiplicity of infection (MOI) of 1.0 were able to demonstrate significant production of extracellular virus (ECV) in each of the cell lines, whereas both cell lines were refractive to an HzSNPV challenge at an MOI of 10. In this study, we have demonstrated both the successful development of a C. cactorum cell line and its ability to support a complete baculovirus infection. The potential is also there to pursue further investigations to determine the susceptibility of the cactus moth cell line to other viruses. Additionally, the availability of a cactus moth cell line will facilitate the analysis of viruses prior to using the more expensive bioassay test. Finally, it is hoped with the knowledge presented here that baculoviruses may also be considered as an alternative biocontrol method for the cactus moth.     

Genetic structure, population size, and seed production of Pulsatilla vulgaris Mill. (Ranunculaceae) in Central Germany

Pulsatilla vulgaris Mill. (Ranunculaceae) is a rare and rapidly declining grassland community species that was once widespread at a time when Central Germany was covered by steppe vegetation. Through the course of this study, the patterns of random-amplified polymorphic DNA (RAPD) variation among 11 populations of varying size were analysed to assess any possible local differentiation, in relation to spatial isolation, resulting from random genetic drift brought on by reduced population size and lack of migration between geographically isolated populations. Following results attained from methods including: multivariate analysis based on asymmetric Soerensen similarity, φ_ST-statistics, and analysis of molecular variance, we were able to conclude that there is a high within-population variability (84.4%) and a weak, but significant, differentiation among populations (φ_ST=0.17). A matrix correlation between genetic and geographical distances revealed that geographical differentiation was reflected in the RAPD profile (Mantel test: r=0.47,p=0.002). Further significant correlations were noted between population size and both percentage of polymorphic loci (p=0.02) and genetic diversity (p=0.03). An additional analysis of seed production showed that mean seed set, seed number, and mean seed mass per population could be attributed to differences in population size, whereas only seed mass was related to genetic variation.     

Vertical transmission and overwintering of microsporidia in the gypsy moth, Lymantria dispar

Vertical transmission and the overwintering success of three different microsporidia infecting Lymantria dispar (Lepidoptera: Lymantriidae) larvae were investigated. Endoreticulatus schubergi, a midgut pathogen, was transmitted to offspring via female and male via the egg chorion (transovum transmission). Between 8% and 29% of the emerging larvae became infected. No spores of E. schubergi were found in surface-washed eggs. Nosema lymantriae, a microsporidium that causes systemic infections, was transovarially transmitted. Between 35% and 72% of the progeny were infected. Vairimorpha disparis, a fat body pathogen, was not vertically transmitted. The infectivity of spores that overwintered in cadavers of infected L. dispar varied by species, placement in the environment, and weather conditions. Spores of E. schubergi were still infective after an eight month exposure period of cadavers on the ground. Spores of N. lymantriae and V. disparis remained highly infective only when cadavers overwintered under a more or less continuous snow cover for four months.     

Characterization of a nucleopolyhedrovirus of Epinotia granitalis (Lepidoptera: Tortricidae)

A nucleopolyhedrovirus (NPV) from the cypress bark moth Epinotia granitalis Butler was partially characterized. Electron microscopy indicated that E. granitalis NPV (EpgrNPV) is a singly embedded nucleocapsid NPV with a relatively small polyhedron, ranging from 480 to 1100nm in diameter. Phylogenetic analysis revealed that EpgrNPV is a Group II NPV that is related to but distinct from Adoxophyes honmai NPV. The LC(50) against third instar E. granitalis larvae was 10(5.5) polyhedral occlusion bodies per gram (OBs/g diet), and the average time to death was 14 days. E. granitalis was the only EpgrNPV-susceptible species in a series of infection tests using insect species belonging to the family Tortricidae. Other test species, including Eucoenogenes ancyrota, A. honmai, Adoxophyes dubia, Pandemis heparana, Homona magnanima, and Archips insulanus, were not susceptible to the infection.     

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%.     

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。     

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.