Efficacy of Heterorhabditis baujardi LPP7 (Nematoda: Rhabditida) applied in Galleria mellonella (Lepidoptera: Pyralidae) insect cadavers to Conotrachelus psidii, (Coleoptera: Curculionidae) larvae

The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil causing severe reduction in fruit quality. We assessed its susceptibility to Heterhorhabditis baujardi LPP7 infective juveniles (IJs) in the greenhouse and under field conditions applying the nematodes in cadavers of seventh instar Galleria mellonella larvae. Field persistence of these nematodes in the soil was evaluated through G. mellonella-baiting. Insect cadaver concentrations of 2, 4 and 6 applied in pots in the greenhouse experiment caused significant mortality compared to the control. Significance differences were observed in the field between control and treatments only when six cadavers per 0.25 m² were applied. Infective juveniles from the cadavers persisted 6 weeks after application in the field, but decreased greatly thereafter. Our work demonstrates that H. baujardi LPP7 IJs emerging from G. mellonella cadavers can be efficacious against guava weevil fourth instar larvae. Also, we demonstrated the long-term persistence of IJs in the soil.     

Comparative study of entomopathogenic nematode isolation using Galleria mellonella (Pyralidae) and Spodoptera littoralis (Noctuidae) as baits

Entomopathogenic nematode (EPN) occurrence in soil from natural areas and crop field edges from La Rioja (northern Spain) was compared using two insects as baits: Galleria mellonella (Lepidoptera: Pyralidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). Both insects trapped Steinernema feltiae, S. kraussei and S. carpocapsae, with G. mellonella being more efficient than S. littoralis recording 5.4 and 2.6% of positive soil samples, respectively. EPN recovery frequency and abundance obtained with G. mellonella were not statistically different between natural and crop field edges values; however, S. littoralis was more successful trapping EPNs from crop field edges. Statistical differences were observed for recovery frequency recorded by both hosts in natural areas. Significant differences in larval mortality between both insects were not observed. The use of S. littoralis in entomopathogenic nematode surveys is discussed.     

Investigations on the efficacy of entomopathogenic nematodes against Spodoptera littoralis (Biosd.) and Galleria mellonella (L.)

Abstract

The infectivity of seven entomopathogenic nematode strains of the families Steinernematidae and Heterorhabditidae were tested against the last instar larvae of the cotton leafworm, Spodoptera littoralis (Biosd.) and the greater wax moth, Galleria mellonella (L.) in a Petri dish and sand column assay. Exposure of the insect species was done at a dose of 100 IJ/ ml. Heterorhabditis sp. ELG., H. indica, Heterorhabditis sp. ELB. were the highest in activity, they gave a 100% mortality to S. littoralis larvae in a Petri dish assay 24 h postexposure. The rest of the strains tested gave mortalities ranged between 50 and 90% at 24 h then 100% after 48 h postexposure . In the sand-column assay strains, Heterorhabditis sp. ELB., S. riobravae and S. carpocapsae were the highest in activity against S. littoralis with 100% mortality within 24 h postexposure. The results of infectivity against G. mellonella showed that H. indica was the highest in the Petri dish assay and H. egyptii was the highest in the sand column assay with 100% mortality after 24 h of exposure. The penetration rate as an indicator of entomopathogenic nematode infection was also evaluated. The highest value was recorded for Heterorhabditis sp. ELB. (62.3%) and S. carpocapsae (61.4%) against S. littoralis in both the Petri dish and sand-column assay, respectively. In the case of G. mellonella, the highest value of penetration was recorded for Heterorhabditis sp. ELG. (68.3%) and S. riobravae (65.00%) in both the Petri dish and sand-column assay, respectively.     

影响大蜡螟幼虫体色的环境因素初探

大蜡螟幼虫体色各式各样 ,通过对比实验分析了温度、湿度、密度、光照和食物等环境因素对大蜡螟幼虫体色的影响。实验结果表明 :不同温度、湿度、密度、光照条件下大蜡螟幼虫的体色均未变化。在研究食物对大蜡螟幼虫体色的影响实验中食蜂巢的白黄色幼虫身体内部发黑 ,这可能是黑色的蜂巢透射出的颜色。实验为进一步研究大蜡螟幼虫体色的遗传机制打下了基础。     

Two strains of Pseudomonas fluorescens bacteria differentially affect survivorship of waxworm (Galleria mellonella) larvae exposed to an arthropod fungal pathogen,Beauveria bassiana

Two strains of Pseudomonas fluorescens were found contaminating a biopesticide used in a previous study against Varroa destructor infestations in honey bee hives. In that study, the biopesticide, a formulation of a fungal pathogen of arthropods, Beauveria bassiana, failed to have any negative impact on the mite infestation despite successful results in previous studies using uncontaminated batches of the same biopesticide. The objective of the present research was to determine whether the bacteria may have interfered with the infectivity and/or virulence of B. bassiana in a simplified system; positive results in that system would then provide a rationale for further work under more complex conditions. Galleria mellonella late instar larvae treated topically with both a bacterial suspension of 6.8 to 7.0×10⁷ cfu/ml and a fungal suspension of 2.5×10⁷ or 2.5×10⁸ B. bassiana conidia/ml showed, in the case of one of the bacterial strains, significantly increased survivorship compared to larvae treated with just the B. bassiana suspension. When larvae were immersed in a bacterial suspension prior to application of B. bassiana suspension using a spray tower, a significant positive effect of the same P. fluorescens strain on larval survivorship was observed at 2.5×10⁸ conidia/ml. Neither the bacterial suspensions alone nor blank control solutions had any effect on larval survivorship. These results show that an interaction between the bacteria and the pathogen may explain some of the results from the prior field trial.     

拟双角斯氏线虫D43品系鞘蛋白对大蜡螟幼虫的免疫抑制作用

侵染期的拟双角斯氏线虫Steinernema ceratophorum D43品系体外都包裹着一个透明的体鞘。为探明体鞘对线虫侵染力的影响, 了解鞘蛋白（sheath proteins, SPs）对大蜡螟Galleria mellonella 幼虫的免疫抑制作用, 本研究通过化学方法使拟双角斯氏线虫D43脱鞘, 以对寄主的致死率和侵入点数量为指标, 与包鞘线虫比较对大蜡螟幼虫的侵染力; 采用乙醇提取的方法获得线虫鞘蛋白, 利用双向电泳和质谱技术对鞘蛋白进行鉴定分析; 从血细胞数量和酚氧化酶活力两个方面评价鞘蛋白对大蜡螟幼虫免疫反应的抑制作用。结果表明： 0.5%次氯酸钠处理20 min可以保证95%以上的线虫存活和脱鞘。与包鞘线虫相比, 脱鞘线虫对大蜡螟幼虫的致死率显著降低, 致死时间延后, 节间膜侵入点数量显著减少。以35%乙醇提取的鞘蛋白提取物可鉴定出6种鞘蛋白, 其中一个被鉴定为丝氨酸蛋白酶。此外, 血腔注射鞘蛋白提取物可导致试虫血细胞数量明显降低, 酚氧化酶活力受到显著抑制。由此说明, 体鞘对拟双角斯氏线虫D43的侵染力具有显著影响, 鞘蛋白在抑制寄主昆虫免疫反应中发挥重要作用。     

Passive vectoring of entomopathogenic fungus Beauveria bassiana among the wax moth Galleria mellonella larvae by the ectoparasitoid Habrobracon hebetor females

Females of the ectoparasitoid Habrobracon hebetor attack and envenomate numerous host individuals during oviposition. The vectoring of the entomopathogenic fungus Beauveria bassiana during the adhesion stage by ectoparasitoid females among the wax moth larvae Galleria mellonella was explored under laboratory conditions. Vectoring occurred both from infected parasitoids to wax moth larvae and from infected to healthy wax moth larvae by parasitoids. The efficacy of vectoring in both cases was dose dependent. Parasitoid females were unable to recognize infected larvae in a labyrinth test. In addition, the presence of H. hebetor females significantly (1.5–13 fold) increased the mycoses level in clusters of G. mellonella, with 40% of the larvae infected with fungal conidia. Envenomation by H. hebetor increased conidia germination on the cuticles of the wax moth larvae by 4.4 fold. An enhanced germination rate (2 fold) was registered in the n‐hexane epicuticular extract of envenomated larvae compared to that of healthy larvae. Both envenomation and mycoses enhanced the phenoloxidase (PO) activity in the integument of G. mellonella and, in contrast, decreased the encapsulation rate in hemolymphs. We hypothesize that changes in the integument property and inhibition of cellular immunity provide the highest infection efficacy of entomopathogenic fungi with H. hebetor.     

大蜡螟幼虫的体色遗传规律

对大蜡螟Galleria mellonella幼虫不同颜色品系的普通遗传学分析表明,大蜡螟幼虫的体色遗传是常染色体遗传且符合复等位基因遗传规律。深黄色基因（AA）对灰黑色基因(BB)和灰色基因(CC)为显性,深黄色基因(AA)对白黄色基因(DD)、灰黑色基因(BB)对白黄色基因(DD)和灰色基因(CC)、灰色基因(CC)对白黄色基因(DD)为不完全显性。基因型为AD、BD、CD的个体,其表现型均为黄色;基因型为AA、BC的个体,其表现型均为深黄色。     

Suboptimal temperature-dependent changes in the brain development and activity in Galleria mellonella larvae

The development of Galleria mellonella larvae is strongly affected by suboptimal temperature (18°C). One-day-old last-instar larvae react to 18°C with the arrest of further development for several months described as facultative larval diapause. The aim of this study was to find what type of changes, if any, in the brain correlate with the larval diapause induced by suboptimal temperature. Morphological analysis demonstrated the gradual inhibition of brain development. Paraldehyde-fuchsin (PAF) staining revealed cyclicity in the activity of the medial neurosecretory cells (M-NSC) in the larval brain. SDS-PAGE was used to examine the brain proteins of larvae reared at 30°C and at 18°C. The rate of protein synthesis in the brain of the last instar larvae kept at 18°C, measured as l -[³⁵S]methionine incorporation during 2-h incubation in vitro, was only about 40% of the value characteristic for this tissue during normal development (at 30°C). Despite decrease in the rate of total protein synthesis, suboptimal temperature induced an increase in the level of two major brain proteins: 112 and 84 kDa. In SDS-PAGE analysis, these two proteins appear 21–28 days after transfer to the lower temperature. Whether these proteins are specific for induction of larval diapause of Galleria mellonella remains to be further investigated. Arch. Insect Biochem. Physiol. 38:66–73, 1998. © 1998 Wiley-Liss, Inc.     

Lysozyme as Pathogen-Recognition Protein in the Hemolymph of Galleria mellonella

ABSTRACT Recognition of invading micro-organisms into hemolymph is a pivotal event for triggering diverse immune mechanisms in insects. It has been known that this recognition was mediated by the binding of hemolymph proteins to pattern-molecules on the cell surface of microbes. Recently, I found that the lysozyme in the G. mellonella hemolymph has binding affinity to cell-walls of Gram (-), (±) bacteria and fungus ( Candida albicans ). After the hemolymph was incubated with heat-killed microbes and treated with acidic buffer containing high concentration of NaCl, several plasma proteins detached from microbes were detected by reverse phase HPLC and SDS-PAGE analyses. Of binding proteins, it was assumed that the major one might be a lysozyme, which was previously characterized in the G. mellonella hemolymph. Furthermore immunoblot analysis performed with antiserum to G. mellonella lysozyme revealed that it was a lysozyme.     

JNK MAP kinase is involved in the humoral immune response of the greater wax moth larvae Galleria mellonella

We investigated the participation of MAP kinases in the response of Galleria mellonella larvae to immune challenge. JNK MAP kinase was activated in fat body 10-15 min after LPS injection in vivo. The level of JNK activation was time- and LPS dosage-dependent. JNK MAP kinase isolated from cell-free extract of fat bodies dissected from immune stimulated larvae phosphorylated c-Jun protein in vitro. The activity of Gm JNK kinase was abolished in the presence of the JNK specific inhibitor SP600125. Our data indicate a correlation between JNK phosphorylation and induction of antimicrobial activity in the insect hemolymph after immune stimulation. Hemolymph from larvae pre-treated with JNK specific inhibitor SP600125 showed a reduced level of antibacterial activity after LPS injection. JNK inhibition by SP600125 abolished antibacterial activity of the in vitro culture of G. mellonella fat body. Finally, we also show a correlation between JNK-dependent immune response of G. mellonella larvae and elevated temperature.     

温度对大蜡螟生长发育和繁殖的影响

为明确不同温度对大蜡螟(Galleria mellonella L.)生长发育和繁殖的影响,在24℃、28℃和31℃三个温度条件下,采用人工饲料饲养观察了大蜡螟的生长发育和繁殖状况。结果表明,温度对大蜡螟发育历期、生长发育速率以及繁殖具有显著影响(P0. 05)。在24～31℃范围内,随着温度升高,各虫态发育历期缩短,幼虫体长增长加快,发育速率加快。在24℃下世代历期最长(47. 96 d),31℃下世代历期最短(33. 68 d)。在同一温度下,雄蛾的寿命明显较雌蛾的寿命长,但成虫寿命、产卵前期和产卵期均随着温度升高而缩短。在24℃下产卵量最高为2 781. 50粒/雌,而在31℃下产卵量最低为1 943. 17粒/雌。     

蜡螟-单梗着色霉Fonsecaea monophora感染模型的构建及其血淋巴功能的变化

暗色真菌单梗着色霉Fonsecaea monophora侵犯皮肤及皮下组织,引起着色芽生菌病;其所引起的皮肤疣状改变或溃疡严重影响患者生活质量,重者可致残甚至发生癌变。近年来,无脊椎动物构建感染模型逐渐兴起,应用范围逐渐扩大。本研究利用蜡螟成功构建了F. monophora体内感染的动物模型,绘制了被感染蜡螟幼虫的生存曲线,观察记录了感染过程中蜡螟幼虫体表及体腔内的变化情况。在此基础上,进一步探索蜡螟幼虫对F. monophora的免疫防御应答,初步研究发现由病原体感染所诱发的血淋巴改变具有一定的抑真菌性,可抑制白念珠菌的生长。     

大蜡螟作为试验昆虫资源的利用现状

随着对资源昆虫的不断认识,人们的目光开始逐渐转到对大蜡螟Galleria mellonella L.的开发利用方面,而不再仅仅局限于对它的防治方面。近些年来,大蜡螟逐渐被作为试验昆虫用于一些生物的研究。文章主要介绍大蜡螟被用于昆虫病原线虫、寄生蜂、新型隐球菌Cryptococcus neoformans、抗菌肽、抗菌免疫机制等方面的研究。     

The effects of boric acid-induced oxidative stress on antioxidant enzymes and survivorship in Galleria mellonella

Larvae of the wax moth, Galleria mellonella (L.), were reared from first instar on a diet supplemented with 156, 620, 1,250, or 2,500 ppm boric acid (BA). The content of malondialdehyde (MDA, an oxidative stress indicator), and activities of the antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione peroxidase (GPx)] were determined in the fat body and hemolymph in the 7th instar larvae and newly emerged pupae. Relative to control larvae, MDA was significantly increased in larval hemolymph, larval and pupal fat body, but decreased in the pupal hemolymph. Insects reared on diets with 156- and 620-ppm BA doses yielded increased SOD activity but 1,250- and 2,500-ppm doses resulted in decreased SOD activity in larval hemolymph. SOD activity was significantly increased but CAT was decreased in the larval fat body. High dietary BA treatments led to significantly decreased GST activity. However, they increased GPx activity in larval hemolymph. Dietary BA also affected larval survival. The 1,250- and 2,500-ppm concentrations led to significantly increased larval and pupal mortality and prolonged development. In contrast, the lowest BA concentration increased longevity and shortened development. We infer that BA toxicity is related, at least in part, to oxidative stress management.     

Effects of Tolypocladium cylindrosporum and its secondary metabolites, efrapeptins, on the immune system of Galleria mellonella larvae

Interactions between Tolypocladium cylindrosporum (Deuteromycetes), its metabolite, efrapeptins, and the insect immune defense were investigated in vivo and in vitro. In the different phagocytosis studies, Bacillus cereus spores which had been labelled with fluorescein-isothiocyanate (FITC) were used. In vitro studies showed that efrapeptins inhibit phagocytic activity of Galleria mellonella (Lepidoptera: Pyralidae) haemocytes. The response was dose-related. Efrapeptins significantly reduced the number of nodules formed in response to an injection of zymosan supernatant. Phenoloxidase (PO) activation system contained in haemocyte lysate (HLS) was not affected by efrapeptins. In vivo studies when larvae were injected with efrapeptins also revealed that efrapeptins did not affect PO activities and total haemocyte count (THC) after 1 and 6 h post-injection. However, 12 h post-injection there was a significant inhibition of PO activities in HLS. There was also no significant reduction of PO activities and THC when larvae were injected with Tolypocladium cylindrosporum spores until 24 h post-injection. However, PO activities were suppressed and THC reduced 48 h post-treatment of larvae with spores. This study suggests that efrapeptins may interfere with the ligand-receptor interactions that are likely to occur at the plasma membrane of specific haemocytes.     

Endocrine alteration and precocious premetamorphic behaviors in the greater wax moth larvae,Galleria mellonella,parasitized by an endoparasitoid,Apanteles galleriae

Parasitization of Galleria mellonella (Lepidoptera: Pyralididae) larvae by a larval endoparasitoid Apanteles galleriae (Hymenoptera: Braconidae) leads to the precocious expression of premetamorphic behavior in the sixth (normally penultimate) instar host larvae prior to the parasitoid's emergence. We investigated the role of parasitization with A. galleriae on the alteration of development and/or behavior of its host. The ecdysteroid titer in the hemolymph of parasitized sixth instar larvae (the last instar of parasitized larvae) was higher than that of unparasitized ones, and the high ecdysteroid concentrations induced premetamorphic behaviors such as wandering and cocoon spinning. However, the epidermis of the parasitized larvae was not pupally committed through this stage. The activity of JH esterase in the parasitized larvae remained low, and application of a JH analogue to these larvae caused the production of a larval-type cocoon. These facts suggest that the parasitization by A. galleriae induces precocious premetamorphic behaviors of G. mellonella larvae by changing host endocrine conditions without causing the typical larval-pupal metamorphosis. Arch. Insect Biochem. Physiol. 34:257–273, 1997. © 1997 Wiley-Liss, Inc.     

Lipophorin uptake by the larval fat body and adult ovary in the wax moth Galleria mellonella

Lipophorin (Lp) acts in the circulation of insects to selectively deliver lipids to target tissues. In the present study, we wanted to show that Lp is taken up into larval fat body cells and the adult ovary in Galleria mellonella. Larval fat body and adult ovary tissues were incubated at room temperature for 30 min with fluorescein isothiocyanate (FITC)‐labeled Lp. Fluorescence microscopy and sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) revealed that fat body and ovary tissues internalize fluorescence‐labeled Lp. The results suggest that both lipids and proteins are taken up by fat body cells and the ovary and also that large amounts of proteins and lipids taken up can serve as building blocks and as a source of energy. Immunological relationships with other insects were investigated using western blotting. The data showed that the Lp of Galleria mellonella is related to that of Hyphantria cunea.     

Novel primers for the amplification of nuclear DNA introns in the entomopathogenic nematode Heterorhabditis bacteriophora and their cross-amplification in seven other Heterorhabditis species

We describe 24 novel primers that amplify intron regions in housekeeping and structural genes of Heterorhabditis bacteriophora. The cross-amplification potential of these primers in seven other Heterorhabditis species was determined. The results obtained showed interspecific nucleotide, length and splice site variability in the sequenced introns and for one gene, an intron gain was observed. These primers will be useful tools for studying population genetics, genetic diversity and intron DNA evolution within the genus Heterorhabditis and other genera of rhabditid nematodes.