Pathology and Epizootiology of Entomophaga maimaiga Infections in Forest Lepidoptera

The insect-pathogenic fungal pathogen Entomophaga maimaiga is endemic to northeastern Asia and was first found in North America in 1989. Due to repeated epizootics and spread within populations of the major forest defoliator in northeastern North America, the gypsy moth (Lymantria dispar), this pathogen has gained much notoriety. Although this pathogen was purposely introduced to North America for biological control of L. dispar in 1910 to 1911, it is questionable whether it became established at the time of release and then remained at innocuous levels until relatively recently. Alternatively, the fungal strain present in North America today could be a more recent accidental introduction. DNA analysis demonstrates that this pathogen differs significantly from North American members of the same species complex (the Lepidoptera-specific Entomophaga aulicae species complex), and, to date, isolates of this introduced pathogen display little heterogeneity in North America. Nonsusceptible lepidopteran larvae have been identified, and either E. maimaiga is unable to penetrate the cuticle or the fungus cannot survive within the hemocoel. In the latter case, although E. maimaiga grows as protoplasts lacking cell walls in the host hemolymph, glycoproteins on plasma membranes of the protoplasts could lead to host recognition. Epizootiological studies demonstrate a clear association between fungal activity and environmental moisture but little association with host density under hypothesized conditions of high fungal density. Prediction of the occurrence of epizootics is not yet possible. E. maimaiga is easily established in new areas by releasing azygospores, but the ability to use this pathogen further for biological control will depend, in large part, on the development of mass production systems.     

Attachment and germination of Entomophaga maimaiga conidia on host and non-host larval cuticle

The lepidopteran-specific fungal pathogen Entomophaga maimaiga is highly virulent against Lymantria dispar (gypsy moth) larvae, and other members of the family Lymantriidae. Numerous species in the subfamily Cuculliinae (Family Noctuidae) are not susceptible to E. maimaiga due to the inability of this fungus to penetrate the larval cuticle. Conidial attachment and germination were compared among five cuculliine species and L. dispar using bioassays and scanning electron microscopy. Although conidia were showered evenly across larvae during bioassays, on L. dispar conidia were most abundant on segments, where they adhered well to the cuticle and germinated at high percentages. Conidia on cuculliine cuticles were predominantly found in large, loose aggregations in intersegmental areas. Few conidia on cuculliine cuticle germinated and scanning electron microscopy revealed a thick film of mucous enveloping conidia. We hypothesize that the conidia on cuculliines become coated by this film and were only loosely attached to the larval cuticle. No such film was seen on L. dispar larvae where individual conidia appeared well attached. On L. dispar larvae many conidia also adhered to setae. To determine if hydrophobicity affected the ability of E. maimaiga conidia to attach and germinate on a substrate, a goniometer was used to determine relative hydrophobicity of larval cuticles. L. dispar cuticle was more hydrophobic than cuculliine cuticle, suggesting that a high level of hydrophobicity could be a required characteristic for hosts. Cuticles from four cuculliine species and L. dispar were sequentially extracted using hexane, chloroform, and methanol. Conidia were showered onto glass slides coated with the different extracts and germination was quantified. Methanol extracts of cuculliine cuticle consistently decreased germination, compared to all extracts of L. dispar cuticle. For all L. dispar extracts, the majority of conidia produced germ tubes, which is a normal prerequisite for cuticular penetration. For the cuculliines, conidia exposed to hexane and chloroform extracts produced secondary conidia as did all controls, but the conidia exposed to cuculliine methanol extracts that germinated produced germ tubes. These studies demonstrated that a range of factors act in concert to prevent E. maimaiga infection of the cuculliine species investigated.     

Diurnal pattern of death and sporulation in Entomophaga maimaiga-infected Lymantria dispar

Entomophaga maimaiga Humber, Shimazu, et Soper (Zygomycotina: Entomophthoraceae) is a naturally occurring obligate fungal pathogen specific to gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae) larvae. This fungus is considered the most important natural enemy of this pest insect in North America and Asia. A critically important step for the development of E. maimaiga epizootics is the transmission of propagules to healthy larvae, a process known to require high humidity. Some pathogens are known to manipulate the time of day that hosts die so that propagules are produced to maximize chances of survival and thus enhance transmission. The objective of this study was to assess whether E. maimaiga manipulates L. dispar to die at a certain time of day. Laboratory bioassays were conducted at 15 and 20 °C to record the 24‐h activity pattern of death and sporulation exhibited under an L14:D10 photoperiod and 100% r.h. by four isolates of E. maimaiga in its host L. dispar. Events were recorded every 4 h. Our results clearly demonstrate that E. maimaiga‐infected L. dispar larvae die mainly in the afternoon and that the fungus sporulates during the night. The rhythm was independent of the fungal isolate tested and type of spores produced after larval death. By raising the temperature from 15 to 20 °C, the peak death time narrowed and sporulation was initiated earlier at night.     

Response of eastern hemlock looper hemocytes to selected stages of Entomophthora egressa and other foreign particles

Indirect evidence for the natural existence of the free-protoplast stage of the fungus Entomophthora egressa in the eastern hemlock looper, Lambdina fiscellaria fiscellaria, is presented. The protoplasts were viable after 72 hr postinjection and subsequent development in the host produced conidia characteristic of E. egressa. The hemocytes studied (plasmatocytes, granular cells, and spherule cells) did not adhere to the protoplasts either in vivo or in vitro. Cells of Escherichia coli and sporangiospores of Absidia repens adhered to the granular cells in vitro. The granular cells adhered to the hyphae of Rhizopus nigricans in vitro. The spherule cells strongly adhered to the hyphae and hyphal bodies of E. egressa in vitro. The protoplasts, hyphae, and conidia of E. egressa and the hemocytes of L. fiscellaria fiscellaria adhered to positively charged DEAE-Sephadex beads and not to negatively charged CM-Sephadex beads. Aspects of active and passive strategies for protoplast evasion of host hemocytes are discussed with some emphasis on hemocyte-protoplast electrostatic repulsion and active secretion of hemocyte inhibitors by the protoplasts.     

Evasion of host defense by in vivo-produced protoplast-like cells of the insect mycopathogen Beauveria bassiana.

In vivo cells (hyphal bodies) of the hyphomycetous insect pathogen Beauveria bassiana collected from host Spodoptera exigua larval hemolymph were osmotically sensitive and lacked a well-defined cell wall. In light and electron microscope studies, a galactose-specific lectin purified from S. exigua hemolymph, concanavalin A (specific for alpha-mannose), and a polyclonal antibody to B. bassiana cell walls all bound to surfaces of in vitro-produced B. bassiana blastospores; however, none of these probes labelled the thin layer of extracellular material covering the plasma membranes of hyphal bodies. These cells were observed freely circulating in S. exigua hemolymph at 36 h postinfection, although immunocompetent hemocytes were known to be present. Additionally, association of hyphal bodies with hemocytes in monolayers was significantly less than for opsonized in vitro blastospores or submerged conidia. The absence of antigenically important galactomannan components on in vivo cells may therefore allow these cells to escape recognition and phagocytosis. Lack of structural components (e.g., chitin, as evidenced by the absence of binding of wheat germ agglutinin) may also be important with respect to evasion of host cellular defense mechanisms. Production of wall material resumed 48 to 60 h postinfection and therefore may coincide with loss of phagocytic capabilities of the hemocytes due to immunosuppressive effects of fungal metabolites. The protoplast-like cells may be formed by the action of hydrolytic enzymes in the hemocytes or by inhibition of fungal cell wall synthetases.     

Distribution of resting spores of the Lymantria dispar pathogen Entomophaga maimaiga in soil and on bark

Cadavers of late instar Lymantria dispar (gypsy moth) larvae killed by the fungal pathogen Entomophaga maimaiga predominantly contain resting spores (azygospores). These cadavers frequently remain attached to tree trunks for several weeks before they detach and fall to the ground. Density gradient centrifugation was used to quantify resting spores in the soil and on tree bark. Titers of resting spores were extremely high at 0–10 cm from the base of the tree and the number decreased with distance from the trunk of the tree. Titers were also highest in the organic layer of the soil with numbers decreasing precipitously with increasing depth in the soil. While resting spores were obtained from tree bark, densities per unit area were much lower than those found in the organic soil layer at the base of the tree. Field bioassays were conducted with caged L. dispar larvae to compare infection levels with distance from the tree trunk as well as on the trunk. Highest infection levels were found at 50cm from the tree base with lowest infection on the tree trunk at 0.5 m height, although we expected the highest infection levels among larvae caged at the bases of trees, where highest spore titers occurred. Laboratory experiments demonstrated that L. dispar larvae exposed to resting spore- bearing soil at the soil surface became infected while larvae exposed to soil with resting spores buried at least 1 cm below the surface did not become infected.     

Virulence and fitness of the fungal pathogen Entomophaga maimaiga in its host Lymantria dispar, for pathogen and host strains originating from Asia, Europe, and North America

In this study, we tested (1) whether non-North American gypsy moth strains are susceptible to North American isolates of Entomophaga maimaiga and (2) the potential for erosion in the efficacy of E. maimaiga in controlling gypsy moth. We used bioassays to assess the variability in virulence (measured as time to death) as well as fitness of the pathogen (measured as spore production) in four gypsy strains challenged with six E. maimaiga isolates, using host and pathogen strains originating from Asia, Europe, and North America. We found that all E. maimaiga isolates tested were pathogenic to all strains of Lymantria dispar, regardless of the geographical origin of the fungal isolate, with at least 86% mortality for all combinations of fungal isolate and gypsy moth strain. We therefore conclude that Asian gypsy moths are susceptible to North American strains of E. maimaiga. No significant interactions between fungal isolates and gypsy moth strains with regard to time to death were found, indicating that each fungal isolate had the same overall effect on all the gypsy moth strains tested. However, fungal isolates differed significantly with regard to virulence, with a Russian isolate being the slowest to kill gypsy moth (5.1+/-0.1 days) and a Japanese isolate being the overall fastest to kill its host (4.0+/-0.1 days). Fungal isolates also differed in fitness, with variability in types of spores produced. These differences in virulence and fitness were, however, not correlated with geographical origin of the fungal isolate. Gypsy moth strains had no or only little effect on fungal virulence and fitness. Based on our studies with laboratory-reared gypsy moth strains, erosion of successful control of gypsy moth by E. maimaiga seems unlikely.     

Introduced pathogens follow the invasion front of a spreading alien host

1.?When an invasive species first colonizes an area, there is an interval before any host-specific natural enemies arrive at the new location. Population densities of newly invading species are low, and the spatial and temporal interactions between spreading invasive species and specific natural enemies that follow are poorly understood. 2.?We measured infection rates of two introduced host-specific pathogens, the entomophthoralean fungus Entomophaga maimaiga and the baculovirus Lymantria dispar nucleopolyhedrovirus (LdNPV), occurring in spreading populations of an invasive forest defoliator, L. dispar (gypsy moth), in central Wisconsin. 3.?Over 3 years, we found that host density was closely associated with the presence and prevalence of both pathogens. The fungal and viral pathogens differed in the sensitivity of their response as E. maimaiga was present in lower-density host population than LdNPV. 4.?We examined the relationship between weather conditions and infection prevalence and found that activity of both the fungus and virus was strongly seasonally influenced by temperature and rainfall or temperature alone, respectively. 5.?Purposeful releases of pathogens (median distances of study sites from release sites were 65·2 km for E. maimaiga and 25·6 km for LdNPV) were not associated with pathogen prevalence. 6.?A generalist fly parasitoid, Compsilura concinnata, also killed L. dispar larvae collected from the study sites, and parasitism was greater when infection by pathogens was lower. 7.?Our results demonstrated that although infection levels were low in newly established host populations, host-specific pathogens had already moved into host populations close behind advancing populations of an invasive host; thus, spreading hosts were released from these enemies for only a relatively short time.     

Deposition and germination of conidia of the entomopathogen Entomophaga maimaiga infecting larvae of gypsy moth,Lymantria dispar

Germination of conidia of Entomophaga maimaiga, an important fungal pathogen of gypsy moth, Lymantria dispar, was investigated on water agar and larval cuticle at varying densities. Percent germination was positively associated with conidial density on water agar but not on larval cuticle. When conidia were showered onto water agar, the rate of germination was much slower than on the cuticle of L. dispar larvae. From the same conidial showers, the resulting conidial densities on water agar were much higher than those on larval cuticle in part because many conidia adhered to setae and did not reach the cuticle. A second factor influencing conidial densities on larval cuticle was the location conidia occurred on larvae. Few conidia were found on the flexible intersegmental membranes in comparison with the areas of more rigid cuticle, presumably because conidia were physically dislodged from intersegmental membranes when larvae moved. Conidia were also exposed to heightened CO(2) to evaluate whether this might influence germination. When conidia on water agar were exposed to heightened CO(2) levels, germinating conidia primarily formed germ tubes while most conidia exposed to ambient CO(2) rapidly formed secondary conidia.     

In vitro formation of resting spores by the insect pathogenic fungus Entomophaga maimaiga

Field-collected resting spores (azygospores) of the fungal pathogen of Lymantria dispar (gypsy moth), Entomophaga maimaiga, have been used to release this biological control agent in areas where this pathogen is not established. We have found that E. maimaiga can produce resting spores in vitro using Grace's insect tissue culture medium (95%) plus fetal bovine serum (5%). The majority of spores become mature between 7 and 21 days after cultures are initiated. Spore production varies by fungal isolate; of 38 isolates tested, 10 produced no resting spores while 7 produced >1000 resting spores/ml. Resting spore production was not affected when isolates were mixed. Glycerol (used for fungal storage), trehalose, and selected amino acids each inhibited resting spore formation. Fetal bovine serum was required for spore production but the presence of >5% yielded lower resting spore densities. A large surface area:volume ratio (12.5 cm(2):ml versus 4.2 cm(2):ml) was required for abundant formation of resting spores. At present, resting spores have only been produced in small volumes with a maximum of 3 x 10(4) resting spores/ml.     

Ultrastructural studies on the fungus,Nomuraea rileyi,infecting the velvetbean caterpillar,Anticarsia gemmatalis

Combined scanning and transmission electron microscopy was used to study the fine structure of the developmental stages of Nomuraea rileyi infecting host larvae of Anticarsia gemmatalis. Larvae were dusted with large numbers of fungal conidia, which germinated and penetrated the cuticle within 6 hr post-treatment. Within 24 hr, penetration hyphae had reached the cuticular epidermis and, via a budding process, initiated the hyphal body stage in the hemocoel. The hyphal bodies, suspended in hemolymph, multiplied and spread throughout the host larvae. By 6–7 days post-treatment, the majority of larvae were mummified. Within 12 hr postmortem numerous conidiophores emerged producing a confluent mycelial mat over the entire cuticular surface. Numerous hydrophobic conidia were formed on phialides present on the aerial conidiophores.     

Comparative analysis of the binding of antibodies prepared against the insect Spodoptera exigua and against the mycopathogen Nomuraea rileyi

Polyclonal antibodies were produced in mice against Spodoptera exigua (beet armyworm) larval hemolymph and hemocytes and against cell wall surfaces of hyphal bodies and hyphae of the entomopathogenic hyphomycete Nomuraea rileyi. In addition to exhibiting strong activity against their original antigenic substrates, all of the antibodies cross-react extensively with other substrates. The hemolymph antibody binds to hemocytes and vice versa, and both antibodies cross-react to the insect fat body basement membrane (extracellular matrix (ECM) and to N. rileyi and Beauveria bassiana (another entomopathogenic fungus) cell wall surfaces (ECM). Likewise, the anti-fungal antibodies cross-react with S. exigua hemolymph and hemocytes, especially the granules that may contain ECM components, and with fat body basement membrane. These cross-reactivities are specific as indicated by negative controls in the microscopy and Western blotting assays. Parallel labeling experiments using Con A suggest that the reactive epitopes contain mannose; however, none of the antibodies bind to mannose residues of nonentomopathogenic Candida albicans or Saccharomyces cerevisiae yeast cells. Thus, these cross-reactivities suggest that the host mimicry expressed by surface components of entomopathogenic fungi represents an important pathogenic determinant.     

莱氏绿僵菌对斜纹夜蛾的致病力及生理效应

【目的】测定莱氏绿僵菌Metarhizium rileyi Nr5772菌株对斜纹夜蛾Spodoptera litura幼虫及蛹的致病能力,研究莱氏绿僵菌侵染后在寄主体内的发育及对寄主的生理效应,探讨莱氏绿僵菌的致病机制。【方法】采用浸渍法测定莱氏绿僵菌孢子对斜纹夜蛾3-6龄幼虫及蛹的致死中浓度(LC_(50))和致死中时(LT_(50))。采用微量注射法接种莱氏绿僵菌虫菌体,在不同时间后采集斜纹夜蛾幼虫血淋巴,在显微镜下检查虫菌体的数量、形态及寄主血细胞数量,并用酶标仪测定寄主血淋巴酚氧化酶(Phenoloxidase,PO)的活性。【结果】M.rileyi孢子对3龄斜纹夜蛾幼虫毒力最强,10 d后LC_(50)=3.12×10~6个孢子/mL,龄期越大,致病力越低;孢子浓度为5×10~9个/mL时,对3龄幼虫的致死速度最快,LT_(50)=4.55 d,致死速度随龄期的增大和浓度的降低逐渐减缓;M.rileyi孢子对蛹的致病力远低于对幼虫的致病力。注射接种虫菌体后,64 h内,虫菌体数量在寄主血腔中以幂函数的形式增长,寄主的血细胞数量没有明显的变化;在侵染初期(接种后44 h内),血淋巴PO活性正常;在侵染后期,虫菌体数量不再增加(55-64 h后),逐渐转化为菌丝体,并快速杀死寄主,PO活性受到抑制。【结论】莱氏绿僵菌Nr5772菌株对斜纹夜蛾幼虫有较强的致病力,应在害虫低龄期应用;莱氏绿僵菌在侵染初期对寄主血细胞和血淋巴PO无影响,后期则完全抑制PO活性。     

Cellular immune responses of spruce budworm larvae to Entomophthora egressa protoplasts and other test particles

The protoplast stage of two isolates of Entomophthora egressa developed normally and eventually produced conidiophores when injected into larvae of the spruce budworm, Choristoneura fumiferana. The spruce budworm hemocytes never made long-term contact with the protoplasts either in vivo or in vitro. The protoplasts made active, short-term contact with spruce budworm granulocytes both in vivo and in vitro. Total larval hemocyte counts (THC) initially declined when larvae were injected with protoplasts, growth medium (MGM), or Escherichia coli. The recovery rate to THC control levels was similar for MGM and protoplasts and supports the concept of nonrecognition of protoplasts by the hemocytes. The granulocytes were important in both nodulation and phagocytosis of E. coli and Bacillus cereus, whereas the plasmatocytes were important in phagocytosis. In in vitro studies, spruce budworm granulocytes did not adhere to rod-shaped hyphal bodies, spherical hyphal bodies, or germinating spherical hyphal bodies of E. egressa, whereas the granulocytes readily encapsulated the hyphae. There was no evidence for the production by the protoplasts of metabolites which might interfere with hemocyte adhesion. When protoplasts contacted Tenebrio molitor granulocytes, the protoplasts reacted by increasing the number of protoplasmic extensions and by granule discharge. The process of granule discharge may be an active protoplast defense mechanism. The sporangiospores of Absidia repens and Rhizopus nigricans adhered to spruce budworm granulocytes; however, the number of A. repens spores per granulocyte and the level of granulocytes with spores decreased in the presence of phenylthiourea. The adhesion of A. repens spores to granulocytes was enhanced by N-acetylglucosamine, whereas glucosamine, sucrose, fucose, fructose, arabinose, and galactose either had no effect on or reduced spore adhesion. Thus, the chitin (or its subunits) in the hyphal wall may initiate the granulocyte response.     

Alterations in carbohydrate and fatty acid levels of Lymantria dispar larvae caused by a microsporidian infection and potential adverse effects on a co-occurring endoparasitoid,Glyptapanteles liparidis

Infection of Lymantria dispar host larvae by the entomopathogenic microsporidium Vairimorpha sp. has a negative impact on the performance of the endoparasitic braconid Glyptapanteles liparidis. To investigate possible causes for this effect, we studied to what extent nutritional host suitability is altered by the microsporidium. Therefore, we analyzed carbohydrates and fatty acids in host larvae after Vairimorpha infection and/or parasitism by G. liparidis. Trehalose levels were significantly reduced in the hemolymph of infected hosts. After day five post infection, it was detected only in traces. Four to six days later, the glycogen resources were depleted in infected larvae. Parasitism by G. liparidis, on the other hand, led to increased hemolymph trehalose levels during the early endoparasitic phase but to a significant decrease at the end of its larval development. No effect of parasitism on the glycogen content was ascertained. Hemolymph levels of the fatty acids analyzed, such as palmitic, stearic, oleic, linoleic, and linolenic acid, were significantly reduced in microsporidia-infected L. dispar. Vairimorpha sp. develops as an intracellular parasite in the fat body of the host larva and synthesis of trehalose and fatty acids may be disturbed. Moreover, microsporidia may also harness metabolites or energy produced by host cells. We conclude that the microsporidia-induced decrease in hemolymph carbohydrates and fatty acids adversely affects growth and development of parasitoid larvae.     

In vivo development of the entomogeneous hyphomycetePaecilomyces farinosus in hostSpodoptera exigua (beet armyworm) larvae

The in vivo development of the entomogenous hyphomycetePaecilomyces farinosus inSpodoptera exigua (beet armyworm) larvae was examined using light and electron microscopic techniques. Blastospores injected into larval hemocoels (500 blastospores/larva) were immediately ingested by phagocytic hemocytes, and no fungal cells were detected in the hemolymph until 36 h post-injection. As indicated by immunocytochemical methods, the in vivo-produced blastosopres, in contrast to in vitro blastospores, lacked a galacto-mannan surface layer required for opsonization by aS. exigua humoral lectin. Therefore, these in vivo cells were not recognized by phagocytic granulocytes and were freely-circulating in the hemolymph. Hyphae differentiating from the blastospores were recognized by the hemocytes and induced formation of multicellular hemocytic nodules. By 72 h post-injection, mycelia were observed emerging from the nodules and by 96 h, larvae had become mummified due to extensive proliferation of the fungus throughout host tissues. Neither phagocytosis of the initially injected in vitro-produced blastospores nor nodule formation around hyphal cells later in the infection process was effective in stopping fungal growth. The in vivo development ofP. farinosus was similar to that of another hyphomycete,Beauveria bassiana except that in the latter case, extensive nodule formation was inhibited by the production of fungal metabolites.     

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.     

颈双缘姬蜂毒液对寄主小菜蛾的免疫抑制作用

对颈双缘姬蜂Diadromus collaris (Gravenhorst)及其毒液引起寄主小菜蛾Plutella xylostella的一些生理效应进行了研究。结果表明,颈双缘姬蜂寄生寄主后可引起寄主小菜蛾蛹总血细胞及浆血细胞和颗粒血细胞数量的上升。寄生后1天观察,血细胞延展行为受到影响,表现在颗粒血细胞放射状丝的产生及浆血细胞伪足的形成受到抑制。通过毒液对寄主离体幼虫血细胞延展行为、形态及活性影响的研究,发现毒液抑制了寄主离体浆血细胞的延展,但对颗粒血细胞的影响不明显;毒液引起寄主浆血细胞和颗粒血细胞的破裂和死亡,毒液对寄主幼虫血淋巴酚氧化酶活性有一定的抑制作用,当反应至40、60及80 min时,毒液处理和未经毒液处理的寄主血淋巴在490 nm处的吸光值差异比较明显。对毒液蛋白成分的聚丙烯酰胺凝胶电泳分析发现,毒液中有9种多肽,分子量介于9～50.2 kD,其中50.2、30.5、28.2、25.1 和12.6 kD的多肽含量较高, 与其他蜂毒液的一些作用已知的蛋白条带相似,因而推测它们同样具有免疫及发育抑制作用。结果证明颈双缘姬蜂毒液能破坏寄主细胞及体液因子调节的免疫反应。     

Different defense strategies of Dendrolimus pini, Galleria mellonella, and Calliphora vicina against fungal infection

The resistance of Galleria mellonella, Dendrolimus pini, and Calliphora vicina larvae against infection by the enthomopathogen Conidiobolus coronatus was shown to vary among the studied species. Exposure of both G. mellonella and D. pini larvae to the fungus resulted in rapid insect death, while all the C. vicina larvae remained unharmed. Microscopic studies revealed diverse responses of the three species to the fungal pathogen: (1) the body cavities of D. pini larvae were completely overgrown by fungal hyphae, with no signs of hemocyte response, (2) infected G. mellonella larvae formed melanotic capsules surrounding the fungal pathogen, and (3) the conidia of C. coronatus did not germinate on the cuticle of C. vicina larvae. The in vitro study on the degradation of the insect cuticle by proteases secreted by C. coronatus revealed that the G. mellonella cuticle degraded at the highest rate. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. Of all the tested species, only plasmatocytes exhibited phagocytic potential. Exposure to the fungal pathogen resulted in elevated phagocytic activity, found to be the highest in the infected G. mellonella. The incubation of insect hemolymph with fungal conidia and hyphae revealed diverse reactions of hemocytes of the studied insect species. The encapsulation potential of D. pini hemocytes was low. Hemocytes of G. mellonella showed a high ability to attach and encapsulate fungal structures. Incubation of C. vicina hemolymph with C. coronatus did not result in any hemocytic response. Phenoloxidase (PO) activity was found to be highest in D. pini hemolymph, moderate in G. mellonella, and lowest in the hemolymph of C. vicina. Fungal infection resulted in a significant decrease of PO activity in G. mellonela larvae, while that in the larvae of D. pini remained unchanged. PO activity in C. vicina exposed to fungus slightly increased. The lysozyme-like activity increased in the plasma of all three insect species after contact with the fungal pathogen. Anti E. coli activity was detected neither in control nor in infected D. pini larvae. No detectable anti E. coli activity was found in the control larvae of G. mellonella; however, its exposure to C. coronatus resulted in an increase in the activity to detectable level. In the case of C. vicina exposure to the fungus, the anti E. coli activity was significantly higher than in control larvae. The defense mechanisms of D. pini (species of economic importance in Europe) are presented for the first time.