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.     

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.     

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocyte phagocytosis of Spodoptera exigua by inhibiting phospholipase A(2)

Phagocytosis is a hemocytic behavior against bacterial infection. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits immune responses of target insects and causes hemolymph septicemia. This study analyzed how X. nematophila could inhibit phagocytosis to increase its pathogenicity. Granular cells and plasmatocytes were the main phagocytic hemocytes of Spodoptera exigua determined by observing fluorescence-labeled bacteria in the cytosol. X. nematophila significantly inhibited phagocytosis of both hemocytes, while heat-killed X. nematophila lost its inhibitory potency. However, co-injection of X. nematophila with arachidonic acid did not show any significant inhibition of hemocyte phagocytosis. In fact, hemocytes of S. exigua infected with X. nematophila showed significant reduction in phospholipase A(2) (PLA(2)) activity. Dexamethasone, a specific PLA(2) inhibitor, significantly inhibited phagocytosis of both cell types. However, the inhibitory effect of dexamethasone was recovered by addition of arachidonic acid. Incubation of hemocytes with benzylideneacetone, a metabolite of X. nematophila, inhibited phagocytosis in a dose-dependent manner. These results suggest that X. nematophila produces and secretes PLA(2) inhibitor(s), which in turn inhibit the phagocytic response of hemocytes.     

Differential pathogenicity of two entomopathogenic bacteria,Photorhabdus temperata subsp. temperata and Xenorhabdus nematophila against the red flour beetle,Tribolium castaneum

Two entomopathogenic bacteria, Photorhabdus temperata subsp. temperata (Ptt) and Xenorhabdus nematophila (Xn), are symbiotically associated with the nematodes, Heterorhabdis megidis and Steinernema carpocapsae, respectively. There is little information on natural host ranges of the nematodes, but a significant difference in pathogenicity was observed between these two bacteria against the red flour beetle, Tribolium castaneum, in which Ptt exhibited more than six times higher pathogenicity than Xn. The pathogenic difference was not due to their inhibitory effect on phospholipase A₂ activity that is required for expression of immune response of T. castaneum. The culture broths of both bacterial species had insecticidal activities when injected into the hemocoel. When the bacterial culture broths were fractionated into aqueous and organic extracts, most insecticidal activity remained in the aqueous extracts. The aqueous extracts of two bacteria contained proteins which showed different profiles.     

Benzylideneacetone, an immunosuppressant, enhances virulence of Bacillus thuringiensis against beet armyworm (Lepidoptera: Noctuidae)

Benzylideneacetone (BZA) is a metabolite of gram-negative entomopathogenic bacterium Xenorhabdus nematophila, and it acts as an enzyme inhibitor against phospholipase A2 (PLA2). PLA2 catalyzes a committed biosynthetic step of eicosanoids, which mediate insect immune reactions to infection by microbial pathogens. This study tested a hypothesis that a putative immunosuppressive activity of BZA may enhance virulence of Bacillus thuringiensis against the fifth instars of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). In in vitro conditions, BZA significantly inhibited hemocyte microaggregation induced by B. thuringiensis and impaired hemocyte-spreading behavior of S. exigua in a dose-dependent manner. Oral administration of BZA gave similar immunosuppressive effect on the hemocytes of the fifth instars. Although BZA itself did not possess any insecticidal activity on oral administration, when BZA was treated in a mixture with a low dose of B. thuringiensis spp. aizawai to fifth instars, the bacterial virulence was significantly enhanced. BZA also enhanced virulence of B. thuringiensis spp. kurstaki, which alone was of limited effectiveness against S. exigua. This study suggests that an immunosuppression by BZA is positively linked to potentiation of B. thuringiensis.     

Phospholipase A2 inhibitors synthesized by two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata

The entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata suppress insect immune responses by inhibiting the catalytic activity of phospholipase A(2) (PLA(2)), which results in preventing biosynthesis of immune-mediating eicosanoids. This study identified PLA(2) inhibitors derived from culture broths of these two bacteria. Both X. nematophila and P. temperata subsp. temperata culture broths possessed significant PLA(2)-inhibitory activities. Fractionation of these bacterial metabolites in the culture broths using organic solvent and subsequent chromatography purified seven potent PLA(2) inhibitors, three of which (benzylideneacetone [BZA], proline-tyrosine [PY], and acetylated phenylalanine-glycine-valine [FGV]) were reported in a previous study. Four other compounds (indole, oxindole, cis-cyclo-PY, and p-hydroxyphenyl propionic acid) were identified and shown to significantly inhibit PLA(2). X. nematophila culture broth contained these seven compounds, while P. temperata subsp. temperata culture broth contained three compounds (BZA, acetylated FGV, and cis-cyclo-PY). BZA was detected in the largest amount among these PLA(2) compounds in both bacterial culture broths. All seven bacterial metabolites also showed significant inhibitory activities against immune responses, such as phenoloxidase activity and hemocytic nodulation; BZA was the most potent. Finally, this study characterized these seven compounds for their insecticidal activities against the diamondback moth, Plutella xylostella. Even though these compounds showed relatively low toxicities to larvae, they significantly enhanced the pathogenicity of Bacillus thuringiensis. This study reports bacterial-origin PLA(2) inhibitors, which would be applicable for developing novel insecticides.     

Eicosanoid biosynthesis is activated via Toll, but not Imd signal pathway in response to fungal infection

Phospholipase A(2) (PLA(2)) catalyzes hydrolysis of phospholipids at sn-2 position and usually releases arachidonic acid, which is oxygenated into various eicosanoids that mediate innate immune responses in insects. PLA(2) activities were measured in both immune-associated tissues of hemocyte and fat body in the beet armyworm, Spodoptera exigua. Upon challenge of an entomopathogenic fungus, Beauveria bassiana, the PLA(2)s were significantly activated in both hemocyte and fat body. The fungal infection also induced gene expression of antimicrobial peptides (AMPs), such as two attacins, cecropin, gallerimycin, gloverin, hemolin, and transferrin of S. exigua. RNA interference of Toll or Imd signal pathway using double-stranded RNAs (dsRNAs) specific to SeToll or SeRelish suppressed specific AMP gene expressions, in which dsRNA specific to SeToll suppressed two attacins, cecropin, gallerimycin, gloverin, hemolin, and transferrin I, while dsRNA specific to SeRelish suppressed only cecropin. Interestingly, dsRNA specific to SeToll also significantly inhibited the activation of PLA(2) in response to the fungal infection, but dsRNA specific to SeRelish did not. Eicosanoid-dependent hemocyte nodulation was inhibited by dsRNA specific to SeToll but was not by dsRNA specific to SeRelish. These results suggest that eicosanoid biosynthesis is activated via Toll, but not Imd signal pathway in response to fungal infection in S. exigua.     

Benzylideneacetone suppresses both cellular and humoral immune responses of Spodoptera exigua and enhances fungal pathogenicity

An entomopathogenic fungus, Beauveria bassiana, had significant insecticidal activity against the beet armyworm, Spodoptera exigua. However, it took almost one week to cause significant mortality. This study used a mixture treatment with an immunosuppressant to enhance the fungal pathogenicity. A bacterial metabolite, benzylideneacetone (BZA), had a significant synergistic effect on the fungal pathogenicity against S. exigua, although it had little insecticidal activity by itself. The mixture treatment shortened median lethal time of B. bassiana by approximately 2 days. The synergistic activity of BZA on the pathogenicity of B. bassiana was induced by its immunosuppressive effects on both cellular and humoral antifungal responses of S. exigua. In response to B. bassiana, S. exigua larvae can form hemocytic nodules. Nodules were significantly suppressed by BZA treatment. Moreover, BZA inhibited expression of some antimicrobial peptide genes of S. exigua in response to fungal challenge. The immunosuppressive condition induced by BZA allowed B. bassiana to easily colonize and multiply in the hemocoel of treated larvae, which resulted in significant enhancement of the pathogenicity of B. bassiana.     

Cellular immune responses and FAD-glucose dehydrogenase activity of Mamestra brassicae (Lepidoptera: Noctuidae) challenged with three species of entomopathogenic fungi

Abstract.  Cellular immune responses and glucose dehydrogenase activity are examined in the larvae of Mamestra brassicae (Lepidoptera: Noctuidae) injected with three entomopathogenic fungi, Beauveria bassiana , Nomuraea rileyi and Paecilomyces tenuipes . Total haemocyte count and the number of spherulocytes increase significantly over 2 h in larvae infected with N. rileyi , the most virulent fungal pathogen of M. brassicae . However, glucose dehydrogenase is not activated in the haemolymph of larvae inoculated with N. rileyi . By contrast, P. tenuipes , the least virulent fungal species, but with the highest proteolytic activity, activates glucose dehydrogenase and the number of nodules formed is significantly larger in larvae inoculated with P. tenuipes . It is suggested that the different virulence of each fungal species is caused by specific immune responses in the larvae. Haemopoiesis is affected by the fungal conidia and this is investigated by culture of the haemopoietic organs of M. brassicae in vitro . The proportion of spherulocytes discharged from the organs is typically high after all of the fungal treatments. In addition, the anterior and posterior haemopoietic organs, according to the histological locations in larva, show different patterns of haemopoiesis in response to fungi.     

An Entomopathogenic Bacterium,Xenorhabdus nematophila K1, Enhances Baculovirus Pathogenicity against Spodoptera exigua and Plutella xylostella

This is a report to demonstrate a pathogenicity enhancement by two microbial agents. Baculovirus pathogenicity was attenuated with larval development of both lepidopteran species: the beet armyworm, Spodoptera exigua, and the diamondback moth, Plutella xylostella. The addition of an entomopathogenic bacterium, Xenorhabdus nematophila Kl, to the baculovirus treatment significantly enhanced the viral pathogenicity in both lepidopteran species. The synergistic pathogenicity may result from their independent pathogenic targets in the insects.     

Phylogenetic relationships among strains of the entomopathogenic fungus, Nomuraea rileyi, as revealed by partial beta-tubulin sequences and inter-simple sequence repeat (ISSR) analysis

AIMS: To elucidate the phyletic relationships among three members of the entomogenous fungal genus, Nomuraea, with an emphasis on N. rileyi. METHODS AND RESULTS: Relationships were evaluated by analysis of the beta-tubulin gene and of inter-simple sequence repeats (ISSR). The amplification product of the partial beta-tubulin gene was larger for N. atypicola than for N. rileyi, and sequencing of this gene fragment confirmed that N. atypicola possesses approximately 25 more nucleotides than N. rileyi and N. anemonoides. Based on neighbor joining and bootstrap analysis of the partial beta-tubulin gene, N. atypicola failed to form a monophyletic grouping with the other two species of Nomuraea. In contrast, the single isolate of N. anemonoides clustered with the N. rileyi isolates, and both taxa grouped with Epichloe typhina (Hypocreales: Clavicipitaceae). Results from this study suggested that N. rileyi and N. anemonoides are closely related to the Clavicipitaceae. In contrast, evidence indicated that N. atypicola is not closely related to this family, and that this taxon is not a Nomuraea. Based on the 83 polymorphic loci of ISSR, it was observed that isolates of N. rileyi from diverse geographical origins were distinctly different from both N. atypicola and N. anemonoides. Considerable heterogeneity was observed in the 18 isolates of N. rileyi tested, and several clusters contained isolates from disparate geographical locations and hosts. However, three isolates from the Philippines (three host species) and three strains isolated from velvetbean caterpillar (Anticarsia gemmatalis) larvae in South America did cluster together. Two other strains from Brazil (isolated from Spodoptera spp.) were distinct from the velvetbean caterpillar isolates from South America. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The beta-tubulin gene was generally too conserved to resolve intraspecies variability. However, ISSR did identify polymorphisms among the isolates of N. rileyi tested. The results of this study indicate that ISSR may be used as robust molecular markers for studying the population genetics of this entomopathogenic fungus.     

几种虫生真菌对斜纹夜蛾的致病性

用布氏白僵菌、球孢白僵菌、玫烟色拟青霉、绿僵菌和莱氏野村菌5种真菌的固体培养物,对斜纹夜蛾2、3龄幼虫进行了毒力测定.结果表明：布氏白僵菌和莱氏野村菌两种菌株对斜纹夜蛾幼虫有明显的致病效果,对2龄幼虫的致死中时(LT₅₀)分别为2.95 d和4.10 d,累计校正死亡率分别为100%和95.2%;对3龄幼虫的致病力低于2龄,致死中时(LT₅₀)分别为19.67 d和19.63 d,累计校正死亡率分别为56.6%和52.2%.玫烟色拟青霉、球孢白僵菌两菌株也有一定的致病力,对2龄幼虫的致死中时(LT₅₀)分别为4.89 d 和6.34 d,累计校正死亡率分别为85.7%和71.4%.     

Inhibition of phagocytic activity and nodulation in Galleria mellonella by the entomopathogenic fungus Nomuraea rileyi

Changes in phagocytic activity and nodulation in the greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae), were examined after treatment with the culture fluid of the entomopathogenic fungus Nomuraea rileyi SH1. When isolated hemocytes of G. mellonella were incubated with the conidia of N. rileyi in vitro, the rates of phagocytosis increased at 4 h after incubation but decreased subsequently. On the contrary, the rates of phagocytosis of isolated hemocytes decreased by 80% after 24 h preincubation with the fungal culture fluid (1/200, 1/100 dilutions). Levels of inhibition of phagocytic activity by the culture fluid depended on dilutions used. Galleria mellonella larvae showed a peak of nodulation at 4 h after injection with conidia. The percentage of nodules in hemolymph did not decrease by preinjection with the culture fluid, whereas the percentage of nodule‐containing conidia decreased, depending on the injected fluid. However, phagocytosis and nodulation in G. mellonella did not change after treatment of the culture fluid with proteinase K, indicating that the culture fluid contained proteinaceous immunosuppressive factors. Electrophoretic analysis of the culture fluid and not the fresh medium without culturing the fungus exhibited protein bands. Therefore, N. rileyi possibly secretes toxic proteins that impair cellular immune responses in G. mellonella larvae.     

转tcdA1B1杀虫基因的阴沟肠杆菌对4种农业害虫的杀虫活性与毒力

tcdA1B1为昆虫病原细菌一发光杆菌Photorhabdus luminescens的杀虫毒素基因。本文测定了转tcdA1B1基因的阴沟肠杆菌对甜菜夜蛾Spodoptera exigua、斜纹夜蛾Spodoptera litura、黄曲条跳甲Phyllotreta striolata和东亚飞蝗Locusta migratoria manilensis4种重要农业害虫的杀虫活性与毒力。在室内条件下,浓度为5．0×108cfu／mL的工程菌悬浮液,采用叶片浸渍法饲喂甜菜夜蛾2龄幼虫、斜纹夜蛾2龄幼虫、黄曲条跳甲成虫、东亚飞蝗2龄若虫,在11d、11d、15d、19d后的致死率分别为80．05％、81．83％、72．31％、39．74％;LT50分别为8．64d,8．38d、11．04d、28．22d。     

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。     

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.     

Xenorhabdus nematophilus inhibits p-bromophenacyl bromide (BPB)-sensitive PLA2 of Spodoptera exigua

Xenorhabdus nematophilus is a Gram-negative symbiotic bacterium of the entomopathogenic nematode, Steinernema carpocapsae. The bacteria delivered into the insect hemocoel by the nematodes cause immunodepression of the target insects to protect host nematodes and themselves from the cellular immune reaction. Previous reports suggest that the immunodepression is caused by inhibition of the eicosanoid pathway that is known to be critically important to mediate cellular immunity. This study focused on the inhibitory effect of X. nematophilus on PLA2 activity of Spodoptera exigua. The PLA2 activity was functionally associated with the activation cascade of prophenoloxidase (pPO). Dexamethasone (DEX), a specific PLA2 inhibitor, inhibited pPO activation completely at the higher doses of approximately 2.4 muM in vitro condition. The inhibitory effect of DEX was reversed by the addition of arachidonic acid, the catalytic product of PLA2. By means of this in vitro PLA2 inhibitor assay system, two different PLA2 inhibitors were used to compare their inhibitory effects on the hemolymph PLA2 of S. exigua. p-Bromophenacyl bromide (BPB), a specific inhibitor of secretory PLA2 (sPLA2), significantly inhibited pPO activation, but methylarachidonyl fluorophosphates (MAFP), a specific inhibitor of cytosolic PLA2 (cPLA2), did not show any inhibitory effect. BPB also inhibited pPO activation of the plasma, though much higher PO activation and its inhibition by BPB was found in the hemocytes. Growth medium of X. nematophilus at the stationary phase had a PLA2 inhibitory effect. Via the in vitro PLA2 inhibitor assay, it was shown that the ethyl ether extract of the medium contained significant PLA2 inhibitor activity. These results indicate that X. nematophilus produces and secretes PLA2 inhibitor, which acts on BPB-susceptible PLA2 of S. exigua.