感染球孢白僵菌的家蚕免疫应答差异表达基因分析

【目的】筛选家蚕Bombyx mori应对白僵菌Beauveria bassiana侵染的应答基因, 以进一步研究家蚕抵御真菌侵染的分子机制。【方法】采用新一代Solexa高通量测序技术对感染白僵菌及未感染白僵菌的对照组家蚕进行了测序分析, 筛选差异表达基因; 结合生物信息学工具分析差异表达基因的功能注释、 分类及涉及的信号通路等; 应用荧光定量PCR技术验证10个基因的差异表达。【结果】通过测序和生物信息学分析共获得377个差异表达基因, 其中表达上调基因236个, 下调基因141个; KEGG通路分析表明, 各通路中既有表达上调的基因, 也有下调基因; 12个上调基因、 26个下调基因参与3个显著性富集的KEGG通路, 即核糖体、 氨酰tRNA生物合成和剪接体通路。定量PCR与测序结果显示, 溶菌酶、 热激蛋白、 谷胱甘肽S-转移酶、 肽聚糖识别蛋白等与免疫应激相关的蛋白基因均呈现表达上调。【结论】本研究筛选获得的差异表达基因, 特别是上调表达的基因可能与家蚕应对白僵菌侵染的应答机制有关, 其中与免疫应激相关的蛋白基因如溶菌酶、 热激蛋白、 谷胱甘肽S 转移酶、 肽聚糖识别蛋白基因等可能直接参与了家蚕对白僵菌的免疫识别和防御, 研究结果为从分子水平阐明家蚕抵御真菌侵染的防御机制和白僵菌对家蚕的致病机理提供新的依据。     

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.     

Gene expression of a novel defensin antimicrobial peptide in the silkworm, Bombyx mori

cDNA encoding a novel defensin (BmDefensinB) was cloned from the fat body of the silkworm, Bombyx mori, and gene expression was analyzed. BmDefensinB showed typical structural characteristics of invertebrate defensins. Phylogenetic and bootstrap analyses indicated that it has no orthologs, whereas previously reported BmDefensinA is the ortholog of Spodoptera frugiperda (Sf)Spodoptericin. The BmDefensinB gene was expressed tissue-specifically in the fat body and was strongly activated by bacteria such as Escherichia coli and Bacillus subtilis, and by an entomopathogenic fungus Beauveria bassiana. In contrast, the BmDefensinA gene was expressed to a much lesser extent. Expression of the BmDefensinB gene was strongly stimulated by B. mori Rel proteins RelB and Relish, supporting the observation that this gene is activated by E. coli, B. subtilis, and B. bassiana. These results suggest that BmDefensinB gene expression is controlled through both the Toll and the Imd pathway, and that this gene plays an important role in B. mori immune reactions against infection by bacteria and fungi.     

Experimental evidence of bark beetle adaptation to a fungal symbiont

The importance of symbiotic microbes to insects cannot be overstated; however, we have a poor understanding of the evolutionary processes that shape most insect–microbe interactions. Many bark beetle (Coleoptera: Curculionidae, Scolytinae) species are involved in what have been described as obligate mutualisms with symbiotic fungi. Beetles benefit through supplementing their nutrient‐poor diet with fungi and the fungi benefit through gaining transportation to resources. However, only a few beetle–fungal symbioses have been experimentally manipulated to test whether the relationship is obligate. Furthermore, none have tested for adaptation of beetles to their specific symbionts, one of the requirements for coevolution. We experimentally manipulated the western pine beetle–fungus symbiosis to determine whether the beetle is obligately dependent upon fungi and to test for fine‐scale adaptation of the beetle to one of its symbiotic fungi, Entomocorticium sp. B. We reared beetles from a single population with either a natal isolate of E. sp. B (isolated from the same population from which the beetles originated), a non‐natal isolate (a genetically divergent isolate from a geographically distant beetle population), or with no fungi. We found that fungi were crucial for the successful development of western pine beetles. We also found no significant difference in the effects of the natal and non‐natal isolate on beetle fitness parameters. However, brood adult beetles failed to incorporate the non‐natal fungus into their fungal transport structure (mycangium) indicating adaption by the beetle to particular genotypes of symbiotic fungi. Our results suggest that beetle–fungus mutualisms and symbiont fidelity may be maintained via an undescribed recognition mechanism of the beetles for particular symbionts that may promote particular associations through time.     

Desiccant dusts synergize the effect of Beauveria bassiana (Hyphomycetes: Moniliales) on stored-grain beetles

Diatomaceous earth (DE) is a desiccant insecticide and most efficacious in low humidity. It acts on insect cuticle by absorbing lipids, and perhaps by cuticular abrasion. Beauveria bassiana (Balsamo) Vuillemin, an entomopathogenic fungus, is most efficacious in high humidity and has a complex interaction with cuticular lipids. Interaction between these materials may enhance insect control performance. Assays with stored-grain beetles were conducted with B. bassiana at rates of 11, 33, 100, and 300 mg of conidia per kilogram of grain with and without single rates of DE that killed 10% or less of the target beetles. The assays revealed synergism in effects on adult Rhyzopertha dominica (F.) and Oryzaephilus surinamensis (L.) at all doses. There was statistically significant synergism for adult Cryptolestes ferrugineus (Stephens) and larval R. dominica but at only one B. bassiana rate for each target. Both amorphous silicon dioxide, a sorptive dust, and diamond dust, an abrasive, showed synergistic interaction with B. bassiana on adult R. dominica. These results may provide a basis for a least-toxic approach to control of stored-product beetles and for efficacy-enhancing formulation of entomopathogenic fungi.     

Toll/IMD signal pathways mediate cellular immune responses via induction of intracellular PLA 2 expression

Phospholipase A₂ (PLA₂) hydrolyzes fatty acids from phospholipids at the sn‐2 position. Two intracellular PLA₂s, iPLA₂A and iPLA₂B, have been found in Spodoptera exigua. Both are calcium‐independent cellular PLA₂. Their orthologs have been found in other insects. These two iPLA₂s are different in ankyrin motif of N terminal region. The objective of this study was to determine whether Toll/immune deficiency (IMD) signal pathways could mediate cellular immune responses via induction of iPLA₂ expression. Both iPLA ₂s were expressed in all developmental stages of S. exigua, showing the highest expression in the adult stage. During larval stage, hemocyte is the main tissue showing expression of these iPLA₂s. Both iPLA₂s exhibited similar expression patterns after immune challenge with different microbial pathogens such as virus, bacteria, and fungi. Promoter component analysis of orthologs encoded in S. frugiperda indicated nuclear factor‐κB‐ and Relish‐responsible elements on their promoters, suggesting their expression in S. exigua under Toll/IMD immune signaling pathways. RNA interference (RNAi) of MyD88 or Pelle under Toll pathway suppressed inducible expression levels of both iPLA₂s in response to Gram‐positive bacteria containing Lys‐type peptidoglycan or fungal infection. In contrast, RNAi against Relish under IMD pathway suppressed both iPLA₂s in response to infection with Gram‐negative bacteria. Under RNAi conditions, hemocytes significantly lost cellular immune response measured by nodule formation. However, addition of arachidonic acid (a catalytic product of PLA₂) rescued such immunosuppression. These results suggest that Toll/IMD signal pathways can mediate cellular immune responses via eicosanoid signaling by inducing iPLA₂ expression.     

Ocurrence of the antibiotic producing bacterium Burkholderia sp. in colonies of the leaf-cutting ant Atta sexdens rubropilosa

Fungus garden material from recently established Atta sexdens rubropilosa colonies (6-12 months old) was sampled to detect antibiotic producing microorganisms that inhibited the growth of pathogens of insects and of the fungus gardens but did not affect their mutualistic fungus. A bacterium with activity against the entomopathogenic fungus Beauveria bassiana was isolated from 56% of the gardens tested (n=57) and identified from its biochemical profile and from 16S and 23S ribosomal DNA sequences as a member of the genus Burkholderia. The ant-associated Burkholderia isolates secreted a potent, anti-fungal agent that inhibited germination of conidia of the entomopathogenic fungi B. bassiana, Metarhizium anisopliae, of the saprophytic Verticillium lecanii, and also of a specialist fungus garden Escovopsis weberi. Growth of the ant's mutualist fungus was unaffected.     

Diversity of entomopathogenic fungi near leaf-cutting ant nests in a neotropical forest, with particular reference to Metarhizium anisopliae var. anisopliae

We investigated the prevalence of entomopathogenic fungi associated with leaf-cutting ant colonies in a small area of tropical forest in Panama. There was a high abundance of Metarhizium anisopliae var. anisopliae near the colonies. Beauveria bassiana was also detected in the soil, Aspergillus flavus in dump material, and six Camponotus atriceps ants were found infected with Cordyceps sp. Based on a partial sequence of the IGS region, almost all of the M. anisopliae var. anisopliae isolates fell within one of the three main clades of M. anisopliae var. anisopliae, but with there still being considerable diversity within this clade. The vast majority of leaf-cutting ants collected were not infected by any entomopathogenic fungi. While leaf-cutting ants at this site must, therefore, regularly come into contact with a diversity of entomopathogenic fungi, they do not appear to be normally infected by them.     

Selection of entomopathogenic fungi for the control of the western corn rootworm Diabrotica virgifera virgifera

Abstract:  The western corn rootworm Diabrotica virgifera virgifera Le Conte (Col., Chrysomelidae), a serious pest of maize, has been recently introduced into Europe. Several approaches for its control are presently under investigation including microbial agents. During a field survey in Hungary in 2005, naturally occurring entomopathogenic fungi were found to attack this pest. These novel isolates together with standard isolates were tested for virulence against D. v. virgifera larvae and adults. Twenty strains of Metarhizium anisopliae , Beauveria bassiana and Beauveria brongniartii were used in bioassays in the laboratory. Larvae and adults were dipped into a spore suspension with a concentration of 1 × 10⁷ conidia (con.)/ml. They were kept for 14 days at 22°C (±2°C) and 70% relative humidity. The number of infected larvae and adults were counted and infection rates were calculated. Adults were significantly more susceptible to entomopathogenic fungi than larvae. The most virulent isolate infected about 47% of larvae ( M. anisopliae Ma2277), whereas the infection rate in adults was up to 97% ( M. anisopliae Ma2275). Isolates of M. anisopliae caused significantly higher mortalities than isolates of B. brongniartii and B. bassiana . Most of the adult beetles were killed within 12 days. Isolates from D. v. virgifera were more virulent than those from other hosts.     

Laboratory Testing of Metarhizium spp. and Beauveria bassiana on Sahelian Non-target Arthropods

Laboratory experiments were conducted to investigate the host specificity of 11 isolates of Metarhizium sp. and one isolate of Beauveria bassiana , all deuteromycete entomopathogenic fungi that are being tested as potential biocontrol agents against locusts and grasshoppers. Metarhizium sp. (isolate IMI 330 189) was applied topically to Pimelia senegalensis and Trachyderma hispida, two Sahelian tenebrionid beetles. No infection was observed at a dose of ca. 2.5 x 106 conidia per insect, much higher than dose rates likely to occur in the field. The hymenopteran parasitoids Bracon hebetor (Braconidae) and Apoanagyrus lopezi (Encyrtidae) were exposed to surfaces treated with the entomopathogens at a rate equivalent to ca. 5 x 1012 spores ha-1, the recommended field rate against locusts. All 12 isolates tested resulted in 100% mortality of the parasitoids. Average survival times ranged from 1.6 to 4.1 days. In all cases there was fungal outgrowth on the cadavers. Exposure time-response experiments further confirmed the pathogenicity of these fungi to the parasitoids. The possibility of these parasitoids being affected in the field is discussed.     

Susceptibility of Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae) and Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) to Entomopathogenic Fungi from Ethiopia

The efficacy of 13 isolates of entomopathogenic fungi belonging to Beauveria , Metarhizium or Paecilomyces spp. was assessed against Sitophilus zeamais (Coleoptera: Curculionidae) and Prostephanus truncatus (Coleoptera: Bostrichidae) using a total immersion bioassay technique in the laboratory. Fungi were applied at concentrations of 1 ×10 7 and 1 ×10 8 conidia mL -1 for P. truncatus and S. zeamais , respectively. All isolates tested were virulent to P. truncatus (98-100% mortality, and median survival time (MST) ranged from 2.85-4.05 days). Metarhizium anisopliae and B. bassiana were also virulent to S. zeamais (92-100% mortality, MST ranged from 3.58-6.28 days). The isolate of Paecilomyces sp. was found to be the least virulent against S. zeamais , causing only 26.32 ±4.29% mortality with MST of 10.38 ±0.29 days. P. truncatus proved more susceptible to the entomopathogenic fungi tested than S. zeamais . One M. anisopliae (PPRC-EE) and three B. bassiana isolates (PPRC-HH, PPRC-9609 and PPRC-9614) were selected for further study and dose-mortality relationships were assessed on S. zeamais . The tested concentrations ranged from 1 ×10 4 -1 ×10 7 conidia mL -1 . M. anisopliae (PPRC-EE) showed the lowest LC 50 (3.39 ×10 5 conidia mL -1 ) followed by B. bassiana PPRC-HH (2.04 ×10 6 conidia mL -1 ). PPRC-9609 and PPRC-9614 showed slight differences in LC 50 but not at LC 90 . The results revealed the higher potency of M. anisopliae as compared with the B. bassiana isolates tested. The study suggests that the use of entomopathogenic fungi may hold promise as an alternative method to control pests of stored-products in Ethiopia.     

Adult polyphagous coleopterans overwintering in cereal boundaries: winter mortality and susceptibility to the entomopathogenic fungus Beauveria bassiana

Autumn-collected Bembidion lampros and Agonum dorsale were kept outdoors under semi field conditions to determine winter mortality. On three occasions (autumn, mid-winter and late winter) sub-samples of the population were incubated in the laboratory at room temperature and mortality was recorded. Generally the mortality was low in autumn and mid-winter, not exceeding 5%. A more pronounced post-winter mortality of up to 30% was observed at the end of hibernation. Only a negligible number were infected by the entomopathogenic fungi Beauveria bassiana and Paecilomyces farinosus. Infection experiments with B. bassiana isolates from Sitona lineatus, Tachyporus hypnorum and three species of ground beetles showed a similarly low susceptibility of B. lampros and A. dorsale to B. bassiana and, although Tachyporus spp. showed a distinct mycophagy and a significantly higher mortality, this could not be related to mycosis.     

In vivo gene expression profiling of the entomopathogenic fungus Beauveria bassiana elucidates its infection stratagems in Anopheles mosquito

The use of entomopathogenic fungi to control mosquitoes is a promising tool for reducing vector-borne disease transmission.To better understand infection stratagems of insect pathogenic fungi,we analyzed the global gene expression profiling of Beauveria bassiana at 36,60,84 and 108 h after topical infection of Anopheles stephensi adult mosquitoes using RNA sequencing(RNA-Seq).A total of 5,354 differentially expressed genes(DEGs) are identified over the course of fungal infection.When the fungus grows on the mosquito cuticle,up-regulated DEGs include adhesion-related genes involved in cuticle attachment,Pthll-like GPCRs hypothesized to be involved in host recognition,and extracellular enzymes involved in the degradation and penetration of the mosquito cuticle.Once in the mosquito hemocoel,the fungus evades mosquito immune system probably through up-regulating expression of |3-l,3-glucan degrading enzymes and chitin synthesis enzymes for remodeling of cell walls.Moreover,six previous unknown SSCP(small secreted cysteine-rich proteins) are significantly up-regulated,which may serve as "effectors" to suppress host defense responses.B.bassiana also induces large amounts of antioxidant genes to mitigate host-generated exogenous oxidative stress.At late stage of infection,B.bassiana activates a broad spectrum of genes including nutrient degrading enzymes,some transporters and metabolism pathway components,to exploit mosquito tissues and hemolymph as a nutrient source for hyphal growth.These findings establish an important framework of knowledge for further comprehensive elucidation of fungal pathogenesis and molecular mechanism of Beauveria-mosqaito interactions.     

MicroRNA通路中的关键因子Dicer-1和Argonaute-1对豌豆蚜免疫防御的影响

【目的】研究microRNA(miRNA)通路中两个关键组分Dicer-1和Argonaute-1(Ago-1)在豌豆蚜Acyrthosiphon pisum抵御细菌和真菌侵染中的作用,加深对豌豆蚜免疫防御系统的了解。【方法】用qRT-PCR检测细菌金黄色葡萄球菌Staphylococcus aureus和大肠杆菌Escherichia coli以及真菌球孢白僵菌Beauveria bassiana分别侵染后豌豆蚜成蚜体内Dicer-1和Ago-1的表达量;通过注射法利用RNAi技术对豌豆蚜成蚜Dicer-1和Ago-1进行沉默后,感染金黄色葡萄球菌、大肠杆菌和球孢白僵菌,检测豌豆蚜体内细菌和真菌的增殖和统计豌豆蚜成蚜存活率。【结果】与0.85% NaCl溶液处理的对照组相比,金黄色葡萄球菌、大肠杆菌和球孢白僵菌侵染豌豆蚜成蚜后,其体内Dicer-1和Ago-1的表达量发生了一定程度的上调。RNAi干扰Dicer-1后,感染金黄色葡萄球菌36 h时豌豆蚜成蚜中细菌菌落数显著高于对照组(注射dsLTA)的,但感染后7 d时的存活率与对照组无显著差异;感染大肠杆菌12和24 h时豌豆蚜成蚜体内的细菌菌落数均显著高于对照组的,但感染后7 d时的存活率显著低于对照组的;感染球孢白僵菌后6 d,豌豆蚜成蚜体内真菌孢子数显著高于对照组的,但感染后7 d时的存活率显著低于对照组的。RNAi敲降Ago-1后感染金黄色葡萄球菌,豌豆蚜成蚜体内细菌菌落数在36 h时显著高于对照组的,但感染后7 d时的存活率显著低于对照组的;感染大肠杆菌后,在24 h时豌豆蚜成蚜体内细菌菌落数显著高于对照组的,感染后7 d时的存活率略低于对照组的,但无显著差异;感染球孢白僵菌后6 d,豌豆蚜成蚜体内真菌孢子数显著高于对照组的,但感染后7 d时的存活率显著低于对照组的。【结论】豌豆蚜miRNA通路中的两个关键组分Dicer-1和Ago-1参与了豌豆蚜抵御细菌和真菌侵染的免疫防御反应,尤其在抵御真菌的防御反应中有重要作用。     

Susceptibility of adult Aethina tumida (Coleoptera: Nitidulidae) to entomopathogenic fungi

Aethina tumida Murray (Coleoptera: Nitidulidae) is an invasive parasite species in populations of honey bees, Apis mellifera L. Aiming toward substitution of chemical control, we here identified a naturally occurring fungal pathogen of adult A. tumida from its endemic range in South Africa [Metarhizium anisopliae (Metschnikoff) Sorokin variety anisopliae strain FI-203]. The susceptibility of adult beetles (n = 400) to this fungus and to three other generalist entomopathogenic fungal isolates [Metarhizium anisopliae, Beauveria bassiana (Balsamo) Vuillemin, and Hirsutella illustris Minter & Brady] was assessed using spore suspension bioassays. The data revealed significantly increased mortality in the B. bassiana (74.00 +/- 8.94%) and M. anisopliae variety anisopliae (28.00 +/- 16.43%) tests but not in the H. illustris (2.00 +/- 4.47%) and M. anisopliae (12.00 +/- 8.37%) groups. The results indicate a potential for entomopathogenic fungi as an alternative control of A. tumida.     

芫菁寄生菌降解斑蝥素

选用2种芫菁的寄生菌——球孢白僵菌Beauveria bassiana和曲霉Aspergillus sp.,进行斑蝥素的抑菌试验和对斑蝥素的降解试验。结果表明:斑蝥素对这2种真菌没有抑制作用,球孢白僵菌可以有效地降解斑蝥素,降解率为90.45%,而曲霉不能降解斑蝥素。