A Proposed Role for the Cuticular Fatty Amides of Liposcelis bostrychophila (Psocoptera: Liposcelidae) in Preventing Adhesion of Entomopathogenic Fungi with Dry-conidia

Maximum challenge exposure of Liposcelis bostrychophila to Beauveria bassiana, Paecilomyces fumosoroseus, Aspergillus parasiticus or Metarhizium anisopliae resulted in no more than 16% mortality. We investigated several of L. bostrychophila's cuticular lipids for possible contributions to its tolerance for entomopathogenic fungi. Saturated C14 and C16 fatty acids did not reduce the germination rates of B. bassiana or M. anisopliae conidia. Saturated C6 to C12 fatty acids that have not been identified in L. bostrychophila cuticular extracts significantly reduced germination, but the reduction was mitigated by the presence of stearamide. Cis-6-hexadecenal did not affect germination rates. Mycelial growth of either fungal species did not occur in the presence of caprylic acid, was reduced by the presence of lauric acid, and was not significantly affected by palmitic acid. Liposcelis bostrychophila is the only insect for which fatty acid amides have been identified as cuticular components. Stearamide, its major fatty amide, did not reduce germination of B. bassiana or M. anisopliae conidia or growth of their mycelia. Adhesion of conidia to stearamide preparations did not differ significantly from adhesion to the cuticle of L. bostrychophila. Pretreatment of a beetle known to be fungus-susceptible, larval Oryzaephilus surinamensis, with stearamide significantly decreased adhesion of B. bassiana or M. anisopliae conidia to their cuticles. This evidence indicates that cuticular fatty amides may contribute to L. bostrychophila's tolerance for entomopathogenic fungi by decreasing hydrophobicity and static charge, thereby reducing conidial adhesion.     

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.     

The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives

BACKGROUND: Beauveria bassiana is an important entomopathogenic fungus currently under development as a bio-control agent for a variety of insect pests. Although reported to be non-toxic to vertebrates, the potential allergenicity of Beauveria species has not been widely studied. METHODS: IgE-reactivity studies were performed using sera from patients displaying mould hypersensitivity by immunoblot and immunoblot inhibition. Skin reactivity to B. bassiana extracts was measured using intradermal skin testing. RESULTS: Immunoblots of fungal extracts with pooled as well as individual sera showed a distribution of IgE reactive proteins present in B. bassiana crude extracts. Proteinase K digestion of extracts resulted in loss of IgE reactive epitopes, whereas EndoH and PNGaseF (glycosidase) treatments resulted in minor changes in IgE reactive banding patterns as determined by Western blots. Immunoblot inhibitions experiments showed complete loss of IgE-binding using self protein, and partial inhibition using extracts from common allergenic fungi including; Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Candida albicans, Epicoccum purpurascens, and Penicillium notatum. Several proteins including a strongly reactive band with an approximate molecular mass of 35 kDa was uninhibited by any of the tested extracts, and may represent B. bassiana specific allergens. Intradermal skin testing confirmed the in vitro results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to B. bassiana. CONCLUSIONS: Beauveria bassiana possesses numerous IgE reactive proteins, some of which are cross-reactive among allergens from other fungi. A strongly reactive potential B. bassiana specific allergen (35 kDa) was identified. Intradermal skin testing confirmed the allergenic potential of B. bassiana.     

三株球孢白僵菌侵染烟粉虱的比较生长动力学及其毒力

同一种虫生真菌的不同菌株对于特定昆虫宿主的毒力可存在显著差异,真菌在昆虫体内的生长能力不同可能是引起这种差异的原因之一。为了探讨真菌在宿主体内的生长动力学与其毒力的关系,本研究用生测法测定了3株球孢白僵菌（GZGY-1-3、SCWJ-2、WLMQ-20）在高剂量（1×108孢子/mL）和低剂量（1×106孢子/mL）下对烟粉虱4龄若虫的毒力,用实时荧光定量PCR技术对真菌在宿主体内的拷贝数进行了定量,用荧光显微方法观察了白僵菌侵染烟粉虱的过程。生物测定实验结果显示：不论在高剂量还是低剂量下,菌株GZGY-1-3杀死烟粉虱所需的时间最短（在高剂量和低剂量下LT50分别为2.29d和6.10d）,其次是菌株SCWJ-2（LT50分别为3.03d和7.38d）,菌株WLMQ-20所需时间最长（LT50分别为4.13d和9.39d）。对于同一菌株,其高剂量下的毒力显著高于低剂量下的毒力。实时荧光定量PCR实验显示,接触高剂量孢子后烟粉虱获得的孢子数远远高于接触低剂量时的孢子数。接种后,每一菌株和每一剂量下的真菌生长都表现出一个相似的模式。在接种24h后,真菌细胞数量显著下降,在接种后24–48h之间是一个简短的恢复阶段,接种48–72h后是真菌的细胞数略有净增长的稳定时期,在宿主死亡前后的阶段,真菌数量急剧增加,与接种后最初24h相比高了近1 000倍。然而,尽管它们的生长模式相似,但是却存在着量上的差异,由致病性强、剂量高的菌株侵染的昆虫体内的最终真菌菌体数高。荧光显微技术观察到的白僵菌对烟粉虱的侵染过程证实了定量PCR的结果。这些结果说明菌株间毒力的差异在一定程度上是由真菌生长动力学的量化差异所决定的。     

球孢白僵菌羧基转运蛋白基因RNA干扰的沉默效应

羧基转运蛋白基因是近年来分离出的新基因,在虫生真菌中可能与穿透昆虫体壁时的能量代谢有关。构建了针对该基因的双链RNA干扰载体,采用芽生孢子转化法将载体质粒转入球孢白僵菌,并通过RT-PCR检测转化前后BbJEN1的表达情况。RT-PCR检测结果显示,球孢白僵菌转化子BbJEN1基因的表达水平显著下降,表明干扰载体具有明显的沉默效应。在两种培养基上,转化子与原始菌株的生长速度和产孢量差异均不显著,而分生孢子萌发则显著滞后于原始菌株。使用马尾松毛虫幼虫对转化子和原始菌株进行生物测定,原始菌株的半致死浓度、半致死剂量和半致死时间分别为2.79×106个孢子/mL、84.12个孢子/mm2和6.49d,而转化子的半致死浓度、半致死剂量和半致死时间则分别增加到1.27×107个孢子/mL、382.92个孢子/mm2和8.09d,毒力显著下降。由此表明BbJEN1基因与球孢白僵菌分生孢子发芽以及毒力均有关。     

Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.     

The effect of the entomopathogenic fungus Conidiobolus coronatus on the composition of cuticular and internal lipids of Blatta orientalis females

The composition of cuticular and internal lipids in females of the cockroach Blatta orientalis L. exposed to the entomopathogenic fungus Conidiobolus coronatus is investigated. The compositions of the fatty acids, n‐alkanes, alcohol, sterols and methyl esters in the lipids are chemically characterized. Although contact with virulent colonies of the fungus does not induce insect mortality, significant changes in the lipid profiles, both cuticular and internal, are found. The cuticular extracts of a control group of B. orientalis females contain 24 compounds varying in carbon chain length from C6 to C22. The main cuticular fatty acids identified are: C16:1, C16:0, C18:1 and C18:0_. The cuticular lipids of B. orientalis females after exposure to C. coronatus contain only 14 free fatty acids from C8 to C20. The highest concentrations identified are C16:0, C18:2 and C18:1. Analysis by gas chromatography‐mass spectrometry identifies the presence of a homologous series of n‐alkanes containing from 25 to 31 carbon atoms. In the case of the insects after fungal exposure, the content of the n‐alkanes in the cuticular lipid is two‐fold higher compared with the controls. Of the cuticular lipids, 11 alcohols are found, ranging from C12:0 to C20:0. There is no presence of alcohols in the internal lipids of the control B. orientalis females and in all of the extracts from the B. orientalis females after fungal exposure. In the samples analyzed, the most common sterol is cholesterol. This is present in the cuticular lipids and the internal lipids of all of the insects sampled. The cuticular and internal lipids of females contain five fatty acid methyl esters, ranging in size from C15 to C19.     

Responses of diamondback moth to diverse entomopathogenic fungi collected from non-agricultural habitats – Effects of dose,temperature and starvation

We evaluated the virulence of Beauveria bassiana and Metarhizium isolates from soil collected across different vegetation types in Queensland, against chlorantraniliprole-resistant and insecticide-susceptible diamondback moth (DBM) larvae. Host insecticide resistance status had no effect on susceptibility to the pathogens when conidia were topically applied to larvae in the laboratory, and one B. bassiana isolate was significantly more virulent to larvae than the others (seven days after inoculation). The influence of temperature (15, 20, 25 or 30 °C): (i) at the point of host inoculation with conidia and (ii) when the pathogens had already initiated infection and were proliferating in the host haemocoel, was determined experimentally for its influence on virulence, disease progression, and sporulation. Temperature at inoculation had a greater effect on host insect mortality than it did when the fungus was already proliferating in the host haemocoel. The rearing temperature of hosts prior to inoculation had a greater effect on host susceptibility to disease than starvation of the larvae at the time of inoculation. Our results also show that each fungal isolate has its own temperature relations and that these can vary considerably across isolates, and at different points in the pathogen life cycle (germination and cuticular penetration versus growth in the host haemocoel). Temperature also had an idiosyncratic effect, across isolates and across the variables typically used to assess the potential of fungal entomopathogens as biological control agents (time to death, mortality and sporulation rates). This study demonstrates that in addition to pathogenicity and virulence, the temperature relationships of each fungal isolate when infecting insects needs to be taken into account if we are to understand their ecology and use them effectively in pest management.     

Laccase2 is required for cuticular pigmentation in stinkbugs

During the maturation of insect cuticle, protein-protein and protein-chitin crosslinkages are formed by the action of diphenoloxidases. Two types of diphenoloxidases, laccases and tyrosinases, are present in the insect cuticle. In coleopteran and hymenopteran insects, laccase2 gene has been identified as encoding an enzyme principally responsible for cuticular pigmentation and hardening, whereas biological roles of laccase genes in hemimetabolous insects remain to be established. Here we identified laccase2 genes from three hemipteran stinkbugs, Riptortus pedestris (Alydidae), Nysius plebeius (Lygaeidae) and Megacopta punctatissima (Plataspidae). In R. pedestris, laccase2 gene was highly expressed in epidermal tissues prior to molting. When the gene expression was suppressed by an RNA interference technique, cuticular pigmentation after molting were blocked depending on the dose of injected double-stranded RNA targeting the laccase2 gene. Similar results were obtained for N. plebeius and M. punctatissima. In all the stinkbug species, injecting 20 ng of double-stranded RNA was sufficient to prevent the cuticular maturation. These results indicate that laccase2 gene is generally required for cuticular pigmentation in different stinkbug families, highlighting its conserved biological function across diverse insect taxa.     

昆虫表皮蛋白基因研究进展

在昆虫表皮的发生、分化和昆虫躯体外部重要部位及器官的构建中,表皮蛋白是不可或缺的组成元素。本文在简要总结了目前昆虫表皮蛋白鉴定与分类方面研究的基础上,重点对近10年来昆虫表皮蛋白基因的时空表达模式、激素及转录因子对表皮蛋白基因表达的调控、表皮蛋白基因功能的研究进展进行了综述,探讨了其在害虫防治中可能的应用前景,旨在为进一步研究昆虫表皮蛋白基因及其潜在利用价值提供参考。目前报道的昆虫表皮蛋白序列已超过1 400条,分为12个家族,如CPR, CPF, CPFL和Tweedle等。经由蜕皮激素激活的相关转录因子（如βFTZ-F1和BR-C等）作用于表皮蛋白基因上游的顺式作用元件,开启或关闭基因,以调控表皮蛋白基因的表达。表皮蛋白基因在昆虫表皮整合,体形塑造,活动能力,抗逆与抗药性,以及先天免疫等生理现象和生理过程中有不可或缺的作用。因此,如果能够通过抑制关键表皮蛋白基因的表达,或将其从基因组中删除,以阻碍昆虫的发育或扰乱昆虫的繁殖能力,或可为害虫防治策略提供参考。     

The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana, are key environmental pathogens of insects that have been exploited for biological control of insect pests. Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth, and pathogenicity, mediating responses to the environment. Bbslt2, encoding for an Slt2 family MAPK, was isolated and characterized from B. bassiana. Gene disruption of Bbslt2 affected growth, caused a significant reduction in conidial production and viability, and increased sensitivity to Congo Red and fungal cell wall degrading enzymes. ΔBbslt2 mutants were altered in cell wall structure and composition, which included temperature dependent chitin accumulation, reductions in conidial and hyphal hydrophobicity, and alterations in cell surface carbohydrate epitopes. The ΔBbslt2 strain also showed hypersensitivity to heat shock and altered trehalose accumulation, which could only be partially attributed to changes in the expression of trehalase (ntl1). Insect bioassays revealed decreased virulence in the ΔBbslt2 strain using both topical and intrahemoceol injection assays. These results indicate that Bbslt2 plays an important role in conidiation, viability, cell wall integrity and virulence in B. bassiana. Our findings are discussed within the context of the two previous MAP kinases characterized from B. bassiana.     

Community composition, host range and genetic structure of the fungal entomopathogen Beauveria in adjoining agricultural and seminatural habitats

Although intensively investigated for biological control of insect pests, little is known about the ecology of the fungal entomopathogenic genus Beauveria in natural or agricultural habitats. In this study, we used molecular phylogenetic and genotypic information to infer species diversity, reproductive potential and genetic structure of Beauveria occurring within a single arable field and bordering hedgerow in Denmark. Isolates were sampled from cultivated field and hedgerow soils, from insects harbouring latent fungal infections, and from the phylloplanes of three plant species common in the hedgerow flora. A nuclear phylogeny of this local Beauveria assemblage resolved seven phylogenetic species, including (i) five phylogenetic species within Beauveria bassiana sensu stricto ; (ii) Clade C, a taxonomically uncharacterized species that is morphologically indistinguishable but phylogenetically distant from B. bassiana s.s. ; and (iii) Beauveria brongniartii. All seven species were present throughout the hedgerow habitat, including as infections in insects. Significantly, only B . bassiana s.s. phylogenetic species Eu_1 was isolated from tilled soils. Mating type polymerase chain reaction assays demonstrated that all five B. bassiana s.s. phylogenetic species possess bipolar outcrossing mating systems. Of these, only the Eu_1 population contained two mating types; however, a 31:2 skew in MAT1:MAT2 mating types suggests a low frequency of sexual reproduction in this population. The four remaining B. bassiana s.s. phylogenetic species were fixed for single mating types and these populations are evidently clonal. Multilocus microsatellite genotyping revealed polymorphism in all five phylogenetic species of B. bassiana s.s. ; however, all show evidence of clonal genetic structure.     

球孢白僵菌对四种十字花科蔬菜害虫的兼控潜力评价

研究了分离自小猿叶甲的一株球孢白僵菌(Beauveria bassiana(SCAU-BB01D))对十字花科蔬菜4种非目标害虫烟粉虱Bemisia tabaci、小菜蛾Plutella xylostella、黄曲条跳甲Phyllotreta striolata和菜缢管蚜Lipaphis erysimi的致病能力。结果表明,在实验室条件下,该菌株对以上4种害虫均有不同程度的侵染力。1×108孢子/mL的白僵菌悬浮液对1,2,3,4龄烟粉虱若虫的侵染率分别为84.88,86.81,55.94和38.78%;对小菜蛾2,3和4龄幼虫的侵染率为67,59和44%;对黄曲条跳甲幼虫和成虫分别为63%和60%;对菜缢管蚜成蚜和若蚜的侵染率分别为44%和35%。通过机率值分析,得出该菌株对烟粉虱1~4龄若虫的LT50分别为4.14,3.78,6.24和7.59 d;对小菜蛾2~4龄幼虫的LT50分别为7.29,8.16和9.82d;对黄曲条跳甲成虫和幼虫的LT50分别为11.22和8.11 d;对菜缢管蚜成蚜和若蚜的LT50分别为11.01和12.15 d。说明该球孢白僵菌菌株对烟粉虱、小菜蛾和黄曲条跳甲3种非目标害虫具有一定的兼控作用,在蔬菜害虫的生物防治中具一定的应用潜力。     

Entomopathogenic Fungi Associated with Natural Populations of the Moroccan Locust Dociostaurus maroccanus (Thunberg) (Orthoptera: Gomphocerinae) and Other Acridoidea in Spain

The entomopathogenic fungi Beauveria bassiana Vuill . and Metarhizium anisopliae (Metschnikoff) have been found in natural populations of the Moroccan locust Dociostaurus maroccanus (Thunberg) and other species of acridoids that cohabit the same locust breeding areas in southern Spain . Infection levels of B. bassiana on insects collected in the field and maintained under laboratory conditions were relatively high (1 . 6 - 20 . 5%) . The prevalence of the disease extended from spring to summer in the three consecutive years monitored . A local isolate of this fungus demonstrated high virulence (LD = 440 conidia / insect) against the 50 locust D. maroccanus in the laboratory bioassay . The relatively wide host range of B. bassiana and its adaptation to the dry and hot conditions dominating the locust breeding area suggest that this isolate could be considered in the development of a biological control programme against D. maroccanus.     

Synergistic effect of entomogenous fungi on some insecticides against Bihar hairy caterpillar Spilarctia obliqua (Lepidoptera: Arctiidae)

A number of fungal parasites infect a wide range of insects and cause epizootics from time to time. Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin are two of the major disease-causing fungi in insects. Investigations were carried out to study the effect of these fungi on the toxicity of endosulfan, imidacloprid, lufenuron, diflubenzuron, dimethoate and oxydemeton methyl against 10-11 days old larvae of Spilarctia obliqua (Walker). For some products the combination treatments showed higher dose mortality response than the sole treatment of fungal conidia or the insecticide. The combination of insecticides with B. bassiana showed 1.26-35.8 fold increase in toxicity of insecticides over sole treatment, while the increase was 1.05-72.0 fold in case of M. anisopliae. Imidacloprid 17.8 SL and oxydemeton methyl 25EC may be used in combination with these fungi for management of S. obliqua.     

Comparative analysis of the production of insecticidal and melanizing macromolecules by strains of Beauveria spp.: in vivo studies

Eleven strains of Beauveria bassiana, and a further five species of Beauveria sp., were tested by injection of 8x10(2) conidia into the haemocoel of the larvae of the lepidopteran Galleria mellonella with the aim of analysing their toxin producing activity in vivo. Although the virulent strains killed 100% of the insects at slightly different rates (4-6 days) there were significant differences in the pattern and intensity of host melanization caused by isolates. The majority of the isolates of Beauveria spp. induced a fast and intense melanization of the cuticle of the integument and of tracheal wall, which followed one of three patterns. Another small group of two B. bassiana strains, isolated from Ostrinia nubilalis, induced very weak or no melanization. Strains 618 and 101 of B. bassiana, were selected as models of "melanizing" and "non-melanizing" strains, respectively. Ultrastructural alterations of cells of hypodermal and tracheal epithelium and of haemocytes, assumed to be at least partially caused by fungal toxins, were revealed in larvae infected by both isolates. However, their effects on the fine structure of the hypodermis were different. Injection of sera obtained from haemolymph of insects infected with B. bassiana 618 showed that they have insecticidal, melanizing, and cytotoxic effects similar to those occurring during mycosis. Chromatographic studies and bioassays with fractions prepared from crude serum have allowed a partial identification of the toxic molecules secreted by the fungus in vivo. They are proteinaceous, as shown by protease treatments, thermolabile, negatively charged, and not glycosylated with alpha-d-mannose or alpha-d-glucose. If strain B. bassiana 618 produces melanizing macromolecules which are vivotoxins secreted during the mycosis, the mode of action of isolate 101 is different. Its capacity to kill the host depends on active mycelial development, and on the production of low molecular weight toxins.