How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions

During the past year, dramatic progress has been achieved in our understanding of Drosophila immune reactions. The completion of the Drosophila genome sequencing project, microarray analysis and the use of genetic screens have led to the identification of several new genes required to combat microbial infection, filling in some important gaps in the understanding of innate immunity. At the same time, this insect was used as a model for the study of host-pathogen interactions. The recent major advances on the mechanisms by which this insect defends itself against intrusion of pathogens are discussed in this review.     

昆虫免疫识别与病原物免疫逃避机理研究进展

昆虫在长期进化过程中形成复杂的天然免疫系统,病原识别是启动下游免疫反应的第一步,这一过程主要是由不同的模式识别蛋白来完成的。目前发现并鉴定的昆虫模式识别蛋白主要包括肽聚糖识别蛋白、类免疫球蛋白、β-1,3-葡聚糖结合蛋白、C型凝集素及具多功能的载脂蛋白等,不同的蛋白种类具有不同的结构、功能及识别对象。与昆虫免疫识别相对应的是,不同昆虫病原物在进化过程中发展出不同策略的免疫逃避能力,以战胜宿主免疫而致病或最终杀死昆虫。本文就昆虫免疫过程中不同模式识别蛋白的结合对象、结构与功能,以及逐渐兴起的病原物通过分子伪装等进行免疫逃避的研究进展进行了综述。并在此基础上,作者就昆虫免疫与昆虫病理研究的发展方向进行了展望,认为只有当两方面研究相结合时,才能更好地揭示昆虫宿主与病原物之间免疫与抗免疫的动态相互作用过程。     

Production and efficiency of protozoa

Despite the chronic and debilitative nature of the infection they cause, several species of microsporidia and neogregarines offer a good potential as microbial control agents, particularly against insect pests of high economic thresholds. Techniques for mass production of protozoa have usually involved per os, inoculation or injection of the protozoa into their usual or alternate hosts. The spores are harvested subsequently from heavily infected host tissues by grinding, filtration, and differential centrifugation. Although fresh spores are used in most field tests, the spores of many species can be stored with high survival either frozen or in water at low temperatures (0–4°C) for up to several months. Sunlight or ultraviolet (UV) radiation is a serious factor limiting spore persistence. However, the protozoa do not appear to be significantly limiting spore persistence. However, the protozoa do not appears to be significantly more susceptible to UV radiation than other insect pathogens and persistence can be prolonged with UV protectants. Most field tests with protozoa have involved the application of spores in sprays and have usually resulted in a high degree of infection in the target host species. The potential for control of few species has been improved by formulation of spores in to baits, and the potential of other species will likely increase if suitable bait formulation can be devised in the future. One species, Nosema locustae, formulated as a bait, has been successfully used to control grasshoppers on rangelands. Limited laboratory and field studies have also suggested that increased short-term control might be obtained if candidate protozoan species can be combined with certain insecticides. While recent and increased efforts have been devoted to assess the potential of protozoa as microbial control agents, potential hazards to nontarget organism have been investigated for only three species. Their close relation taxonomically to protozoa pathogenic for mammals will necessitate careful evaluation of the safety of candidate control species for nontarget organisms.     

Response of rhizosphere microbial communities associated with Antarctic hairgrass (<Emphasis Type="Italic"> Deschampsia antarctica</Emphasis><Emphasis Type="Underline">)</Emphasis> to UV radiation

The response of rhizosphere microbial communities associated with natural populations of Deschampsia antarctica growing on Léonie Island (67°36S, 68°21W, Antarctic Peninsula) to UV radiation was investigated. UV radiation was controlled in the field using Perspex VA screens (UV-B opaque) which transmit little radiation below 380 nm but allow penetration of approximately 92% of radiation above 400 nm, and Perspex OXO2 screens (UV-B transparent) which transmit approximately 70% of radiation at 280 nm, rising to 90% at 300 nm and above. Reducing ambient UV radiation altered the phenotypic profile of the rhizosphere microbial community. This alteration was expressed as enhanced carbohydrate and carboxylic acid utilisation by the rhizosphere micro-organisms. It is hypothesised that ambient levels of UV radiation indirectly affect rhizosphere micro-organisms by influencing the quality or quantity of root exudates.     

A method of visualizing and assessing deposits of aerially sprayed insect microbes

A technique is described for visualizing and assessing deposits of various types of insect microbes on membrane filter surface. It is proposed as an accurate method of determining coverage of entomopathogenic microorganisms sprayed in a tower or from an aircraft in microbial control operations. The actual numbers of spores and/or crystals and number of International Units deposited per unit area, spore: crystal ratio, microbial impact droplet size, and spore viability of Bacillus thuringiensis have been determined with simulated aerial applications. The morphological integrity of different types of insect pathogens is easily distinguishable with the technique.Field trials with nuclear polyhedrosis virus of the spruce budworm, Choristoneura fumiferana, demonstrate the utility of the method in insect pest control by aerially applied microbial agents.     

Genome-wide screening and identification of antigens for rickettsial vaccine development

The capacity to identify immunogens for vaccine development by genome-wide screening has been markedly enhanced by the availability of microbial genome sequences coupled to proteomic and bioinformatic analysis. Critical to this approach is in vivo testing in the context of a natural host–pathogen relationship, one that includes genetic diversity in the host as well as among pathogen strains. We aggregate the results of three independent genome-wide screens using in vivo immunization and protection against Anaplasma marginale as a model for discovery of vaccine antigens for rickettsial pathogens. In silico analysis identified 62 outer membrane proteins (Omp) from the 949 predicted proteins in the A. marginale genome. These 62 Omps were reduced to 10 vaccine candidates by two independent genome-wide screens using IgG2 from vaccinates protected from challenge following vaccination with outer membranes (screen 1) or bacterial surface complexes (screen 2). Omps with broadly conserved epitopes were identified by immunization with a live heterologous vaccine, A. marginale ssp. centrale (screen 3), reducing the candidates to three. The genome-wide screens identified Omps that have orthologs broadly conserved among rickettsial pathogens, highlighted the importance of identifying immunologically subdominant antigens, and supported the use of reverse vaccinology approaches in vaccine development for rickettsial diseases.     

一种体内研究病原体致病机理的新方法——信号标签诱变技术

信号标签诱变技术是以整个基因组为基础的研究病原体致病机制,可在体内对毒力基因进行高通量筛选的一种新方法。近几年应用该技术已对十多种病原微生物进行了筛选。这些筛选中除了找到已知的毒力基因外,还都鉴定到了未知的毒力因子。本文就该技术的原理、优缺点、应用的必要条件、技术的改进及应用该技术鉴定到的毒力基因等作一综述。 A Novel Approach to Study Pathogenesis of Pathogens in vivo——Signature-tagged Mutagenesis YAO Xiao1,2,HUANG Liu-yu1,YANG Bo-lun2,SU Guo-fu1 1.Beijing Institute of Biotechnoloy,Beijing 100071,China; 2.College of Environmental and Chemical Engineering,Xi'an Jiaotong University,Xi'an 710049,China Abstract:Signature-tagged mutagenesis (STM) is a novel approach to study pathogenesis of pathogens and to screen virulence genes with high throughput in vivo,which is based on whole genome of pathogen in question.In resent years,more than ten species of microbial pathogens have been screened with this technology.There are also unknown virulence factors being identified with exception of known virulence genes identified in all these screens.This article reviews the principle,advantages and current limitations,the requirements,modifications of STM,and to date virulence genes identified by this technology. Key words：signature-tagged mutagenesis;virulence genes;pathogens;in vivo     

Group‐specific polymerase chain reaction amplification of SSU rRNA‐encoding gene fragments from 12 microbial taxa

Starting from an alignment of all known representatives in GenBank, we designed group specific primers targeting SSU rRNA‐encoding sequences of 12 microbial taxa known to contain insect pathogens and symbionts. We tested the specificity of these primers using representative species of all 12 groups as control templates. Polymerase chain reaction amplification conditions were modified until only group‐specific templates yielded a positive signal. The presented primer pairs thus allow for the amplification of SSU rRNA‐encoding sequences representing specific microbial groups directly from the environment (a social insect host in our study). We discuss possible applications of the identified molecular tools.     

Signals involved in Arabidopsis resistance to Trichoplusia ni caterpillars induced by virulent and avirulent strains of the phytopathogen Pseudomonas syringae

Plants have evolved different but interconnected strategies to defend themselves against herbivorous insects and microbial pathogens. We used an Arabidopsis/Pseudomonas syringae pathosystem to investigate the impact of pathogen-induced defense responses on cabbage looper (Trichoplusia ni) larval feeding. Arabidopsis mutants [npr1, pad4, eds5, and sid2(eds16)] or transgenic plants (nahG) that are more susceptible to microbial pathogens and are compromised in salicylic acid (SA)-dependent defense responses exhibited reduced levels of feeding by T. ni compared with wild-type plants. Consistent with these results, Arabidopsis mutants that are more resistant to microbial pathogens and have elevated levels of SA (cpr1 and cpr6) exhibited enhanced levels of T. ni feeding. These experiments suggested an inverse relationship between an active SA defense pathway and insect feeding. In contrast to these results, there was increased resistance to T. ni in wild-type Arabidopsis ecotype Columbia plants that were infected with P. syringae pv. maculicola strain ES4326 (Psm ES4326) expressing the avirulence genes avrRpt2 or avrB, which elicit a hypersensitive response, high levels of SA accumulation, and systemic acquired resistance to bacterial infection. Similar results were obtained with other ecotypes, including Landsberg erecta, Cape Verdi Islands, and Shakdara. When infected with Psm ES4326(avrRpt2) or Psm ES4326(avrB), nahG transgenic and npr1 mutant plants (which are more susceptible to virulent and avirulent P. syringae strains) failed to show the increased insect resistance exhibited by wild-type plants. It was surprising that wild-type plants, as well as nahG and npr1 plants, infected with Psm ES4326 not expressing avrRpt2 or avrB, which elicits disease, became more susceptible to T. ni. Our results suggest two potentially novel systemic signaling pathways: a systemic response elicited by HR that leads to enhanced T. ni resistance and overrides the SA-mediated increase in T. ni susceptibility, and a SA-independent systemic response induced by virulent pathogens that leads to enhanced susceptibility to T. ni.     

台湾的微生物防治

化学防治自1940年代后虽然为农业和医界害虫防治带来许多的经济效益。相对的。因其对非标的生物(有益生物,野生生物．家畜及农场操作人员)有毒害作用．及其残留量(residue)可在空气中．土壤,水中发现．又可经作物带给消费。也使我们得考虑其安全性．     

Physical stress primes the immune response of Galleria mellonella larvae to infection by Candida albicans

Larvae of the greater wax moth (Galleria mellonella) that had been subjected to physical stress by shaking in cupped hands for 2 min showed reduced susceptibility to infection by Candida albicans when infected 24 h after the stress event. Physically stressed larvae demonstrated an increase in haemocyte density and elevated mRNA levels of galiomicin and an inducible metalloproteinase inhibitor (IMPI) but not transferrin or gallerimycin. In contrast, previous work has demonstrated that microbial priming of larvae resulted in the induction of all four genes. Examination of the expression of proteins in the insect haemolymph using 2D electrophoresis and MALDI TOF analysis revealed an increase in the intensity of a number of peptides showing some similarities with proteins associated with the insect immune response to infection. This study demonstrates that non-lethal physical stress primes the immune response of G. mellonella and this is mediated by elevated haemocyte numbers, increased mRNA levels of genes coding for two antimicrobial peptides and the appearance of novel peptides in the haemolymph. This work demonstrates that physical priming increases the insect immune response but the mechanism of this priming is different to that induced by low level exposure to microbial pathogens.     

Pathogen incidence and their potential as microbial control agents in IPM of maize stem borers in West Africa

A review of the existing basis for maize stem borer IPM is given and the role of pathogens in the system is evaluated. Survey work outlining the major groups of insect pathogens is described; fungi (Beauveria bassiana and Metarhizium anisopliae), bacteria (Bacillus thuringiensis and Serratia marcesens), and viruses (granuloviruses and cytoplasmic polyhedroviruses) were identified. The presence of other unidentified protozoans, nematodes, fungi and viruses was noted. The virulence of some of the more promising known insect pathogens was explored in preliminary bioassays. Considering the cryptic habits of the insects, and the low input agriculture practiced by the majority of maize farmers in sub-Saharan Africa, Beauveria bassiana isolates possessing the capacity to grow systemically in the maize plant are considered one of the more interesting candidates for development as microbial control agents despite limited control in preliminary trials. Further work should also investigate the potential of pathogens of moderate virulence, such as the protozoans and CPVs.     

Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens

Conventional assays for quantifying the virulence of microbial pathogens and mutants have traditionally relied upon the use of a range of mammalian species. A number of workers have demonstrated that insects can be used for evaluating microbial pathogenicity and provide results comparable to those that can be obtained with mammals since one component of the vertebrate immune system, the innate immune response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity.     

Insect immune responses to nematode parasites

Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites.     

Cell wall-bound ultraviolet-screening compounds explain the high ultraviolet tolerance of the Antarctic moss, Ceratodon purpureus

* Studies of ultraviolet (UV) light-induced DNA damage in three Antarctic moss species have shown Ceratodon purpureus to be the most UV tolerant, despite containing lower concentrations of methanol-soluble UV-screening compounds than the co-occurring Bryum pseudotriquetrum. * In this study, alkali extraction of cell wall-bound phenolics, combined with methanol extraction of soluble phenolics, was used to determine whether cell wall-bound UV screens explain the greater UV tolerance of C. purpureus. * The combined pool of UV screens was similar in B. pseudotriquetrum and C. purpureus, but whilst B. pseudotriquetrum had almost equal concentrations of MeOH-soluble and alkali-extractable cell wall-bound UV-screening compounds, in C. purpureus the concentration of cell wall-bound screening compounds was six times higher than the concentration of MeOH-soluble UV screens. The Antarctic endemic Schistidium antarctici possessed half the combined pool of UV screens of the other species but, as in C. purpureus, these were predominantly cell wall bound. Confocal microscopy confirmed the localization of UV screens in each species. * Greater investment in cell wall-bound UV screens offers C. purpureus a more spatially uniform, and potentially more effective, UV screen. Schistidium antarctici has the lowest UV-screening potential, indicating that this species may be disadvantaged under continuing springtime ozone depletion. Cell wall compounds have not previously been quantified in bryophytes but may be an important component of the UV defences of lower plants.     

The rhizosphere microbiome and plant health

The diversity of microbes associated with plant roots is enormous, in the order of tens of thousands of species. This complex plant-associated microbial community, also referred to as the second genome of the plant, is crucial for plant health. Recent advances in plant-microbe interactions research revealed that plants are able to shape their rhizosphere microbiome, as evidenced by the fact that different plant species host specific microbial communities when grown on the same soil. In this review, we discuss evidence that upon pathogen or insect attack, plants are able to recruit protective microorganisms, and enhance microbial activity to suppress pathogens in the rhizosphere. A comprehensive understanding of the mechanisms that govern selection and activity of microbial communities by plant roots will provide new opportunities to increase crop production.     

Glyphosate inhibits melanization and increases susceptibility to infection in insects

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin’s broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate—the most widely used herbicide globally—inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate’s mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation–reduction balance. Overall, these findings suggest that glyphosate’s environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

Glyphosate, the most commonly used herbicide in the world, inhibits the production of melanin. Melanin is an important pigment and a key component of the insect immune system; this study shows that glyphosate weakens insects’ melanin-based immune system and makes them more vulnerable to infections, including with the human malaria parasite Plasmodium falciparum.     

Enhanced UV Resistance and Improved Killing of Malaria Mosquitoes by Photolyase Transgenic Entomopathogenic Fungi

The low survival of microbial pest control agents exposed to UV is the major environmental factor limiting their effectiveness. Using gene disruption we demonstrated that the insect pathogenic fungus Metarhizium robertsii uses photolyases to remove UV-induced cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) photoproducts [(6-4)PPs] from its DNA. However, this photorepair is insufficient to fix CPD lesions and prevent the loss of viability caused by seven hours of solar radiation. Expression of a highly efficient archaeal (Halobacterium salinarum) CPD photolyase increased photorepair >30-fold in both M. robertsii and Beauveria bassiana. Consequently, transgenic strains were much more resistant to sunlight and retained virulence against the malaria vector Anopheles gambiae. In the field this will translate into much more efficient pest control over a longer time period. Conversely, our data shows that deleting native photolyase genes will strictly contain M. robertsii to areas protected from sunlight, alleviating safety concerns that transgenic hypervirulent Metarhizium spp will spread from mosquito traps or houses. The precision and malleability of the native and transgenic photolyases allows design of multiple pathogens with different strategies based on the environments in which they will be used.     

昆虫hemocytin蛋白研究进展

本课题组前期研究发现,绿僵菌素A与家蚕Bombyx mori的hemocytin蛋白互作,强烈抑制血淋巴免疫,预示hemocytin可能成为一种杀虫剂的新型作用靶标。因此,进一步了解hemocytin十分必要。Hemocytin是昆虫血淋巴免疫的重要因子,作为一种凝集素,介导血淋巴中的凝血、结节和囊胞化过程,防止表皮破损造成的血淋巴外溢和微生物入侵,并参与对已入侵病原的固定与清除。昆虫的hemocytin一般由3 000～4 000个氨基酸组成,是一个巨大的多结构域蛋白,含有多个重复排列的结构域,包括FA58C (coagulation factor 5 or 8 C-terminal), VWD (von Willebrand factor type D), TIL (trypsin inhibitor like cysteine rich), VWC (von Willebrand factor type C), CT (C-terminal cystine knot-like), C8 (8 conserved cysteine residues), ChtBD2 (chitin...