昆虫体内的天然屏障——围食膜

昆虫围食膜是由昆虫中肠上皮细胞分泌的非细胞薄膜状结构,主要成份是几丁质、蛋白质和多糖,是昆虫抵御外界侵害的第一道天然屏障,能够保护中肠上皮细胞不受机械损伤并且能够抵御病毒、细菌及其他有害物质,防止化学损伤.昆虫病毒增效蛋白、荧光增白剂和几丁质酶等生物防治促进因子通过与围食膜上特异位点的结合,能够破坏围食膜结构,加速病原微生物对害虫的感染进程.就围食膜组分、结构、功能以及与害虫防治的关系等方面的研究进展进行综述,并且论述了以围食膜为害虫生物防治靶标的应用前景.     

围食膜: 害虫生物防治的潜在靶标

围食膜是昆虫中肠细胞分泌的一层特有的非细胞结构,由蛋白质、粘多糖和几丁质组成,是昆虫中肠细胞抵御随食物摄入的病原微生物入侵的第一道天然屏障。昆虫病毒增效蛋白、几丁质酶、荧光增白剂和外源凝集素等生物防治促进因子通过与围食膜上特异位点的结合,可破坏围食膜结构,改变其通透性,促进病原微生物对害虫的感染。该文综述了与昆虫围食膜密切相关的生防促进因子的增效活性及其作用机理,阐明了以围食膜为害虫生物防治靶标的应用前景。     

昆虫围食膜的研究进展

围食膜是大多数昆虫中肠内的半透性薄膜 ,主要由几丁质、蛋白质构成。依据其形成的方式分 :Ⅰ型围食膜 ,由整个中肠细胞分泌形成多层管状膜 ;Ⅱ型围食膜由中肠前端特殊的细胞分泌成连续的套筒管状膜。由于位于食物与中肠上皮细胞间而在中肠生理中起重要作用 ,围食膜保护中肠上皮免于机械损伤以及病原菌、毒素的入侵 ;作为半透膜以及将中肠分为不同的区室而在营养物质的消化和吸收中具有重要作用。该文综述了有关围食膜结构、组分、功能、通透性以及与害虫防治的关系等方面的研究进展。     

昆虫天然免疫反应分子机制研究进展

昆虫体内缺乏高等脊椎动物所具有的获得性免疫系统, 只能依赖发达的天然免疫系统抵抗细菌、 真菌、 病毒等外源病原物的侵染。本文概括了昆虫天然免疫反应发生和作用的分子机制相关进展, 重点阐述了重要免疫相关因子在昆虫天然免疫反应中的功能和作用机制。昆虫天然免疫反应分为体液免疫和细胞免疫两种, 二者共同作用完成对病原物的吞噬 (phagocytosis)、 集结 (nodulation)、 包囊 (encapsulation)、 凝结 (coagulation)和黑化（melanization）等。当昆虫受到外界病原物的侵染时, 首先通过体内的模式识别蛋白（pattern recognition proteins/receptor, PRPs）识别并结合病原物表面特有的模式分子（pathogen-associated molecular pattern, PAMPs）, 继而一系列包括丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂在内的级联激活反应被激活和调控, 产生抗菌肽、 黑色素等免疫效应分子, 清除或杀灭外源物。抗菌肽是一类小分子量的阳离子肽, 具有广谱抗菌活性, 针对不同类型的病原物, 抗菌肽的产生机制也不尽相同。昆虫体内存在着两种信号转导途径调节抗菌肽的产生： 一是由真菌和大部分革兰氏阳性菌激活的Toll途径; 二是由革兰氏阴性菌激活的Imd途径（immune deficiency pathway）。这两个途径通过激活不同转录因子调控不同抗菌肽基因的表达参与昆虫体内的天然免疫反应。     

Molecular structure of the peritrophic membrane (PM): identification of potential PM target sites for insect control

Peritrophic membranes (PMs) are an invertebrate-unique structure that lines the digestive tract, playing important roles in facilitating food digestion and providing protection to the gut epithelium. The importance of PMs in insects has been recognized ever since its presence was identified 200 years ago. In the last 5 years, significant progress towards understanding the PM molecular structure and the mechanism for PM formation has been made. Recent studies on Type 1 PMs from lepidopteran larvae have suggested a model for the PM molecular structure and formation. The important physiological functions of the PM suggest that PMs can be a significant structural target for insect control and the current understanding of the structure of lepidopteran larval PMs has provided us with potential opportunities for targeting the PM by various mechanisms.     

Disruption of the peritrophic matrix by exogenous chitinase feeding reduces fecundity in Lutzomyia longipalpis females

Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.     

The peritrophic membrane of the gastropod Megathura crenulata: structure,composition, and site of formation

Peritrophic membranes (PMs) are acellular layered structures secreted around ingested materials by the gut epithelium. Most studies on PMs have focused on those of insects and crustaceans due to their potential ability to block the movement of pathogens from ingested materials into the body, and their possible use as unique targets relevant to pest management. While PMs are known to occur in other taxa, their distribution is spotty and little is known about their role in these other species. The gastropod Megathura crenulata produces a true PM, which has a chitinous matrix that makes up nearly half its wet weight. Unlike arthropod PMs, which are released by delamination from the microvilli of their gut cells, the chitinous matrix of the M. crenulata PM is secreted from epithelial cells lining most regions of its gut. Although its mode of synthesis is unique, it may serve the same functions as proposed for other PMs, including regulating diffusion, binding metabolites, restricting protease activity, blocking pathogens, and providing lubrication. In arthropods, numerous proteins with chitin‐binding specificities have been identified, consistent with the proposed functions. Analysis of PMs in M. crenulata showed several integral proteins associated with the membrane, suggesting that the PM in this mollusc may be involved in complex functions like those seen in the arthropods.     

植物病媒昆虫的翅型分化

翅多型现象是昆虫非遗传多型性的一种表现,包括不具飞行能力的短翅型或无翅型,以及可以进行长距离迁飞的长翅型或有翅型。翅多型现象常发生在可以携带病原并将其传播给植物宿主的媒介昆虫中,对植物病害的时空分布与暴发有重要影响。本文从翅型分化的遗传规律、诱导因素、分子机制和伴随翅型分化的其他生理表现4个方面,对植物病原主要传播媒介蚜虫和飞虱的翅型分化研究进行综述和梳理。昆虫翅型分化的诱导因素主要包括温度、湿度和光周期等非生物因素以及虫口密度、宿主营养、病毒等生物因素;而其内在的分子机制大多是通过胰岛素/胰岛素样生长因子信号(IIS)通路、c-Jun氨基末端激酶(c-Jun NH 2-terminal kinase,JNK)信号通路、Wingless和嗅觉受体SaveOrco等调控。翅型分化的同时伴随着生理状态的变化,表现为短翅型具有更强的繁殖能力和长翅型含有更丰富的飞行肌结构成分。目前,昆虫翅型分化的研究尚不够完善,有许多需要解答的问题,如找到胰岛素/胰岛素样生长因子信号通路中真正发挥功能的靶基因,JNK如何调控翅型分化以及虫媒病毒影响媒介昆虫翅型的分子机理。本综述可为控制虫媒病原的传播以及其他昆虫翅多型的研究提供参考。     

The origin and functions of the insect peritrophic membrane and peritrophic gel

There is a a fluid (peritrophic gel) or membranous (peritrophic membrane, PM) film surrounding the food bolus in most insects. The PM is composed of chitin and proteins, of which peritrophins are the most important. It is proposed here that, during evolution, midgut cells initially synthesized chitin and peritrophins derived from mucins by acquiring chitin-binding domains, thus permitting the formation of PM. Since PM compartmentalizes the midgut, new physiological roles were added to those of the ancestral mucus (protection against abrasion and microorganism invasion). These new roles are reviewed in the light of data on PM permeability and on enzyme compartmentalization, fluid fluxes, and ultrastructure of the midgut. The importance of the new roles in relation to those of protection is evaluated from data obtained with insects having disrupted PM. Finally, there is growing evidence suggesting that a peritrophic gel occurs when a highly permeable peritrophic structure is necessary or when chitin-binding molecules or chitinase are present in food.     

Trafficking at the host cell surface during plant immune responses

The plasma membrane (PM) of eukaryotic cells is not only an outermost covering to contain and protect inner molecules required for cell viability but also a place where communications dynamically occur with adjacent cells and environments including pathogens. However the selective permeability limits the free translocation of information across the PM between cells. Therefore, eukaryotic cells have invented an elaborate machinery to safely export and import proteins and small molecules within a membrane-wrapped container called a vesicle. Upon infection, a host plant cell also actively interacts with a phytopathogen to achieve its goal, defense to frustrate the pathogen attempt. To understand communications between pathogens and plants, hence this review is mainly focused on molecular transport events that occur at the host PM during plant immune responses.     

棉铃虫围食膜的蛋白质组鉴定

【目的】围食膜(peritrophic membrane, PM)是昆虫抵御随食物摄入的病原微生物入侵的第一道天然屏障。本研究旨在鉴定出农业重大害虫棉铃虫Helicoverpa armigera围食膜的总蛋白成分,为进一步揭示昆虫围食膜的形成机制及研发新颖的害虫控制策略奠定基础。【方法】剥离棉铃虫5龄幼虫PM,用三氟甲磺酸(trifluoromethane sulfonic acid, TFMS)处理,采用液质联用技术(LC-MS/MS)鉴定围食膜蛋白质组,然后对鉴定结果进行生物信息学分析。【结果】本研究共鉴定出棉铃虫幼虫围食膜蛋白质169个,是目前鉴定最多的棉铃虫围食膜蛋白。通过GO分析,可以将这些鉴定的蛋白分为细胞组分、分子功能和生物学过程三大类;KEGG富集结果显示,鉴定蛋白可以富集在12条代谢通路中;蛋白互作分析(protein protein interaction, PPI)结果表明,以ACC和CG3011等蛋白为核心可以形成蛋白互作网络。【结论】本研究鉴定了169个棉铃虫幼虫围食膜蛋白质,并对其进行了GO, KEGG和PPI分析,结果有助于人们全面理解昆虫围食膜的分子结构和功能。     

Entomopathogenic fungi and insect behaviour: from unsuspecting hosts to targeted vectors

The behavioural response of an insect to a fungal pathogen will have a direct effect on the efficacy of the fungus as a biological control agent. In this paper we describe two processes that have a significant effect on the interactions between insects and entomopathogenic fungi: (a) the ability of target insects to detect and avoid fungal pathogens and (b) the transmission of fungal pathogens between host insects. The behavioural interactions between insects and entomopathogenic fungi are described for a variety of fungal pathogens ranging from commercially available bio-pesticides to non-formulated naturally occurring pathogens. The artificial manipulation of insect behaviour using dissemination devices to contaminate insects with entomopathogenic fungi is then described. The implications of insect behaviour on the use of fungal pathogens as biological control agents are discussed.     

Spindles of an entomopoxvirus facilitate its infection of the host insect by disrupting the peritrophic membrane

The mode of action by which entomopoxvirus (EPV) spindles, proteinaceous crystalline bodies produced by EPVs, enhance EPV infection has not been clarified. We fed Anomala cuprea EPV (AcEPV) spindles to host insects; subsequent scanning electron microscopy revealed the disruption of the peritrophic membranes (PMs) of these insects. The PM is reportedly a barrier against the infection of some insects by viruses. Quantitative PCR of AcEPV DNA in the ectoperitrophic area revealed that PM disruption facilitated the passage of EPVs through the PM toward the initial infection site, the midgut epithelium. These results indicate that EPV spindles enhance infection by EPVs by disrupting the PM in the host insects. Fusolin is almost exclusively the constituent protein of the spindles and is the enhancing factor of the infectivity of nucleopolyhedroviruses (NPVs) and possibly that of EPVs. Spheroid is another type of proteinaceous crystalline structure produced by EPVs. Pseudaletia separata EPV (PsEPV) spheroids reportedly contain considerable amounts of fusolin and enhance NPV infection. We assessed the ability of AcEPV spheroids to enhance EPV infectivity and their effect on the PM and carried out immunological experiments; these experiments showed that AcEPV spheroids contain little or no fusolin and are biologically inactive, in contrasts to the situation in PsEPV.     

Interactions among fire, insects and pathogens in coniferous forests of the interior western United States and Canada

1 Natural and recurring disturbances caused by fire, native forest insects and pathogens have interacted for millennia to create and maintain forests dominated by seral or pioneering species of conifers in the interior regions of the western United States and Canada. 2 Changes in fire suppression and other factors in the last century have altered the species composition and increased the density of trees in many western forests, leading to concomitant changes in how these three disturbance agents interact. 3 Two‐ and three‐way interactions are reviewed that involve fire, insects and pathogens in these forests, including fire‐induced pathogen infection and insect attack, the effects of tree mortality from insects and diseases on fuel accumulation, and efforts to model these interactions. 4 The emerging concern is highlighted regarding how the amount and distribution of bark beetle‐caused tree mortality will be affected by large‐scale restoration of these fire‐adapted forest ecosystems via prescribed fire. 5 The effects of fire on soil insects and pathogens, and on biodiversity of ground‐dwelling arthropods, are examined. 6 The effects of fire suppression on forest susceptibility to insects and pathogens, are discussed, as is the use of prescribed fire to control forest pests.     

Molecular characterization of a peritrophic membrane protein from the silkworm, Bombyx mori

The peritrophic membrane lines the gut of most insects at one or more stages of their life cycles. It facilitates the digestive processes in the guts and protects from invasion by pathogens or food particles. In the current study, a novel PM protein, designated as BmMtch, was identified from the silkworm, Bombyx mori. The open reading frame of BmMtch is 888 bp in length, encoding 295 amino acid residues consisting of two domains (Mito_carr domains) and three transmembrane regions. They are localized on the 11th chromosome as single copy with one exon only. Quantitative real time PCR analysis (qRT-PCR) revealed that BmMtch was mainly expressed in larval fat bodies, Malpighian tubules, testis and ovaries, and could be detected through all stages of the life cycle of silkworm. Immuno-fluorescence analysis indicated that BmMtch was localized within the goblet cell of larval midgut. Western blotting analysis showed that BmMtch were detected in total proteins of PM and larval midgut. The characteristics of BmMtch indicated that BmMtch represents a novel member of insect PM proteins, without chitin-binding domains.     

One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus,Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%–4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.     

Chitin is a necessary component to maintain the barrier function of the peritrophic matrix in the insect midgut

In most insects, the peritrophic matrix (PM) partitions the midgut into different digestive compartments, and functions as a protective barrier against abrasive particles and microbial infections. In a previous study we demonstrated that certain PM proteins are essential in maintaining the PM's barrier function and establishing a gradient of PM permeability from the anterior to the posterior part of the midgut which facilitates digestion (Agrawal et al., 2014). In this study, we focused on the effects of a reduction in chitin content on PM permeability in larvae of the red flour beetle, Tribolium castaneum. Oral administration of the chitin synthesis inhibitor diflubenzuron (DFB) only partially reduced chitin content of the larval PM even at high concentrations. We observed no nutritional effects, as larval growth was unaffected and neutral lipids were not depleted from the fat body. However, the metamorphic molt was disrupted and the insects died at the pharate pupal stage, presumably due to DFB's effect on cuticle formation. RNAi to knock-down expression of the gene encoding chitin synthase 2 in T. castaneum (TcCHS-2) caused a complete loss of chitin in the PM. Larval growth was significantly reduced, and the fat body was depleted of neutral lipids. In situ PM permeability assays monitoring the distribution of FITC dextrans after DFB exposure or RNAi for TcCHS-2 revealed that PM permeability was increased in both cases. RNAi for TcCHS-2, however, led to a higher permeation of the PM by FITC dextrans than DFB treatment even at high doses. Similar effects were observed when the chitin content was reduced by feeding DFB to adult yellow fever mosquitos, Aedes aegypti. We demonstrate that the presence of chitin is necessary for maintaining the PM's barrier function in insects. It seems that the insecticidal effects of DFB are mediated by the disruption of cuticle synthesis during the metamorphic molt rather than by interfering with larval nutrition. However, as DFB clearly affects PM permeability, it may be suitable to increase the efficiency of pesticides targeting the midgut.     

六类常见泌蜡昆虫的蜡腺和蜡泌物研究进展

泌蜡昆虫体表除了具有和其他昆虫一样的薄蜡层之外,还具有特殊的蜡腺,在生长发育过程中能够分泌蜡质覆盖于体表,从而有利于昆虫防止体内水分过度蒸发、抵御外界多种不利条件、阻碍病原物入侵和躲避天敌寄生或捕食,对昆虫的生存和繁衍具有重要的保护作用。本文概述了蚧虫、蚜虫、蜡蝉、木虱、粉虱和粉蛉等6类常见泌蜡昆虫的蜡腺和蜡泌物的超微形态结构、蜡泌物的主要化学组成及泌蜡的分子机制等方面的研究进展,以期为今后其在昆虫分类学和系统发育学的研究以及在害虫防治中的应用提供参考。