昆虫血细胞功能、形态及细胞免疫反应研究前沿

细胞免疫反应是昆虫先天免疫系统的重要组成部分,与体液免疫反应共同作用以防御外源物。不同类群的昆虫其血细胞种类不同,空间形态及免疫应答功能也各具特征,但在细胞免疫中大都发挥着吞噬、结节与包囊作用。本文根据国内外的研究,对昆虫血细胞的类型、功能、形态、吞噬过程、细胞表面吞噬受体、以蚜虫为代表的不完全变态昆虫免疫学和影响蚜虫免疫系统的共生体等研究动态进行了综述,以期为害虫防治提供思路和防治策略。     

栉孔扇贝血细胞吞噬和包囊化作用实验方法的改进

为了建立一种快速、准确观察血细胞吞噬和包囊化作用的实验方法,通过抽取栉孔扇贝(Chlamysfarreri)血淋巴,与杆菌或茶花花粉作用30 min,制片,吖啶橙染色,用荧光显微镜观察吞噬或包囊化现象。结果表明,在荧光显微镜下可以明显看到扇贝血细胞呈现绿色,杆菌和茶花花粉呈现红色,两者颜色反差大,易于观察和计数。是研究血细胞吞噬和包囊化作用一种效果更好的实验方法。     

类花生酸在昆虫细胞免疫中的作用研究进展

类花生酸包括前列腺素类,凝血恶烷类和白细胞三烯类等,在高等动物中的作用研究较多,后来有许多学者发现类花生酸在昆虫的细胞免疫反应中发挥了重要作用。本文就类花生酸在昆虫清除细菌、真菌、寄生物及病毒等过程中发挥的作用进行了综述,并就深入研究方向进行了展望。     

棉铃虫血细胞对葡聚糖凝胶珠的包囊反应

研究了棉铃虫Helicoverpa armigera(Hübner)血细胞对葡聚糖凝胶珠的包囊反应。结果表明:棉铃虫血细胞对不同类型的葡聚糖凝胶珠具有不同的包囊能力,其中对SephadexA-25葡聚糖凝胶珠具有很强的包囊能力;棉铃虫血细胞启动对SephadexA-25葡聚糖凝胶珠的包囊反应是较迅速的,而包囊的完成大概是在凝胶珠进入棉铃虫体内12h之后;在离体的情况下,棉铃虫血细胞仍然能包囊葡聚糖凝胶珠。     

重金属Cd2+对棕尾别麻蝇血细胞数量、包囊作用和形态结构的影响

为了揭示重金属对昆虫细胞免疫的影响及其机理,本文采用血细胞计数、台盼蓝染色、延展与包囊率测定, 以及显微与超微结构观察等方法,以经重金属Cd²⁺（浓度为150 μg/g）处理的棕尾别麻蝇Boettcherisca peregrina及其对照为研究对象,分别测定了处理组与对照组血细胞的数量与存活率、延展与包囊率,观察其形态结构,并比较了两组间的差异。结果表明： 棕尾别麻蝇初产幼虫经连续喂饲含Cd²⁺的饲料24,48,72和96 h后,与对照相比其血细胞总数和存活率显著下降,而血细胞的延展率和包囊作用的显著下降则分别出现于取食72和48 h之内,此后则下降不显著;由Cd²⁺处理幼虫发育形成蛹的血细胞包囊作用也显著低于对照。形态结构观察结果表明,Cd²⁺处理对幼虫的原血细胞、浆血细胞、颗粒血细胞和类绛色血细胞的显微形态影响不大,但可导致部分浆血细胞不能产生伪足;但均能导致各类血细胞的超微结构发生不同程度的变化,其中主要变化包括：细胞膜受损或破裂,染色质呈现凝聚, 线粒体和内质网等细胞器明显减少,以及胞质内出现空囊泡。结果说明重金属Cd²⁺对棕尾别麻蝇的血细胞可具有毒害作用。     

包囊游仆虫包囊形成和解脱过程中大、小核的研究

包囊游仆虫形成包囊时大核除经历形态大小的变化外,大核DNA含量也低于正常大核水平;细胞脱包囊前,在大核一端或两端发生染色质粗浓集,是大核DNA复制的结果;染色质粒在整个大核内浓集时,大核DNA含量已达到正常大核水平,此时DNA复制结束,细胞脱包囊。小核在游仆虫形成包囊和脱包囊过程中,其形态大小、DNA含量等无明显变化。     

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。     

昆虫造血作用和造血干细胞研究进展

昆虫血细胞（insect hemocyte）在昆虫代谢、 发育变态以及先天免疫等方面承担着重要的作用。昆虫只有先天免疫系统, 血细胞所行使的免疫功能对于昆虫对抗外源病菌尤为重要。本文主要介绍了昆虫血细胞类型、 造血作用、 造血干细胞及造血相关因子的相关研究。通过特殊染色和形态学观察, 果蝇Drosophila血细胞主要由3类细胞组成, 而鳞翅目等大部分昆虫血细胞由5类细胞组成。昆虫血细胞主要存在于循环血液环境及造血器官内, 而在这两个系统中都存在有进行复制的血细胞, 这为研究昆虫造血干细胞特性和其定位提供了一个很好的系统。果蝇血细胞祖细胞来自于胚胎中胚层细胞, 然后再分化为各种血细胞, 这一系列分化过程由造血因子所调控。     

两种金小蜂毒液对菜粉蝶蛹血细胞延展、存活及包囊作用的影响

活体微注射测定结果表明,将0.5毒囊当量（venom reservoir equivalent, VRE）的蝶蛹金小蜂毒液注射于其寄主菜粉蝶蛹体内,注射后4～24 h寄主浆血细胞和颗粒血细胞的延展、存活和对Sephadex A50微珠的包囊能力显著下降;以0.002～0.02 VRE/μL的该蜂毒液处理其离体寄主血细胞均能产生同样的生理效应。该毒液抑制90%菜粉蝶蛹浆血细胞和颗粒血细胞延展的浓度各为0.00076 VRE/μL和0.00804 VRE/μL。可见,蝶蛹金小蜂毒液能显著抑制其寄主血细胞的延展和包囊作用,并导致血细胞的死亡。然而,同样条件下丽蝇蛹集金小蜂毒液对其非自然寄主菜粉蝶蛹的血细胞延展、存活和包囊作用则无任何效应。可见,寄生蜂毒液的生理作用具有明显的寄主特异性。     

The insect cellular immune response

The innate immune system of insects is divided into humoral defenses that include the production of soluble effector molecules and cellular defenses like phagocytosis and encapsulation that are mediated by hemocytes. This review summarizes current understand- ing of the cellular immune response. Insects produce several terminally differentiated types of hemocytes that are distinguished by morphology, molecular and antigenic markers, and function. The differentiated hemocytes that circulate in larval or nymphal stage insects arise from two sources： progenitor cells produced during embryogenesis and mesodermally derived hematopoietic organs. Regulation of hematopoiesis and hemocyte differentiation also involves several different signaling pathways. Phagocytosis and encapsulation require that hemocytes first recognize a given target as foreign followed by activation of downstream signaling and effector responses. A number of humoral and cellular receptors have been identified that recognize different microbes and multicellular parasites. In turn, activation of these receptors stimulates a number of signaling pathways that regulate different hemocyte functions. Recent studies also identify hemocytes as important sources Of a number of humoral effector molecules required for killing different foreign invaders.     

Studies on the in vivo cellular reactions of insects: Clearance of pathogenic and non-pathogenic bacteria in Galleria mellonella larvae

The fate of various doses of bacteria of different pathogenicities injected into Galleria mellonella larvae was monitored over time, from haemocyte and bacterial counts, phagocytic responses and the speed and extent of formation of melanized cell aggregates (nodules). An initial haemocytopenia was recorded in all larvae, probably as a result of wound healing, an increased stickiness of the haemocytes for host tissues and/or cell clump or nodule formation. The results also showed that phagocytosis is the primary cellular defence reaction of this insect for doses of bacteria below ca. 10³ μl^?1 haemolymph while above this level phagocytosis and bacterial clearance are usually rapidly augmented by nodule formation. The extent to which these processes are elicited depends greatly upon the nature of the bacteria injected. In general, the more pathogenic strains produced greater responses than the relatively non-pathogenic forms. This enhanced cellular reactivity was, however, soon overcome by the pathogens which rapidly induced a secondary bacteraemia, a huge drop in haemocyte numbers and death of the larvae. The relative importance of phagocytosis and nodule formation in dealing with various doses of bacteria of differing pathogenicities is discussed.     

白蛾周氏啮小蜂毒液对美国白蛾蛹血细胞免疫的影响

【目的】明确白蛾周氏啮小蜂Chouioia cunea毒液对其寄主美国白蛾Hyphantria cunea蛹细胞免疫的影响。【方法】采用Na_2-EDTA分离美国白蛾蛹颗粒细胞,尼龙毛法分离浆血细胞,再利用细胞离体培养法,测评了白蛾周氏啮小蜂毒液对寄主美国白蛾两种血细胞包囊作用和吞噬作用的影响。【结果】美国白蛾颗粒细胞的包囊指数强于浆血细胞。白蛾周氏啮小蜂毒液对颗粒细胞和浆血细胞的包囊指数均有明显的抑制作用,毒液浓度越大,抑制作用越强,两种血细胞的包囊作用均呈先增长后降低的趋势。在所有浓度毒液处理下,颗粒细胞的包囊指数在12 h时最强。未经小蜂毒液处理的浆血细胞包囊指数在15 h时达到最强,但经浓度为0.01~0.03 VRE/μL的毒液处理后浆血细胞的包囊指数在12 h时达到最强,而经浓度为0.04~0.10 VRE/μL的毒液处理后包囊指数在9 h时最强。白蛾周氏啮小蜂毒液对美国白蛾蛹颗粒细胞的吞噬作用强于浆血细胞。毒液对两种血细胞的吞噬能力均有明显的抑制作用,但毒液处理对浆血细胞的吞噬作用影响较小。【结论】白蛾周氏啮小蜂毒液可以抑制美国白蛾蛹颗粒细胞和浆血细胞的包囊作用和吞噬作用,且随着毒液浓度的增加,两种血细胞的免疫作用显著下降。     

Aspergillosis in German cockroach Blattella germanica (L.) (Blattoidea: Blattellidae)

Natural infection of Aspergillus flavus was observed in adults of Blattella germanica. Though the adult insects exhibited no external symptoms, they became hypoactive and later died. The dead and experimentally infected insects repeatedly yielded Aspergillus flavus in culture on Czapek's medium. Direct microscopic observation of the tissues of infected insects revealed fungal material. The blood films stained with Giemsa stain showed granulocytes (GRs) engulfing fungal hyphae. A remarkable increase in GR and plasmatocyte (PL) counts occurred in differential haemocyte counts (DHCs) of the infected insects. Two main types of immunological responses of the insect noticed were phagocytosis and encapsulation. DHC showed maximum involvement of GRs and PLs in immune mechanism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphofunctional characterization of hemocytes in black soldier fly larvae

In insects, the cell-mediated immune response involves an active role of hemocytes in phagocytosis, nodulation, and encapsulation. Although these processes have been well documented in multiple species belonging to different insect orders, information concerning the immune response, particularly the hemocyte types and their specific function in the black soldier fly Hermetia illucens, is still limited. This is a serious gap in knowledge given the high economic relevance of H. illucens larvae in waste management strategies and considering that the saprophagous feeding habits of this dipteran species have likely shaped its immune system to efficiently respond to infections. The present study represents the first detailed characterization of black soldier fly hemocytes and provides new insights into the cell-mediated immune response of this insect. In particular, in addition to prohemocytes, we identified five hemocyte types that mount the immune response in the larva, and analyzed their behavior, role, and morphofunctional changes in response to bacterial infection and injection of chromatographic beads. Our results demonstrate that the circulating phagocytes in black soldier fly larvae are plasmatocytes. These cells also take part in nodulation and encapsulation with granulocytes and lamellocyte-like cells, developing a starting core for nodule/capsule formation to remove/encapsulate large bacterial aggregates/pathogens from the hemolymph, respectively. These processes are supported by the release of melanin precursors from crystal cells and likely by mobilizing nutrient reserves in newly circulating adipohemocytes, which could thus trophically support other hemocytes during the immune response. Finally, the regulation of the cell-mediated immune response by eicosanoids was investigated.     

Humoral and cellular immune memory to four COVID-19 vaccines

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Maintenance of redox balance by antioxidants in hemolymph of the greater wax moth Galleria mellonella larvae during encapsulation response

The lipid peroxidation process in hemocytes, activities of phenoloxidase and key enzymatic antioxidants (superoxide dismutase, glutathione‐S‐transferase, catalase) and nonenzymatic antioxidants (thiols, ascorbate) in hemolymph of the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) were studied during the encapsulation process of nylon implants. It has been established that as soon as 15 min after piercing a cuticle with the implant, a capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. During the first hours after incorporating the implant, an increase in phenoloxidase activity and lipid peroxidation in the insect hemocytes has been revealed. Adhesion and degranulation on the surface of foreign object lead to the depletion of total hemocytes count (THC). Our results indicated that thiols and ascorbate molecules take part in the immediate antioxidant response, during later stages of encapsulation process hemolymph glutathione‐S‐transferase detoxifies and protects insect organism thereby restoring the internal redox balance. We suggest that nonenzymatic and enzymatic antioxidants of hemolymph plasma play a key role in the maintenance of redox balance during encapsulation of foreign targets.     

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.     

结核分枝杆菌海藻糖磷酸磷酸酶诱导小鼠体液和细胞免疫应答

目的 研究结核分枝杆菌(M．tuberculosis)海藻糖磷酸磷酸酶(TPP)诱导小鼠体液和细胞免疫。方法 差速离心分离结核分枝杆菌H37Rv和卡介苗(BCG)的各细胞组分,通过Western杂交检测抗原TPP在结核分枝杆菌H37Rv和BCG中的亚细胞定位情况。分别用5×10~6CFU的BCG和50μg的TPP蛋白免疫C57BL/6小鼠,检测小鼠血清中抗TPP的IgG1和IgG2a抗体效价。取免疫小鼠的脾细胞,体外抗原刺激,用酶联免疫斑点试验(ELISPOT)检测γ干扰素(IFN-γ)分泌细胞。结果 TPP亚细胞定位于结核分枝杆菌H37Rv和BCG的胞壁和细胞膜组分。TPP蛋白免疫后小鼠产生的TPP特异性IgG1和IgG2a抗体效价明显高于BCG免疫小鼠,并且IgG2a的抗体效价高于IgG1。体外抗原刺激TPP蛋白和BCG免疫小鼠的脾细胞,都能诱导较高的IFN-γ分泌。结论 结核分枝杆菌细胞壁蛋白TPP能诱导小鼠Ⅰ型辅助性T细胞介导的免疫反应,可作为抗结核疫苗的候选抗原。