不同培养基上繁殖的昆虫病原线虫格氏线虫表皮蛋白的差异

表皮蛋白(surface coat proteins, SCPs)是格氏线虫Steinernema glaseri克服寄主免疫系统的关键因素, 能够抑制昆虫免疫反应, 促进线虫的侵染。为了进一步研究表皮蛋白抑制昆虫免疫的作用机理和线虫与寄主间的相互关系, 我们比较分析了不同培养基繁殖的格氏线虫表皮蛋白的差异。我们用人工培养基和大蜡螟Galleria mellonella末龄（5龄）幼虫分别培养得到格氏线虫侵染期幼虫, 利用酒精提取表皮蛋白。SDS-PAGE和非变性PAGE分析显示, 大蜡螟来源的格氏线虫的表皮蛋白具有更多的蛋白条带。体外和体内的裂解血细胞实验表明, 大蜡螟来源的格氏线虫的表皮蛋白表现出强烈的裂解血细胞活性, 而人工培养基来源的格氏线虫的表皮蛋白活力不明显; 且具有裂解血细胞活性的表皮蛋白为诱导表达型蛋白。不同培养基来源的格氏线虫的表皮蛋白组成的差异和活性的不同, 表明格氏线虫表皮蛋白的产生受线虫培养条件影响较大。     

小卷蛾斯氏线虫侵染对草地贪夜蛾幼虫天然免疫反应的影响

【目的】探讨昆虫病原线虫小卷蛾斯氏线虫Steinernema carpocapsae All侵染对草地贪夜蛾Spodoptera frugiperda幼虫天然免疫反应的影响。【方法】借助倒置显微镜观察和鉴定草地贪夜蛾幼虫的血细胞类型,并对小卷蛾斯氏线虫侵染后不同时间的草地贪夜蛾幼虫血细胞总数目进行统计;通过倒置显微镜观察草地贪夜蛾幼虫对侵入的小卷蛾斯氏线虫的包囊反应;利用倒置荧光显微镜观察小卷蛾斯氏线虫侵染后的草地贪夜蛾幼虫血细胞对金黄色葡萄球菌Staphylococcus aureus的吞噬活性;检测小卷蛾斯氏线虫侵染后的草地贪夜蛾幼虫血淋巴中酚氧化酶(phenoloxidase, PO)活性、体内抗菌肽基因相对表达水平以及血浆的抗菌活性。【结果】从草地贪夜蛾幼虫体内共发现5种不同类型的血细胞,分别为原血细胞、粒细胞、类绛色细胞、珠血细胞和浆血细胞。注射1 μL侵染期(infective juveniles, IJs)小卷蛾斯氏线虫(3 IJs/μL)后9和12 h,草地贪夜蛾幼虫的血细胞总数目显著增多。草地贪夜蛾幼虫的血细胞不能包囊活的以及冷处死的小卷蛾斯氏线虫,但可以包囊热处死的线虫。活的小卷蛾斯氏线虫会显著抑制草地贪夜蛾幼虫血细胞对金黄色葡萄球菌的吞噬活性,但冷处死和热处死的线虫不能。注射1 μL(3 IJs/μL)小卷蛾斯氏线虫后,草地贪夜蛾幼虫血淋巴PO活性总体呈“下降 升高 下降”变化趋势;体内抗菌肽基因Attacin-A2, Attacin-B1, Cecropin-B3, Cecropin-D, Gallerimycin, Gloverin-3以及Lebocin-2的表达水平在线虫侵染后12 h时显著上调,24 h时恢复到对照水平或低于对照水平;血淋巴抗菌活性水平在小卷蛾斯氏线虫侵染后12 h时显著升高,24 h时与对照无显著差异。【结论】小卷蛾斯氏线虫在侵入早期会抑制草地贪夜蛾幼虫的天然免疫反应来建立感染;随后草地贪夜蛾的免疫系统会被激活试图抵御小卷蛾斯氏线虫的侵染;后期随着线虫的成功定殖,草地贪夜蛾的免疫系统最终被抑制或破坏。本研究所得结果为进一步揭示线虫 草地贪夜蛾的免疫互作机理奠定了基础,也为改善昆虫病原线虫对草地贪夜蛾的防治效果提供了理论依据。     

嗜线虫致病杆菌血腔毒素Tp40对大蜡螟幼虫体内酶活和中肠组织的影响

嗜线虫致病杆菌Xenorhabdus nematophila在侵入到寄主昆虫血腔后能够成功地逃避或抑制寄主昆虫的免疫反应并快速杀死昆虫。为深入了解嗜线虫致病杆菌的杀虫机理,明确关键的致病因子,作者应用盐析和制备型非变性凝胶电泳等方法,从嗜线虫致病杆菌HB310菌株的细胞内分离纯化了一种新的杀虫蛋白——Tp40,该蛋白对大蜡螟Galleria mellonella具有高血腔注射活性,对大蜡螟5龄幼虫的LD₅₀为68.54 ng/头。本文检测了该毒素对大蜡螟幼虫的致病特性,注射Tp40毒素后,大蜡螟幼虫表现出兴奋和痉挛等症状,当以不低于（70±0.02）ng/头的剂量注射Tp40,大蜡螟幼虫均在20 min内死亡,但试虫的体色 、血淋巴的颜色以及血细胞的形态没有发生明显的变化。对大蜡螟体内酶活性的测定结果显示,在注射LD₅₀剂量的Tp40蛋白后,试虫体内羧酸酯酶和乙酰胆碱酯酶活力都明显的高于对照（P<0.05）,而酚氧化酶活力显著低于对照（P<0.05）。对大蜡螟幼虫中肠的组织病理学研究显示：这种42 kDa蛋白能够破坏试虫的中肠组织,导致其肠壁细胞出现排列紊乱、脱落和围食膜消失。据此推测,Tp40与嗜线虫致病杆菌对寄主昆虫的免疫抑制有关,寄主中肠组织可能是其作用靶标之一。     

斯氏线虫对皮暗斑螟控制能力的研究

本文报导了应用科学斯氏线虫属（Steinernema spp.）8个品系室内外控制木麻黄皮暗斑螟的试验结果。通过对8个线虫品系在不同温度下皮暗斑螟的侵染力测定,筛选出Steinernema feltima Agriotos及Steinernema feltima,Beijing为最佳品系,能寄生低、中、高龄幼虫及蛹,3-4天幼虫死亡率达100%;温度20～30℃不影响线虫对寄生的致死效果,只是致死速度不同,在25℃时侵染力最强;以100条侵染期线虫/害虫为最适剂量。林间以200条线虫/ml的Steinernema feltiae,Agriotos及Steinerneama feltiae,Beijing两品系线虫液喷洒虫巢,皮暗斑螟幼虫的平均致死率分别为72.0%、69.9%。     

昆虫病原线虫感染寄主行为研究进展

昆虫病原线虫斯氏属Steinernema和异小杆属Heterorhabditids线虫是新型的生物杀虫剂。其感染期幼虫是惟一能够侵染寄主昆虫的虫态。这类线虫感染寄主的行为分为寻找寄主、识别寄主和侵染寄主。文章综述昆虫病原线虫感染寄主昆虫的行为以及在感染寄主过程中的影响因素。     

芜菁夜蛾线虫和嗜菌异小杆线虫寄生特性的比较

对2种昆虫病原线虫(芫菁夜蛾线虫Steinernema feltiae Filipjev和嗜菌异小杆线虫Heterorhaditis bacteriphora Poinar)的生物学特性进行研究。结果表明:在25℃下,在大蜡螟Galleria mellonella L.幼虫体内完成1个侵染周期芫菁夜蛾线虫Otio品系需220～230h,嗜菌异小杆线虫15-2品系需200～210h。15-2品系比Otio品系具有较强的繁殖力。2种线虫侵染大蜡螟幼虫的温度范围均为20～30℃,在此温度范围内,Otio品系的侵染力和运动能力均强于15-2品系。当土壤含水量(w/w)为5%～15%,线虫的侵染活性最高,随湿度降低线虫的侵染活性明显降低。     

昆虫病原线虫斯氏线虫和异小杆线虫对长角血蜱雌蜱的致病力

用昆虫病原线虫小卷蛾斯氏线虫（Sc BJ）、夜蛾斯氏线虫（Sf Otio）、拟双角斯氏线虫（Sc D43）、格氏斯氏线虫（Sg NC32）和嗜菌异小杆线虫（Hb E-6-7）对长角血蜱雌蜱进行感染试验,所用线虫剂量为4 000 Ijs/dish。结果表明,5种线虫均对长角血蜱雌蜱有致死效应。Hb E-6-7和Sc BJ两种线虫对雌蜱各发育期致病力最强,导致雌蜱的累积死亡率和半致死时间分别为饥饿雌蜱82.5%,9.0天和75.0%,8.8天;吸血雌蜱90.0%,8.0天和82.5%,8.0天;饱血雌蜱93.3%,7.3天和86.7%,7.3天。Sc D43对饱血雌蜱有较高的致死效应,为80.0%,但半致死时间较长,为11.7天。Sf Otio和Sg NC32对长角血蜱雌蜱的致死效应较低。饱血雌蜱较饥饿雌蜱和吸血雌蜱更易被线虫感染。     

昆虫病原线虫对黄地老虎致病力的研究

为了探索应用昆虫病原线虫防治黄地老虎Agrotis segetum的可行性,室内生测比较了9个昆虫病原线虫品系对黄地老虎4龄幼虫的致病力。生测结果表明,9个不同昆虫病原线虫品系对黄地老虎4龄幼虫的致病力差别很大,其中格氏线虫Steinernema glaseri ib(Sgib)是对黄地老虎幼虫致病力最强的昆虫病原线虫品系,侵染24、36和72 h黄地老虎幼虫的校正死亡率均高于其他线虫品系。此外,还测定了不同温度、土壤湿度以及线虫剂量对格氏线虫Sgib致病力的影响,结果表明,在21℃-27℃、20%土壤湿度以及侵染期线虫与黄地老虎幼虫数量比不少于10∶1的条件下,格氏线虫Sgib对黄地老虎幼虫的致病效果最好。田间小区实验表明,格氏线虫Sgib对黄地老虎幼虫的防效达到96.67%。     

4种昆虫病原线虫对草地贪夜蛾的致死作用

【目的】探讨昆虫病原线虫对草地贪夜蛾幼虫的侵染效果。【方法】在室内条件下采用生测试验法,测定了小卷蛾斯氏线虫AⅡ、长尾斯氏线虫X-7、夜蛾斯氏线虫SN和嗜菌异小杆线虫H06等4种昆虫病原线虫对草地贪夜蛾2龄和5龄幼虫的致死作用。【结果】小卷蛾斯氏线虫AⅡ与草地贪夜蛾数量比为50∶1时,36 h后草地贪夜蛾2龄和5龄幼虫的死亡率分别为92%和100%。长尾斯氏线虫X-7与草地贪夜蛾数量比为50∶1时,36 h后草地贪夜蛾2龄幼虫的死亡率为80%;在数量比30∶1时,36 h后草地贪夜蛾5龄幼虫死亡率为100%。夜蛾斯氏线虫SN与草地贪夜蛾数量比为400∶1时,36 h后草地贪夜蛾2龄和5龄幼虫的死亡率分别为88%和85%。嗜菌异小杆线虫H06与草地贪夜蛾在400∶1时,36 h后草地贪夜蛾2龄和5龄幼虫的死亡率分别为32%和67.5%。【结论】小卷蛾斯氏线虫AⅡ具有较好的草地贪夜蛾生物防治潜质,其次是长尾斯氏线虫X-7。     

拟双角斯氏线虫生物学特性的研究

本文对拟双角斯氏线虫Ｄ－４－３品系的生物学特性进行了研究。     

Rapid age-related changes in infection behavior of entomopathogenic nematodes

Nonfeeding infective juvenile (IJ) entomopathogenic nematodes (EPNs) are used as biological agents to control soil-dwelling insects, but poor storage stability remains an obstacle to their widespread acceptance by distributors and growers as well as a frustration to researchers. Age is one factor contributing to variability in EPN efficacy. We hypothesized that age effects on the infectiousness of IJs would be evident within the length of time necessary for IJs to infect a host. The penetration behavior of "young" (<1-wk-old) and "old" (2- to 4-wk-old) Heterorhabditis bacteriophora (GPS 11 strain), Steinernema carpocapsae (All strain), and Steinernema feltiae (UK strain) IJs was evaluated during 5 "exposure periods" to the larvae of the wax moth, Galleria mellonella. Individual larvae were exposed to nematode-infested soil for exposure periods of 4, 8, 16, 32, and 64 hr. Cadavers were dissected after 72 hr, and the IJs that penetrated the larvae were counted. Larval mortality did not differ significantly between 72- and 144-hr "observation periods," or points at which larval mortality was noted, for any age class or species. However, age and species effects were noted in G. mellonella mortality and nematode penetration during shorter time periods. Initial mortality caused by S. carpocapsae and H. bacteriophora IJs declined with nematode age but increased with S. feltiae IJ age. Young S. carpocapsae IJs penetrated G. mellonella larvae at higher rates than old members of the species (27-45% vs. 1-4%). Conversely, old S. feltiae IJs had higher penetration rates than young IJs (approximately 8 to 57% vs. 4 to approximately 31%), whereas H. bacteriophora IJs had very low penetration rates regardless of age (3-5.6%). Our results show that the effect of age on IJ infectiousness can be detected in IJs aged only 2 wk by a 4-hr exposure period to G. mellonella. These results have important implications for storage and application of EPNs and suggest the possibility of shortening the time required to detect nematodes in the soil.     

Comparison of Bioassays to Measure Virulence of Different Entomopathogenic Nematodes

Five bioassays were compared for their usefulness to determine the virulence of four nematode strains. The objective of this study was to develop standard assays for particular nematode species. In all assays, the nematodes Steinernema feltiae (strain UK), S. riobravis, S. scapterisci Argentina and Heterorhabditis bacteriophora HP88 were exposed to Galleria mellonella larvae. All bioassays except the sand column assay were conducted in multi-well plastic dishes. In the penetration rate assay, the number of individual nematodes invading the insect was determined after a 48-h exposure to 200 infective juveniles (IJs). In the one-on-one assay, each larva was exposed to an individual nematode for 72 h before insect mortality was recorded. In the exposure time assay, insect mortality was recorded after exposure to 200 IJs for variable time periods. The dose-response assay involved exposing larvae to different nematode concentrations over the range 1-200 IJs/insect and recording mortality every 24 h for a 96-h period. In the sand columns assay, insects were placed in the bottom of a plastic cylinder filled with sand. Nematodes were applied on top of the sand and insect mortality was determined after IJs had migrated through the cylinder. The highest mortality level in the sand column assay was obtained with IJs of S. feltiae followed by H. bacteriophora; treatments with S. riobravis and S. scapterisci produced low levels of insect mortality. In the other four assays, S riobravis was the most virulent, followed by S. feltiae, H. bacteriophora and S. scapterisci. In the exposure time assay, rapid mortality was achieved when the insects were exposed to S. feltiae and S. riobravis. For these nematode species, a gradual increase in the number of individuals which penetrated into cadavers was recorded. Conversely, the number of nematodes in the cadavers of insects infected by H. bacteriophora and S. scapterisci remained low during the entire exposure period. In this assay, exposing the insects to these nematodes resulted in a gradual increase in mortality. In the dose-response assay, complete separation among nematode species was obtained only after 48 h of incubation at a concentration of 15 IJs/insect. LD and LD values were calculated from 50 90 dose-response assay data. However, these values did not indicate differences among the different nematode species. The present study demonstrated the variation in entomopathogenic nematode performance in different bioassays and supports the notion that one common bioassay cannot be used as a universal measure of virulence for all species and strains because nematodes differ in their behavior. Furthermore, particular assays should be used for different purposes. To select a specific population for use against a particular insect, assays that are more laborious but which simulate natural environmental conditions (e.g. the sand column assay) or invasion by the nematode (e.g. the penetration rate assay) should be considered. In cases where commercial production batches of the same nematode strains are compared, simple and fast assays are needed (e.g. the one-on-one and exposure time assays). Further studies are needed to determine the relationships between data obtained in each assay and nematode efficacy in the field.     

Food preference of Sancassania polyphyllae (Acari: Acaridae): living entomopathogenic nematodes or insect tissues?

Sancassania polyphyllae (Acari: Acaridae) adult female mites will feed on insect cadavers and infective juveniles (IJs) of entomopathogenic nematodes. Our objective was to determine whether S. polyphyllae has a food preference when offered a choice between tissues of Polyphylla fullo (Coleoptera: Scarabaeidae) or Galleria mellonella (Lepidoptera: Pyralidae), or IJs of Steinernema feltiae (Rhabditida: Steinernematidae) or IJs of Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae). When offered a choice between no food and one of the different food sources, P. fullo, G. mellonella or S. feltiae IJs, S. polyphyllae had a significant preference for food sources compared to no food. When it was offered either no food or H. bacteriophora, there was no significant difference in the mite distribution. When offered two different food choices, P. fullo or G. mellonella, P. fullo or S. feltiae, and P. fullo or H. bacteriophora, the mite showed significant preferences for P. fullo larvae. In S. feltiae vs. G. mellonella and S. feltiae vs. H. bacteriophora experiments, S. polyphyllae showed significant a preference for S. feltiae. In three-choice experiments, S. polyphyllae had a preference for P. fullo, followed by S. feltiae, G. mellonella and H. bacteriophora, respectively. Our data confirm, in part, our hypothesis that when offered different food choices, this mite species prefers tissues of its phoretic host, P. fullo over lepidopteran host tissues or living IJs. Based on these laboratory data, H. bacteriophora should be used as a biological control agent against P. fullo over a Steinernema species.     

斜纹夜蛾抗菌肽基因Moricin和Cecropin时空表达模式及微生物敏感性的分析

抗菌肽是一类具有广谱抗菌活性的小分子多肽, 包括抗细菌、 病毒和真菌。本研究以斜纹夜蛾Spodoptera litura (Fabricius)为研究对象, 利用Real-time PCR和半定量RT-PCR检测了其体内两个重要抗菌肽基因Cecropin和Moricin的时空表达模式及其对微生物的敏感性。Real-time PCR检测结果表明, 抗菌肽基因Cecropin和Moricin在斜纹夜蛾各个龄期（1, 2, 3, 4, 5和6龄幼虫、 蛹、 成虫）都有表达, 随着龄期的增长, 表达量逐渐增加, 6龄幼虫中表达量达到最高。 半定量RT-PCR检测表明, 抗菌肽基因Cecropin和Moricin在7个不同组织（气管、 卵巢、 马氏管、 体壁、 中肠、 脂肪体和血细胞）中都有表达, Cecropin在脂肪体中表达量最高, 而Moricin在血细胞中表达量相对较高; RT-PCR检测表明, Cecropin和Moricin的表达在大肠杆菌Escherichia coli诱导后迅速上升, 其中诱导后8 h达到高峰期, 一直持续到48 h。不同病原微生物（大肠杆菌E. coli、 金黄色葡萄球菌Staphyloccocus aureus和白僵菌Beauveria bassiana）分别诱导后, Real-time PCR 检测表明, 斜纹夜蛾抗菌肽基因Cecropin和Moricin对大肠杆菌的刺激最为敏感。本研究结果为进一步研究斜纹夜蛾抗菌肽基因Cecropin和Moricin的功能奠定基础。     

Influence of Insect Larvae and Plant Roots on the Host-finding Behaviour of Heterorhabditis megidis

We studied the host-finding and dispersion behaviour of Heterorhabditis megidis (strain NLHE 87.3) in the presence of Galleria mellonella or Otiorhynchus sulcatus larvae and strawberry roots. In large Petri dishes (19 cm diameter) filled with moist sand (8% w/w), and incubated at 15°C over 24 h, infective juveniles (IJs) responded positively to the presence of G. mellonella , to roots of a single strawberry plant and to O. sulcatus larvae in direct contact with roots of a single strawberry plant. A neutral or negative response was observed when IJs were presented with only O. sulcatus larvae or a combination of several strawberry plants with O. sulcatus larvae, either in contact or not in contact with the roots. IJs responded strongly to the combination of plant roots and feeding larvae indicating that the tritrophic interaction formed by IJs - O. sulcatus larvae - strawberry plants may be an infochemical-mediated interaction.     

Effects of Density, Age and Host Cues on the Dispersal of Heterorhabditis megidis

Agar plate assays were used to assess the effect of density, incubation time and age of nematodes and the presence of insect hosts on the dispersal of infective juveniles (IJs) of Heterorhabditis megidis (strain NLH-E87.3). IJs dispersed faster and further at high densities than at low densities. Dispersal was also influenced by the age of the IJs. Individuals stored for a period of 1.5 and 4.5 weeks showed to be more active than those stored for 2.5 and 3.5 weeks. The presence of a host insect enhanced the dispersion of nematodes. The increasing in the incubation period showed that IJs responded positively to host cues from Galleria mellonella but poorly to cues from Otiorhynchus sulcatus larvae.     

Factors influencing the infectivity of a Canadian isolate of Steinernema kraussei (Nematoda: Steinernematidae) at low temperature

The effectiveness of Canadian isolate 76 of Steinernema kraussei, at 10 degrees C, in penetrating Galleria mellonella larvae (percentage parasitism and number of IJs developed to adult nematodes) was measured at different host densities (differing number of larvae and size of experimental arena) and for different durations of exposure. The greater the size of the inoculum of infective juvenile nematodes per unit area and the longer the duration of exposure, the greater the number of larvae that were killed and the larger the number of mature nematodes in the larval host. The infection rate (alpha) and the adjusted infection rate (beta) were determined using the modified Anderson model. This model successfully described the behavior of the S. kraussei-G. mellonella interaction.     

Skin penetration by ensheathed third-stage infective larvae of Necator americanus, and the host's immune response to larval antigens.

In vitro experiments were conducted to assess skin penetration by ensheathed third-stage infective larvae (L3) of Necator americanus. The fact that only a small proportion of larval sheaths was recoverable from the outer skin surface suggested that some larvae penetrate mouse skin without undergoing exsheathment. Penetration by ensheathed larvae was confirmed visually using a novel fluorescein isothiocyanate (FITC)-labelling technique in which viable ensheathed larvae were fluoresceinated, applied onto intact mouse skin, and their progress monitored in frozen skin sections. This direct observation that the L2-derived sheath can present antigens to the host's immune system was also monitored by immunoassay to provide confirmatory information regarding skin penetration by ensheathed larvae. Sera from humans infected with Necator americanus were shown to react in ELISA against antigens stripped by detergent (cetyltrimethylammonium bromide) from the sheath surface, and with antigens contained in L3-exsheathing fluid. These data suggest that the host's immune response, as a result of antigenic stimulation by the cast sheath and exsheathing fluid, could in fact be diverted away from the potentially vulnerable L3 stage.     

Entomopathogenic Nematode Infectivity Assay: Comparison of Penetration Rate into Different Hosts

Penetration rate (the percentage of the initial infective juvenile inoculum that invades an insect host) was tested as an indicator of entomopathogenic nematode infectivity. Several host-parasite-substrate combinations were evaluated for penetration rate. Four steinernematids, Steinernema carpocapsae, S. glaseri, S. feltiae, S. riobravis and two strains of Heterorhabditis bacteriophora were tested in a contact bioassay against the wax moth, Galleria mellonella, the yellow meal worm, Tenebrio molitor, the beet armyworm, Spodoptera exigua, the black cutworm, Agrotis ipsilon, and the European corn borer, Ostrinia nubilalis. The insect larvae were confined individually in sand and filter paper arenas and exposed to 200 infective juveniles. After incubation, dead insects were dissected in order to count the nematodes penetrated. The data were analyzed for the effects of nematode strain and substrate on penetration rate. The bioassay substrate had a variable effect depending on the insect species. The nematode effect was highly significant for all insects tested. The penetration rate therefore allowed comparisons among nematode strains invading a host. Nematode ranking for infectivity differed according to the insect tested.     

Different defense strategies of Dendrolimus pini, Galleria mellonella, and Calliphora vicina against fungal infection

The resistance of Galleria mellonella, Dendrolimus pini, and Calliphora vicina larvae against infection by the enthomopathogen Conidiobolus coronatus was shown to vary among the studied species. Exposure of both G. mellonella and D. pini larvae to the fungus resulted in rapid insect death, while all the C. vicina larvae remained unharmed. Microscopic studies revealed diverse responses of the three species to the fungal pathogen: (1) the body cavities of D. pini larvae were completely overgrown by fungal hyphae, with no signs of hemocyte response, (2) infected G. mellonella larvae formed melanotic capsules surrounding the fungal pathogen, and (3) the conidia of C. coronatus did not germinate on the cuticle of C. vicina larvae. The in vitro study on the degradation of the insect cuticle by proteases secreted by C. coronatus revealed that the G. mellonella cuticle degraded at the highest rate. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. Of all the tested species, only plasmatocytes exhibited phagocytic potential. Exposure to the fungal pathogen resulted in elevated phagocytic activity, found to be the highest in the infected G. mellonella. The incubation of insect hemolymph with fungal conidia and hyphae revealed diverse reactions of hemocytes of the studied insect species. The encapsulation potential of D. pini hemocytes was low. Hemocytes of G. mellonella showed a high ability to attach and encapsulate fungal structures. Incubation of C. vicina hemolymph with C. coronatus did not result in any hemocytic response. Phenoloxidase (PO) activity was found to be highest in D. pini hemolymph, moderate in G. mellonella, and lowest in the hemolymph of C. vicina. Fungal infection resulted in a significant decrease of PO activity in G. mellonela larvae, while that in the larvae of D. pini remained unchanged. PO activity in C. vicina exposed to fungus slightly increased. The lysozyme-like activity increased in the plasma of all three insect species after contact with the fungal pathogen. Anti E. coli activity was detected neither in control nor in infected D. pini larvae. No detectable anti E. coli activity was found in the control larvae of G. mellonella; however, its exposure to C. coronatus resulted in an increase in the activity to detectable level. In the case of C. vicina exposure to the fungus, the anti E. coli activity was significantly higher than in control larvae. The defense mechanisms of D. pini (species of economic importance in Europe) are presented for the first time.