小菜蛾幼虫血淋巴中β-1,3-葡聚糖结合蛋白的分离及其对根虫瘟霉侵染的免疫活性

用亲和沉淀法从小菜蛾(Plutella xylostella)幼虫血淋巴中分离获得2种β-1,3-葡聚糖结合蛋白,分子质量分别为75.9 ku和83.2 ku,主要存在于幼虫血浆中,但血细胞中未检出.研究表明,β-1,3-葡聚糖结合蛋白能特异性地识别β-1,3-葡聚糖,并显著激活幼虫血淋巴中的酚氧化酶原（ProPO）,与昆布多糖共存时所激活的酚氧化酶（PO）活性显著高于两者单独存在时的PO活性.与4株根虫瘟霉(Zoophthora radicans)菌丝裂解液共存时,β-1,3-葡聚糖结合蛋白能激活幼虫血淋巴中的ProPO,使PO活力显著高于该菌原生质体裂解液所激活的PO活性.显然,β-1,3-葡聚糖结合蛋白只有特异性地识别根虫瘟霉细胞壁中的β-1,3-葡聚糖后才能激活幼虫血淋巴中的ProPO,表明虫霉原生质体可逃避寄主免疫反应.此外,β-1,3-葡聚糖结合蛋白对不同菌株所激活的PO活性存在差异,各菌株所激活的PO活性由高到低依次为：ARSEF1342＞ARSEF2699＞F99101＞ARSEF1100,这与各菌株对小菜蛾的毒力强弱相一致,即菌株逃避寄主免疫识别的能力与其毒力相关.     

不同寄主来源的根虫瘟霉菌株对小菜蛾幼虫的毒力比较

在不同寄主来源的4株根虫瘟霉Zoophthora radicans对小菜蛾 Plutella xylostella 2龄幼虫的生物测定中,发现源于小菜蛾的菌株ARSEF1100毒力最强,在0.53～319.32/mm²的孢子剂量下,接种后第8 天累计死亡率为2.38%～97.44%,虫尸全部表现典型的虫瘟霉症状;源于叶蝉的ARSEF2699和F99101菌株的同日累计死亡率分别为2.38%～50.00% (剂量为1.56～314.84/mm²孢子)和2.38%～57.89% (剂量为1.84～484.08/mm²的孢子);而源于菜粉蝶的ARSEF1342菌株在3.54～633.0/mm²的孢子剂量下只引发6.52%～13.63%的累计死亡率,后3个菌株致死的小菜蛾幼虫仅部分表现典型症状。所获数据经时间剂量-死亡率模型模拟分析,剂量效应参数依次为ARSEF1100 (1.89) > F99101 (1.48) > ARSEF2699 (1.23) > ARSEF1342 (0.37),相互间差异均达极显著水平。接种后4～8 天内,ARSEF1100的LD₅₀值分别为231.68、113.08、71.41、40.87和35.30/mm²的孢子,其毒力远高于其余3个菌株;ARSEF2699的相应LD₅₀值为1344.43、922.39、555.58、410.06和397.07/mm²的孢子;F99101的LD₅₀值为666.86、451.64、413.82、350.65和332.57/mm²的孢子,而ARSEF1342的毒力太弱难以估计。这些结果表明,ARSEF1100菌株最有希望用于小菜蛾的微生物防治。     

绿僵菌侵染对椰心叶甲酚氧化酶活性的影响

对椰心叶甲Brontispa longissima(Gestro)成虫血淋巴中酚氧化酶的特性进行分析,并研究绿僵菌(Metarhizium anisopliae)侵染对血浆甲酚氧化酶活性的影响。结果显示,椰心叶甲成虫的血浆及血细胞裂解液中均检测到酚氧化酶活性,且昆布多糖及胰蛋白酶可显著提高其活性。绿僵菌MA-4侵染组在侵染后第1至第5d的血浆酚氧化酶活性高于未侵染组(P<0.05),但是椰心叶甲成虫体内注射10μg昆布多糖后,侵染组的酚氧化酶活性显著低于未侵染组(P<0.05),表明绿僵菌一方面对可激活椰心叶甲的酚氧化酶原激活系统,另一方面又可抑制昆布多糖对椰心叶甲酚氧化酶原激活系统的诱导作用。     

棉铃虫血淋巴酚氧化酶活性的微量测定

昆虫血淋巴黑化的形成由激活酚氧化酶原的级联系统所引发 ,酚氧化酶在昆虫体液免疫中起着重要作用。用抗凝剂从棉铃虫血淋巴中分离获得了血浆及完整的血细胞 ,以L DOPA为底物 ,牛胰蛋白酶为激活剂 ,测定了血浆及血细胞裂解液中酚氧化酶及酚氧化酶原的活性。结果表明 ,血浆及血细胞中两者都有一定量的分布。这一昆虫血淋巴酚氧化酶的微量测定方法 ,所需样品量少 ,耗时短 ,简便易行。     

亚洲玉米螟血淋巴中酚氧化酶的研究

酚氧化酶(Phenoloxidase,简称PO)大量存在于亚洲玉米螟(Ostrinia furnacalis)幼虫的血浆中,血细胞中甚少.该酶的活力与幼虫的发育期相关,在蜕皮和化蛹前后活力达高峰.PO可以粘附在酵母菌细胞表面,但未发现具有调理作用.以Sephadex G-200层析,对PO进行了部分纯化,比活力可提高56倍.试虫感染酵母菌后,活体内血浆PO活力即刻下降;体外证明,感染血浆可抑制正常血浆PO活力,以胰蛋白酶和昆布多糖测试以及行耐温实验,表明该抑制物为蛋白质性质.对PO的防卫作用进行了简要的讨论.     

几种化学因子对三种赤眼蜂离体酚氧化酶活性的影响

为比较不同赤眼蜂酚氧化酶（PO）活性的大小, 明确赤眼蜂种间免疫防御能力强弱, 本研究通过研磨、离心提取松毛虫赤眼蜂Trichogramma dendrolimi、螟黄赤眼蜂T. chilonis、玉米螟赤眼蜂T. ostriniae匀浆液, 首次测定了几种化学因子（Ca²⁺、昆布多糖、纤维素、右旋糖酐、脂多糖和丝氨酸蛋白酶抑制剂）对3种供试赤眼蜂匀浆液中酚氧化酶原激活系统（proPO-AS）的激活程度。结果表明: 玉米螟赤眼蜂匀浆液PO活性最高, 螟黄赤眼蜂次之, 松毛虫赤眼蜂最低。Ca²⁺是激活供试赤眼蜂proPO的必需因子, 且在0.03 mol/L浓度下激活作用最强; 昆布多糖和脂多糖也能有效激活proPO, 0.01及0.05 mg/mL昆布多糖能强烈激活PO活性; 纤维素和右旋糖酐对PO活性存在显著抑制作用。结果证明了赤眼蜂种间存在明显的免疫能力差异。本研究建立了小型昆虫proPO-AS研究体系, 为研究Wolbachia-小型昆虫的免疫互作体系奠定了基础。     

注射亲水性硅珠对棉铃虫血浆酚氧化酶活性的影响

昆虫通过细胞免疫和体液免疫的协同作用对入侵的异物做出防御反应。在不同时间向棉铃虫体内注射亲水性硅珠后,测定了血浆中酚氧化酶（PO）的活性,同时研究了不同抑制剂和激活剂对注射硅珠后的酚氧化酶活性的影响。结果表明,注射亲水性硅珠后,棉铃虫血浆中酚氧化酶的活性明显升高。分别以牛胰蛋白酶和昆布多糖作为酚氧化酶原（proPO）的激活剂,发现两者都可激活注射硅珠后血浆中的proPO。以牛胰蛋白酶激活时,随着注射硅珠后时间的延长,PO活性逐渐增高;而用昆布多糖激活后PO活性也明显升高,但注射硅珠后不同时间proPO被昆布多糖激活的情况基本相似。这些结果表明,在异物入侵后酚氧化酶原有很大程度的积累,并能被激活,协同细胞免疫抵御异物入侵。P-NPGB和PTU几乎能完全抑制酚氧化酶的活性。     

亚洲玉米螟免疫细胞中一种细胞粘附因子的研究

以亚洲玉米螟Ostrinia furnacalis为试虫,发现血细胞裂解液被昆布多糖或低浓度钙离子激活后,能粘附浆细胞,使浆细胞伸展。昆布多糖处理的血细胞裂解液,经硫酸铵沉淀、羧甲基纤维素离子交换层析、ConA-Sepharose 4B亲合层析,提纯到一种细胞粘附因子。细胞粘附因子是分子量为64KD的蛋白质。以同样方法在血浆中纯化出一种分子量为390KD的蛋白质,推测其可能是血浆凝固原.经53%PercOll浓度梯度分离纯化了两类免疫细胞(颗粒细胞和浆细胞)。证明细胞粘附因子仅存在于颗粒细胞中。     

棉铃虫不同虫态及虫龄血淋巴中酚氧化酶活力的比较

分别测定了棉铃虫Helicoverpa armigeraHübner不同虫态及虫龄血清和血细胞中酚氧化酶(phenoloxidase,PO)的活力。结果显示血清和血细胞中都有酚氧化酶活性,且血细胞中高于血清中。不同虫态及虫龄的血清和血细胞中酚氧化酶活力有很大的不同,血清和血细胞中酚氧化酶活力变化规律一致。3龄幼虫酶活力最高,5龄幼虫最低。酶活力大小依次为:3龄幼虫>预蛹>4龄幼虫>蛹>5龄幼虫     

大菜粉蝶根虫瘟霉菌株转染小菜蛾后侵染力变化及其与寄主血淋巴酚氧化酶活性的关系

源于大菜粉蝶Pieris brassicae的根虫瘟霉Zoophthora radicans菌株R₀通过反复转染小菜蛾Plutella xylostella而分别获得转染菌株R₁、R₃和R₅。用这些菌株对小菜蛾2龄幼虫进行生物测定,发现菌株对寄主的侵染力有随转染次数增加而增强的趋势。接种后第1～6 天,R₀的LC₂₀（孢子数/mm²）分别为14.7、14.5、9.0、7.1、6.0和5.5;R₁的LC₂₀分别为9.6、5.0、4.2、3.6、3.1和3.0;R₃的LC₂₀分别为4.6、2.9、2.8、2.5、2.4和2.2; R₅的LC₂₀分别为5.2、3.7、3.2、2.8、2.6和2.6,接种后同一天菌株 R₃的LC₂₀值最小即侵染力最强。各菌株感染小菜蛾幼虫后可显著激活寄主血淋巴中的酚氧化酶活性,但R₁、R₃和R₅对酚氧化酶的激活程度显著低于原始菌株R₀。各菌株对小菜蛾的侵染力强弱指标log₁₀ (LC₂₀)与其侵染后寄主血淋巴酚氧化酶活性呈明显正相关（0.852<0.95）,表明R0在对新寄主转染过程中逐渐获得了逃避或克服新寄主免疫防御的能力,从而增强对新寄主的侵染力。     

根虫瘟霉转寄主过程中毒力相关胞外蛋白酶系的诱导表达

根虫瘟霉Zoophthoraradicans是寄主范围较广的专性昆虫病原真菌。在该菌胞外蛋白酶系与毒力变化关系的研究中,不同寄主来源的根虫瘟霉菌株产胞外蛋白酶水平与其对小菜蛾(Plutellaxylostella)的毒力间无明显的相关性。但转寄主各代菌株随毒力逐步上升产胞外蛋白酶水平也有所增加,两者间有一定的相关性。经活性电泳检测显示,ARSEF1342原始菌株(R0)有分子量分别为148kD,153kD和162kD的三条蛋白酶条带,但转寄主传代菌株(R1~R4)的148kD蛋白酶条带突然消失,而153kD蛋白酶条带则随转寄主传代数增加逐步趋于明显,相关性分析发现153kD和148kD与毒力间具有较好的相关性。这表明根虫瘟霉菌株在转寄主过程中逐渐增加了对新寄主具有较高基质特异性的胞外蛋白酶的诱导表达,从而使转寄主菌株更适应对新寄主的入侵及致病。     

Conflicts between a fungal entomopathogen, Zoophthora radicans, and two larval parasitoids of the diamondback moth

Zoophthora radicans (Zygomycetes: Entomophthorales), Diadegma semiclausum (Hymenoptera: Ichneumonidae), and Cotesia plutellae (Hymenoptera: Braconidae) are all natural enemies of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). Adult C. plutellae are not susceptible to Z. radicans infection but the pathogen can infect and kill adult D. semiclausum. Infection of adult D. semiclausum prior to exposure to P. xylostella host larvae significantly reduced the number of parasitoid cocoons subsequently developing from the host larvae. Although Z. radicans infection of P. xylostella larvae prior to parasitism by D. semiclausum or C. plutellae always resulted in the death of the immature parasitoids, neither species discriminated between healthy and Z. radicans-infected host larvae in an oviposition choice experiment. However, host larvae recently killed by Z. radicans were always rejected by D. semiclausum but sometimes accepted by C. plutellae. At 20 degrees C, egg to pupa development took 6.7 and 7.8 days for D. semiclausum and C. plutellae, respectively. C. plutellae parasitism significantly increased host instar duration but D. semiclausum parasitism did not. Cadavers of P. xylostella larvae parasitized 1 day prior to fungal infection showed no reduction in Z. radicans conidia yield. However, cadavers of larvae parasitized 3 days prior to fungal infection demonstrated a marked decrease in Z. radicans conidia yield. Z. radicans infection of P. xylostella larvae < or = 4 days after parasitism resulted in 100% parasitoid mortality; thereafter, the reduction in parasitoid cocoon yield decreased as the time between parasitism and initiation of fungal infection increased. The extended duration of the host larval stage induced by C. plutellae parasitism increased the availability of the parasitoid to the pathogen. Estimates of interspecific competition indicated a similar pattern for the interaction between Z. radicans and each species of parasitoid.     

Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs

Phenoloxidase (PO) activity was studied in larval and juvenile homogenates and in the plasma and haemocytes of adult Crassostrea gigas, Argopecten ventricosus, Nodipecten subnodosus, and Atrina maura. Samples were tested for the presence of PO activity by incubation with the substrate L-3, 4-dihydroxyphenylalanine using trypsin, alpha-chymotrypsin, laminarin, lipopolysaccharides (LPS), and sodium dodecyl sulphate (SDS) to elicit activation of prophenoloxidase (proPO) system. PO activity was not detected in larval homogenate. In juvenile homogenate, PO activity was found only in C. gigas and N. subnodosus. PO activity was present in adult samples and was enhanced by elicitors in the plasma of all species tested, but in haemocyte lysate supernatant (HLS) of only N. subnodosus. Activation of proPO by laminarin was suppressed by a protease inhibitor cocktail (P-2714) in plasma and HLS of all species tested.     

Insect haemolymph: Cooperation between humoral and cellular factors in Locusta migratoria

In Locusta migratoria, prophenoloxidase is present in the plasma and serum, but in reduced amounts relative to the haemocytes. This phenoloxidase activity cannot be induced by either heating or freezing and thawing and it is lost by heating at 70°C for 30 min. Both lipopolysaccharides and laminarin can elicit the prophenoloxidase activating system. These elicitors of prophenoloxidase activation are active in haemocyte lysate and in serum but never induce any phenoloxidase activity in plasma. In haemocyte lysate, the activating system is not heat resistant, and heating at 56°C for 30 min prior to incubation with laminarin or lipopolysaccharide precludes any phenoloxidase activity. Plasma contains a strong inhibitor of the prophenoloxidase activating system but serum does not. This inhibitor does not affect the phenoloxidase enzyme itself. The possible role of the activating system in immune recognition and the strategies evolved by parasites or pathogens to escape being recognized by their host are discussed.     

Demonstration of a true phenoloxidase activity and activation of a ProPO cascade in Pacific oyster, Crassostrea gigas (Thunberg) in vitro

The prophenoloxidase (ProPO) system is the origin of melanin production and is considered to be an innate defence mechanism in invertebrates. In different bivalve species, phenoloxidase (PO) is present in the haemolymph as an inactive form of ProPO. The present study focuses on the Pacific adult oyster, Crassostrea gigas, an economically important bivalve species along French coasts. The results indicate that many factors may inhibit the PO-like activity. These include: phenylthiourea (PTU), sodium diethylthiocarbamate (DETC), beta-mercaptoethanol and tropolone, which repressed the spontaneous PO activity. The activation of PO-like activity in C. gigas acellular fraction by lipopolysaccharide (LPS) involved participation of other factors, including at least one serine protease. PO was present as proPO in the acellular fraction of haemolymph and haemocytes of C. gigas and could be activated by an exogenous protease (trypsin-N-tosyl-l-phenylalanine chloromethyl ketone) when used at 1 gL(-1). Treatment of acellular fractions with other modulators/activators namely LPS (1 gL(-1)), zymosan (0.6 gL(-1)) or laminarin (0.6 gL(-1)) also increased PO-like activity but to a less important way. The study demonstrated the evidence of a true phenoloxidase activity in Pacific oyster, C. gigas (Thunberg). The activation of a proPO system by non-self molecules suggests the role played by PO in vivo in the internal defence mechanisms. Understanding the activation of the ProPO system could enable the evaluation of the health of oyster stocks.     

Pathogenicity of Paecilomyces fumosoroseus isolates to diamondback moth, Plutella xylostella: correlation with spore size, germination speed, and attachment to cuticle

Infectivity to larvae of the diamondback moth, Plutella xylostella, was compared among eight Paecilomyces fumosoroseus isolates. Isolate infectivity was assessed for correlation with spore length and germination speed. Four isolates applied to P. xylostella cuticle were also compared for number of spores remaining on the cuticle after washing and for percentage germination after 36 h. Infection of larvae inoculated with the different isolates at an average dosage of 4000 conidia/cm2 ranged from 2 to 47%. The correlation of infectivity with spore length and germination speed in broth was highly significant. Fewer spores of the least infective isolate, ARSEF 1576, attached to larval cuticle compared to spores of the more infective isolates ARSEF 3682, 4461, and 4482 (P < 0.05). After 36 h on larval cuticle, the percentage of spores germinated for isolates 1576 and 3682 was 3 and 95%, respectively. Spores of isolate 1576 were smaller, germinated more slowly, and attached to cuticle in smaller numbers than spores of the more infective isolates. Further research will expand our understanding of the mechanisms of virulence among isolates of P. fumosoroseus.