土壤含水量对华北大黑鳃金龟生长发育的影响

室内试验研究土壤含水量对华北大黑鳃金龟Holotrichia oblita Faldermann生长发育的影响。结果表明,土壤含水量对华北大黑鳃金龟的影响因其发育阶段的不同而异。卵期适宜土壤含水量为10%～20%,幼虫期、蛹期适宜土壤含水量分别为10%～15%和15%～20%。土壤含水量为10%～15%时,成虫性比接近1∶1。土壤含水量为15%时,世代存活率最高。     

华北大黑鳃金龟幼虫对3种寄主植物根系分泌物的趋性反应

华北大黑鳃金龟幼虫是我国北方地区为害植物根系的重要土壤害虫,探索寄主根系分泌物对其引诱作用,可以为开发大黑鳃金龟幼虫引诱剂等绿色防控提供理论基础.以纯净空气作为对照,利用“Y”型嗅觉仪分别测定了3龄幼虫对花生、大豆、玉米根系的趋性反应.以正己烷为对照,采用GC-MS分析鉴定3种根系分泌物的成分,观测试虫对其主要成分的嗅觉反应.结果表明: 华北大黑鳃金龟幼虫对花生、大豆和玉米根系都具有显著的趋向性.通过GC-MS分析鉴定发现,根系分泌物主要成分在3种植物中均有20种及以上,而且在不同植物之间除丙三醇、十二醇和乙苯3种共有组分之外,其余组分存在明显差异.分泌物的趋性反应结果显示,供试幼虫对分泌物同一组分不同浓度的趋性反应程度明显不同.在浓度为40和80 μg·mL^-1时,2-丁烯酸、甲基丁二酸、肉豆蔻酸、乙酸和邻苯二甲酸等的引诱虫数显著多于对照;在浓度为100、200 μg·mL^-1时,化合物十四烷和十六烷等的引诱虫数显著多于对照.在浓度为300、500 μg·mL^-1时,供试幼虫对对二甲苯、邻二甲苯和棕榈酸甘油酯等有显著的趋向性反应.可见,植物根系分泌物的主要成分对华北大黑鳃金龟幼虫具有显著的引诱作用.     

华北大黑金龟幼虫对注射细菌的免疫反应

在华北大黑金龟三龄幼虫体腔内分别注射病原菌Bacillus popilliae及非病原菌Bacillus megaterium,观察血细胞总数(THC)和血淋巴蛋白质组分的变化。注射后15分钟,两者使THC都下降50%左右,1小时后开始回升。这时,注射非病原菌的幼虫THC上升到最高峰,约超过正常的86%。此后10小时内又经过三次下降和回升。用聚丙烯酰胺凝胶电泳测定表明,注射病原菌的幼虫血淋巴会产生蛋     

华北大黑鳃金龟对桃树挥发物的电生理反应

【目的】华北大黑鳃金龟Holotrichia oblita是一种分布范围广,寄主种类多的农业害虫。本研究鉴定了桃树顶空挥发物中对华北大黑鳃金龟雌虫具有触角电生理活性的化合物,并探索桃树挥发物活性成分的剂量对华北大黑鳃金龟雌、雄虫触角电生理反应的影响。【方法】采用顶空吸附法收集桃树枝叶挥发物,用气相色谱 触角电位联用技术(GC-EAD)和气相色谱 质谱联用技术(GC-MS)鉴定有电生理活性的桃树挥发物成分,最后用触角电位(electroantennograph, EAG)技术测试这些挥发物在0.01,0.1, 1, 10和100 μg剂量下引起的华北大黑鳃金龟雌、雄成虫EAG反应。【结果】桃树挥发物中能引起华北大黑鳃金龟雌成虫触角电生理反应的成分有7种,包括Z-β-罗勒烯、顺-3-己烯乙酸酯、顺-3-己烯醇、壬醛、水杨酸甲酯以及2种未知化合物。所测试的对华北大黑鳃金龟雌成虫触角具有电生理活性的5种桃树挥发物成分在各剂量下都能引起雌、雄成虫明显的EAG反应。雌、雄虫对同种物质的EAG反应随着剂量的增加而有增大的趋势,并且雌虫的EAG反应显著高于雄虫。【结论】桃树挥发物中,罗勒烯、顺-3-己烯乙酸酯、顺-3-己烯醇、壬醛、水杨酸甲酯均能引起华北大黑鳃金龟雌、雄成虫的剂量依赖性的触角电生理反应,而且存在显著的性别差异。可利用这些化合物开展行为反应和田间诱集试验,进一步验证其对华北大黑鳃金龟的引诱活性。     

昆虫病原线虫Steinernema longicaudum X-7增效药剂的筛选

测试了19种昆虫生长调节剂、植物源或病毒类药剂对昆虫病原线虫Steinernema longicaudumX-7的存活和侵染率的影响。筛选9种对线虫存活和侵染率均无影响的药剂,测定其与线虫混用对黄粉虫Tenebrio molitorL.9~11龄幼虫的交互作用。结果显示,各种参试药剂和线虫组合均表现为相加作用。为了进一步确定参试药剂与线虫混用对目标昆虫卵圆齿爪鳃金龟Holotrichia ovataChang 3龄幼虫的交互作用,本研究选择氟虫脲、除虫脲、灭幼脲、苦参碱、氟啶脲和虫酰肼在低浓度下与线虫混用,除虫脲与线虫混用对测定昆虫处理7 d后表现为增效作用,其它药剂与线虫则表现为相加作用;各种参试药剂与线虫组合处理14 d后均表现为相加作用。     

华北大黑鳃金龟两种围食膜蛋白cDNA的分子克隆与序列分析

本文以华北大黑鳃金龟Holotrichia oblita中肠为材料,依据Stratagene公司文库构建试剂盒方法,构建其中肠cDNA表达文库,该文库滴度为1.9×106 pfu/mL,重组率为99.97%。依据现代免疫学原理,利用棉铃虫Helicoverpa armigera围食膜蛋白多克隆抗体筛选文库,得到两个编码华北大黑鳃金龟围食膜蛋白的cDNA克隆Ho-Peritrophin1与Ho-Peritrophin2,其cDNA长分别为2 385 bp和1 633 bp,在PolyA末端上游各有3个多聚腺苷酸信号序列AATAAA,最长开放阅读框(ORF)分别编码729个和477个氨基酸,与粉纹夜蛾Trichoplusia ni CBP2(chitin binding protein 2)的相似性最高,分别为21.9%和19.1%。结构域分析表明,Ho-Peritrophin1与Ho-Peritrophin2分别具有9个和6个几丁质结合功能域,只含有较少的O-糖基化位点,不含有类粘蛋白结构域。胰蛋白酶和胰凝乳蛋白酶对两种蛋白的作用位点主要位于几丁质结合功能域(chitin binding domain, CBD)内部,而因受几丁质结合功能域保护,这两种蛋白能够抵抗这些蛋白酶的降解。与正常CBD比较,这两种蛋白C端的CBD只含有4个Cys,只在第1与第3、第4与第5个Cys之间形成两对二硫键,缺少由第2与第6个Cys形成的二硫键。推测其N端还应包括信号肽序列和几丁质结合功能域的未知序列。     

华北大黑鳃金龟引诱剂配方筛选及引诱效果评价

【目的】筛选可有效引诱华北大黑鳃金龟Holotrichia oblita成虫的引诱剂配方用于生态防控。【方法】选取5种植物挥发物、2种华北大黑鳃金龟聚集信息素和1种性信息素,根据各化合物单体最佳引诱浓度进行两两混配,得到植物挥发物+聚集信息素、植物挥发物+性信息素和聚集信息素+性信息素共17种二元引诱剂配方。通过触角电位(EAG)反应和趋性行为反应逐步筛选出引诱效果较好的配方。将所筛选配方与其单一组分通过趋性行为反应进行引诱效果比较,并通过室内笼罩诱捕检验配方对华北大黑鳃金龟成虫的引诱效果。【结果】通过EAG反应及趋性行为反应从17种二元引诱剂配方中逐步筛选出了对华北大黑鳃金龟成虫具有较强引诱作用的3种配方,即顺-3-己烯基乙酸酯+顺-9-十八烯乙酸酯,1-己醇+顺-9-十八烯乙酸酯和邻苯二甲酸二丁酯+甘氨酸甲酯,它们引起的华北大黑鳃金龟雌虫EAG反应相对值分别为5 981%, 5 796%和5 722%,引起的雄虫EAG反应相对值分别为3 983%, 4 712%和3 809%。趋性行为反应试验中,引起华北大黑鳃金龟成虫选择反应率最高的为配方邻苯二甲酸二丁酯+甘氨酸甲酯,成虫选择反应...     

嗜菌异小杆线虫侵染后暗黑鳃金龟和大黑鳃金龟幼虫脂肪体和中肠组织超微结构观察

【目的】探究昆虫病原线虫嗜菌异小杆线虫沧州品系 Heterorhabditis bacteriophora strain Cangzhou侵染对蛴螬脂肪体和中肠的影响,进一步明确其对蛴螬的致病机理。【方法】采用透射电镜技术,观察暗黑鳃金龟 Hololtrichia oblita (Faldermann)和大黑鳃金龟 H. parallela Motschulsky 2龄幼虫被嗜菌异小杆线虫沧州品系侵染后其脂肪体和中肠组织的病理变化。【结果】血腔注射感染期病原线虫嗜菌异小杆线虫沧州品系悬浮液24和48 h后,观察发现暗黑鳃金龟和大黑鳃金龟2龄幼虫脂肪体和中肠的组织结构均按时序逐渐发生变化,起初表现为脂肪球变形或变小,颜色变浅,脂肪体细胞和中肠细胞内质网、线粒体肿胀,中肠微绒毛变形脱落等现象,48 h后包裹脂肪球的膜结构破裂,脂肪体细胞和中肠细胞线粒体破裂,内质网数量减少,中肠微绒毛大量脱落,同时核内染色质大量解离,核膜破裂。【结论】经昆虫病原线虫嗜菌异小杆线虫沧州品系处理后,暗黑鳃金龟和大黑鳃金龟两种金龟甲2龄幼虫脂肪体和中肠细胞均出现明显的病理变化过程,这是嗜菌异小杆线虫高效致死蛴螬的原因之一。本研究可为昆虫病原线虫作为一种生物防治手段在蛴螬的综合防治中更好地发挥作用提供理论依据。     

Laboratory evaluation of entomopathogenic fungi against the white grubs,Holotrichia oblita and Anomala corpulenta (Coleoptera: Scarabaeidae) from the field of peanut,Arachis hypogaea

We evaluated the pathogenicity of nine isolates of entomopathogenic fungi, including six isolates of Metarhizium anisopliae, one of Beauveria bassiana, and two of Beauveria brongniartii, against eggs and various larval instars of two scarab-beetle species, Holotrichia oblita and Anomala corpulenta, under laboratory conditions. The fungal isolates differed in pathogenicity. Generally, the isolates were more pathogenic to A. corpulenta than to H. oblita. Some of the isolates prevented egg hatching and caused larval death. Isolates M2-2 and Br5-8 caused 39 and 27% egg mortality, respectively, and produced 23 and 24% viewable fungal-infection rates in H. oblita. Three isolates had no significant effect on egg hatchability. Three isolates (Br5-8, Br232818, and M200614) caused about 40% mortality in H. oblita first instars. In A. corpulenta, all isolates except M200614 caused more than 60% egg mortality, and M2-2, Br232818 and Br5-8 caused egg-infection rates greater than 50%. M2-2 caused 47% infection and 100% mortality in first-instar larvae of A. corpulenta, while Br5-8 and Br232818 yielded over 80% mortality of the larvae. The three most virulent isolates, M2-2, Br232818 and Br5-8, were selected for further bioassays against second- and third-instar larvae. In addition, seven graduated concentrations of a Br5-8 conidial suspension were assayed against H. oblita second instars. Larval mortality was positively correlated with fungal dosage, and the LC₅₀ was 4.49×10⁶ conidia/mL. These three virulent isolates may be used to prevent H. oblita and A. corpulenta larval infestations in field crops.     

华北大黑鳃金龟中肠丝氨酸蛋白酶cDNA克隆、 序列分析及表达

丝氨酸蛋白酶是昆虫体内一类重要的消化酶, 为了了解该类酶的分子特性及功能, 本研究利用粉纹夜蛾Trichoplusia ni围食膜蛋白多克隆抗体筛选华北大黑鳃金龟Holotrichia oblita中肠cDNA表达文库, 首次得到编码华北大黑鳃金龟丝氨酸蛋白酶cDNA序列, 命名为HoSP1（GenBank登录号为FJ573146）。序列分析表明, 该基因长902 bp, 开放阅读框（ORF）长783 bp, 编码260个氨基酸, 推测分子量和pI值分别为26.7 kDa和4.19, 不含有N-糖基化位点, 但在Thr₁₅₇处有一个O-糖基化位点, 含有6个保守的半胱氨酸残基, 组成3对二硫键, 对于维持蛋白质的三级结构起着重要的作用。通过与几种丝氨酸蛋白酶的比对发现, 该酶具有组氨酸（His）、 天冬氨酸（Asp）、 丝氨酸（Ser）催化中心, 与褐新西兰肋翅鳃金龟Costelytra zealandica的14种丝氨酸蛋白酶有明显的相似性, 其中与CzSP3的序列一致性最高, 为52.47%。把该基因与pET21b载体重组后, 进行体外表达, 以BTEE为底物, 测得该酶的活力为0.0378 μmol/mg·min。HoSP1基因的克隆及体外表达为进一步研究该酶在华北大黑鳃金龟体内的表达及功能提供了依据。     

Interaction between halofenozide and the entomopathogenic nematode Heterorhabditis marelatus for control of Japanese beetle (Coleoptera: Scarabaeidae) larvae

Japanese beetle, Popillia japonica Newman is a major pest of turf and ornamentals. Laboratory bioassays were conducted to evaluate the potential interactions between a biological control agent, Heterorhabditis marelatus (Nematoda: Heterorhabditidae) IN strain and the insecticide halofenozide against both overwintered and nonoverwintered 3rd instars of Japanese beetle. Treatments consisted of all combinations of 2 rates of halofenozid with H. marelatus nematodes Imidacloprid was used as a standard. Percentage larval mortality was evaluated at 7, 14, and 21 d after treatment. No deleterious effects were observed. The nematode treatments generally produced significantly greater larval mortality relative to both chemical treatments. Twenty-one days after treatment, both rates of nematodes resulted in 100% mortality, whereas insecticide treatments did not surpass 60% mortality. No synergism was detected in any of the combination treatments. There were no significant differences in nematode reproduction in larvae exposed to halofenozide and nematodes versus larvae exposed to only nematodes.     

铜绿丽金龟和黄褐丽金龟幼虫感染Bt HBF-1菌株后的病症及中肠组织病理变化

研究了丽金龟科(Rutelidae) 铜绿丽金龟Anomala corpulenta和黄褐丽金龟A. exoleta幼虫感染苏云金芽孢杆菌Bacillus thuringiensis HBF-1菌株后的病症,并采用组织切片的方法研究了感染HBF-1菌株后中肠的组织病理变化。结果表明： 丽金龟科幼虫感染HBF-1菌株后,初期幼虫无明显感病症状,随着感染时间的延长,逐渐出现反应迟钝、麻痹、丧失条件反射能力等症状,最终虫体变黑、伸直或是收缩,直至死亡,时间稍长便会呈腐稠状。在相差显微镜下观察中肠组织切片发现,感染3 d时,肠壁细胞出现变形及空洞;7 d时,细胞破坏更加严重,甚至无法辨认细胞形状;10 d时,肠壁细胞开始脱离底膜;新鲜死虫,肠壁细胞连同细胞内含物全部脱离,仅留底膜。     

粘虫颗粒体病毒对苏云金杆菌的增效特性及对Bt毒蛋白的降解活化作用

以小菜蛾Plutella xylostella为试虫,采用生物测定方法测定了粘虫颗粒体病毒（Pseudaletia unipuncta granulosis virus,PuGV-Ps）对苏云金杆菌（Bacillus thuringiensis,Bt）的增效作用。结果表明：不同配伍PuGV-Ps和Bt间的共毒系数在105.3至195.0之间, PuGV-Ps对Bt毒力具有增强作用,其中以Bt∶PuGV-Ps为4∶1增效作用最明显,72 h LC₅₀为0.039 mg/mL。不同温度和pH值都影响PuGV-Ps对Bt的增效作用,16℃～20℃增效程度明显高于24℃～32℃,而碱性条件下（pH 8～9）增效作用更显著。PuGV-Ps对Bt的增效作用因小菜蛾龄期不同而变化,2、3龄幼虫死亡率较单独使用Bt分别提高了50%和30.31%,而作用于低龄（1龄）和高龄（4龄）幼虫时对Bt的增效作用不显著。PuGV-Ps饲喂2 h后再接毒Bt,小菜蛾死亡率明显提高,48 h死亡率达66.67％,较直接饲喂Bt+PuGV-Ps处理死亡率提高了53.87％,差异极显著。SDS-PAGE表明PuGV-Ps具有碱性蛋白酶的活性,离体条件下能促进δ-内毒素酶解为47 kD,60 kD和61 kD的毒性肽。     

Characterization and phylogenetic analysis of an entomopathogenic Bacillus cereus strain WGPSB-2 (MTCC 7182) isolated from white grub, Anomala dimidiata (Coleoptera: Scarabaeidae)

White grubs (Coleoptera: Scarabaeidae) are cosmopolitan and polyphagous insect pests of agricultural crops, forests and pastures around the world. The lack of an environmentally sound approach for white grub management has prompted the exploration and detection of a novel microbial biocontrol agent against these sub-terranean insect pests. In this study we describe the isolation, establishment of pathogenesis, biochemical characterization and phylogenetic analysis of an entomopathogenic Bacillus cereus strain WGPSB-2 (MTCC 7182), isolated from an atrophied pupa of Anomala dimidiata, collected from the N.W. Indian Himalayas. The sequencing and subsequent comparison of the 16S rDNA revealed that the strain has100% similarity with Bacillus cereus sequences. Phylogenetic analysis of the 16S rDNA sequence revealed that the isolate is closely related to Bacillus thuringiensis and Bacillus sphaericus. In vitro bioassays showed that the isolate was able to infect and cause 92 and 67% mortality in second instar larvae of Anomala dimidiata and Holotrichia seticollis, respectively. The infected larvae exhibited bacterial septicemia like symptoms and mortality occurred between the third and ninth weeks after inoculation. The culture has been granted the accession number MTCC 7182 by the Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India.     

Bacillus thuringiensis serovar israelensis is highly toxic to the coffee berry borer, Hypothenemus hampei Ferr. (Coleoptera: Scolytidae)

A native collection of Bacillus thuringiensis strains was screened, once a reliable bioassay technique to assess the toxicity against the coffee berry borer (CBB) first-instar larvae was developed. A first round of bioassays with 170 strains indicated that the great majority of them showed no or very little insecticidal activity and that very few showed significant levels of toxicity. Interestingly, only those strains that had previously been associated with mosquitocidal activity were also toxic to CBB. Qualitative bioassays (using one high dose) were carried out only with those native mosquitocidal strains, corroborating their significant toxicity towards the CBB first-instar larvae. Most of these strains belong to serovar israelensis. In a second approach, strains from the Institut Pasteur type collection, whose mosquitocidal activity had been previously demonstrated, were also subjected to bioassays. Only those strains that showed a comparable protein content in their parasporal crystals to the israelensis type strain also showed high levels of toxicity towards CBB. Finally, an accurate LC(50) was estimated, using purified parasporal crystals from B. thuringiensis serovar israelensis type strain, at 219.5 ng cm(-2) of diet. All the statistical requirements for a reliable estimator were fulfilled. This is the first report of B. thuringiensis serovar israelensis being active against a coleopteran species.