桑天牛卵啮小蜂基因组DNA的提取及RAPD反应体系的优化

桑天牛卵啮小蜂是抑制桑天牛的有效天敌。为使天敌能大量人工繁殖,从分子生物学角度对单头桑天牛卵啮小蜂基因组DNA的提取方法及RAPD反应条件进行了探索。结果表明,采用改良的SDS方法,提取的DNA质量较高,适宜于RAPD-PCR扩增分析。在25μL反应体系中,RAPD分析的优化反应体系为：1．5mmol/L Mg~(2+)、2μL的dNTPs(2．5mmol/L)、320nmol/L随机引物、80ng模板DNA、0．8U TaqDNA聚合酶。     

桑天牛长尾啮小蜂对桑枝挥发物的触角电位和嗅觉反应

桑天牛长尾啮小蜂(Aprostocetus fukutai)是蛀干害虫桑天牛(Apriona germari)的重要卵寄生蜂之一,桑天牛危害的桑枝挥发物在该寄生蜂寄主生境定向搜寻过程中起着至关重要的作用。采用触角电位(EAG)仪测定了桑天牛长尾啮小蜂对8种桑枝挥发物的电生理反应,发现该寄生蜂对这8种桑枝挥发物都能产生电生理反应,且随着化合物浓度的增加电生理反应增强。利用Y型嗅觉测定仪测定了桑天牛长尾啮小蜂对不同浓度挥发物的趋性行为,结果显示0.01%月桂烯、0.01%里那醇和1%Z-乙酸-3-己烯酯对该寄生蜂均具有显著的引诱作用。研究结果对揭示桑天牛长尾啮小蜂寄主定向的化学本质具有重要意义。     

桑天牛长尾啮小蜂产卵及寄主识别行为的观察与研究

桑天牛长尾啮小蜂(Aprostocetus fukutai)是桑天牛(Apriona germari)卵期寄生蜂,研究其寄生机制对桑天牛的生物防治有重要意义。该寄生蜂的产卵行为主要包括桑天牛刻槽的定向搜寻、刻槽表面的检验、寄主卵检验、产卵寄生、标记等,其中刻槽表面检验的时间较长。选择试验显示,正常刻槽产卵桑枝对寄生蜂的引诱作用明显高于正常桑枝和取食桑枝。在刻槽未产卵桑枝、刻槽产卵未涂抹分泌物桑枝和正常刻槽产卵桑枝中,正常刻槽产卵桑枝对寄生蜂的引诱作用最强,并且刻槽检验时间、寄主卵检验率和寄生率等都显著高于前两个处理。已寄生刻槽对长尾啮小蜂的引诱作用明显低于未寄生刻槽。     

桑天牛危害和外源茉莉酸诱导桑树防御反应的研究

通过选择试验研究了桑天牛危害和外源茉莉酸(JA)对于诱导桑树产生防御反应的影响。桑天牛危害诱导的结果表明:在危害诱导后的第3天,受害桑枝被桑天牛继续取食的面积明显少于对照,而同株系统枝被取食的面积与对照间的差异不明显;危害诱导后的第1天,被害桑枝对桑天牛卵啮小蜂的引诱活性显著高于健康桑枝,此后其引诱作用逐渐下降,第3天时与健康桑枝间的差异消失。JA诱导的结果表明:JA处理3 d后,桑枝被桑天牛取食的面积也明显少于对照;JA处理1 d后,桑枝对桑天牛卵啮小蜂的引诱作用明显高于健康桑枝,JA处理2 d后的桑枝对寄生蜂的引诱作用达到了极显著的程度,第3天时与健康桑枝的差异消失。此外,无论是桑天牛危害诱导还是JA处理诱导,对桑天牛的产卵数量均未产生影响。分析认为:桑天牛危害和外源JA均能诱导桑树通过减少桑天牛取食桑枝的面积产生直接防御,但这种抗虫性仅局限于被害桑枝;桑天牛危害和外源JA还通过增强桑枝对桑天牛卵啮小蜂的引诱作用使桑树产生间接防御能力。     

两种桑天牛卵寄生蜂触角感器超微结构的比较

利用扫描电镜观察了同属的桑天牛卵啮小蜂(Aprostocetus prolixus)和桑天牛长尾啮小蜂(Aprostocetus fukutai)的触角感器,对这两种寄生蜂触角感器的种类、形态、数量及分布进行了描述,并对雌雄两性触角之间的差异做了比较。结果发现,在两种寄生蜂的触角上着生着6种感器,分别为指形感器、板状感器、毛形感器、坛形感器、刺形感器、蒲姆氏鬃;两种桑天牛卵寄生蜂触角感器的种类、数量及分布模式在种间没有明显差异,但在种内雌雄个体间差异较为显著。研究结果将有助于揭示桑天牛卵寄生蜂的寄生机制。     

桑枝和桑天牛虫粪挥发物对桑天牛长尾啮小蜂的引诱作用

桑天牛长尾啮小蜂(Aprostocetus fukutai)是蛀干害虫桑天牛(Apriona gernari)重要的卵期寄生蜂,开展该寄生蜂趋性行为与其寄主昆虫(桑天牛)和寄主植物(桑树)挥发物关系的研究对桑天牛的综合防治具有重要意义。嗅觉测定表明,经不同处理的桑枝和桑天牛虫粪对桑天牛长尾啮小蜂都具有显著的引诱作用,而且咬食桑枝和产卵桑枝的引诱活性高于正常桑枝。通过热脱附/气相色谱-质谱联用仪(TCT/GC-MS)对桑枝和桑天牛虫粪挥发物进行分析鉴定的结果表明:正常桑枝挥发物主要为酯类物质,咬食桑枝和产卵桑枝挥发物主要为萜烯类物质,且大多数挥发物的释放速率高于正常桑枝,咬食桑枝挥发物的数量多于正常桑枝而少于产卵桑枝;桑天牛虫粪挥发物的数量少于桑枝,主要物质为1,6-庚二炔。     

桑天牛卵啮小蜂Aprostocetus prolixus的寄生生物学研究

桑天牛卵啮小蜂 (Aprostocetusprolixus)是一个新种 ,对桑天牛有较高的寄生率。观察发现该寄生蜂 1年 2～ 3代 ,老熟幼虫以休眠状态越冬。 6月下旬越冬代的成虫开始羽化 ;第 1代成虫 7月下旬开始羽化 ,8月上中旬为盛期 ;第 2代幼虫一部分以老熟幼虫直接越冬 ;第 2代成虫 8月下旬至 9月中旬羽化 ,8月底与 9月初为盛期 ,所产小蜂幼虫从 9月中下旬开始陆续以老熟幼虫在寄主体内越冬。探明了该寄生蜂的羽化时间、交配、产卵、寄生等行为习性。     

桑天牛分泌物及虫粪中的信息化合物对桑天牛卵啮小蜂寄主识别的影响

来源于寄主昆虫的信息化合物对寄生蜂的寄主识别和趋性反应有较强的调控作用。桑天牛卵啮小蜂(Aprostocetus prolixus)是桑天牛(Apriona germari)的卵期寄生蜂。初步研究了雌性桑天牛成虫口腔分泌物和产卵后的肛门分泌物以及裸卵等单一信息化合物对桑天牛卵啮小蜂的寄主识别影响,结果表明这些单一信息化合物对该寄生蜂的寄主识别均没有影响。采用有机溶剂萃取法提取桑天牛成虫粪便中的信息类化合物,并且对不同提取溶剂获得的提取液进行了寄生蜂寄主趋性反应和寄主识别行为的测定,结果表明:二氯甲烷溶剂获得的提取液对该寄生蜂的趋性反应有显著影响,正己烷、甲醇、丙酮以及蒸馏水4种溶剂获得的提取液对寄生蜂的趋性反应均无影响;正己烷、二氯甲烷、甲醇3种有机溶剂获得的提取液对寄生蜂的寄主识别行为有影响,证明有机溶剂能够有效提取存在于桑天牛成虫粪便中的该寄生蜂的接触性信息化合物。利用硅胶柱层析的方法对提取液分离纯化,层析液对寄生蜂寄主识别影响的测定结果显示,用正己烷作溶剂的层析液对该寄生蜂的寄主识别有一定的调控作用;而二氯甲烷、甲醇、丙酮和蒸馏水作溶剂的4种层析液则没有调控作用。     

桑天牛卵啮小蜂不同地理种群间遗传关系的RAPD及酯酶分析

利用随机扩增多态性DNA标记和酯酶同工酶电泳技术,对分别属于河北、山东、湖北3个省区的6个桑天牛卵啮小蜂种群进行了遗传关系的分析。根据扩增结果构建了种群间的聚类树状图,山东泰安种群与河北各种群聚为一类,并与河北易县种群为同一亚类,其结果与形态学上的分类有些不同。而酯酶同工酶电泳图谱的结果显示,泰安种群与河北和湖北种群都有差别,与形态学分类的结果较一致。     

桑天牛卵啮小蜂对已被寄生桑天牛刻槽的识别

为了探究桑天牛卵啮小蜂对已被寄生桑天牛刻槽(被寄生蜂产卵并被涂抹产卵分泌物的刻槽)的识别行为,通过选择和非选择性试验,调查桑天牛卵啮小蜂对不同处理刻槽的行为反应.结果 显示,选择性试验中,寄生蜂首先接触到已被寄生刻槽(自己寄生或它蜂寄生刻槽)的次数显著少于正常刻槽.无论在选择还是非选择条件下,寄生蜂接触到已被寄生刻槽的时间显著多于正常刻槽,而在已被寄生刻槽上搜索的时间显著少于正常刻槽,且对已被寄生刻槽寄生的比率也显著低于正常刻槽.无论在选择还是非选择性试验中,雌蜂对它蜂寄生刻槽和自己寄生刻槽的行为反应无明显差异.选择性试验中,雌蜂对正常刻槽和它蜂搜索过的刻槽的行为参数间无显著差异.与此类似,雌蜂对已被寄生蜂产卵但未涂抹产卵分泌物的刻槽及正常刻槽的行为参数间也不存在显著差异.研究结果表明该寄生蜂在产卵完成后,通过其产卵器在刻槽表面涂抹产卵分泌物即寄主标记信息素对刻槽进行标记,使寄生蜂通过在近距离感知该标记信息素来识别已被寄生刻槽,该结果有望为揭示该寄生蜂的寄主识别机制以及实现桑天牛的生物防治提供理论依据.     

Toxicity of Different Diets Contaminated with Various Fungi to Rice Moth Larvae (Corcyra Cephalonica St.)

Growth studies of rice moth larvae (Corcyra cephalonica st) have been carried out in groundnut meal and wheat bran contaminated with A. flavus, A. oryzae, P. purpurogenus and P. rubrum. It was observed that the diets contaminated with A. flavus only are toxic to these larvae. Wheat bran contaminated with A. flavus is more toxic than contaminated groundnut meal. The higher toxicity of wheat bran contaminated diet has been discussed. Aflatoxins produced in different substrata are shown to differ when analysed chromatographically. Growth studies of rice moth larvae have also been carried out with aflatoxin and the susceptibility of these larvae has been established.     

A Study of Krebs Citric Acid Cycle Enzymes in Rice Larvae (Corcyrace phalonica St) During Mycotoxicosis

Krebs citric acid cycle enzymes have been studied in rice moth larvae (Corcyra cephalonica St) reared in groundnut meal control and contaminated with A. flavus, wheat bran control and wheat bran contaminated with A. flavus and also wheat bran containing aflatoxin. It was observed that the activity of enzymes other than succinic oxidase, succinic dehydrogenase and isocitric dehydrogenase were reduced significantly in larvae reared in contaminated groundnut meal when compared with the control. In the case of larvae reared in contaminated wheat bran all the enzymes except succinic oxidase were inhibited when compared to the control larvae. It was also observed that the inhibition of these enzymes is greater in the case of larvae reared in contaminated wheat bran than in contaminated groundnut meal. The higher toxicity of wheat bran has been discussed.     

Immune response to and tissue localization of the Wolbachia surface protein (WSP) in dogs with natural heartworm (Dirofilaria immitis) infection

Human and animal parasitic filarial nematodes, including the agent of canine and feline heartworm disease Dirofilaria immitis, harbour intracellular bacteria of the genus Wolbachia (Rickettsiaies). It is thought that these bacteria play an important role in the pathogenesis and immune response to filarial infection. Immunoglobulin G (total IgG, IgG1, IgG2) production against and immunohistochemical staining of tissues for the Wolbachia surface protein (WSP) from dogs with natural heartworm infection were evaluated. All infected dogs had significant total anti-WSP IgG levels compared to healthy controls. Interestingly, WSP was recognized by the IgG2 subclass in both microfilariemic dogs and in dogs with no circulating microfilariae (occult infection). However, microfilariemic dogs also produced gG1 antibodies. Positive staining for WSP was observed in lungs, liver and kidneys, in particular in glomerular capillaries of naturally infected dogs who had died from heartworm disease. Our results show for the first time that Wolbachia is recognized specifically by D. immitis--infected dogs and that the bacteria is released into host tissue. Furthermore, microfilariemic status appears to effect immune responses to this endosymbiont.     

Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia surface protein activates canine neutrophils with production of IL-8

Filarial nematodes, including Dirofilaria immitis and D. repens, harbour intracellular bacteria belonging to the genus Wolbachia. These bacteria have been implicated in the pathogenesis of filarial diseases, possibly through their endotoxins. Recent studies have shown that a major surface protein of Wolbachia (WSP) induces a specific IgG response in hosts infected by D. immitis. WSP from the Wolbachia of D. immitis was produced in recombinant form. The purified protein was used in stimulation assays on canine neutrophils. The assays performed using a modified Boyden chamber showed that WSP stimulates neutrophil chemokinesis. In addition, RT-PCR revealed increased production of chemokine IL-8 by cells incubated with this protein. Neutrophils have been shown to play a major role in the pathogenesis of river blindness, and to accumulate in the nodules of onchocerciasis patients. In dogs infected by D. immitis, neutrophils accumulate in kidneys and in the wall of pulmonary arteries. As shown by our studies, Wolbachia could contribute to these inflammatory phenomena through its surface protein WSP, independently from its endotoxin component.