尘螨连续石蜡切片的制备及染色技术

研究尘螨连续石蜡切片的制备技术。利用琼脂预包埋后再以塑化石蜡包埋,经切片和HE染色,获得结构完整、定位准确、染色清晰的连续切片。探讨制片过程中一些步骤的改进和注意事项,为尘螨显微结构的形态学研究提供可能,也为免疫组化、原位PCR及过敏性疾病尘螨特异性变应原的定位等研究奠定良好的基础。     

蚊虫连续石蜡切片制备及染色

本研究利用塑化石蜡进行了吸血前后白纹伊蚊整个蚊虫的石蜡包埋、制片及 HE染色 ,获得了染色清晰 ,组织完整的连续石蜡切片 ,并讨论了有关蚊虫石蜡包埋及制片染色的一些注意事项。为进一步利用免疫组化、原位 PCR等技术研究虫媒病毒在蚊虫体内的分布规律提供了可能     

一株新的致倦库蚊细胞系的建立与鉴定

【目的】以致倦库蚊Culex pipiens quinquefasciatus初孵幼虫组织为材料建立细胞系并对其进行分子鉴定。【方法】将实验室筛选的对硫磷抗性的致倦库蚊深桂品系的初孵幼虫剪碎后放入添加20%胎牛血清的改良M199培养基原代培养,并对原代细胞用胰蛋白酶传代培养成细胞系;通过显微镜观察其生物学特性以及DNA扩增指纹图谱和内转录间隔区序列鉴定其组织来源。【结果】该细胞系Cxq-1在M199完全培养基中生长良好,已成功传代培养12个月,并于液氮中冻存,并多次成功复苏。通过DNA扩增指纹图谱鉴定,Cxq-1与实验室培养的其他昆虫细胞DNA指纹图谱的条带各异,表明确为同源昆虫致倦库蚊细胞系。通过内转录间隔区序列测定技术鉴定,Cxq-1的r DNA-ITS1和r DNA-ITS2序列与Gen Bank中致倦库蚊相应核酸序列的一致性大于99%,表明其来源组织确为致倦库蚊。【结论】新建立的蚊初孵幼虫细胞系Cxq-1确为致倦库蚊细胞系。该细胞系为实验室今后开展杀虫剂抗性分子机制、蚊媒病毒、寄生虫等研究提供了重要平台。     

有机磷抗性致倦库蚊种群中酯酶基因扩增的定量分析

致倦库蚊Culex qinquefasciatus是丝虫病的主要传染媒介。通过生物测定、单个蚊虫酯酶α2和β2基因拷贝数分析和酯酶β基因序列比较, 分析了抗性水平、抗性相关基因在种群中的分布及其基因拷贝数等的抗性分子特征。应用快速PCR仪（realtime quantitatIve PCRs）直接检测库蚊中酯酶基因和mRNA拷贝数。结果显示：上海致倦库蚊对对硫磷的抗性LC₅₀为8.12, 酯酶活性升高是上海致倦库蚊种群对有机磷杀虫药剂产生抗性的主要机理。编码致倦库蚊酯酶β的氨基酸序列同编码尖音库蚊酯酶B1的氨基酸序列相比同源性为98%;同致倦库蚊酯酶B2氨基酸序列相比同源性为100%,同环蹶库蚊酯酶B3氨基酸序列相比同源性为90%, 上海致倦库蚊中酯酶α和β基因均扩增。有机磷抗性的上海和PellRR蚊虫种群中单个蚊虫酯酶α2 和β2定量基因拷贝数均不同,其同一蚊虫个体的酯酶α2 比酯酶β2基因的拷贝数高,但没有明显的规律性,酯酶结构基因的扩增是上海致倦库蚊种群对有机磷杀虫药剂抗性的主要机理,估计在野外种群的杂合个体中存在多种调控机制。     

热带无爪螨体内特异性变应原定位

【目的】探讨热带无爪螨Blomia tropicalis特异性变应原在体内的定位。【方法】制作热带无爪螨石蜡切片, HE染色后光镜观察其内部形态结构。选用对尘螨过敏患者的阳性血清特异性IgE 抗体作探针,免疫组织化学染色分析其特异性抗原存在位置。【结果与结论】免疫组织化学染色可见热带无爪螨中肠、盲囊、结肠的肠壁和内容物及生殖腺等均有阳性反应。其中肠壁组织和肠腔内容物呈强阳性反应,提示肠为特异性抗原的主要存在部位。     

基于改良石蜡切片技术的飞蝗胚胎浆膜表皮发育模式及形态变化观察

【目的】通过优化飞蝗Locusta migratoria胚胎石蜡切片制备技术,研究飞蝗胚胎发育时期浆膜表皮变化规律。【方法】通过优化飞蝗胚胎固定前处理、洗涤、脱水和透明等步骤,改良胚胎期石蜡切片制备技术;制备飞蝗胚胎发育第3-10天的石蜡切片,通过HE染色观察浆膜表皮发育规律;通过制备飞蝗发育第6-8天从卵孔端至卵尾端的纵切切片,观察飞蝗胚胎转旋时期的形态变化。【结果】飞蝗胚胎期石蜡切片技术优化后的操作步骤为:固定前Na Cl O预处理和打孔、洗涤30min、脱水30 min和透明30 min。通过此方法可获得表皮结构完整且清晰的胚胎期组织切片。在30℃条件下,飞蝗胚胎浆膜表皮及几丁质沉积形成于胚胎发育的第5天,在第8天时开始降解。飞蝗胚胎的转旋发生在第6-7天,伴随着浆膜和浆膜表皮的形态变化。【结论】本研究优化了飞蝗胚胎石蜡切片制备步骤,揭示了飞蝗胚胎浆膜表皮及胚胎转旋的发育模式,为昆虫胚胎发育的研究提供了基础资料。     

武汉地区蚊类群落结构、时空动态及多样性研究

【目的】掌握武汉地区蚊类群落结构、时空动态及多样性特征,为蚊媒防治提供科学依据。【方法】2007-2016年每月采用灯诱法调查该地区5种生境蚊类群落结构和密度的时空动态,测定群落物种丰富度、多样性指数、均匀度指数及优势度指数。【结果】2007-2016年武汉市蚊平均密度为58.54只/(灯·夜),隶属2亚科5属8种,优势蚊种为致倦库蚊Culexpipienspallens和三带喙库蚊Culex tritaeniorhynchus。季节消长呈单峰,高峰出现在6月。牲畜棚、农户生境与居民区、公园、医院生境在蚊类丰富度、多样性指数、均匀度指数和优势度指数方面均有差异。【结论】武汉地区不同季节和不同生境蚊虫种类、数量和多样性有差异,应根据具体情况采取有针对性的蚊媒防治措施。     

致倦库蚊饲养驯化的初步体会

致倦库蚊是丝虫病、流行性乙型脑炎、东方马脑炎等多种疾病的传播媒介,对人类健康和工农业生产的发展危害甚大。为测试蚊虫对各种杀虫剂的敏感性等生物和化学测定,需人工饲养和驯化大量的致倦库蚊,得到实验室品系。我们自1999年以来开展了实验室饲养驯化致倦库蚊的工作,已连续数年饲养致倦库蚊达60多代。各个世代蚊虫生长发育良好,种群情况稳定。现将初步体会简报如下。     

树脂包埋半薄切片在视神经组织学和免疫组织化学研究中的应用

目的探讨半薄切片在视神经组织学和免疫组织化学研究中的应用,寻找一种视神经形态学研究和疾病诊断的有效方法。方法健康Sprague-Dawley大鼠视神经分别行树脂包埋制成半薄切片与石蜡包埋制成石蜡切片,分别采用HE、甲苯胺蓝染色及髓鞘碱性蛋白(myelin basic protein,MBP)免疫组织化学方法染色,光学显微镜下观察并比较半薄切片与石蜡切片染色结果的差异。结果石蜡切片HE和甲苯胺蓝染色均显示视神经髓鞘不着色,MBP免疫组织化学染色示MBP阳性着色较重,着色较紊乱,髓鞘着色不清晰。半薄切片HE及甲苯胺蓝染色均显示髓鞘呈环形,着色清晰,颜色对比鲜明;MBP免疫组化染色结果可见髓鞘呈环形,非特异性染色不明显,阳性对比显著,可清晰显示髓鞘结构。结论半薄切片在视神经组织学和免疫组织化学研究中优于石蜡切片,可用于视神经疾病的诊断和研究。     

Bt新菌株资源的采集与鉴定

【目的】寻找对致倦库蚊高效的苏云金芽胞杆菌(Bacillus thuringiensis,Bt)杀蚊菌株新资源。【方法】从福建省的武夷山自然保护区、建阳、建瓯、浦城等多个地区采集土壤样品,采用热处理法从土壤中分离Bt菌株,并测定其对致倦库蚊活性的效果。【结果】从125份土壤样品中分离出71株Bt菌株,经生物测定得到4株对致倦库蚊有效菌株(QQ13、QQ42、QQ66和QQ92)。其中,QQ66和QQ92有较高的毒性,均有几丁质酶基因,没有检测到cry1、cry1Ⅰ、cry2、cry4、cry5、cry6、cry7、cry8、cry9、cry10和cry11基因,在75～100 ku处各有一条杀虫晶体蛋白条带。【结论】采集和鉴定到的Bt新菌株资源将对致倦库蚊的生物防治起到促进作用。     

小蓬草精油对两种蚊虫的毒杀活性和成分分析

【目的】明确小蓬草Conyza canadensis ( L.) Cronq.精油的杀虫潜力及其活性成分。【方法】通过浸虫法和密闭熏蒸法测试了小蓬草精油对白纹伊蚊Aedes albopictus和致倦库蚊Culex pipiens quinquefasciatus幼虫及成蚊的毒杀活性, 并利用气相色谱 质谱联用仪（GC-MS）对精油的挥发性成分进行了定性分析。【结果】 小蓬草精油对白纹伊蚊1-4龄期幼虫及蛹的24 h LC50值分别为25.01, 45.88, 56.94, 64.60和346.23 μg/mL; 对致倦库蚊幼虫1-4龄期幼虫及蛹的24 h LC₅₀值分别为9.16, 8.65, 32.12, 43.68和197.83 μg/mL。在剂量分别为48, 64, 80, 96, 112和128 μg/cm3时, 小蓬草精油对白纹伊蚊成蚊的KT₅₀值分别为28.81, 22.31, 20.38, 17.05, 13.92和9.74 min; 对致倦库蚊的KT50值分别为34.90, 32.97, 23.97, 19.60, 15.20和10.34 min。 24 h熏蒸对白纹伊蚊和致倦库蚊成蚊的LC50值分别是75.46和99.19 μg/cm³。小蓬草精油的GC MS定性分析共分离鉴定出31种化合物, 其中单萜类物质6种, 倍半萜烯类物质17种, 含氧萜烯类6种。【结论】结果表明小蓬草精油对这两种蚊虫的毒杀活性较高, 具有深开发潜力。     

Effects of blood meal source on the reproduction of Culex pipiens quinquefasciatus (Diptera: Culicidae)

Culex pipiens quinquefasciatus were fed blood meals from a live chicken (LC), chicken blood in Alsever's (AC) solution, defibrinated bovine blood (DB), or bovine blood in citrate (CB) and incubated at 28° C. The effects of different blood meal sources were evaluated with respect to rates of blood feeding and reproduction (i.e., fecundity and fertility) over two gonotrophic cycles. Mosquitoes that fed on the first blood meal were subjected to a second blood meal as follows (first blood meal / second blood meal): LC/LC, LC/DB, DB/DB, CB/CB, AC/AC. Fecundity and fertility of Cx. p. quinquefasciatus were significantly (P < 0.05) greater in mosquitoes fed LC blood; however, fecundity and fertility in different treatment groups varied by gonotrophic cycle. These results contribute to our understanding of the impact of blood meal source on feeding and reproduction in Cx. p. quinquefasciatus. The potential impacts of blood meal source on virus transmission experiments are discussed.     

中国尖音库蚊复合组支序系统学的研究

本文应用支序系统学原理和方法,采用形态和生态性状,对尖音库蚊复合组的４个成员进行了支序系统学研究,提出在尖音库蚊复合组中,尖音库蚊是原始的类群,骚扰库蚊为较进化的类群,而致倦库蚊和淡色库蚊是最为进化的类群。     

BIOSYSTEMATICS OF CULEX PIPIENS COMPLEX IN CHINA

Abstract The present paper is the results of the biosystematic studies of Culex pipiens complex in China, including hybridization, mathematical analyses of morphological characters and GC analyses of culticular hydrocarbons. On the bases of the results of these studies, in addition to their allopatric distribution, it can be concluded that Cx. pipiens complex in China consists of four subspecies, including Cx. p ipiens molestus recently discovered in Beijing. Cx. pipiens quinquefasciatus can not be considered as a spearate species and Cx. pipiens pallens is not an intermediate form as claimed by some western workers.     

致倦库蚊对登革Ⅱ型病毒的中肠感染屏障作用

为探讨致倦库蚊对登革Ⅱ型病毒的中肠感染屏障作用,通过病毒分离、逆转录聚合酶链反应、透射电镜等技术进行了相关研究。结果表明：吸食感染性血液后,登革Ⅱ型病毒能侵染白纹伊蚊中肠上皮细胞并大量复制,但不能侵染致倦库蚊中肠上皮细胞。以上研究证明致倦库蚊对登革Ⅱ型病毒存在中肠感染屏障。     

竹叶花椒果实精油对两种蚊虫的毒杀活性研究

采用浸液法测试了竹叶花椒果实的水蒸汽蒸馏精油对白纹伊蚊和致倦库蚊幼虫的毒杀效果,并用三角瓶密闭熏蒸法研究了精油对这两种成蚊的熏杀活性;此外,采用GC-MS分析了该精油的化学成分。研究结果:(1)精油对白纹伊蚊和致倦库蚊的Ⅰ、Ⅱ、Ⅲ、Ⅳ龄期幼虫及蛹的LC50值分别为25.634/61.472、31.763/76.431、52.356/110.172、258.497/121.884和198.263/162.048mg.L-1;(2)精油对白纹伊蚊和致倦库蚊成蚊的LC50值分别为24.957和29.517μg.cm-3;(3)在147.52μg.cm-3熏杀剂量下,精油对白纹伊蚊和致倦库蚊成蚊的KT50值分别为3.493和2.993min,24h致死率均为100%;(4)共鉴定出18种化合物,其中萜烯类10种,占精油总量的67.122%,为竹叶花椒果实精油的主要成分。竹叶花椒果实精油对白纹伊蚊和致倦库蚊均有明显的致死作用,且作用速度快,具有开发为植物源杀蚊剂的潜力。     

Stempellia milleri sp. n. (Microsporida: Nosematidae) in the Mosquito Culex pipiens quinquefasciatus Say*

Stempellia milleri sp. n. was found in blood cells and the adipose tissue of field-collected larvae of Culex pipiens quinquefasciatus Say. Its development is different from other Stempellia species described in that it is dimorphic, producing two types of spores in adipose cells. Both were studied with the electron microscope. One spore is thin-walled and uninucleate; the other is thick-walled and binucleate. The latter spores are produced from sporonts that are somewhat similar to those of Nosema. Sporoblasts and spores of S. milleri were compared to those of Stempellia magna Kudo, and Stempellia lunata Hazard and Savage in both light and electron microscope preparations. S. milleri was transmitted experimentally to Culex pipiens pipiens, C. p. quinquefasciatus, C. salinarius, C. tarsalis, and C. territans. Transmission of S. magna to its host and other mosquito species was not possible. Although, generally low numbers of test larvae became infected with the pathogen, heavy infections were seen occasionally in individual specimens of its natural host, C. p. quinquefasciatus. Species of Aedes, Anopheles, Culiseta, Psorophora, and Uranotaenia exposed to spores of S. milleri were not susceptible to the disease.     

Swarming behaviour of the mosquito Culex pipiens quinquefasciatus: a quantitative analysis

ABSTRACT. Analysis of video recordings of Culex pipiens quinquefasciatus Say swarms showed that the overall swarming patterns of males and virgin females are similar, even though the short-term characteristics of their flight, such as speed and turning angle, differ. This suggests they have the same response to the visual cue of the swarm 'marker'. The swarming pattern of an individual consists of elliptical loops which, in the short-term, define an area smaller than the swarm as a whole. The foci of these ellipses drift gradually with respect to the marker; individuals do not seem to have preferred positions within the swarm, but drift at random. Male mosquitoes identify and locate females by the sound of their wing-beats. Diffuse sound at the wing-beat frequency of female C.p. quinquefasciatus (500–600 Hz) caused males within the swarm to slow down significantly. Their turning behaviour remained unchanged, so the ellipses decreased in size. The swarm as a whole collapsed into a smaller volume, centred over the marker. Apparently, the response of males to sound consists of at least two parts: initially they alter their flight speed, and then alter their turning behaviour once they have located the source of the sound.     

Effects of insect hormones on hemagglutination activity in two members of the Culex pipiens complex

Effects of methoprene and 20-hydroxyecdysone on the development and hemagglutination activity (HA) were studied in both sexes of two members of the Culex pipiens complex-anautogenous C. p. quinquefasciatus and autogenous C. p. molestus. Juvenile hormone analogue (methoprene) and 20-hydroxyecdysone caused developmental changes in both mosquito strains. High larval mortality, prolongation of intermolt period in each larval instar and in the pupal stage, and morphological changes in the larval-pupal and pupal-adult transformations were also observed. Developmental changes were accompanied with some differences in the HA. HA was found in both sexes of both experimental mosquito strains. The juvenile hormone analogue used in the larval stage caused significant decrease of HA in the gut of adults of both sexes. On the other hand, 20-hydroxyecdysone decreased HA only in the female gut. Results obtained indicate that HA depends on the sex, the studied organ, and the level of hormones.     

Binding Properties of Bacillus thuringiensis Cry4A Toxin to the Apical Microvilli of Larval Midgut of Culex pipiens

Cry4A is a dipteran-specific δ-endotoxin produced by Bacillus thuringiensis, and toxic to Culex pipiens (mosquito) larvae. The immunohistochemical staining of the midgut sections of C. pipiens larvae revealed that Cry4A bound in vitro and in vivo to the microvilli of the epithelial cells of posterior midgut and gastric caecae. The binding of digoxigenin-labeled Cry4A (DIG-Cry4A) to the apical microvilli was almost abolished in the presence of excess unlabeled Cry4A, suggesting that the binding of Cry4A to the microvilli was specific. Several Cry4A-specific binding proteins were detected using the ligand blotting technique with DIG-Cry4A. Moreover, an insertion assay was done, where the binding of DIG-Cry4A to the BBMVs was completely irreversible and did not compete with excess unlabeled Cry4A. On the basis of these results, we propose a schematic interpretation for the binding process of Cry4A.