用免疫金颗粒标记鉴定舞毒蛾核型多角体病毒的抗原

用免疫电镜金颗粒标记技术准确、快速地对舞毒蛾(Lymantria dispar)核型多角体病毒抗原进行了定位和鉴定.舞毒娥病毒的多克隆抗体与同源的多角体抗原之间存在着强烈的亲和性,但与不同源的松柏锯角叶蜂(Neodiprion sertifer)病毒多角体抗原仅有极微弱的交叉反应.舞毒蛾病毒粒子和核衣壳抗原也能与同源的多克隆抗体作用.在被病毒感染的舞毒蛾脂肪体细胞核中,成熟的多角体被金颗粒重重标记,其外缘的游离病毒粒子和核衣壳亦被标记,但亲和力较弱.在被感染的脂肪体细胞核和质内发现一种与多角体蛋白晶体不同源的菱形结晶体.     

绿原酸提高舞毒蛾核型多角体病毒(LdNPV)的致病力

【目的】明确绿原酸(chlorogenic acid,CA)对舞毒蛾核型多角体病毒Lymantria dispar necleopolyhydrovirus(Ld NPV)致病力的影响,为舞毒蛾的防治提供参考依据。【方法】采用食料给毒法进行生物测定,测定舞毒蛾2龄幼虫对单独Ld NPV及添加绿原酸的Ld NPV(CA+Ld NPV)的剂量及致死时间响应。【结果】CA+Ld NPV与单独Ld NPV对舞毒蛾2龄幼虫的剂量及时间响应间有显著差异,二者对舞毒蛾2龄幼虫的致死中浓度(LC50)分别为161.8 OBs/μL(95%的置信区间为105.6~235.3 OBs/μL)和264.4 OBs/μL(95%的置信区间为178.6~384.0 OBs/μL),前者对舞毒蛾的致病力较后者强。CA+Ld NPV对舞毒蛾2龄幼虫的致死中时间(LT_(50))较Ld NPV的短,当Ld NPV浓度为590 OBs/μL,CA+Ld NPV及Ld NPV对舞毒蛾2龄幼虫的LT_(50)分别为9.9 d和12.3 d;当Ld NPV浓度为5 900 OBs/μL时,则LT_(50)分别为6.9 d和8.0 d。绿原酸降低了Ld NPV对舞毒蛾幼虫的致死中浓度,缩短了致死中时间。【结论】绿原酸可提高Ld NPV对舞毒蛾2龄幼虫的致病力,其机理有待进一步研究。     

舞毒蛾几丁质酶基因的真核表达与重组病毒的获得

几丁质是昆虫外骨骼和围食膜的重要组成部分,鉴于几丁质酶在昆虫生长发育过程中发挥着举足轻重的作用,应用昆虫几丁质酶为探索新的生物防治害虫的方法提供了途径。本文分别根据苜蓿银纹夜蛾Autographa californica核型多角体病毒多角体蛋白基因序列和编码舞毒蛾Lymantria disparⅠ型几丁质酶基因的开放阅读框设计引物,使用聚合酶链反应扩增出以上两个基因,全长分别为783 bp和1 737 bp。构建重组质粒pFastBac-LdCht和pFastBac-AcPH-LdCht,转化大肠杆菌DH10Bac后获得重组穿梭载体,通过脂质体介导转染Sf9细胞产生重组杆状病毒AcMNPV-AcPH--LdCht和AcMNPV-LdCht,分别用于表达蛋白和获得重组病毒。细胞成功表达出有活性的舞毒蛾几丁质酶,并在棉铃虫体内扩增得到重组病毒。研究为深入了解昆虫几丁质酶性质提供依据,并为应用重组病毒奠定基础。     

木毒蛾复合病毒制剂研究

通过木毒蛾质型多角体病毒、核型多角体病毒和颗粒体病毒,对木毒蛾幼虫的复合感染试验,以及三种病毒最佳配比的筛选试验,发现木毒蛾质型多角体病毒、核多角体病毒和颗粒体病毒,按6：3：1混合后防治木毒蛾效果最佳。同时通过添加适量936乳油和人工大田繁殖木毒蛾病毒,较好地解决了病毒制剂的室温保存问题和毒源生产问题。复合病毒制剂的林间大面积防治试验结果表明,该制剂防治效果好,配制容易,成本低廉,不污染环境,是一种优良的生物制剂。     

蜀柏毒蛾核型多角体病毒的分离鉴定

本文报道了新分离的一株核型多角体病毒:蜀柏毒蛾核型多角体病毒(Paroceneria orient Nuclear Polyhedrosis Virus)。其多角体为四边形、五边形、大小在1.06—2.42μm。病毒粒子杆状,大小为385×55nm。室内感染蜀柏毒蛾幼虫其死亡率达9S％具较强的毒力。     

木毒蛾核型多角体病毒的PCR快速检测技术

【目的】木毒蛾是福建沿海地区防护林树种木麻黄的主要害虫之一,具有成为国际危险性有害生物的潜在可能性。木毒蛾核型多角体病毒(Lymantria xylina multiple nucleopolyhedrovirus,LyxyMNPV)是高效控制木毒蛾的优良天敌资源,具有高度安全性。建立LyxyMNPV的PCR快速检测技术,有利于该虫防治技术及木毒蛾核型多角体病毒的进一步研究。【方法】根据LyxyMNPV基因组中的特有基因gp131设计特异性引物,利用PCR法扩增该目的基因片段并测序检验,建立LyxyMNPV的分子快速检测技术体系。【结果】以GAL为引物的PCR检测技术对LyxyMNPV基因组的灵敏度可达1 fg·mL^-1,对多角体悬液浓度检测最低量可达5.0 PIB·mL^-1,可明确区分LyxyMNPV和其他7种昆虫核型多角体病毒。该体系同时适用于感病木毒蛾的不同虫态(卵、幼虫、蛹和成虫)的检测,以及环境样本(包括土壤、树枝和寄生蜂)的检测。【结论】成功建立了具有高度特异性、灵敏性的LyxyMNPV分子快速检测技术,为LyxyMNPV的快速检...     

柏毒蛾核型多角体病毒的分离鉴定

柏毒蛾核型多角体病毒的分离鉴定李崇荣,彭辉银,周显明,陈新文,谢天恩（贵州省铜仁地区林科所,铜仁５５４３００）（中国科学院武汉病毒研究所,武汉４３００７１）（贵州省林业科学研究院,贵阳５５００１１）关键词柏毒蛾,核型多角体病毒柏毒蛾（Ｐａｒｏｃｅｎｅ...     

舞毒蛾Ldcht10基因序列的克隆及其功能分析

【目的】研究舞毒蛾Lymantria dispar几丁质酶基因的时空表达特性及其在蜕皮发育过程中的生物学功能,筛选在舞毒蛾发育过程中致死性的几丁质酶基因,为实现基于RNAi的舞毒蛾有效控制提供重要的基础数据。【方法】设计简并引物克隆舞毒蛾几丁质酶基因Ldcht10关键序列,通过使用实时荧光定量PCR方法检测该基因在舞毒蛾不同龄期与组织中的相对表达量,选取部分序列的双链RNA(ds RNA)研究该基因功能。【结果】本研究克隆了长度为2 057 bp的舞毒蛾几丁质酶基因Ldcht10序列;各组织与不同龄期RT-PCR结果表明Ldcht10的时空表达特性存在明显差异,Ldcht10在各个龄期均表达活跃且在前肠与后肠中的表达水平最高;Ldcht10的RNA干扰试验表明:注射Ldcht10 ds RNA后Ldcht10的表达受到极大抑制,该基因被沉默后24.3%的舞毒蛾幼虫因无法完成蜕皮或化蛹而死亡。【结论】舞毒蛾中几丁质酶基因Ldcht10在各个龄期与组织中的表达存在差异,且在舞毒蛾蜕皮过程中具有十分重要的生物学功能,该基因被沉默后部分舞毒蛾因无法完成蜕皮而导致死亡。     

黑龙江省农药防治舞毒蛾研究

舞毒蛾是世界性重要食叶害虫,主要危害阔叶树和针叶树。此害虫在黑龙江省每年均大量发生,给林业生产带来了严重的影响。本文分析了舞毒蛾的生物学特性,在防治上分析了5种农药的毒杀幼虫效果,筛选出了2种农药作为防治常用药剂。     

荧光增白剂UBL对蜀柏毒蛾核型多角体病毒增效作用研究

用1%UBL荧光增效剂作为蜀柏毒蛾核型多角体病毒的增效剂,对蜀柏毒蛾2龄幼虫进行室内毒力测定,结果表明1%UBL对Parocneria orienta NPV有较强的增效作用。     

Food utilization efficiency in fifth instar larvae of Antheraea mylitta (Lepidoptera: Saturniidae) infected with Nosema sp. and its effect on reproductive potential and silk production

Antheraea mylitta, a sericigenous insect of economical importance is often infected with an intracellular parasite of the genus Nosema. This pathogen is known to cause fatal pebrine disease and is considered as an important factor that strongly influences the development of the host. Larvae developed from the eggs laid by a female infected with Nosema sp. showed extended development period. The increment in the larval weight declined significantly in infected larvae in comparison to uninfected ones. Food consumption, digestion, relative consumption rate (RCR), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD), and relative growth rate (RGR) values declined significantly, but at the same time a significant increase in approximate digestibility (AD) was also observed. Silk production declined in infected larvae. Silk gland weight and shell weight also significantly declined following infection over uninfected larvae. The reproductive potential in adults declined significantly (P<0.001) with decrease in ovary weight (31.6%), fecundity (54.1%), and fertility (34.9%). Egg chorionation was also affected in adults, which developed from infected larvae. The maternal infection level in one generation (10.4 x 10(6) spores/female) decreased significantly in the next generation (8.0 x 10(6) spores/female).     

Sex-specific occurrence of androgen receptors in rat brain.

The metabolism and binding of [1, 2, 6, 7-3H] testosterone in male and female rat brain has been studied in an attempt to find an explanation for the relative androgen unresponsiveness characterizing the female hypothalamo-pituitary axis involved in regulation of hepatic steroid metabolism. The most significant sex differences in the pattern of [3H] testosterone metabolites recovered from several brain regions (including pituitary, pineal gland, and hypothalamus) after intraperitoneal administration of [3H] testosterone were the predominance of testosterone and androstenedione in male brain compared to the quantitative importance of 5alpha-androstane-3alpha, 17beta-diol, 5alpha-androstane-3beta, 17beta-diol, epitestosterone, and dihydroepitestosterone in female brain. One possible explanation for the androgen unresponsiveness of female rats is, therefore, the faster metabolism of testosterone to inactive compounds in female brain. Experiments both in vivo and in vitro showed the presence of high affinity, low capacity binding sites for [3H] testosterone in male pituitary, pineal gland, and hypothalamus (Kd values in the region of 1 X 10(-10) to 1 X 10(-9) M and number of binding sites 1.0 to 1.4 X 10(-14) mol per mg of protein). The steroid - macromolecular complexes generally had a pI of 5.1, were excluded from Sephadex G-200, were heat-labile, and were sensitive to protease. Competition experiments indicated the following order of ligand affinities: testosterone is greater than 5alpha-dihydrotestosterone and estradiol is greater than androstenedione is greater than corticosterone. No steroid-binding proteins of similar nature were found in pituitary, pineal gland, or hypothalamus from female rats. On the basis of these results it is suggested that the androgen unresponsiveness of female rats referred to above relates to the absence of receptor protein for androgens in female rat brain. In support of this hypothesis, 28-day-old female rats, which are known to be affected by androgens with regard to liver enzyme activities, were shown to contain receptor proteins for androgen in the brain. In conclusion, the relative androgen unresponsiveness of the female hypothalamo-pituitary axis is probably explained by the absence of receptor proteins for androgen in female hypothalamus and pituitary. The fast metabolism of testosterone in female rat brain also serves to decrease the availability of active androgen to potential receptor sites. It may be speculated that the presence of androgen receptors in male brain is the result of neonatal programming ("imprinting") by testicular androgen.     

Prolactin evokes lactational transmission of larvae in mice infected with Toxocara canis

We investigated the trans-lactational maternal–neonatal transmission of Toxocara canis larvae in mice, with particular interest in the role of prolactin in their migration to the mammary gland. Two female mice were infected with 300 T. canis eggs soon after delivery of 27 offspring. After 1 week of breast-feeding, seven larvae were recovered from 4 of 13 offspring. After 2 weeks of lactation, 101 larvae were recovered from all the remaining offspring. Daily prolactin administration (5 μg) was performed 2 weeks before T. canis infection and continued until 2 weeks after infection in six non-pregnant female mice, which resulted in larval accumulation in the mammary gland. Furthermore, prolactin administration in female mice that had been infected with T. canis 4 weeks prior to prolactin treatment induced migration of larvae into the mammary gland. These findings suggest that prolactin is a promoting factor contributing to lactational transmission of T. canis larvae in mice.     

The anatomy and development of the rhizocephalan barnacle Clistosaccus paguri Lilljeborg and relation to its host pagurus bernhardus (L.)

The anatomy of all developmental stages of Clistosaccus paguri Lilljeborg including internal primordia only 200 μm across has been studied by conventional and ultrastructural techniques. The earliest primordia lack any roots and organs. A receptacle and ovary arise as separate structures. The colleteric gland develops by infolding of the mantle cavity epithelium. The primordium becomes external during a host intermoult. The externa is apparently female. It is proposed that male cyprids, through the integument, have implanted cells found in the mantle of the externa. These male cells travel through the mantle into the single receptacle, where they proceed with spermatogenesis. Externae lacking male cells will not grow. The larvae are released as cyprids, in which no preformed kentrogon is present. About half of the infected hosts carry more than one externa. Multiple externae are separate and may represent different invading female cyprids. C. paguri infects all sizes of its host, and castrates it. The effect of the parasite on the host is discussed. The life span of the parasite seems to be one year and the parasite population shows an annual cycle.     

基于双元转基因CRISPR/Cas9系统的家蚕pax3基因功能分析

【目的】本研究旨在以家蚕Bombyx mori为研究模型探索pax3基因在鳞翅目昆虫中的生物学功能。【方法】利用PCR扩增验证家蚕Bmpax3外显子序列;利用qRT-PCR检测Bmpax3在5龄第3天家蚕幼虫头、表皮、脂肪体、中肠、马氏管、前部丝腺、中部丝腺、后部丝腺和生殖腺(包括精巢和卵巢)中的表达谱;利用双元转基因CRISPR/Cas9系统构建Bmpax3敲除突变体,分析Bmpax3突变对家蚕幼虫存活、体节分化及性别差异的影响。【结果】Bmpax3在家蚕5龄第3天幼虫头、中肠和丝腺中均有表达,其中在前部丝腺表达量最高。Bmpax3突变体的卵孵化率约为90%,但约有80%的突变体在1龄幼虫期死亡,有将近10%的突变个体能幸存并发育到成虫阶段,并且存活成虫数存在性别差异,雄性显著多于雌性。在幸存的成虫中,约有将近1/2的个体腹部末端体节分节异常,表皮条纹混乱,腹节腹板部分缺失,生殖器官及其周围的其他辅助器官出现发育缺陷。【结论】Bmpax3发生突变后会对家蚕的生存及形态发育产生较大的影响,提示Bmpax3可能参与了家蚕的生长发育过程。     

Host sex preferences and transmission success by the water mite Unionicola foili (Acari: Unionicolidae) parasitic on the midge Chironomus tentans (Diptera: Chironomidae)

This study examined whether ecoparasitic larval Unionicola foili exhibited a sex bias when infecting laboratory populations of the host insect Chironomus tentans and whether an association with male or female midges increased the likelihood of larval mites returning to the aquatic habitat. When laboratory populations of C. tentans were exposed to larval U. foili, there was a higher prevalence of mites among female hosts at emergence (17 of 30 males vs. 25 of 30 females infected by mites). However, there was no significant difference in the distribution or abundance of larvae among infected male (mean = 2.3 larvae per host) and female (mean = 2.6 larvae per host) midges. Larval mites parasitizing both male and female chironomids were more likely to return to water than could be expected by chance. Mite larvae infesting female C. tentans were more likely to return to water when female hosts deposited egg masses in water, suggesting that oviposition plays an important role in cueing larvae parasitizing female midges to detach. The mechanism responsible for increasing the likelihood that mites parasitizing male hosts return to water remains unclear. Future studies will address the possibility of parasite-mediated changes in host behavior.     

Mechanisms of parasite-induced sex reversal in Gammarus duebeni

The amphipod Gammarus duebeni is host to the feminising microsporidian parasite Nosema granulosis that converts males into functional females. To test the hypothesis that the parasite acts through endocrine disruption we compared the morphology of the gonad and activity of the androgenic gland, which coordinates male sexual differentiation, in infected and uninfected animals. Male gonad consisted of testis, seminal vesicle and vas deferens that was anchored to the genital papilla on segment 7. The androgenic gland was associated with the distal end of the vas deferens. In female and intersex animals the bi-lobed ovary opened into the oviduct at segment 5, vestigial vas deferens and vestigial androgenic gland were retained. The majority of parasitised individuals (38/39) were either phenotypic females or intersexes with fully developed ovaries and an undifferentiated androgenic gland. Our data suggest that the parasite prevents differentiation of the androgenic gland. In further support of this hypothesis, mass spectrometry of a single androgenic gland from males revealed a dominant molecular ion with a mass/charge ratio of 4818.4+H, corresponding to a peptide of androgenic gland hormone from Armadillidium vulgare. In contrast the vestigial androgenic gland from parasitised and unparasitised females showed only low intensity peaks. Our observations demonstrate that the parasite manipulates host sex by preventing androgenic gland differentiation, androgenic gland hormone production and consequently male differentiation. This is in agreement with observations of A. vulgare with inherited Wolbachia infection, suggesting that phylogenetically distant feminisers manipulate hosts through a common mechanism. The high frequency of infection in intersexes (89.3%) suggests that this phenotype results from incomplete feminisation by the parasite.     

赤链蛇不同组织Sox基因表达的RT-PCR分析

采用RT-PCR技术,研究了赤链蛇不同组织Sox基因的表达。通过PCR产物直接克隆法和SSCP技术筛选阳性克隆,分析了雄性睾丸和雌性卵巢组织中的Sox基因序列。结果显示,在赤链蛇雌雄成体组织中,Sox基因在睾丸、卵巢、脑和脾组织中均有不同程度的表达,而在雌雄成体肌肉组织中均无表达,显示该基因表达有一定的组织特异性。序列分析显示睾丸组织中表达的是DRSox3,卵巢组织中表达的是DRSox22。在Sox家族中,Sox3表达于中枢神经系统和尿生殖嵴的发育过程中;Sox22则表达于多种组织和神经系统中,可横跨CNS和PNS的整个过程。此结果表明Sox基因不仅在性别决定中起作用,还可能在胚胎发育过程中担负重要功能。     

Studies on the impact of two Nosema isolates from Bulgaria on the gypsy moth (Lymantria dispar L.)

We investigated host-parasite interactions of two Nosema-type microsporidian isolates recovered from populations of Lymantria dispar L. in northwestern Bulgaria, one near Veslec and one near Levishte. Bioassay studies produced information on development, stage specific mortality, pupation, and adult eclosion of infected individuals. Horizontal transmission of the two isolates was investigated in a second set of experiments. At dosages ranging from 2 x 10(2) to 5 x 10(4) spores/microl, the infection rates varied between 77 and 100% for the isolate from Veslec and between 92 and 99% for the Levishte isolate. The Veslec isolate caused a slightly higher mortality rate and the median time to death was shorter compared to the isolate from Levishte. The total mortality for both isolates varied between 79 and 99%, independent of spore dosages. A lower relative growth rate was recorded for male and female L. dispar larvae infected with either isolate during the third larval instar and a higher relative growth rate during the fourth instar compared to the control groups. Pupal weight did not differ significantly among females, but male infected pupae were heavier than the controls. Nosema sp. [Veslec] was as efficiently transmitted as Nosema sp. [Levishte]; 42% of the susceptible larvae became infected with the Veslec isolate when uninfected larvae were exposed to infected larvae; 43% of larvae became infected with the Nosema sp. [Levishte]. The latency period varied between 7 and 8 days for both isolates.     

体外培养日本血吸虫成虫生殖器官超微结构的观察

将日本血吸虫成虫于851培养基中培养23天后,对其生殖器官进行透射电镜观察。观察结果显示,雌虫卵巢内卵母细胞出现不同程度的变性、坏死;成熟卵黄细胞的卵黄小滴融合,脂质小滴数量增多、体积增大;培养后期卵壳形成发生障碍,最终导致无活性、无卵壳的畸形卵形成。超微结构观察首次显示,体外初产期虫卵卵壳中有条带状低电子密度区和高电子密度区相间排列。