中华卵索线虫雌雄寄生后期幼虫基因差异表达分析

索科线虫是一类具有很大生防潜力的昆虫(如棉铃虫等)天敌资源,但由于其体外培养尚未获得成功,阻碍了商业化生产和大规模应用,究其原因主要是体外培养的线虫不能完成雌雄性别分化。因此,研究该类线虫性别分化机制成为近年本领域的热点课题。该文以中华卵索线虫为实验材料,采用mRNA差异显示的方法,分析了中华卵索线虫性别分化关键时期雌、雄寄生后期幼线虫的基因表达差异。在雌雄线虫体内得到具有表达差异的基因片段20条。其中8条在雄虫体内特异性表达,12条在雌虫体内特异性表达。利用信息生物学技术对所分离到的差异片段进行了序列分析。其中,Ensembl分析发现4个片段与秀丽线虫X染色体具有可匹配片段,推测这些片段可能是影响中华卵索线虫性别分化的重要基因。这些结果将为后续研究提供思路。     

李拭库蠓生活史的研究(双翅目:蠓科)

在重庆地区所作有关李拭库蠓生活史的研究,观察在实验条件结合自然情况下进行。设计了虫尸培养法,在虫尸培养内幼期处于严格控制的条件下,可以确切地观察卵的孵化、幼虫蜕皮、化蛹和羽化等现象,解决了库蠓变态发育的规律。 李拭库蠓的主要动物宿主是牛和家猪,雌虫吸取血液后糖类对卵巢的发育并不是必需的。9—11.5℃时胃血消化和卵巢发育仍能缓慢地进行。雌虫在20—28℃时一般在吸血后第3—4日间产卵,产卵数为40—213。 幼期发育的时间随温度和食物而改变。虫尸培养,在27±1℃时自卵至成虫为28—44天(卵期3天:第一龄幼虫6—8天:第二龄幼虫5—10天;第三龄幼虫6—9天:第四龄幼虫5一11天;蛹期3天)。当6.8—22℃时,共需101—212天(卵期3—8天;第一龄幼虫9—31天;第二龄幼虫27—35天;第三龄幼虫34—48天:第四龄幼虫25—80天:蛹期3—10天)。夏季室温条件下泥土培养基内自卵至成虫为22—32天。     

我国结膜吸吮线虫在中间宿主——变色纵眼果蝇体内发育过程的研究

以结膜吸吮线虫产在家兔眼分泌物内的初产蚴和解剖雌虫子宫内的幼虫,喂饲实验室繁殖的变色纵眼果蝇进行感染,证明了变色纵眼果蝇可作为我国结膜吸吮线虫的中间宿主。以终宿主眼分泌物中的初产蚴喂饲感染果蝇,结果在感染后第16天,于存活的11只果蝇中,在2只雄蝇的口器和头部检出感染期幼虫9条。用取自雌虫体内的幼虫,平均每蝇5条幼虫的比例喂饲感染608只果蝇,结果检出阳性蝇106只,其感染率为17.4％。并发现幼虫在果蝇体内发育主要侵入雄蝇睾丸外层组织和雌蝇的血腔壁形成“虫泡囊”,幼虫在囊内发育至腊肠蚴蜕1次皮,进入感染前期发育阶段,再行第2次蜕皮,而发育成感染期幼虫。发育速度与外界温度密切相关,当外界温度为26.1—31.8℃,发育到感染期幼虫最早天数需要17天。     

中华卵索线虫的研究与应用

中华卵索线虫Ovomermis sinensis Chen etal.作为一种昆虫病原线虫,在农业害虫的生物防治中起着重大作用。文章综述90年代以来我国有关中华卵索线虫的生物学特性、田间应用、体内外培养以及生化与分子生物学研究进展,并对中华卵索线虫今后的研究方向提出建议。     

中华卵索线虫雌雄成虫可溶性蛋白双向电泳分析

昆虫寄生线虫 (又称虫生线虫、昆虫病原线虫)是本世纪发展起来的一种有潜能的生物防治因子 ,它寄主范围广、能主动寻找寄主、对人畜及环境安全无毒。中华卵索线虫是昆虫寄生线虫的一种 ,由我国学者发现并命名的新种 (陈果等 ,1 9 91 ) ,具有广泛的寄主范围 ,可杀灭粘虫 (任慧芳等 ,1 989)、烟青虫、烟蚜 (侯茂林等 ,2 0 0 2 )及棉铃虫 (陈果 ,1 994)等危害十分严重的害虫 ,其寄生率等于宿主的死亡率。因此在生物防治手段日益突出的今天 ,中华卵索线虫无疑有着广泛的应用前景。然而 ,在线虫培养的过程中 ,无论是体内培养还是体外培养 ,我们发…     

蜕皮激素在银盾革蜱生殖滞育中的作用(英文)

用高效液相色谱法和放射免疫测定法测定了银盾革蜱滞育雌虫的合神经节、血淋巴、卵巢和整体中蜕皮激素的含量,并与正常发育的雌虫做比较。结果表明,滞育雌虫血淋巴和卵巢中蜕皮激素的含量在饱血后前10天与正常发育雌虫基本上相同。饱血10天以后,滞育雌虫蜕皮激素的含量下降,较正常发育雌虫少得多。蜕皮激素的缺乏影响了卵母细胞的发育。为阐明蜕皮激素对滞育雌虫的影响,在不同时间向银盾革蜱滞育雌虫体内注入不同剂量的β-蜕皮素。刚饱血者注入大剂量(1.375和10000ng/蜱)的β-蜕皮素引起雌虫死亡。饱血后20—30天,注入一定剂量(50,70,100ng/蜱)的β-蜕皮素可以促进卵黄形成,并解除了雌虫的生殖滞育,但其所产的卵量明显少于正常发育的雌虫。应用外源蜕皮激素解除硬蜱雌虫的生殖滞育现象尚属首次报道。     

猪后圆线虫的发育观察

1.猪后圆线虫在蚯蚓体中的发育时间长短,随温度高低不同。自感染发育成为感染期幼虫,在月平均室温10.6和13.8下不能发育;在14—21℃(平均室温17.5℃)下需1个月;在24—30℃仅需8天。 2.幼虫在蚯蚓体中的第一期幼虫需时较长,第二期幼虫时间甚短,经常检得第一次蜕皮的外鞘尚未脱去,又进入第二次蜕皮,形成感染期幼虫。 3.通过闽侯、晋江、闽北三专区19个养猪场的调查,有14种蚯蚓充为猪后圆线虫的中间宿主,其主要的是湖北环毛蚓,这种蚯蚓生活在猪场中,感染率和感染度均甚高,为传播猪后圆线虫的主要种类。 4.幼虫在蚯蚓体中寄生的部位,随各种类不同,参环毛蚓主要寄生在胃壁中,湖北环毛蚓则寄生在胃壁和肠管前段肠壁中,暗灰异唇蚓主要在食道壁,少数在胃壁中。 5.第一期幼虫在外界潮湿的环境中可生存半年,感染期幼虫在水中可生存2—4个月。在环毛蚓体中可生存一年半以上。 6.幼虫侵入猪体后,于1—5天内在盲肠壁,大肠前段肠壁和肠淋巴结中行第三、四次蜕皮,然后穿过肠壁经淋巴系统移行到肺。雌虫发育较快,于第4天便移行到肺、雄虫经6天才在肺中检得。经23天发育成熟排出虫卵。从耳静脉注射入幼虫发育与口吞食感染相同。 7.猪后圆线虫在小白鼠、猕猴、山羊、幼狗和豚鼠体中,仅能短时间的发育生存,不能发育为成熟成     

日本血吸虫在离体培养中的产卵和虫卵发育过程的研究

1.本文证明日本血吸虫卵在适合的条件下,在离体培养中亦能发育至成熟,并孵出毛蚴,而并不一定需要经过一个宿主组织内发育阶段。日本血吸虫卵在离体培养中自产出到完全发育成熟所需的时间最少为12—13天。 2.日本血吸虫卵在离体培养中发育至成熟的必需条件为:培养液中必需有血清及红血细胞的存在,其中尤以红血细胞的存在更为重要。 3.本文应用相比差显微镜初步但较系统地观察和描写了血吸虫卵细胞的分裂发育过程。证明血吸虫卵细胞的分裂过程亦属不等裂的类型,与其它吸虫卵细胞分裂发育的过程类似。为了便于统计,本文把血吸虫卵的发育过程分为以下四个时期:1)单细胞期。2)细胞分裂期。3)胚胎发育期。4)毛蚴成熟期。本文并对发育各期的出现时间及增减趋势进行了初步观察。 4.本文证明凡是在宿主体内曾经合抱过的发育成熟的血吸虫雌虫,不论它在离体培养时是否处于合抱状态,雌虫均能产卵。其所产出的虫卵数量的多少与所用培养液的种类及雌虫在培养液中的存活时间有关。同时亦证明了血吸虫在离体培养的环境中不仅能将其子宫中已形成的虫卵排出,而且还有新的虫卵的形成。其产卵数量和培养时间有关,其产卵高峰期为培养后的第2—7天。     

电刺激家兔卵母细胞孤雌活化的研究

采用电刺激使家兔卵母细胞孤雌活化,并对电刺激参数和电刺激介质进行选择,发现在电刺激电压为1.5kV/cm,脉冲持续时间为80us,电刺激3次,电刺激介质为M16的条件下,兔卵母细胞孤雌活化效果最好,可使95％的兔卵母细胞激活,体外培养有89.5％的激活卵发育到正常的8—16细胞期,12％的激活卵发育到囊胚,在体内培养的条件下,26％的激活卵发育到囊胚。采用不同的电刺激介质,卵母细胞最佳激活条件和发育情况不同,表明电刺激介质中Ca~(++)和Mg~(++)浓度似乎与卵母细胞的孤雌活化和维持早期发育有关。家兔在注射LH后17—19小时取卵,电刺激效果最好,激活卵在含细胞松弛素B的培养基中培养3小时,电镜观察已有原核形成,细胞膜附近的细胞器向中央移动。光镜检查表明:约有85％的激活卵具有二个原核,约15％的激活卵有四个原核。     

棉铃虫感染中华卵索线虫后血淋巴酚氧化酶活性的变化及其分离纯化

研究了中华卵索线虫Ovomermis sinensis感染棉铃虫Helicoverpa armigera幼虫后宿主体内酚氧化酶活性的变化。研究结果表明,在感染后的第1天,中华卵索线虫的侵入引起酚氧化酶活性的增加,感染组酶活性是同期对照组的1.12倍; 但在随后的寄生期间,中华卵索线虫抑制了宿主的酚氧化酶活性,其中以第5天的抑制最为强烈： 同期对照组酶活性是感染组的1.52倍。对酚氧化酶进行了初步的分离纯化,纯化倍数为41.5倍,酶得率为12.7%,比活力为4 030.6 U/mg。     

Potentialities of mermithid nematodes for the biocontrol of blackflies (Diptera: Simuliidae)--a review

Mermithids comprise a family of nematodes which invariably kill/sterilize their insect host(s). These nematodes have considerable potential as biocontrol agents of agricultural insect pests and medically important insect vectors. More specifically, mermithid nematodes appear to regulate natural population of blackflies. The taxonomy of the Mermithidae has been only partially evaluated and the taxonomic status of many representatives is uncertain. At least three mermithid genera and species parasitize North American blackflies, although a more varied mermithid fauna probably exists. The host specificity of mermithid parasites of simuliids is variable, but these nematodes do not appear to infect other stream fauna. The sporadic distribution of mermithid parasites of simuliids among potential biotopes may be associated with a relatively inefficient mode of dispersal for such nematodes. Detailed information is lacking concerning stages in the life cycles of these pathogens and their synchronization with the simuliid host. Mermithids cause pathogenic effects upon several blackfly tissues, although no information is available concerning physiological manifestations of mermithid parasitism in blackflies. A brief review of the present state of knowledge of simuliid taxonomy and bionomics is presented. The physiology of blackflies and their mermithid parasites has been largely ignored. The potentialities of mermithid nematodes for the biocontrol of blackflies are assessed and a feasible research programme presented, in relation to the present state of knowledge of mermithid-simuliid interrelationships and related areas of insect nematology.     

Heydenius dominicus n. sp. (Nematoda: Mermithidae), a Fossil Parasite from the Dominican Republic

Heydenius dominicus n. sp. is described as a new species of fossil mermithid nematode from Dominican Republic amber. The species is represented by two specimens of parasitic juveniles that left their insect host and became embedded in the resin. The nematodes are associated with an adult male limoniid (Diptera: Limoniidae) and an adult female mosquito (Diptera: Culicidae). The parasites are thought to have emerged from the mosquito host. This is the first report of a fossil mermithid from a Neotropical area.     

Insect protein digestion improves purity of Steinernema carpocapsae in vitro culture and reduces culture period

Insect protein, used for in vitro culture media for entomopathogenic nematode, produces nematodes of high quality. However, the time-consuming culture and poor purity of nematodes hinder the commercial application of insect protein media. We show that hydrolyzed insect protein improves nematode purity in in vitro culture. The results revealed that nematode purity was increased by more than 90 %, and the culture period was reduced by 6 days. Estimated economic efficiency of using hydrolyzed insect protein medium was increased by 44.25 % over that obtained with non-hydrolyzed insect medium.     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

Heydenius araneus n.sp. (Nematoda: Mermithidae), a parasite of a fossil spider, with an examination of helminths from extant spiders (Arachnida: Araneae)

Abstract. A mermithid, a parasite of a spider (Araneae: Thomisidae) in Baltic amber (40 mya), is described as Heydenius araneus n.sp. (Nematoda: Mermithidae) and represents the first fossil record of a nematode parasite of an arachnid. After a critical examination of reports of naturally occurring helminths of extant spiders, I conclude that although mermithid parasitism is well established in this host group, previous reports of hairworm parasites of spiders are "nomina dubia," putative records, or refer to mermithid nematodes.     

Mermithid Nematodes: Physiological Relationships with their Insect Hosts

This paper assesses our state of knowledge of physiological processes involved in the relationships between insects and their mermithid nematode parasites. Three major components of the host-parasite relationship(s) are reviewed: effects of mermithids on host physiology, effects of host physiology on mermithids, and the physiology of the nematodes themselves. Mermithids induce an array of changes in host physiology, and the effects on host metabolism and endocrinology are discussed at some length. Few studies have been done to ascertain the effects of the host on the parasites from a physiological standpoint. Whereas host immunity mechanisms against mermithids have been described at the ultrastructural level, the physiological basis of such responses is not known. Mermithids are atypical nematodes, both structurally and physiologically. In the absence of a functional gut, nutrients are absorbed across the outer cuticle and stored in a trophosome. The transcuticular mode of feeding, storage within the trophosome, and metabolism of storage products are discussed. The usefulness of physiological information toward expediting in vitro culture of these nematodes is discussed, and problems that need to be addressed are defined.     

Possible commercial formulations of insect-parasitic nematodes

At least two experimental nematode biological insecticide formulations are currently being produced in small quantities around the world, These formulations are efficacious and people are willing to pay money for them. Advantages for the use of these parasites can be: nonpolluting, self-perpetuating, safe, and not harmful to beneficial organisms. The disadvantages are: relatively short shelf-life, acceptability, moisture, price, and dispersal techniques. One of the preparations is a nematode that vectors a bacterium. It can be produced at the rate of 100 × 10⁶ infective nematodes/2 liter container at a cost of 2 cents/10⁶. It can kill over 1000 species of insects. The other product kills over 60 species of mosquitoes and has been sold as mermithid nematode eggs in moist sand. This mosquito parasite can be established in the new site and self-perpetuate to suppress mosquito parasite can be established in the new site and self-perpetuate to suppress mosquito populations. It kills the mosquito before pupating. Increased research activity, using these parasites in the last ten years, has stimulated interest in these organisms for pest insect control.     

Bioassays for comparative infectivity of mermithid nematodes (Romanomermis iyengari,Romanomermis culicivorax and Strelkovimermis spiculatus) for culicine mosquito larvae

Two improved bioassays were developed to establish infectivity baselines for selection experiments using mermithid nematode variants. Comparative infectivity of Romanomermis iyengari, Romanomermis culicivorax and Strelkovimermis spiculatus using larvae of three mosquito spp. Aedes sierrensis, Aedes aegypti and Culex pipiens were evaluated with “plate” and “tray” bioassays at selected intensity of infections. Using the “plate” bioassay, single mosquito larvae were immersed in 2 ml of water within individual depressions of 12-well, polystyrene tissue culture plates. One, three, or five preparasitic juveniles (J2) were added to each well. In the “tray” bioassay, polyethylene trays containing 500 ml water and 100 mosquito larvae were exposed to 500 (5:1, nematode:insect host) or 1000 (10:1) J2s. Percentage infection (PINF, infectivity) and intensity of infection (IINF, #nematodes per infected larvae) number were determined only after emergence of post-parasitic J3 juveniles. Under the bioassay conditions, all three species of nematodes resulted in infections in all mosquito hosts, but R. iyengari exhibited better effectiveness in the parasitism of mosquito larvae. The three species of mosquitoes presented high levels of susceptibility to each of the three species of nematodes, but in general Cx. pipiens and Ae. sierrensis were slightly more susceptible than Ae. aegypti. The “plate” bioassay was more efficient in measurement of infectivity of the mermithid species and in establishing baseline characteristics for these mosquito-parasitic nematodes. The “tray” bioassay was an effective bioassay for large cohorts of both infective juveniles and host larvae and, potential for field interactions.     

Laboratory Culture and Life History of Heleidomermis magnapapula in Its Host, Culicoides variipennis (Diptera: Ceratopogonidae)

The mermithid parasite Heleidomermis magnapapula was maintained in larvae of the midge Culicoides variipennis for 20 months in enamel pans containing nutrient-rich water and polyester pads as a substrate. Inseminated female mermithids were introduced to the pad surface when the host was in the late second or early third-instar. Host larvae were harvested from the pans 9 days after exposure and held in tap water for nematode emergence. Preparasite yield was positively correlated with female nematode size and averaged 1,267 preparasites/female. Male and female nematodes emerged an average of 12.2 and 13.4 days after host exposure, respectively. Supplemental host food (Panagrellus) during the final days of parasitism did not alter time of emergence. Parasites emerging singly were 64% females, whereas superparasitized hosts yielded males (up to nine/host). Nematode carryover into the adult midge normally occurred at a level of 0.5-2.5%. Parasite load (nematodes/ parasitized individual) in midge adults was lower than that of larvae from the same cohort, and adult midges were more likely to harbor female parasites. Exposure of fourth-instar host larvae resulted in higher levels of adult parasitism (up to 17%).