Ecdysteroids in the pycnogonid Pycnogonum litorale (STROM) (Arthropoda, Pantopoda)

In order to study the environmental conditions and the endocrine mechanism which induce neotenous characteristics, the fine structure of the thyroid glands of larvae of the salamander Hynobius retardatus were examined. The thyroid glands of larvae exposed to a low temperature (4°) were compared with those of larvae exposed to a higher temperature (20°). At 4°, the follicular lumen was large and its contents of low electron density; the epithelial cells were flat or cuboidal. In contrast, the 20° larvae showed a small dense lumen and tall cylindrical epithelial cells, which may indicate a higher level of activity. No apical vesicles were found in the epithelial cells of the 4° larvae, although they were present in 20° larvae. On the other hand, in 4° larvae the chromatin in the nuclei was scattered, while in 20° larvae it was condensed. In addition, large dense granules occupied 2.4% of the epithelial cytoplasm in 4° larvae and as much as 7.3% in 20° larvae. The structural features observed in larvae exposed to the cold resembled those reported for thyroid glands made inactive by a lack of thyrotropin.     

Dipetalonema viteae (Filarioidea): development of the infective larvae in vitro

The development of Dipetalonema viteae third stage larvae was attempted in vitro. A monophasic culture system consisting of BHK-21 medium supplemented with 10% tryptose-phosphate broth and 15% fresh jird serum allowed the growth of previously in vivo triggered larvae, but not of those isolated from ticks. The larvae could complete their third moult and grew on a fourth stage larvae up to 5 mm. This development was comparable to that observed in vivo. The presence of an irradiated hamster kidney cell feeder layer could replace an in vivo trigger. The development of the infective larvae took place up to the third moult. Some larvae completed the moult to the fourth stage and reached a maximum length of 3 mm after 14 days. These culture systems offer the possibility to study in vitro the complete development of the infective larvae to fourth stage larvae.     

Antibody-mediated in-vivo cytotoxicity to Trichinella spiralis newborn larvae in immune rats

The antibody-dependent cell-mediated larvicidal response of AO rats against Trichinella spiralis newborn larvae was studied in vivo. Rats were immunized with 2000-3000 muscle larvae orally and then challenged 6-20 days later with 10,000-20,000 newborn larvae intraperitoneally. Newborn larvae recovery from the peritoneal cavity decreased significantly and was accompanied by cuticular cell adherence and killing of newborn larvae by day 9 of infection. Similar effects were observed when newborn larvae were incubated with blood obtained from immunized rats. The cell adherence and larvicidal responses reached their peak by day 16 of the primary infection. Passive transfer experiments demonstrated that newborn larvae infectivity was substantially impaired once cell adherence occurred. Cuticular adherence took place in vitro only when immune serum was added to the incubation medium. Complete destruction of newborn larvae in vivo after passive transfer, as measured by muscle larvae burden was only evident after exposure to both immune serum and immune cells, not to either alone. Non-specific stimulation of the peritoneal cavity with a sterile intestinal infection failed to induce cuticular adherence or larval killing in these rats. We conclude that a stage-specific antibody-dependent cell-mediated larvicidal response is rapidly generated in vivo after the host is exposed to newborn larvae. It is a systemic response which impairs the infectivity of newborn larvae and can destroy them before they reach muscle tissue.     

旋毛虫成虫、新生幼虫和肌幼虫抗原的保护性免疫研究

目的：比较旋毛虫成虫抗原和肌幼虫抗原接种小鼠诱导产生的保护性免疫。方法:检查肠道成虫、肌幼虫和血液中嗜酸性细胞数;用ELISA测血清中抗旋毛虫肌幼虫IgG抗体滴度。结果：成虫、新生幼虫和肌幼虫抗 免疫组的成虫减虫率分别为82.19％、72.31％和42.88％;肌幼虫减虫率分别为68.83％、78.19％和51.96％。血清IgG抗体滴度明显升高,成虫、新生幼虫和肌幼虫抗原免疫组的GMRT分别为对照组的7.46倍、5.28倍和4.92倍。血液嗜酸性粒细胞明显增多。结论：旋毛虫成虫抗原、新生幼虫抗原均为激发特异性液免疫和细胞免疫,诱导小鼠对攻击感染产生保护性。其中,成虫抗原和新生幼虫抗原显示出更好的免疫原性,而成虫抗原有可能成为较理想的免疫疫苗来源。     

Oviposition behavior of female Anopheles gambiae in western Kenya inferred from microsatellite markers

Anopheles gambiae females in a relatively isolated hut and all larvae from larval habitats within 100 m of the hut were collected in August 2001 in western Kenya. Among 42 aquatic habitats, 16 had A. gambiae larvae. Two hundred fifty larvae and 58 adults were genotyped using nine microsatellite markers to infer sibling relationship between the larvae and maternity between the females and larvae. The pairwise genetic relatedness of A. gambiae larvae per habitat ranged from -0.4112 to 0.9375, indicating that full siblings, half siblings, and genetically unrelated individuals presented at those habitats with multiple larvae. From a likelihood analysis, it was estimated that 56.6% of females had larvae in multiple habitats. These results substantiate that one A. gambiae female uses multiple breeding sites for oviposition, and thus, average genetic relatedness for breeding sites with high larval populations tends to be low.     

Predatory activity of Rhantus sikkimensis and larvae of Toxorhynchites splendens on mosquito larvae in Darjeeling, India

BACKGROUND AND OBJECTIVES: Predation potential of the dytiscid beetle, Rhantus sikkimensis Regimbart 1899 and the larvae of Toxorhynchites splendens Wiedemann 1819 occurring along with the larval stages of the mosquitoes in the annual lentic water bodies of Darjeeling was evaluated using the larvae of Culex quinquefasciatus Say 1823 as preys, in the laboratory under simulated natural conditions. METHODS: Field collected R. sikkimensis and larvae of Tx. splendens were offered IV instar larvae of Cx. quinquefasciatus to observe the rate of predation, at varying prey and predator densities. Based on the data obtained on the predation for a period of three consecutive days, two indices of predation, predatory impact (PI) and clearance rate (CR) values were estimated, and compared between the predator species. RESULTS: The rate of predation of IV instar Cx. quinquefasciatus larvae by R. sikkimensis ranged between 21.56 and 86.89 larvae per day, depending on the prey and predator densities. The PI value remained between 18.67 and 35.33 larvae/day depending on prey densities, while the CR ranged between 2.21 and 2.23 larvae litres/day/predator. Compared to these, the Tx. splendens larvae consumed the prey larvae at the rate of 0.67 to 34.22 larvae per day, depending on the prey and predator densities. The PI value ranged between 7.67 and 11.33 larvae/day, and the CR value ranged between 1.41 and 1.76 larvae litres/day/predator. The rate of predation, CR values and PI values of R. sikkimensis and Tx. splendens varied significantly. INTERPRETATION AND CONCLUSION: Both the predators R. sikkimensis and larvae of Tx. splendens can consume a good number of mosquito larvae, though the rate of consumption between the two predators vary owing to the difference in the life history traits and features. It can be assumed that these predators play an important role in larval population regulation of mosquitoes and thereby impart an effect on species composition and interactions in the aquatic insect communities of Darjeeling Hills, India.     

A behavioral screen for isolating zebrafish mutants with visual system defects.

Optokinetic and phototactic behaviors of zebrafish larvae were examined for their usefulness in screening for recessive defects in the visual system. The optokinetic response can be reliably and rapidly detected in 5-day larvae, whereas the phototactic response of larvae is variable and not robust enough to be useful for screening. We therefore measured optokinetic responses of mutagenized larvae as a genetic screen for visual system defects. Third-generation larvae, representing 266 mutagenized genomes, were examined for abnormal optokinetic responses. Eighteen optokinetic-defective mutants were identified and two mutants that did not show obvious morphological defects, no optokinetic response a (noa) and partial optokinetic response a (poa), were studied further. We recorded the electroretinogram (ERG) to determine whether these two mutations affect the retina. The b-wave of noa larvae was grossly abnormal, being delayed in onset and significantly reduced in amplitude. In contrast, the ERG waveform of poa larvae was normal, although the b-wave was reduced in amplitude in bright light. Histologically, the retinas of noa and poa larvae appeared normal. We conclude that noa larvae have a functional defect in the outer retina, whereas the outer retina of poa larvae is likely to be normal.     

The parasitoid Apanteles kariyai inhibits pupation of its host, Pseudaletia separata, via disruption of prothoracicotropic hormone release

When the parasitoid Apanteles kariyai laid eggs into host Pseudaletia separata larvae, before prothoracicotropic hormone (PTTH) was released in the last instar preparatory to metamorphosis, the host did not pupate and the larvae of the wasps emerged. The ecdysteroid titer of unparasitized intact larvae increased up to 1 microgram/ml 1 day before pupation, whereas the titer of parasitized larvae was maintained at a low level without the surge. Isolated prothoracic glands from intact larvae synthesized much more ecdysone than those of parasitized larvae both in vivo and in vitro. Administration of exogenous PTTH caused the activation of the prothoracic glands seen during parasitization. Injection of 20-hydroxyecdysone (20-HE) into the parasitized larvae caused by host's pupation, but did not affect the development of the wasp larvae. However, the sensitivity of the integument to 20-HE was lower in parasitized than in unparasitized larvae. Injection of a mixture of adult wasp calyx and venom fluids into last instar unparasitized larvae delayed their pupation, suggesting that calyx and venom fluids are factors contributing to disturbance of the normal function of brain-prothoracic gland system. These results show that parasitization inhibits secretion and/or synthesis of PTTH and also delays the larval-pupal commitment of the integument by keeping the ecdysteroid level low.     

旋毛虫肌幼虫体外生存状况实验研究

目的观察旋毛虫肌幼虫在干燥和生理盐水环境中的生存状况。方法90只昆明小鼠被随机分为正常对照组、生理盐水组和干燥组,生理盐水组和干燥组分别再分为4组,共9组,每组10只。正常对照组每鼠经口感染200条收集的肌幼虫。生理盐水组分为4组：①肌幼虫0℃放置10 d,②肌幼虫20℃放置10 d,③肌幼虫0℃放置20 d,④肌幼虫20℃放置20 d;干燥组分组与生理盐水组相同。然后将8组小鼠每鼠经口感染200条处理的肌幼虫。感染后30 d处死小鼠,取膈肌压片镜检,并将全部肌肉人工消化后计数幼虫数。结果在生理盐水和干燥环境中放置10 d的肌幼虫分别感染小鼠后,压片法和人工消化法镜检,感染小鼠的幼虫检出率均为100%;在生理盐水和干燥环境中放置20 d的肌幼虫分别感染小鼠后,两种方法镜检,感染小鼠均未检出幼虫。结论在生理盐水和干燥环境中,肌幼虫能生存较长时间,但不超过20 d。     

Trichinella spiralis: Intestinal expression of systemic stage-specific immunity to newborn larvae

Rats immunized with newborn Trichinella spiralis larvae i.v. were found to confer a specific anti-newborn larvae immunity in the small intestine. In rats immunized with newborn larvae i.v. and then challenged with adult worms intraintestinally, total newborn larvae recovery was reduced by 75-90% in thoracic duct lymph and in hepatic portal vein blood. No newborn larvae were found in the peritoneal fluid of immunized rats. In addition to an absolute reduction in number, larval migration from the small intestine to the thoracic duct was delayed by 6-12 h and migration to the portal vein was inhibited for at least 8 h. The establishment of adult worms in the small intestine and female worm fecundity were not affected by anti-newborn larvae immunity. Identical quantitative effects on newborn larvae migration from the small intestine were achieved by homologous transfer of anti-newborn larvae immune serum i.v. into naive recipient rats.     

Ts21基因在旋毛虫不同期虫体的表达及特性的研究

目的观察旋毛虫Ts21基因在不同发育期虫体的表达及其特性。方法应用RT-PCR检测旋毛虫Ts21基因在成虫、新生幼虫、感染后15d的成囊前幼虫及感染后42d的成囊幼虫(肌幼虫)的转录情况;应用抗Ts21重组蛋白血清通过间接荧光抗体试验(IFA)对成囊前幼虫及肌幼虫冰冻切片抗原进行检测。应用抗Ts21重组蛋白血清对旋毛虫肌幼虫粗(可溶性)抗原及排泄分泌(ES)抗原进行Western-blot分析。结果RT-PCR发现旋毛虫成虫与感染后42d肌幼虫均扩增出一条分子量约540bp的特异性条带(Ts21基因目的条带),而新生幼虫与感染后15d成囊前幼虫则未扩增出目的条带;IFA结果表明抗Ts21重组蛋白血清只与感染后42d成囊幼虫抗原发生阳性反应,而不与感染后15d的成囊前幼虫抗原发生阳性反应。Western-blot结果显示抗Ts21重组蛋白血清只与旋毛虫肌幼虫粗抗原和ES抗原中Mr 21 000的单一蛋白条带发生阳性反应。结论旋毛虫Ts21基因的表达具有期特异性,Ts21蛋白是旋毛虫肌幼虫ES抗原中的一部分。     

Influence of cadmium on the course of intestinal and muscular phases in mice infected with Trichinella spiralis]

At the first experiment 20 Swiss male mice were infected with 100 larvae and 20 mice with 500 larvae T. spiralis per mouse. Two days after infection (d.a.i.) mice orally received 1.5 mg Cd (water solution CdCl2) each. 40 mice were infected only T. spiralis as control. At the second experiment muscle larvae used were isolated from mice (from the first experiment) which received Cd. 20 mice were infected with 100 larvae and 20-with 500 larvae per mouse. Two d.a.i. mice received 1.5 mg Cd. Mice from both experiments were killed at 5, 10, 20 and 42 d.a.i. Total number of adult T. spiralis worms present in the small intestine and muscle larvae were recovered by conventional technique. Results of the first experiment: the number of adult worms and muscle larvae recovered from mice received Cd. were statistically significant lower as in control. Results of the second experiment: the mean number of adult worms in experimental group and in control were the same but the mean number of larvae per gram of mice muscle were significant higher as in control.     

Immediate and cell-mediated reactions in parasitic infections by Anisakis simplex

BACKGROUND: Anisakis simplex is responsible for allergic symptoms after repeated ingestion or contact with parasitized fish. OBJECTIVE: To further analyze type I and IV immunologic mechanisms in a group of patients with allergic reactions to A simplex, we performed prick-by-prick testing with A simplex larvae and patch tests with live, cooked, or frozen larvae of the A simplex parasite. METHODS: Thirty-eight patients underwent skin prick test and radioallergosorbent test with inhalant allergens, foods, and A simplex. Prick-by-prick tests with A simplex and patch tests with live, cooked, and frozen larvae were carried out in 10 patients with evidence of allergy to A simplex. RESULTS: Prick-by-prick testing yielded a positive result in 100% of cases with live larvae and in 70% with cooked and frozen larvae. Patch tests with A simplex were positive in 8 patients for live larvae, in 3 patients for frozen larvae, and in 1 patient for cooked larvae. CONCLUSION: Our data emphasize that A simplex is responsible for both immediate allergic reactions and cell-mediated (delayed) reactions, in particular in subjects with occupational exposure. In addition, our data demonstrate that not only live but also frozen and cooked larvae can induce sensitization. This observation may be explained by differences in the allergenic proteins involved, and further studies will be required to address this possibility.     

The development of normal and gamma-irradiated Litomosoides carinii larvae in cotton rats

The development of infective, stage 3 larvae of Litomosoides carinii irradiated at either 2.7, 5.4, 10.8, 16.2, 21.6 or 40 krads in a cobalt 60 source was compared with that of normal larvae following injection into cotton rats. Worm recovery decreased as the radiation dose increased, male worms being more affected than female worms. Worms developing from irradiated larvae were shorter than normal and at 16.2 krads and above they failed to develop into adults despite reaching the pleural cavity and moulting to stage 4 larvae. Development had ceased by 35 and 20 days post infection for larvae irradiated at 21.6 and 40 krads respectively. 10 krads and below allowed larvae to moult into adults but they were stunted and retarded developmentally. Microfilariae were absent from the pleural cavity and peripheral circulation of all cotton rats injected with irradiated larvae.     

不同旋毛虫粗抗原对BALB/c小鼠体内SP2/0肿瘤抑制作用观察

目的研究不同旋毛虫抗原对BALB／c小鼠体内的SP2／0骨髓瘤的抑制作用。方法对BALB／c小鼠经腹腔和肌肉注射不同抗原，3d后对小鼠荷瘤，30d后观察小鼠体内肿瘤的大小，检测小鼠机体的免疫状态，评价不同旋毛虫抗原对小鼠体内的肿瘤的抑制作用。结果腹腔注射肌幼虫可溶性粗抗原、成虫可溶性粗抗原和新生幼虫可溶性粗抗原组的抑瘤效果略好于肌肉注射组。肌幼虫ES抗原组的抑瘤效果最好，成虫ES抗原组次之，但比3种虫体抗原组的抑瘤效果好，3种虫体粗抗原组的抑瘤效果间没有明显差别。结论不同的旋毛虫抗原都有一定的抑制肿瘤的作用，腹腔注射肌幼虫ES抗原能够很好的抑制小鼠体内肿瘤的生长。     

Cryopreservation of third-stage larvae of Brugia malayi and Dipetalonema viteae

Methods are described for the cryopreservation of third-stage larvae of Brugia malayi. Optimum conditions utilized larvae free from the mosquito host frozen at the rate of -1 degree or -0.8 degrees C per min in medium containing 9% dimethyl sulfoxide and 0.004 M polyvinylpyrrolidone. Nonfrozen or thawed larvae were inoculated intraperitoneally into jirds (Meriones unguiculatus), the thawed larvae after cryogenic storage for 5-378 days. In general, the percentage of adult worms recovered at necropsy was comparable between the two groups and ranged from a mean of 6-9% of the larval inoculum. In addition, three of four patas monkeys (Erythrocebus patas) inoculated with thawed B. malayi larvae developed patent infections. The cryopreservation of third-stage larvae of Dipetalonema viteae also is discussed.     

阿苯达唑和氟苯达唑对旋毛虫作用的形态学、组织学及组织化学的观察

应用形态学、组织学及组织化学方法,观察比较阿苯达唑、氟苯达唑对旋毛虫幼虫的杀虫作用机制。实验结果表明两种药物作用机制相同,均使旋毛虫囊包破坏,虫体破损,囊包炎性细胞浸润,终至虫体被完全机化;对糖代谢有明显干扰作用,对酸性磷酸酶、碱性磷酸酶活性无变化。     

旋毛虫肌幼虫囊包染色标本制作方法的改进

目的探讨旋毛虫肌幼虫囊包染色标本的制作方法。方法采用醋酸卡红染色法制作旋毛虫肌幼虫囊包标本。结果醋酸卡红染色的标本,囊包轮廓清晰,囊内幼虫透明度及清晰度高,体态自然,立体感较强。结论醋酸卡红染色可用于旋毛虫肌幼虫囊包永久标本的制作。     

Repeated infections of Brugia pahangi in the jird, Meriones unguiculatus.

Male jirds, Meriones unguiculatus, were subcutaneously inoculated in the groin with 1 to 5 doses of infective-stage larvae of Brugia pahangi at weekly or monthly intervals. When a dose of either 25 or 75 larvae or 4 weekly doses of 25 larvae were given, 15-16% of the larvae were recovered as adults approximately 4 to 7 months post inoculation. Only 8-10% of the larvae were recovered if 4 weekly or 5 monthly doses of 75 larvae each were given. After an inoculation of 75 larvae, 25% of the worms were recovered at 30 days. The 30 day-old population consisted of an average of 10 female and 8.8 male worms. Jirds previously inoculated with 4 weekly or 5 monthly doses were challenged with an additional 75 larvae 30 days prior to necropsy. An average of only 4.5 thirty day-old female worms were recovered in these cases, presenting a 55% decrease as compared to the single inoculation situation. There was some decrease in the mean length of female worms in multiply-inoculated jirds, but no difference in the mean lengths of the male worm population from singly or multiply-inoculated jirds was observed. No differences in prepatent periods or in patterns of microfilaraemia were observed in singly or multiply-inoculated jirds.     

Laboratory transmission of lymphatic filariasis by vector mosquitoes

Aedes togoi and Ae. aegypti were used to examine the transmission potential of Brugia pahangi to one of its natural hosts, the domestic cat. Although a larger proportion of microfilariae taken in by Ae. togoi developed into infective larvae, the total number of B. pahangi larvae recovered from a cat exposed to Ae. aegypti was larger than from a cat exposed to Ae. togoi. Factors influencing the transmission dynamics included: development of microfilariae to infective larvae; survival of mosquitoes; willingness to take repeated blood meals; and proportion of infective larvae that egress from mosquitoes during the feeding process. From 19 to 25% of infective larvae were transferred to a susceptible host. The feasibility of using a Brugia-cat model to do comparative vector efficiency studies was demonstrated.