摘要
在研究中华卵索线虫的体外培养方法的同时 ,对其在不同培养基中的生长发育情况进行了观察。结果表明 :以培养基TC 199加 2 0 %热灭活胎牛血清的培养效果较为理想 ,大多数线虫可存活 3个月 ,最大虫体长5 5 1mm、宽 2 0 4 13μm ,其发育程度大致与该种索线虫在宿主粘虫体内寄生 8～ 9天的情况相近。培养期间观察到 2次蜕皮 :第一次蜕皮在卵内 ,第二次在培养 6～ 8天之后。口针消失 ,虫体内滋养物体发育明显 ,尾部附器已经形成 ,没有观察到生殖原基的发育。
The insect parasitic nematode, Ovomermis sinensis Chen et al ., 1991 has the potential for the biological control of a range of insect pests due to many insect sepecies are susceptible to this nematode. For example Leucania separata, Heliothis armigera, Prodenia litura, Spodoptera exigua, Plutella xylostella, Pieris rapae, Agrotis ypsilon, Euxoa segetum etc. The insect host invariably dies when the juvenile nematode completes its parasitic development and exits from the host's hemocoel. There is an important significance for studying the mass cultivation of O. sinensis for field trials. This paper describes the techniques of in vitro cultivation of O. sinensis. Gravid females of O. sinensis collected from sand were washed three times with an antibiotic solution of 200 U penicillin/ml and 200 μg/ml streptomycin/ml per ml solution. Surface washed gravid female were placed in 25 ml culture tubes containing 1～10 ml deionized water, 100 U penicillin/ml, and 100 μg streptomycin /ml for oviposition. Eggs were collected and their surface were sterilized by immersing them for 5～10 min in 0 13%～0 5% sodium hypochlorite solution and rinsing four times with sterile distilled water. Surface sterilized eggs were incubated at 26±1℃ in the culture media. The preparasites which emerged from those eggs were utilized for culture work. The four media were tested and they were TC 199, TC 199+MK, TC 100 or Grace supplemented with 20% heat inactivated fetal bovine serum respectively. The pH of the media were adjusted to pH6 8～7 0. The culture medium was changed or replenished every 3～6 days by withdrawing the supernatant and replacing it with a portion of fresh medium. In vitro growth of O. sinensis parasites was monitored in TC 199 supplemented with 20% heat inactivated fetal bovine serum under differing condition of crowding. Crowded cultures (150～200 parasites per tube) were set up in early days of in vitro (length of nematodes<10 mm), whereas crowed culture (50～70 parasites per tube) and uncrowded cultures (2～7 parasites per tube) were tested in the late days of in vitro cultivation (length of nematodes>10 mm). Cultures were examined daily with an inverted microscope for nematode growth and activity. The results showed that the best growth obtained among the test media was TC 199 supplemented with 20% heat inactivated fetal bovine serum. In this medium, most nematodes can grow for up to three months, reaching a development stage similar to 8～9 day old parasites grown in vivo in the armyworm ( Leucania separata walker). Maximum size of the juveniles was 55 1 mm in length and 204 13 μm in width at the widest point and the results to date have yielded a 20 fold increase in body volume from the preparasitic to the parasitic stage. Two molts were observed in vitro , with the first occurred within the egg, and the second occurred after 6～8 days in culture. The stylet was lost. Trophosome developed prominently, and some of the nematodes developed a caudal appendage. However, no gonadial development was observed. Population density were investigated. In vitro length of nematodes (<10 mm) that had developed in the early days of cultivation under crowed condition was significantly greater than that of uncrowed nematodes. In the late stage of cultivation growth of uncrowded nematodes was greater than that of nematodes of crowded nematodes. The experiment proved that terrestrial mermithid nematode, O. sinensis can live and oviposit in water. The idea that terrestrial mermithid nematode cannot survive in water because lack of O\-2 is not supported.
出处
《动物学报》
SCIE
CAS
CSCD
北大核心
2001年第2期235-239,T001,共6页
ACTA ZOOLOGICA SINICA
基金
国家自然科学基金资助项目!(NO .3 95 70 10 1)&&
关键词
中华卵索线虫
索科
体外培养
Ovomermis sinensis ,Mermithid, In vitro cultivation
