Gouge Dawn H, Snyder Jennifer L
University of Arizona, MAC, 37860 West Smith-Enke Road, Phoenix, AZ 85239, USA.
J Invertebr Pathol. 2006 Mar;91(3):147-57. doi: 10.1016/j.jip.2005.12.003. Epub 2006 Jan 31.
Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.
将大蜡螟（Galleria mellonella L.）幼虫用三种斯氏线虫属（Steinernema spp.）线虫（七个菌株）或三种异小杆线虫属（Heterorhabditis spp.）线虫（三个菌株）进行感染。将感染后的幼虫在22、27和32摄氏度下孵育。在48小时内，每隔6小时对幼虫进行背部解剖。收集血淋巴并划线接种于胰蛋白胨大豆琼脂平板上。从感染昆虫尸体中鉴定出几种非共生细菌物种：杰氏肠杆菌（Enterobacter gergoviae）、弧菌属（Vibrio spp.）、C型荧光假单胞菌（Pseudomonas fluorescens type C）、粘质沙雷氏菌（Serratia marcescens）、弗氏柠檬酸杆菌（Citrobacter freundii）和变形斑沙雷氏菌（Serratia proteomaculans）。在孵育18 - 24小时时，几乎只出现与线虫相关的共生菌。其他相关细菌通常在18 - 24小时这个时间段之外出现。对斯氏夜蛾线虫（Steinernema feltiae）（Filipjev）（27）、里奥布拉夫斯氏线虫（Steinernema riobrave）卡瓦尼利亚斯、波伊纳尔和劳尔斯顿（Oscar）或小卷蛾斯氏线虫（Steinernema carpocapsae）（Weiser）（Kapow）的感染性幼虫不进行处理，或用硫柳汞进行表面消毒，然后在无菌条件下吸取到胰蛋白胨大豆琼脂平板上。与从感染的大蜡螟中分离出的细菌种类范围较窄相比，用这种方法鉴定出了几种额外的相关细菌物种。从未经消毒或表面消毒的线虫中鉴定出的细菌种类没有差异，这表明鉴定出 的细菌要么源自线虫内部，要么源自第二和第三阶段幼虫表皮之间。从田间样本中分离出斯氏夜蛾线虫（Cowles）、小卷蛾斯氏线虫（Cowles）和食菌异小杆线虫（H. bacteriophora）波伊纳尔（Cowles）的感染性幼虫。线虫用次氯酸钠进行表面消毒后，与大蜡螟血淋巴混合，然后吸取到Biolog BUG（含血液）琼脂上。从有限的可用样本中仅分离出了相关的共生菌。然后将线虫在实验室中培养14个月（在大蜡螟中传代培养7次）。之后可以从斯氏线虫属线虫中分离出其他肠杆菌科细菌，包括粘质沙雷氏菌、沙门氏菌属和杰氏肠杆菌，这表明线虫在实验室培养中能够与其他细菌建立联系。
