Establishment of Monoxenic Inocula for Scaling Up in Vitro Cultures of the Entomopathogenic Nematodes Steinernema Spp. and Heterorhabditis Spp

Abstract

Mass production of entomopathogenic nematode-bacterium complexes (Steinernema spp./Heterorhabditis spp. - Xenorhabdus spp.) for biological control of insect pests require scaling up of truly monoxenic cultures. Attempts to establish monoxenic cultures of Steinernema feltiae by addition of surface-sterilized dauer juveniles to bacterial lawns of Xenorhabdus boaienii always resulted in polyxenicity of the cultures. Therefore, methods were developed to obtain bacteria-free first stage nematode juveniles either by surface sterilization of nematode eggs or by alkaline lysis of gravid females. The procedures described allow control of the success of the axenization before monoxenic cultures are established. All tested Steinernema spp. and strains (18) reproduced under axenic conditions on untreated sterile rat kidney. The axenic culture of S. carpocapsae reached a density of over 18,000 nematodes/ml in a spinner flask filled with 200 ml liquid medium. Monoxenic cultures of steinernematids were obtained by addition of axenic nematodes to cultures of their specific bacterial symbiont. Attempts to initiate the reproduction of Heterorhabditis spp. in axenic cultures by addition of different growth factors to hatched juveniles were unsuccessful. Hence, monoxenic cultures were established by the combination of bacteria-free first stage juveniles with their specific symbiotic bacterium. They were successful with all tested strains of H. bacteriophora. North-West European Heterorhabditis strains could only be axenized and subsequently cultured under monoxenic conditions when their eggs were obtained by alkaline lysis. Hatching of juveniles after alkaline lysis of Heterorhabditis sp. (strain HSH) and H. bacteriophora (strain HD01) was not influenced by the liquid media used but depended on the state of development of the gravid hermaphrodites on the day of egg isolation. The survival of the juveniles was significantly reduced when the osmolarity of the medium was low. To establish monoxenic liquid cultures of both strains, bacterial suspensions in different growth media were added to the axenic first stage juveniles. Strain HSH developed and reproduced in only one medium and HD01 in 3 of 4 media tested. Under liquid conditions monoxenic cultures of Heterorhabditis spp. were successful only when the juveniles developed to hermaphrodites.