Entomopathogenic nematodes (EPNs) suffer from trait deterioration, a potential problem when these antagonists are transferred into artificial environments for mass production. In order to improve beneficial traits of EPN genetic selection and hybridization has been successfully carried out. Should these selected strains deteriorate during serial culturing the efforts would be in vain. Inbreeding might offer a possibility to stabilize traits but can also result in inbreeding depression. This study attempted to increase heat tolerance of Heterorhabditis bacteriophora by selective breeding for seven cycles either with nematodes propagated in vivo in Galleria mellonella or with in vitro propagated nematodes which were exposed to heat stress in monoxenic liquid culture. After release of the selection pressure, the tolerance was monitored over 15 additional reproductive cycles to compare the stability of the trait. Virulence of the selected strains was assessed to check for negative tradeoff effects. Heat tolerance was successfully increased in vivo (from 39.03 to 40.85°C) and in vitro (from 39 to 40°C) propagated H. bacteriophora, but could only be maintained in populations which were serially reared in liquid culture. When H. bacteriophora is cultured in vivo, reproduction by cross fertilization is possible. In in vitro culture male and female cannot mate and reproduction is solely by self-fertilizing hermaphrodite resulting in homozygous inbred lines. Trait deterioration seems to be restricted to in vivo propagated H. bacteriophora, whereas monoxenic liquid cultures handling large numbers of inbred lines provided genetically stable and virulent nematode populations. Selection using liquid culture technology is thus superior over in vivo propagation to sustain beneficial traits in H. bacteriophora not only for selective breeding but also for mass production.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is used in biological plant protection to control pest insects. In the past, several attempts targeted at an enhancement of the desiccation tolerance of EPN by genetic selection in order to improve their storage stability. The subsequent loss of improved beneficial traits after release of selection pressure has often been reported. In order to stabilize progress of selective breeding, selection during liquid culturing was tested against propagation in host insects. After release of the selection pressure, the tolerance was monitored over additional reproductive cycles in vivo and in vitro to compare the stability of the trait. Furthermore, it was tested whether the virulence of the selected strains would be impaired. Exposure to desiccation stress prior to propagation, in vivo or in vitro, both resulted in increasing desiccation tolerance. When selection pressure was released, the gained tolerance was lost again during in vivo production, whereas the tolerance was maintained at a high level when EPNs were cultured in liquid culture. In Heterorhabditis sp., liquid culture conditions produce highly homozygous, genetically stable inbred lines. The investigation provides easily applicable methods to improve and stabilize beneficial traits of heterorhabditid EPNs through selective breeding in liquid culture. Compared to nematodes from in vivo propagation, production in liquid media yielded EPN of higher virulence.

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