In vitro liquid culture and optimization of Steinernema jeffreyense using shake flasks Murray D. Dunn . Prasanna D. Belur . Antoinette P. Malan Received: 31 May 2019 / Accepted: 11 October 2019 Ó International Organization for Biological Control (IOBC) 2019 Abstract Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are efficient biological control agents against impor- tant insect pests. In vitro liquid culture production technology is a key factor in the success of imple- menting EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate, Steinernema jeffreyense, in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria, Xenorhabdus khoisa- nae. After the successful in vitro production of S. jeffreyense-inoculum, different parameters for opti- mizing infective juvenile (IJ) recovery (developmen- tal step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml -1 after 14 days, in compar- ison to 75% and 99,875 IJs ml -1 respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml -1 ), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo. Keywords Entomopathogenic nematode Á In vitro culture Á Pathogenicity Á Shake flask Á Steinernema jeffreyense Introduction Entomopathogenic nematodes (EPNs) of the genus Steinernema (Rhabditida: Steinernematidae) have a symbiotic relationship with bacteria of the genus Xenorhabdus (Boemare et al. 1993; Ehlers et al. 1988). This bacto-helminthic complex is a safe biological control agent (Ehlers and Hokkanen 1996) and has been widely studied as a biological control agent against a wide range of insects (Kaya and Gaugler Handling Editor: Ralf Ehlers M. D. Dunn Á A. P. Malan (&) Department of Conservation Ecology and Entomology, Faculty of AgriSciences, Stellenbosch University, Stellenbosch, South Africa e-mail: apm@sun.ac.za P. D. Belur Department of Chemical Engineering, National Institute of Technology Karnataka Surathkal, Mangalore 575025, India 123 BioControl https://doi.org/10.1007/s10526-019-09977-7