Monitoring life cycle stages of axenic cultures of in vitro produced Oscheius L8 MCB in semi-solid state and liquid state and comparative efficacy studies between in vitro and in vivo nematode production against Galleria mellonella

Abstract

The emergence of entomopathogenic nematodes as biocontrol agents of insect pests has caused a shift of focus towards the use of in vitro methods for commercial production of these organisms for use in agricultural settings. In this study, Oscheius L8 MCB, an insecticidal nematode isolated from Brits in the North West Province of South Africa, was produced in vitro via axenic means in liquid and semi-solid states. Light microscopy was used to monitor and enumerate life cycle stages of this isolate, with special focus given to the yield of the insecticidal Infective juvenile (IJ) stage. Comparative dose–response assays were used to determine the efficacy of in vitro production of Oscheius L8 MCB versus in vivo production against Galleria mellonella. In vitro produced Oscheius L8 MCB consisted of four juvenile stages, with one of the juvenile stages being the non-feeding IJ stage. Adults stages comprised amphimictic males and females, and female hermaphrodites. The semi-solid state produced IJs in three yield peaks of 2.33 × 104 IJs/ml (SEM ± 1922 IJs/ml), 1.93 × 104 IJs/ml (SEM ± 2455 IJs/ml) and 1.92 × 104 IJs/ml (SEM ± 3542 IJs/ml) at days 6, 9 and 11 respectively. In contrast, the liquid state produced a single peak of 3.6 × 104 IJs/ml (SEM ± 1178 IJs/ml) at day 12. In vivo produced Oscheius L8 MCB established at least 50% G. mellonella larvae mortality 24 h post infection, at an infective IJ concentration of 25 IJ/100 µl compared to 0% (semi-solid) and 16.67% (liquid) larvae mortality established by in vitro produced Oscheius L8 MCB at the same concentration after 24 h. The number of IJs emerging from a larvae cadaver may be indicative of field coverage, efficacy, and field persistence. Larvae cadaver infected with in vivo produced IJs produced a maximum of 8.36 × 104 IJs/larvae (SEM ± 3900 IJs/larvae) in comparison to larvae cadaver infected with in vitro produced IJs, which produced maximums of 5.50 × 104 IJs/larvae (SEM ± 2527 IJs/larvae) and 4.44 × 104 IJs/larvae (SEM ± 1803 IJs/larvae) for liquid and semi-solid states respectively. The mortality and IJ emergence in larvae infected with semi-solid state and liquid state Oscheius L8 MCB varied according to infective IJ concentration. However, in conclusion, axenic in vitro culturing of Oscheius L8 MCB may reduce its efficacy against insect hosts.