Abstract
The objective of this research is to develop fermentation methodology for the production of the biocontrol agent <i>Heterorhabditis bacteriophora</i>. Deployment of this organism will reduce the use of chemical insecticides which threaten the environment. This study shows how to produce the entomopathogenic nematode (EPN) <i>Heterorhabditis bacteriophora</i> and its bacterial symbiont <i>Photorhabdus luminescens</i> utilizing an <i>in vitro</i>, monoxenic liquid culture. EPNs were cultured in three different bioreactor working volumes of 1.5, 4 and 7 liters with initial nematode inoculation concentrations of approximately 2x10<sup>3</sup>/mL. Liquid nematode media was conditioned with the bacterial symbiont 24 hours prior to nematode inoculation. Within three days after inoculation, infective juveniles (IJs) developed into self-fertilizing hermaphrodites and eventually produced IJ offspring. Maximum nematode densities were obtained seven days post-nematode inoculation. All three working volumes (1.5, 4 and 7 liters) produced final yields of 4.6x10<sup>4</sup> ± 2000 IJs/mL, 4.2x10<sup>4</sup> ± 2200 IJs/mL and 3.9x10<sup>4</sup> ± 2000 IJs/mL, respectively. <i>In vitro</i> scale-up technology can be further optimized for production of this biocontrol agent by improving media formulation, process parameters, bioreactor design and inoculation times that will maximize nematode yield.
Highlights
Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis have long been recognized as effective biocontrol agents [1, 2]
The last instars of the Lepidopteron insect Galleria mellonella obtained from Carolina Biological Supply Company (Burlington, NC USA) and the nematode Heterorhabditis bacteriophora obtained from Gardening Zone (Camearillo, CA USA) were used throughout this study
Adults which had not laid eggs into the production media commensed intra-uterine birth causing maternal death, where all eggs developed into infective juveniles (IJs) within the maternal female adult body
Summary
Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis have long been recognized as effective biocontrol agents [1, 2] These microscopic round worms may be used to control a wide variety of economically important agricultural pests and act as potent bioinsecticides in an effectual, environmentally suitable approach. Their potency relies upon a symbiotic partnership between a fatal insect pathogen bacterium and the hostseeking nematode [3]. The bacterium Photorhabdus luminescens is symbiotically associated with Heterorhabditis bacteriophora [4] Upon penetration of these EPN into a host insect body via the insect’s mouth, anus, spiracles, or integument, they release their symbiont into insect hemolymph where the bacteria are proliferated [5, 6].
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