Effect of inoculum age and physical parameters on in vitro culture of the entomopathogenic nematode Steinernema feltiae

Authors: LEITE, LUIS - Instituto Biologicio - Brazil; Shapiro Ilan, David; HAZIR, SELCUK - Adnan Mederes University; Jackson, Mark

Submitted to: Journal of Helminthology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/7/2016
Publication Date: 12/20/2017
Citation: Leite, L., Shapiro Ilan, D.I., Hazir, S., Jackson, M.A. 2017. Effect of inoculum age and physical parameters on in vitro culture of the entomopathogenic nematode Steinernema feltiae. Journal of Helminthology. 91(6):686-695. https://doi:10.1017/S0022149X16000821.
DOI: https://doi.org/10.1017/S0022149X16000821

Interpretive Summary: Entomopathogenic nematodes, also known as beneficial nematodes, are small worms that can be used as environmentally-friendly bio-insecticides. One barrier to wider usage of these nematodes in insect pest management is the high costs associated with producing the nematodes. Therefore, we are studying methods to improve nematode production and reduce costs. Most nematodes are grown in liquid fermentation tanks but also a solid fermentation method is used in crumbled foam. In this study, we investigated the effects nematode age used in the starting culture. We also investigated the effects of some physical parameters such as viscosity flask size, and agitation speed (which affects aeration). Older nematodes (up to 28 days) in the starter culture increased final nematode yield in liquid fermentation, and increased agitation and viscosity also were beneficial. Additional research will be conducted to improve nematode nutrition and quality during liquid fermentation.

Technical Abstract: Entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae have a symbiotic association with bacteria which makes them virulent against insects. EPNs have been mass produced using in vivo and in vitro methods including both solid and liquid fermentation. This study assessed the effect of nematode inoculum age on the production of S. feltiae in liquid, solid and biphasic processes. Several physical parameters were also assessed: the effect of medium viscosity, flasks size and aeration speed on the recovery and yield of infective juveniles (IJs). Inoculum age treatments included inoculum liquid cultures that were 7, 14, 21 and 28 days old. Nematodes from the same inoculum were added to one liquid medium (Liquid culture), one solid medium with bacteria previously grown in sponge (Solid culture, i.e., the Bedding Method), and a variation of the solid medium (a Biphasic culture), in which the bacteria were first grown in liquid and, then, soaked into the sponges with the purpose to providing a more homogenous bacterial culture before nematode inoculation. Experiments were conducted in Erlenmeyer flasks. Eight treatments were established involving combinations of three variables: two media (with and without 0.2% agar), two flask sizes (250 and 150 ml), and two agitation speeds (180 and 280 rpm). The study showed increases in nematode yield for liquid cultures but not for solid or biphasic cultures with the advance of the inoculum age up to 28 days of growth. Furthermore, the addition of 0.2% agar in the liquid medium and increasing aeration rate by using larger flasks with higher agitation speed may increase nematode recovery and final yield. The experiments were conducted using shake flasks but the results may also be applicable for bioreactors conditions.