Heterorhadbitis indica sl0708 hermaphrodite and axenic egg isolation

In Colombia, Heterorhabditis indica SL0708 is an entomopathogenic nematode (EPN) used for effective biological control of insect pests. The purpose of this study was to standardize hermaphrodite and axenic egg extraction, in addition to describe egg developmental stages to optimize in vitro infective juvenile egg production for future pest control. Thus, the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae), in its last larval instar was dissected at 90, 95, 100, 105, 110, and 115 h post-infection. Hermaphrodites were counted and egg stage within the uterus was characterized. Fertilized H. indica SL0708 egg presented 9 developmental stages; stage 1: maternal pronucleus migrates to the posterior pole, forming pseudoclivage segmentation until reaching stage 9 with a moving J1 formed. At 95 h post-infection, the greatest number of hermaphrodites, at stage 9, was observed. Not all hermaphrodites presented the same number of eggs and stage of development within their uterus. Subsequently, a protocol to rupture hermaphrodites and sterilize eggs was evaluated, assaying three different exposure times and two egg incubation periods. A hermaphrodite exposure to 0.1 M NaOH and 1% NaOCl solution for 15 min to allow obtaining viable axenic eggs, facilitating laboratory tests with nematode H. indica SL0708 free of Photorhabdus luminescens SL0708 or other bacteria that could introduce variability in future assays was established.