Abstract
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.
Highlights
The entomopathogenic nematode, Heterorhabditis indica SL0708 is a eumetazoan, ecdysozoan protostomate, and an obligate and lethal parasite of insects, as a consequence of its symbiotic association with enteric bacteria that kill the insect host
H. indica SL0708 hermaphrodite quantification and egg development characterization The largest hermaphrodite population was obtained at 105 h (31 hermaphrodites) and 115 h (24 hermaphrodites) (Fig. 1) with significant differences post-infection (P = 0.001, df = 5, F = 16.969)
The nine stages of egg cleavage were observed within H. indica SL0708 uterus
Summary
The entomopathogenic nematode, Heterorhabditis indica SL0708 is a eumetazoan, ecdysozoan protostomate, and an obligate and lethal parasite of insects, as a consequence of its symbiotic association with enteric bacteria that kill the insect host. Fertilization occurs in hermaphrodite adults, with sperm and eggs generated in the ovotestis. H. indica SL0708 is protoandrous, namely, sperm is produced before eggs, resulting in self-fertilization (Johnigk and Ehlers 1999a, b and Sáenz and Lopez 2011). H. indica SL0708 exhibits rotational holoblastic egg cleavage, in an asymmetric fashion along the anterior-posterior axis, near of what is to become the posterior pole. Each asymmetrical division results in a large AB founder cell, with a smaller posterior stem cell of P1 lineage.
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