Investigating the Foraging Behaviour of Entomopathogenic Nematodes Cultured in Vitro

Abstract

Synthetic pesticides have provided a cost-effective approach for the control and protection of agricultural and horticultural crops against insect pests and diseases since their introduction and success in the mid-1940s. Despite the noticeable benefits of synthetic pesticides, an inconsiderable amount of evidence has shown that their overuse poses several adverse effects, including the development of resistance, crop contamination and the unconscious reduction of non-target organisms. Therefore, safer yet effective techniques for managing insect pests are required. Insect parasitic nematodes such as entomopathogenic nematodes (EPNs) are widely studied for their use as potential biological control agents (BCAs) against various economically important insect pests. EPNs are classified into three foraging groups namely cruise, ambush, and intermediate foragers which influence their soil depth distribution and host range. Upon their emergence from host cadavers or inundative release into soil, EPNs experience a variety of environmental and host-associated cues which significantly affect their foraging behaviour and, ultimately, their pathogenicity, survival, and persistence. Therefore, understanding the foraging behaviour of EPNs following their exposure to abiotic and chemotactic stimuli is essential for classifying EPNs into foraging groups and their assignment to specific hosts. As such, this study aimed to isolate EPNs from soil samples collected from Mahobong Leribe in Lesotho and investigate their foraging behaviour by exposing the nematodes to abiotic factors and chemical cues that influence their host-seeking behaviour. Nematodes were recovered from collected soil samples and infected Tenebrio molitor cadavers using the soil baiting technique and White trap method, respectively. PCR amplification and Sanger sequencing of the 18S and 16S rDNA regions were used to identify the phylogenetic relationships of the isolated nematode and its potential bacterial symbiont using the GenBank sequence database, respectively. The isolated nematode had a high affinity to Cruznema sp. isolate 734C-1 (OR606776.1), whereas the potential bacterial symbiont had a high affinity to Enterobacter ludwigii strain EN-119 (NR 042349.1). The foraging behaviour of Cruznema sp. NTM-2021 was studied by investigating the influence of soil moisture and the nematode's response to a distal chemotactic cue. After 7 days, insect mortality observed in the 2D sand substrate assays was 0%, 23.3%, 40%, 46.7% and 66.7% for 10%, 12.5%, 15%, 17.5% and 20% soil moisture levels, respectively. This overall trend suggests that insect mortality increases with an increase in soil moisture. After 7 days, the vertical sand column assay revealed that at 10% soil moisture levels, the IJs travelled to a maximum depth of 8 cm and caused <12% insect mortality at this depth. At 12.5% soil moisture, the IJs travelled to a maximum depth of 14 cm and caused >85% insect mortality at this depth. In soil moisture levels ranging between 15-17.5%, IJs reached depths of 32cm, causing >85% insect mortality at all depths along the column. Interestingly, at a soil moisture level of 20%, IJs reached lengths of 26 cm; however, they could only parasitise <15% of the insect larvae, suggesting that higher moisture levels may restrict the vertical dispersal of nematodes. The PF127 gel assay revealed that the IJs of Cruznema sp. NTM-2021 were strongly repelled by prenol, suggesting its use as a distal chemotactic cue. Based on the IJs ability to move to depths of 32 cm and its response to prenol, Cruznema sp. NTM-2021 may be classified as a cruise forager. Overall, the results of this study not only classified the foraging behaviour of a potential entomopathogenic nematode but also characterised the nematode according to its moisture requirements to optimise its performance in field applications since it showed great promise as a biological control agent.