AbstractEntomopathogenic nematodes (EPNs) employ a sophisticated chemosensory apparatus to detect potential hosts. Understanding the molecular basis of relevant host-finding behaviours could facilitate improved EPN biocontrol approaches, and could lend insight to similar behaviours in economically important mammalian parasites. FMRFamide-like peptides are enriched and conserved across the Phylum Nematoda, and have been linked with motor and sensory function, including dispersal and aggregating behaviours in the free living nematode Caenorhabditis elegans. The RNA interference (RNAi) pathway of Steinernema carpocapsae was characterised in silico, and employed to knockdown the expression of the FMRFamide-like protein 21 (GLGPRPLRFamide) gene (flp-21) in S. carpocapsae infective juveniles; a first instance of RNAi in this genus, and a first in an infective juvenile of any EPN species. Our data show that 5 mg/ml dsRNA and 50 mM serotonin triggers statistically significant flp-21 knockdown (-84%***) over a 48 h timecourse, which inhibits host-finding (chemosensory), dispersal, hyperactive nictation and jumping behaviours. However, whilst 1 mg/ml dsRNA and 50 mM serotonin also triggers statistically significant flp-21 knockdown (-51%**) over a 48 h timecourse, it does not trigger the null sensory phenotypes; statistically significant target knockdown can still lead to false negative results, necessitating appropriate experimental design. SPME GC-MS volatile profiles of two EPN hosts, Galleria mellonella and Tenebrio molitor reveal an array of shared and unique compounds; these differences had no impact on null flp-21 RNAi phenotypes for the behaviours assayed. Localisation of flp-21 / FLP-21 to paired anterior neurons by whole mount in situ hybridisation and immunocytochemistry corroborates the RNAi data, further suggesting a role in sensory modulation. These data can underpin efforts to study these behaviours in other economically important parasites, and could facilitate molecular approaches to EPN strain improvement for biocontrol.Author summaryEntomopathogenic nematodes (EPNs) use a range of behaviours in order to find a suitable host, some of which are shared with important mammalian parasites. The ethical burden of conducting research on parasites which require a mammalian host has driven a move towards appropriate ‘model’ parasites, like EPNs, which have short life cycles, can be cultured in insects or agar plates, and have excellent genomic resources. This study aimed to develop a method for triggering gene knockdown by RNA interference, a biochemical pathway involved in gene regulation. Through knocking down the expression of a target gene we can then study the function of that gene, helping us to understand the molecular basis of behaviour. Here we have characterised the RNAi pathway of Steinernema carpocapsae through analysing the genome sequence for relevant genes, and have successfully knocked down the neuropeptide gene flp-21 in S. carpocapsae infective juveniles. We find that it is involved in the regulation of behaviours which rely on sensory perception and relate to host-finding. We have localised the gene and mature neuropeptide, and find them to be expressed in paired anterior neurons, which is in broad agreement with our behavioural observations following RNAi. Our observations are relevant to interactions of S. carpocapsae with two insect hosts, the waxworm Galleria mellonella, and the meelworm, Tenebrio molitor. We identified the volatile compounds relating to both insects, and find that there are both shared and unique compounds to both species; EPNs use volatile compound gradients, as well as other physical cues in order to find and invade a host. This study provides a method for employing RNAi in a promising model parasite, and characterises the molecular basis of host-finding behaviours which could be relevant to economically important mammalian parasites. EPNs are also used as bioinsecticides, and so understanding their behaviour and biology could have broad benefits across industry and academia.
A survey of entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae (Nematoda: Rhabditida) in the north-west of Iran