Plant parasitic nematodes (PPNs) seriously threaten global food security. Conventionally an integrated approach to PPN management has relied heavily on carbamate, organophosphate and fumigant nematicides which are now being withdrawn over environmental health and safety concerns. This progressive withdrawal has left a significant shortcoming in our ability to manage these economically important parasites, and highlights the need for novel and robust control methods. Nematodes can assimilate exogenous peptides through retrograde transport along the chemosensory amphid neurons. Peptides can accumulate within cells of the central nerve ring and can elicit physiological effects when released to interact with receptors on adjoining cells. We have profiled bioactive neuropeptides from the neuropeptide-like protein (NLP) family of PPNs as novel nematicides, and have identified numerous discrete NLPs that negatively impact chemosensation, host invasion and stylet thrusting of the root knot nematode Meloidogyne incognita and the potato cyst nematode Globodera pallida. Transgenic secretion of these peptides from the rhizobacterium, Bacillus subtilis, and the terrestrial microalgae Chlamydomonas reinhardtii reduce tomato infection levels by up to 90% when compared with controls. These data pave the way for the exploitation of nematode neuropeptides as a novel class of plant protective nematicide, using novel non-food transgenic delivery systems which could be deployed on farmer-preferred cultivars.

Entomopathogenic 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 peptide 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.

(Fig. 1A). Glucose (125.1% ± 5.5; P <0.001) and fructose (124.8% ± 5.4; P 61 <0.001) also triggered an elevated level of serotonin-triggered stylet thrusting in 62 treated juveniles; xylose failed to trigger any significant response (99.36% ± 10.87; P 63 >0.05) when compared with control treatments (Fig. 1B). Globodera pallida infective 64 stage juveniles were mildly repelled by glucose (CI: -0.23 ± 0.09; P >0.05), and did 65 not respond to fructose (CI: 0.15 ± 0.08; P >0.05), or xylose (CI: -0.19 ± 0.09; P 66 >0.05) as compared with control treated worms (Fig. 1C). Glucose (118.6% ± 9.7; P 67 >0.05), fructose (107.2% ± 7.3; P >0.05), or xylose (119.6% ± 8.6; P >0.05) had no 68 significant impact on the frequency of serotonin-triggered stylet thrusting in G. pallida 69 infective juveniles when compared with control treatments (Fig. 1D) Fig. 2A). 80Corresponding reductions in glucose and fructose exudate concentration were 81 observed for both STP1 (5.10 µg/ml ± 1.31; P <0.01 and 3.14 µg/ml ± 0.92; P <0.01, (Fig. 3B). 93When exoRNAi-treated seedlings were challenged by M. incognita infection, 94 significant reductions in percentage infection levels relative to control (neo) dsRNA 95 treatment were observed for both STP1 (14.15% ± 4.77; P <0.01) and STP2 96(27.08% ± 7.32; P <0.05) dsRNA treatments (Fig. 3C) (Fig. 3D). 100These data demonstrate that the exogenous application of aqueous dsRNA 101 onto tomato seedlings is sufficient to trigger specific gene knockdown. However, we 102 found that different experimental populations of tomato seedlings could display wide

16Entomopathogenic nematodes (EPNs) employ a sophisticated chemosensory apparatus to 17 detect potential hosts. Understanding the molecular basis of relevant host-finding behaviours 18 could facilitate improved EPN biocontrol approaches, and could lend insight to similar 19 behaviours in economically important mammalian parasites. FMRFamide-like peptides are 20 enriched and conserved across the Phylum Nematoda, and have been linked with motor and 21 sensory function, including dispersal and aggregating behaviours in the free living nematode 22Caenorhabditis elegans. The RNA interference (RNAi) pathway of Steinernema 23 . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/061101 doi: bioRxiv preprint first posted online Jun. 28, 2016; carpocapsae was characterised in silico, and employed to knockdown the expression of the 24 FMRFamide-like protein 21 (GLGPRPLRFamide) gene (flp-21) in S. carpocapsae infective 25 juveniles; a first instance of RNAi in this genus, and a first in an infective juvenile of any EPN 26 species. Our data show that 5 mg/ml dsRNA and 50 mM serotonin triggers statistically 27 significant flp-21 knockdown (-84%***) over a 48 h timecourse, which inhibits host-finding 28 (chemosensory), dispersal, hyperactive nictation and jumping behaviours. However, whilst 1 29 mg/ml dsRNA and 50 mM serotonin also triggers statistically significant flp-21 knockdown (-30 51%**) over a 48 h timecourse, it does not trigger the null sensory phenotypes; statistically 31 significant target knockdown can still lead to false negative results, necessitating appropriate 32 experimental design. SPME GC-MS volatile profiles of two EPN hosts, Galleria mellonella 33and Tenebrio molitor reveal an array of shared and unique compounds; these differences 34 28, 2016; basis of behaviour. Here we have characterised the RNAi pathway of Steinernema 51 carpocapsae through analysing the genome sequence for relevant genes, and have 52 successfully knocked down the neuropeptide gene flp-21 in S. carpocapsae infective 53 juveniles. We find that it is involved in the regulation of behaviours which rely on sensory 54 perception and relate to host-finding. We have localised the gene and mature neuropeptide, 55 and find them to be expressed in paired anterior neurons, which is in broad agreement with 56 our behavioural observations following RNAi. Our observations are relevant to interactions 57 of S. carpocapsae with two insect hosts, the waxworm Galleria mellonella, and the 58 meelworm, Tenebrio molitor. We identified the volatile compounds relating to both insects, 59is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint . http://dx.doi.org/10.1101/061101 doi: bioRxiv preprint first po...

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