Exploiting Entomopathogenic Nematode Neurobiology to Improve Bioinsecticide Formulations

Abstract

Entomopathogenic nematodes (EPNs) are soil dwelling organisms that target and kill insects. They have sophisticated chemosensory apparatus, with which they perceive host associated cues to locate them. Once a suitable host is located, they employ a diverse range of host finding behaviours to facilitate contact with a host, a prerequisite for infection. These EPNs are described as beneficial nematodes as they are employed as biocontrol agents for crop protection against insect pests. Insects cause devastating damage to annual crop yields. Many chemical pesticides have been banned across the world due to their devastating effects on the environment and the health of animals, therefore, a safer alternative is essential to protect crops from pests. EPNs have proven that they can provide effective protection against insects in both field and glass house trials, however, there have been reports of inconsistencies in biocontrol efficiency. This could be attributed to our limited knowledge of their biology post-application, and the molecular mechanisms that drives their host finding behaviours. 
Some insight into the genes that regulate these diverse host finding behaviours in EPNs could be used to enhance their biocontrol efficiency. Small interfering RNAs can silence genes post transcriptionally. One method to achieve gene knockdown with small interfering RNAs is RNA interference (RNAi). RNAi is a powerful tool to determine the function of a gene, as it can be tailored to target a specific gene of interest, silence the gene in vivo, and observe the effect this has on the organism. 
Functional studies using RNAi have identified the role of many genes in the model organism Caenorhabditis elegans, including many genes involved in sensory perception. In this study, the neuropeptide encoding gene flp-21, will be targeted using RNAi in Steinernema carpocapsae to observe its effect on their host finding behaviours. 
This study is the first instance of RNAi in a Steinernema species of EPN, whereSc-flp-21 was suppressed by 84 % (P<0.0001) by soaking S. carpocapsae IJs in a high amount of dsRNA for 48 hours with 50 mM serotonin. The suppression of Sc-flp-21 had significant effects on their host finding behaviours through impeding olfactory perception in these nematodes. Another method used to identify genes that regulate intraspecific phenotypic differences between populations is RNA-sequencing. Sequencing the transcriptome allows observation of the gene expression pattern of a single cell or entire organism at one time. Differentially expressed genes between phenotypically different populations can indicate the genes responsible for the intraspecific differences observed between populations. Transcriptome analysis identified differentially expressed genes between S. carpocapsae strains that showed phenotypic differences with regards to their host finding behaviours. Nictation behaviour in S. carpocapsae may be heavily regulated by Sc-daf-2 and Sc-daf-7, which has been described in C. elegans previously. Host finding behaviour was enhanced in Breton strain, and transcriptome analysis revealed many GPCRs and neuropeptides to be upregulated in Breton, relative to the other two strains. Functional studies would need to take place before these genes’ roles can be confirmed. 
Natural EPN isolates were collected from Northern Irish soil, and challenged through host finding assays, along with various other trait assessments, to investigate their natural diversity. Ten isolates were collected, spanning at least four EPN species: S. feltiae, S.affine, Heterorhabditis downesi and other, unidentified Heterorhabditis species. All isolates tested, including some laboratory strains, showed phenotypic variation in their host finding behaviours. Intraspecific phenotypic variation was also observed between S. feltiae strains. The large phenotypic variation observed in natural populations of EPN may allow biocontrol formulations to be tailored to the user’s needs, and facilitate artificial selection for enhanced EPN strains. This thesis focuses on the molecular basis of host finding behaviours in EPNs, and exploring their natural diversity to enhance their biocontrol efficiency for crop protection.

Date of Award	Jul 2020
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Aaron Maule (Supervisor) & Johnathan Dalzell (Supervisor)