Interactions of entomopathogenic fungi and other control agents: mechanism and field potential against Hylobius abietis larvae

Biological control is the beneficial application of natural enemies such as pathogens, predators and parasites in managing pests and their damage. Entomopathogenic fungi (EPF) have a crucial role in natural ecosystems and are being developed as alternative control agents for insect pests. Both Beauvaria bassiana and Metarhizium anisopliae have been proven to be effective biological control agents against a range of pests and are commercially produced. Advantages of using EPF for biological pest control include their degree of specificity, absence of effects on mammals, reduced probability of insects developing resistance and they may persist for long periods in some environments which could provide long term control effects. Disadvantages include that it takes EPF longer to kill insects than their chemical counterparts, application needs to be timed for high relative humidity and low pest numbers, and efficacy varies among different insect species. If a combination of treatments resulted in a synergistic interaction then the efficacy of these biopesticides would be increased. A number of laboratory and field studies have used combinations of entomopathogenic nematodes (EPN) and EPF against insect pests with resulting interactions ranging from antagonistic to synergistic. In the case of synergism resulting from combined applications it is suggested that EPF may make the host more susceptible through suppressing its immune system. To understand how this putative synergistic interaction between control agents could occur mechanistically, the effect of EPF supernatant was tested on the immune response of the forestry pest, Hylobius abietis, to screen for species with immunomodulating properties. The potential of the commonly used model organism, the greater wax moth, Galleria mellonella, as a model for the study of the immune response of H. abietis to pathogens was also explored. Hylobius abietis, the large pine weevil, is a major pest of reforestation in Europe. It is estimated that H. abietis costs the forestry industry approximately €140 million/year. Current control measures rely heavily on the synthetic chemical cypermethrin. However, due to concerns over its environmental impact cypermethrin is being phased out across Europe. Therefore there is an interest in the use of entomopathogens as biological control agents in integrated pest management. Thus one objective of this work was to assess the efficacy of EPF, EPN and EPF-EPN combinations for H. abietis suppression in the field in order to ultimately determine if the treatments used exert synergistic control over H. abietis. The effect of EPF supernatant on the immune response of insects was assessed through a number of bioassays that investigated the effect of EPF on haemocyte densities and yeast proliferation in the insect haemocoel, as well as testing whether pre-treatment with EPF increases larval susceptibility to subsequent pathogens. The effect of EPF supernatant on the humoral immune response was investigated by subjecting larval haemolymph to label free quantitative (LFQ) proteomic analysis. To enable proteomic investigations into the effects of EPF on the immune response of H. abietis and to compensate for the lack of genomic information for H. abietis; a de novo transcriptome study of H. abietis larvae was performed with Beijing Genomics Institute (BGI, Hong Kong). Bioassays indicated that M. anisopliae, B. bassiana and Beauveria caledonica, demonstrated immunomodulating effects on H. abietis larvae, while M. anisopliae and B. caledonica modulated the immune response of G. mellonella making the insect more susceptible to subsequent pathogens. LFQ analysis on larval haemolymph showed that in response to EPF supernatant both insect species displayed altered abundance of proteins involved in antimicrobial defence, the prophenoloxidase cascade, detoxification and detection and sensing. These patterns of alteration may be integral to the modulation of the host immune response by EPF. A major difference observed between the proteomic profiles of H. abietis and G. mellonella haemolymph was that H. abietis injected with B. caledonica supernatant had a major alteration in metabolic proteins involved in cellulose cleavage, reflective of its wood based diet. It was concluded G. mellonella may have an application as a model for looking for secondary metabolites or natural products that display immunomodulating properties so that EPF isolates could be screened for production of these products. However G. mellonella are not a substitute for the target pest for proteomic analysis. Moreover laboratory bioassays with either G. mellonella or H. abietis are not predictive of whether synergy will occur in the field as there are many more factors involved than just the ability of EPF to modulate the immune response. To investigate the ability of EPF and EPN to suppress H. abietis populations in the field, three field studies were carried out over three consecutive years. Treatments were applied to tree stumps harbouring H. abietis developmental stages. The efficacy of EPF and EPN was investigated alone and in combination through emergence trapping and destructive sampling. Three EPF strains were utilised in these field studies, commercial strains of M. anisopliae and B. bassiana and a strain of B. caledonica native to Ireland. Two EPN species were utilised in these field studies, Steinernema carpocapsae and Heterorhabditis downesi, the latter is native to Ireland. In this work EPN were found to offer superior control over H. abietis in the field than EPF, with all treatments that caused significant reduction in adult emergence being EPN alone or EPN in combination with EPF. Ultimately EPF are not suited to control of H. abietis using this strategy. Synergy between EPF-EPN was not achieved in any of the three field studies with all combinations tested giving additive results. The final aim of this work was to investigate if the little-studied native fungus B. caledonica produces immunomodulating compounds active against H. abietis. Identification, large scale production and structural determination of an abundant secreted natural product of B. caledonica was carried out using High Performance Liquid Chromatography (HPLC) and Nuclear magnetic resonance (NMR) spectroscopy, and the metabolite of interest was found to be oosporein. Subsequently assessment of the potential insecticidal, anti-feedant and immunomodulating effects of oosporein was carried out. Oosporein was identified as an abundant metabolite in B. caledonica supernatant that displayed immunomodulating properties in H. abietis larvae as well as being a feeding stimulant to H. abietis adults. The work presented throughout this thesis offers the first report of the effect of EPF on the cellular and humoral immune system of H. abietis. It presents a critical assessment of G. mellonella as a model organism for immune studies of H. abietis. It highlights that EPN are a superior biocontrol agent for the control of H. abietis in the context of the current approach used. However the native B. caledonica isolate tested throughout this thesis presents an interesting avenue for future research into the utilisation of EPF as biocontrol agents in a wider sense than researched in this work