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    Pheromones in entomopathogenic nematodes: effects on dispersal and reproductive behaviours

    Hartley, Cathryn J. (2017) Pheromones in entomopathogenic nematodes: effects on dispersal and reproductive behaviours. PhD thesis, National University of Ireland Maynooth.

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    Entomopathogenic nematodes (EPN) are increasingly gaining favour as model organisms for studying traits relevant to parasitism and for their commercial value as biocontrol agents. The overall project aim is to explore the role of pheromones in coordinating a variety of important behaviours in entomopathogenic nematodes, particularly Steinernema, both as primers (longer term developmental effects) and releasers (short term behavioural effects). I investigate both sex pheromones and chemical signals produced by and affecting the infective juvenile (IJ) stage. In this thesis, I both test central hypotheses, and attempt the identification of key pheromone components, using assay-guided fractionation. As lethal parasites of insects, EPN have been successfully commercialised for pest control, however greater understanding of their ecology and biology is required to improve their efficacy in the field, which is often inconsistent. Infective juveniles (IJs) actively search for hosts within the soil. The IJs enter the host and release symbiotic bacteria which both kill the insect and convert it into a nutritive “soup”. The nematodes develop and reproduce inside, and eventually hundreds of thousands of newly-produced IJs emerge and disperse from the spent cadaver. It has previously been shown that chemicals within the spent cadaver stimulate IJ dispersal. In Chapter 3, I show that IJs themselves may also produce chemical signals which promote dispersal in both conspecific and heterospecific IJs. Behavioural assays confirmed that IJs of three species of Steinernema (S. carpocapsae, S. longicaudum and S. feltiae) and one species of Heterorhabditis (H. megidis) disperse more when exposed to cadaver extract of pre-infected hosts. More interestingly, dispersal is also promoted by water “conditioned” for 1 or 7 days with IJs which had recently vacated a spent host, suggesting that they release cues which modify dispersal behaviours. Furthermore, it was found that these cues are not species-specific, with IJs dispersal enhanced by water conditioned with their own species and other species (even between families) to a similar extent. In a longer term storage experiment with S. carpocapsae and H. megidis, I showed that replacing the water in which IJs were stored reduced the dispersal of these stored IJs when assayed, indicating that chemicals released into the medium by IJs maintained high dispersal. Parallell assays showed that replacing the water did not affect the virulence of the stored IJs. Chapter 4 addresses the role of chemical signals in mate attraction in dioecious Steinernema carpocapsae and Steinernema longicaudum adults. It was found that males are attracted to exudates of conspecific females, but not to exudates from females of the other species. The sexual history of the females dictated their level of attractiveness. Exudates of virgin females were highly attractive to males, whereas attractiveness was lost just 1 h post-mating and the loss of attractiveness was sustained for 48 h post-mating. Assay-guided fractionation of female exudates was used to give insight into the chemical properties of the as yet uncharacterised attractant. Males were less attracted to extracted material (using C18 Sep-Pak columns) in comparison to un-extracted female exudates. They also showed some degree of attraction to column flow-through, which indicates that some components may not have bound to the column during the extraction process, perhaps due to high polarity. Furthermore, recombination of column retentate and flow-through did not restore full bioactivity of the original sample, indicating that volatile molecules may be part of the attractant composition and were lost during the extraction process. Chapter 5 investigated the role of female-emitted chemical cues in sexual priming of males, (inducing production of sperm). This phenomenon, unusual amongst animals, whereby female presence or odour is essential for sexual maturation of males, was previously described in S. longicaudum. Here I show that the same is true for a second species, S. carpocapsae. As with female-produced male attractants, maturation signals were also shown to be species-specific, with males only producing sperm on exposure to exudates from females of their own species. Bioassay of C18 extracted female exudates again showed some degree of physiological response to both column retentate and column flow-through, again indicating that some components of the pheromonal blend were not retained by the C18 column resin during extraction. However, unlike in attraction, recombination of these two constituents fully restored bioactivity to the level of the original unextracted female exudates, indicating that there was no essential volatile component involved. Assay-guided fractionation of female exudates showed that the early-eluting fractions (10-30% methanol) were the most biologically active with respect to inducing maturation in males. This finding was supported by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis which revealed the presence of numerous ascarosides, the family of molecules known to be involved in nematode communication, in active fractions. S. carpocapsae was previously reported to produce ascarosides at both juvenile and adult stages of the life-cycle; however this study is the first report of ascarosides produced by S. longicaudum. Ascr#10 was the dominant ascaroside in both species, with several others found in much lower relative abundance. Although similarities in ascaroside profiles between species were seen (both S. carpocapsae and S. longicaudum produced ascrs # 9, #1, #14 and #10), differences in ratios and the presence of ascr#14 exclusively in S. longicaudum female exudates may contribute to the species-specificity of Steinernema sex pheromones. LC-MS/MS analysis also revealed the presence of a number of female-specific non-ascarosidal molecules in biologically active fractions. Although these were not structurally characterised, their detection may direct future research in the potential role of non-ascarosidal components of chemical signals in nematodes. The work of this thesis supported two of the three hypotheses proposed: Hypothesis 1, that chemical signals differ between species where recognition is important (sex pheromones) but coincide where different species have a common aim (dispersal from overcrowded conditions), and Hypothesis 2, that chemical signals exuded by IJs after emergence from the cadaver continue to affect IJ behaviour. Full support was not obtained for Hypothesis 3, that the same chemical blend produced by female Steinernema is responsible for both behavioural (attraction) and organisational (gonad maturation) effects in males.

    Item Type: Thesis (PhD)
    Keywords: Pheromones; entomopathogenic nematodes; effects; dispersal; reproductive; behaviours;
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 13837
    Depositing User: IR eTheses
    Date Deposited: 15 Jan 2021 12:19
      Use Licence: This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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