Lillis, Peter E. (2021) The Molecular Signatures of Adaptive Plasticity in Parasitic Nematodes. PhD thesis, National University of Ireland Maynooth.

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Abstract

Entomopathogenic nematodes (EPN; Steinernema spp. and Heterorhabditis spp.) are parasites which kill and reproduce within insects. The infective juvenile (IJ) is a developmentally arrested, nonfeeding, stress tolerant stage which forages in the soil for new insects to infect. IJs carry symbiotic bacteria which aid killing the insect and converts the host’s tissues to a nutritive liquid which the EPN can consume. EPN are promising biocontrol agents for the control of insect pests and are also model organisms for the study of parasitism. The objective of this project was to characterise the plasticity of IJ behaviour, and to investigate some of the associated molecular processes. There are two broad components: firstly, the identification of molecules (ascarosides) externally secreted by IJs and their effects on dispersal, and secondly changes in behaviour and stress tolerance of IJs over time during storage at different temperatures, and associated changes in the proteome. When the host cadaver becomes overcrowded, there is a build-up of signals which encourage newly formed IJs to disperse, thus reducing competition between the many IJs arising from the same cadaver. Among these chemicals are members of a class of pheromones, ascarosides, which have profound effects on IJ behaviour and development. Whether or not these IJs continue producing ascarosides into the surrounding medium, the composition of the mixture, the abundance of these pheromones and whether they affect the behaviour of conspecifics and heterospecifics was investigated. The IJs of four EPN species, Steinernema carpocapsae, Steinernema longicaudum, Steinernema feltiae, and Heterorhabditis megidis, were collected and stored in water at 5000 IJs/ml. The worm conditioned water was analysed with LC-MS/MS after storage in 20°C for specific timepoints. Various ascarosides were detected, generally increasing in abundance the longer the IJs were stored in the water, indicating that these IJs secrete ascarosides consistently after emergence from the host. The composition of ascarosides detected was species specific however the most abundant ascaroside detected in all species was Ascr#9, similar to many other insect-associated species. The worm-conditioned water from both conspecifics and heterospecifics was shown to induce dispersal behaviour in each of the species in agar plate assays. The species-specificity and complexity of ascaroside secretion, despite the similar effects on dispersal, implies that it has other ecological or biological functions. The second part of the thesis investigates the effects of time and temperature on stress tolerance, behaviour (chemotaxis) and the proteome of S. carpocapsae and H. megidis IJs. Previously it has been shown that conditioning IJs in low temperatures (<10°C) enhances not only their freezing tolerance, but their tolerance to other abiotic stressors. This cross tolerance is presumed to be due to the accumulation of non-specific protectants within the IJs. The IJs of H. megidis and S. carpocapsae were stored in 20°C and 9°C for up to 12 weeks, and at specific timepoints, assays were performed, and their proteins were extracted and analysed via LC-MS/MS. More proteins were detected within S. carpocapsae IJs (2422) than in H. megidis IJs (1582). The S. carpocapsae proteome was more strongly affected by low temperature storage (9°C), whereas the H. megidis proteome changed over time in a similar manner, less influenced by the ambient temperature. Compared to freshly emerged IJs, the highest abundance proteins detected within S. carpocapsae IJs at 20°C were proteins related to the cytoskeleton, cell signalling, and infection proteins such as proteases, protease inhibitors and a chitinase. The proteins that were detected at the highest abundance after conditioning at 9°C were late embryogenesis abundant proteins, heat shock proteins, and proteins related to stress tolerance. The proteins which were decreased in abundance to the greatest extent after conditioning at 20°C and 9°C were those related to the cytoskeleton and stress related proteins. The highest abundance proteins detected in H. megidis after conditioning in both 20°C and in 9°C were those related to the cytoskeleton, cell signalling, and carbon metabolism. The proteins decreased to the greatest extent after conditioning the IJs in 20°C and 9°C were those related to metabolism, heat shock proteins and ribosomal proteins. Storage of IJs at low temperatures prolongs their survival. S. carpocapsae IJs exhibited increased molecular chaperones over time, and to a greater extent in 9°C. The H. megidis IJs exhibited decreased abundance of proteins associated with translation over time. These two responses may represent a species-specific response to proteostatic collapse as the IJs age. Proteostatic collapse is one of the consequences of aging in cells and likely a contributing factor to nematode longevity, as misfolded and damaged proteins and toxic aggregations of proteins begin to accumulate within the cells. Similarly, as nematodes age, reactive oxygen species (ROS) accumulate. Both species demonstrated an increase in abundance of proteins which enhance ROS tolerance, and to a greater extent in 9°C. Storage of EPN IJs in low temperatures also affects various behaviours such as dispersal, infectivity, and response to host volatiles. If these behaviours are adversely affected, then cold storage of nematodes will be a trade-off between longevity of the nematodes in storage and the efficacy of these parasites when applied to the field as biocontrol agents. After conditioning at 20°C, IJs of both species were attracted to hexanol, methyl salicylate and acetone, and were repelled by prenol (an odour associated with infected hosts). The chemotaxis responses of H. megidis to each odorant was enhanced after storage in 9°C, becoming more strongly attracted or repelled to each odour than after storage at 20°C. S. carpocapsae IJs tended to act in the opposite manner after exposure to low temperatures, becoming repelled by attractants and strongly attracted to the repellent, prenol. Storage of H. megidis IJs at temperatures below the culture temperature of 20°C resulted in IJs with a gradual change in chemotaxis from 9°C to 12°C to 15°C, whereas S. carpocapsae showed a binary response, as all temperatures below culture temperature tested resulted in IJs with similar responses. To investigate whether IJs retain these altered chemotaxis responses following a return to culture temperature, and how long a period of cold it takes to induce them, IJs from both species were stored at 9°C for brief periods, 3 hours, 1 day and 1 week, and were transferred to 20°C for the duration of the experiment (3 weeks). At the 3-week timepoint, all of the conditioned IJs were exposed to a strong attractant or a strong repellent, and their responses were compared to that of IJs which were stored at 9°C or 20°C for the full 3 weeks. H. megidis did not retain cold-altered chemotaxis responses after transfer to 20°C, while S. carpocapsae IJs stored at 9°C and transferred to 20°C after just 3 hours retained their altered chemotaxis responses for 3 weeks. Storage at 9°C for a week enhanced the freezing (-10°C for 6 hours) and desiccation (75% relative humidity) tolerance of S. carpocapsae IJs, and this enhanced stress tolerance was retained by those IJs which were transferred to 20°C when tested after 2 weeks at the temperature. I next explored whether the cold-induced changes in the S. carpocapsae proteome were also maintained following return to 20°C, as was found for behaviour and stress tolerance. IJs which were stored at 9°C for the full 3 weeks, and those stored for just for 1 week and then transferred to 20°C for 2 weeks had similar proteomes and were both different to that of IJs stored at 20°C for the full 3 weeks. The first two groups displayed hundredfold increases in the abundance of many molecular chaperones when compared to IJs that were stored at 20°C for the full duration. Conversely, the highest abundance protein in the IJs stored at 20°C for 3 weeks relative to the IJs stored at 9°C was a chitinase, implicated in many roles in nematodes including fungal defence and infection. The plasticity of EPN IJs enables them to adapt to diverse and changing environments. Proteomic profiling is a useful guide for further elucidating the molecular mechanisms behind these phenotypes, which will facilitate their use as biocontrol agents, and as models for parasitism.

Item Type:	Thesis (PhD)
Keywords:	Molecular Signatures; Adaptive Plasticity; Parasitic Nematodes;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	16527
Depositing User:	IR eTheses
Date Deposited:	19 Sep 2022 13:50
URI:
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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