Maher, Abigail M.D. (2014) Co-existence and niche separation of two subspecies of Photorhabdus temperata associated with Heterorhabditis downesi in a dune grassland. PhD thesis, National University of Ireland Maynooth.

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Abstract

Entomopathogenic nematodes belonging to the families Heterorhabditidae and Steinernematidae form symbiotic relationships with entomopathogenic bacteria from the genera Photorhabdus and Xenorhabdus, respectively. Previous studies showed that Heterorhabditis downesi was associated with two different colour variants (pur and yel) of its Photorhabdus bacterial symbiont in sand dunes on North Bull Island in Dublin Bay, with evidence of the pur variant being more common to the front of the dunes. The main objectives of this study were to confirm the distribution of both colour variants of the nematode-bacterium complex on North Bull Island, identify them, investigate the specificity of the nematode-bacterium relationship and ascertain characteristics of the bacterial variants that might explain their distribution. A section of the dune system on North Bull Island was surveyed in 2008 and resurveyed in 2012 (Chapter 2). In 2008 the colour variants were spatially segregated, with nematodes associated with the pur variant recovered from the front section of the dunes only and nematodes associated with the yel variant recovered from the rear section only. In 2012 nematodes associated with each colour variant were recovered from the front section of the dunes and only nematodes associated with the yel variant were recovered from the rear section of the dunes. The nematode-pur complex was more abundant than the nematode-yel complex in sections where both combinations co-occurred. Three laboratory lines of each nematode and its associated bacterial colour variant were established for further study. A phylogenetic analysis of the nematode ITS region showed that all the nematode isolates were monophyletic, with no difference between the isolates carrying the pur or yel bacterial symbionts. On the other hand, a phylogenetic analysis of a portion of the gyrB gene of the bacterial isolates showed that the two colour variants were distinct subspecies, Photorhabdus temperata subsp. temperata (yel) and Photorhabdus temperata subsp. cinerea (pur). This confirms Photorhabdus temperata subsp. temperata as the bacterial partner in the YEL H. downesi – Photorhabdus complex. In addition, this is the first report of Photorhabdus temperata subsp. cinerea in Ireland and also one of the few reports of the co-occurrence of two different symbiotic bacteria subspecies with a single nematode species (Chapter 2). In antibiosis tests against a range of organisms the P. temperata subsp. cinerea isolates had a greater inhibitory effect than the P. temperata subsp. temperata isolates. Only one pair of isolates, yel1 and pur2, showed any antibiotic activity against the other subspecies and this pair were mutually inhibitory (Chapter 2). Phenotypic tests indicated that the subspecies had different growth requirements as the pur isolates grew better on MacConkey agar than on nutrient agar while there was no difference between the two media for the yel isolates. Biochemical characterisation was carried out using API 20E test strips. There was variability both within and between the colour variants, however a notable difference was that only the yel isolates were able to utilise trehalose, a major component of insect hemolymph (Chapter 2). The cross-compatibility of the two different bacterial subspecies with the heterogeneous nematode isolates was tested by injecting Galleria mellonella larvae with 10,000 cells of either the pur1 or yel3 isolate or both and then infecting them with H. downesi nematodes carrying either pur1 or yel3 bacteria. The emerging nematodes carried the bacterial subspecies that had been injected into the host, irrespective of which bacterial subspecies the infecting nematode had carried and hybrid lines of nematode-bacterium complex were successfully maintained for the duration of this study (Chapter 3). In the case of cadavers infected with both bacterial types all the emerging nematodes tended to carry a single subspecies. However, between 20 % and 30 % of the cadavers produced some nematodes carrying the pur1 and some carrying the yel3 isolate, (indicating that both bacterial subspecies were colonising the insect host and subsequently colonising the developing nematodes prior to emergence) and where this occurred a higher proportion of the nematodes carried the yel3 isolate. Furthermore, there was an apparent shift in the colonisation of the nematodes within the host cadaver the proportion of nematodes carrying the yel3 isolate was higher in later emerging nematodes (Chapter 3). To test the hypothesis that the distribution of the colour variants was due to host specialisation (H. downesi complex differs from front to back of the dune system) the pathogenicity of the homogenous and heterogenous combinations against Hylobius abietis larvae was investigated. The LD50 was significantly higher for the homogenous YEL/yel combination compared to the heterogeneous YEL/pur combination. There was no difference in reproduction rate of the nematodes carrying either subspecies in either H. abietis or G. mellonella and this was backed up by tests of all three isolates of each colour type. All six isolates from North Bull Island were tested against insects from three orders (Lepidoptera, Coleoptera (2) and Diptera), with no differences in mortality or reproduction rate. This is the first report of EPN being able to use the novel host kelp fly (Coelopa spp.). While there was no difference in mortality rates between the PUR and YEL strains in this host there did appear to be a difference in the rate of progression of the infection with nematodes carrying the yel bacteria emerging earlier than those carrying the pur bacteria (Chapter 4). To test the hypothesis that the bacteria influenced nematode survival in desiccated hosts G. mellonella larvae were infected with the homogenous and heterogeneous combinations and stored in dry sand or at 0% relative humidity. There was no difference due to bacteria type in the number of cadavers with emergence from those stored in dry sand for 28 days. There was a highly significant difference due to bacteria type in the number of cadavers with emergence for cadavers stored at 0 % relative humidity for 28 days, with emergence from more cadaver infected with the pur1 isolate. Overall, more nematodes emerged from cadavers infected with the pur1 isolate following 28 days storage in dry sand or at 0 % relative humidity (Chapter 5). Heterorhabditis downesi is associated with two different subspecies of Photorhabdus temperata on North Bull Island: Photorhabdus temperata subsp. temperata and Photorhabdus temperata subsp. cinerea and the association of the nematode with a particular subspecies appears to vary with the soil conditions prevalent in the front and the rear sections of the dunes: Under normal conditions P.t. temperata appears to outcompete P.t. cinerea in forming a symbiotic association with the nematode when both subspecies are present in the same insect host, resulting in the H. downesi-P.t. temperata association predominating in the rear section of the dunes, where the increased organic content of the soil favours moisture retention. On the other hand the drier conditions that predominate in the sandier soil towards the front of the dune system favour H. downesi-P.t. cinerea association.

Item Type:	Thesis (PhD)
Keywords:	Co-existence; niche separation; two subspecies; Photorhabdus temperata; Heterorhabditis downesi; dune grassland;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	7759
Depositing User:	IR eTheses
Date Deposited:	16 Jan 2017 11:54
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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