Katie Marshall

Intertidal invertebrates often adopt freeze tolerance as a strategy to survive winter low tides, but the physiological mechanisms by which intertidal species survive freezing is not well understood. Ice-binding proteins (IBPs) may play an important role, but their occurrence throughout the intertidal zone worldwide is not well-catalogued. Here I survey species in the intertidal zone of Vancouver, BC to assess IBP activities and the possible role they play in freeze tolerance. I conducted targeted assays in multiple intertidal species to measure three distinct activities of IBPs: ice nucleation (IN), ice recrystallization inhibition (IRI), and thermal hysteresis (TH), and showed that some activities were decreased when proteins were denatured. I also exposed intertidal species to a cold exposure mimicking a freezing event in the intertidal and measured survival post-exposure. I found that species with the highest degree of freeze tolerance display IN activity attributable to proteins. I also identified three species –Mytilus trossulus, Magallana gigas, and Lottia persona – that showed evidence of containing IRI proteins. I found no measurable TH in any species, but there is evidence that TH proteins may be present in at least five species as evidenced by characteristic ice shaping activity seen in the nanoliter osmometer. I also found that species that live in the high intertidal appear to show more IBP activity than species that live lower in the intertidal. This thesis significantly increases the number of known intertidal species with IBPs, and provides a framework for sampling new species for IBP activity in the intertidal and elsewhere.

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Molluscs live in diverse habitats, but the physiological mechanisms enabling their wintersurvival in temperate and polar environments remain poorly understood. Here I investigated thecold tolerance of two molluscan species: the terrestrial slug Ambigolimax valentianus and theintertidal bay mussel Mytilus trossulus. My first objective was to understand the cold toleranceof A. valentianus, an invasive slug that has established populations worldwide. To do this, Iacclimated A. valentianus to different environmental conditions (differing day lengths andtemperatures), then exposed them to sub-zero temperatures and measured survival. Then, Imeasured low molecular weight metabolites using ¹H NMR to see if they play a role in their coldtolerance as they do in other invertebrate species. I found that A. valentianus is not freezetolerant but does become more cold-hardy after acclimation to shorter day lengths. I also foundthat low molecular weight metabolites were not upregulated in response to winter conditions, andinstead I saw evidence of metabolic suppression leading up to winter. My second objective wasto better understand the freeze tolerance of M. trossulus through investigating biologicalmolecules and whole-body ice formation. I predicted that aquaporin water channels would play arole in freeze tolerance, that ubiquitin would exhibit seasonal differences in expression, and thatice formation would occur in a consistent way throughout the body. To test these predictions, Ifirst used western blotting to measure aquaporin expression after a 3-hour freeze, and then useddot blotting to measure ubiquitin concentrations to estimate protein damage from musselscollected from different tidal heights across eight months. I then imaged whole-body iceformation using a thermal infrared camera. I found aquaporins were not up-regulated afterfreezing, and that protein damage increased in thermally stressful months in both summer andwinter. I also found that mussels began ice formation at the anterior end of their body. Taken together, these findings contribute to a deeper understanding of how molluscs respond to cold stress at physiological and molecular levels, highlighting the role of environmental and species-specific factors in shaping the mechanisms underlying cold tolerance.

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The mechanisms behind freeze tolerance in intertidal invertebrates is poorly understood. Due to differences in habitat and physiology, many biochemical processes utilized by terrestrial freeze tolerant organisms are not possible for intertidal invertebrates. Here I investigate the potential role of ice-binding proteins (IBPs) in the freeze tolerance of intertidal invertebrates. I first used bioinformatics to determine if there is molecular evidence for IBPs in intertidal invertebrates. I found a significant overrepresentation of putative IBPs in intertidal invertebrates relative to invertebrates from other habitat types, with no taxonomic patterns. These putative IBPs had high sequential similarity to type II antifreeze proteins from fish and antifreeze glycoproteins from both fish and ticks. Using some basic gene mapping I was also able to investigate the potential evolutionary origin of one of these putative IBPs from a mussel species (Mytilus coruscus), finding that a duplication and neofunctionalization event likely occurred. Knowing this I investigated the role of IBPs in the freeze tolerance of the local mussel species M. trossulus, a species that is more freeze tolerant in individuals from high shore positions during the winter months. I predicted that IBP activity would be measured in the protein extract of the species and that said activity would be greatest in winter individuals from high shore heights. Using a series of freezing assays and chemical treatments, I was able to find ice nucleation activity in M. trossulus and show strong evidence the activity was mediated by a protein, which I interpreted as IBPs. IBP activity did not vary by season or by shore height. This means IBPs may play a role in the year-long freeze tolerance of the species, but other mechanisms must explain the seasonal and tidal patterns in their freeze tolerance. In all, this thesis expands on our knowledge of intertidal freeze tolerance and provides the groundwork for future research into IBPs in multiple intertidal species.

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Many intertidal invertebrates are freeze tolerant, meaning that they can survive ice formation within their bodies when exposed to freezing air temperatures during low tides. In my thesis I addressed two key questions regarding intertidal invertebrate freeze tolerance using the intertidal mussel Mytilus trossulus. First: What biochemical mechanisms enable freeze tolerance in intertidal invertebrates? Second: How do sublethal single and repeated freeze exposures negatively impact intertidal invertebrates? To address the first question, I investigated the role of osmolytes in mussel freeze tolerance, which may be cryoprotective by mitigating osmotic stress caused by freezing. I sought to determine if different osmolytes are interchangeable cryoprotectants (acting as colligative cryoprotectants), or if each osmolyte has unique a cryoprotective role, beyond just contributing to increased intracellular osmolarity (and thus act as non-colligative cryoprotectants). I did this by manipulating the composition of mussels’ intracellular osmolyte pools, and then testing how mussel freeze tolerance changed. I found that mussel freeze tolerance did not change after taurine and betaine increased in concentration, significantly decreased after alanine and glycine increased in concentration, and increased with increasing TMAO concentrations, indicating that TMAO may be cryoprotective. Overall, my findings indicate that osmolytes are non-colligative cryoprotectants. Next, I explored how mussels are impacted by sublethal freezing. I found that mussels do not filter feed for the first four hours post-freeze, but resume filter feeding 24 hours after freezing, which corresponds to my microscopic examinations of mussel gill tissues after freezing which reveal freeze-related damage. I also found that freezing decreased mussel posterior adductor strength, although this effect did not lead to an increase in mussel susceptibility to sea star predation. Finally, I found that mussels survived shorter, repeated freezes (where mussels received 1 day for recovery between freezes) better than prolonged freezes, when total time frozen is held constant. Thus, mussels are well-adapted to survive the short freezing events which they regularly encounter in their habitat, and one mechanism behind this survival could be TMAO accumulation. Further, the effects of sublethal freezing on mussel performance are limited, although how these effects scale up to entire mussel beds remains unknown.

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Of all abiotic factors that drive range boundaries, temperature is the best studied because of its pervasive influence on biological processes. For populations at high-latitudes, extreme cold and the populations’ cold-hardiness set the range boundary. Phenotypic plasticity, where a single genotype results in differentiated phenotypes under differential environmental conditions, can assist populations in managing changing temperatures. Local adaptation in phenotypic plasticity, which results in different responses in different populations, can assist with the variability in temperature a species can experience across its range, especially at range boundaries. I used the eastern spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae) as a model system for exploring local adaptation and phenotypic plasticity of insect cold-hardiness. The species is one of the most destructive forest pests in North America, therefore accurately predicting its range and population growth is essential for management. In this thesis, I show that there is no transgenerational plasticity in cold-hardiness. However, I found a fitness cost associated with repeated cold exposures. Additionally, across the species’ range, I found both local adaptation of seasonal cold-hardiness and short-term plasticity of this trait. Therefore, the findings of this thesis provide evidence for including phenotypic plasticity and local adaptation when modelling species distributions under climate change.

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