Jeffrey Richards

Professor

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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Adaptive variation of mitochondrial function in response to oxygen variability in intertidal sculpins (Cottidae, Actinopterygii) (2018)

Variation in environmental oxygen (O₂) poses a significant physiological challenge to animals, not only because of the impact on aerobic metabolism, but also because it can lead to generation of potentially harmful reactive oxygen species (ROS). In this thesis, I aimed to investigate the interplay between two aspects of O2 use at the mitochondria, aerobic respiration and ROS metabolism, using species of intertidal sculpins (Cottidae, Actinopterygii) which are distributed along the marine intertidal zone, exposed to varying O2 conditions and vary in their tolerance to low O₂ (hypoxia). I first hypothesized that there would be a relationship between whole animal hypoxia tolerance and mitochondrial and cytochrome c oxidase (COX) O₂-binding affinity, whereby hypoxia tolerant sculpins would have higher mitochondrial and COX O₂-binding affinity than less hypoxia tolerant sculpins. This hypothesis was supported with functional analysis. In silico modelling of the COX catalytic core revealed that the variation in O₂ binding was related to interspecific differences in the interaction between COX3 and membrane phospholipid, cardiolipin, which could impact O₂ diffusion to its binding site. I then investigated whether intact mitochondria from hypoxia tolerant sculpins were able to use O₂ more efficiently such that phosphorylation efficiency was improved and ROS generation was reduced compared to mitochondria from less hypoxia tolerant sculpins. Although there were relationships between hypoxia tolerance and complex I and II dependencies, there were no interspecies differences in phosphorylation or mitochondrial coupling that would indicate differences in aerobic metabolism. Moreover, mitochondria from hypoxia tolerant sculpins generated more ROS under resting conditions and were more perturbed by in vitro redox and anoxia-recovery challenges. Finally, I confirmed consistent responses of mitochondria to in vivo responses with a whole animal study comparing ROS metabolism (redox status, mitochondrial H₂O₂, oxidative damage and scavenging capacity) between two sculpin species with different hypoxia tolerance to hypoxia, hyperoxia, with normoxia-recovery exposures. Taken together, my thesis demonstrates that hypoxia tolerance is associated with improved O₂ binding at the mitochondria and COX. Further, hypoxia tolerance in sculpins is associated with higher ROS generation compared to less tolerant species, suggesting a potentially important role of ROS in mediating hypoxia tolerance.

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The hypoxic metabolic response : how time and PO₂ shape the way fishes combine aerobic, anaerobic and depressed metabolism in hypoxic environments (2017)

Animals rely on O₂ to balance cellular ATP supply and demand. In O₂-limited hypoxic environments, survival depends on the maintenance of this balance and is accomplished through some combination of aerobic metabolism, anaerobic metabolism and metabolic rate depression (MRD). My thesis studied how fishes combine these three metabolic strategies as a total hypoxic metabolic response (HMR) to survive hypoxic environments that vary in O₂ level (PwO₂) and duration. Calorimetry is required to accurately measure the metabolic rates (MR) of hypoxia (or anoxia)-exposed fishes that are partially reliant on anaerobic glycolysis and/or MRD. Thus, I started by building a novel calorespirometer that simultaneously measures indices of aerobic metabolism, anaerobic metabolism and MRD, and used it for the remainder of my thesis projects. Using goldfish, I found that time influences how PwO₂ affects HMR. Under acute and continually decreasing PwO₂ conditions, goldfish maintained routine O₂ uptake rates (ṀO₂) to ~3.0 kPa PwO₂ (i.e., Pcrit), but sustained routine MR to 0.5 kPa by up-regulating anaerobic glycolysis. Under constant hypoxia (1 or 4 h) at a variety of PwO₂s, however, goldfish maintained routine ṀO₂ to ~0.7 kPa and consequently reduced their reliance on anaerobic glycolysis. I confirmed this rapidly enhanced O₂ uptake ability in subsequent experiments by using different rates of hypoxia induction (RHI) to vary the amount of time goldfish spent at hypoxic PwO₂s. Gradual RHIs yielded greater lamellar surface areas, haemoglobin-O₂ binding affinities, and subsequently, lower Pcrits than rapid RHIs. However, goldfish only induced MRD below 0.7 kPa. To test the idea that MRD is reserved for extreme hypoxia, I compared two threespine stickleback populations from two isolated lakes: one that experiences deep, long-term hypoxia due to winterfreeze (Alta Lake), and the other that does not (Trout Lake). The two populations did not differ in Pcrit or capacities for anaerobic metabolism, but Alta Lake sticklebacks, which were 2-fold more hypoxia-tolerant than Trout Lake sticklebacks, employed hypoxia-induced MRD while Trout Lake sticklebacks did not. My results reveal that the HMR varies with an animal’s biology and the abiotic aspects of its natural hypoxic environment in a way that may optimize hypoxic survival.

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Biochemical and molecular traits underlying hypoxia tolerance in sculpins (2015)

Variation in environmental conditions can sometimes impose severe limitations on organismal survival and reproductive success and organisms that live in variable environments have evolved complex traits to cope with or buffer against the environmental conditions. In my dissertation I examined how adaptive evolution and phenotypic plasticity play a role in the ability of nearshore species of sculpin to tolerate low levels of O₂ or hypoxia. I showed that constitutively expressed biochemical traits in the brain correlated with hypoxia tolerance among the different species independent of phylogenetic relationships, suggesting that these traits may have evolved via natural selection in response to hypoxia. A similar correlation was not seen in either the liver or the white muscle. Next, I showed that transcriptionally mediated phenotypic plasticity is likely associated with the difference in hypoxia tolerance between two species of sculpin. The hypoxia-tolerant tidepool sculpin (Oligocottus maculosus) did not alter gene transcription during the ecologically relevant time-frames of hypoxia exposure (up to 8 hours), in contrast to the hypoxia-intolerant silverspotted sculpin (Blepsias cirrhosus). This suggests that the tolerant species may not rely on phenotypic plasticity during a typical environmental hypoxia exposure and instead may rely on constitutively expressed or fixed traits for survival. Only if hypoxia persists do the hypoxia tolerant tidepool sculpins alter gene transcription, for which a large set of genes showed transcriptional patterns that were divergent to the hypoxia intolerant silverspotted sculpin. Lastly, I examined if similar transcriptional responses occur among three species of sculpin all with the same measured hypoxia tolerance. While a high proportion (65%) of clones showed similar transcription patterns among the species, a majority of genes associated with metabolism and protein production showed differences in both short and long exposures to hypoxia. As metabolism and protein production both play a major role in hypoxic survival, transcriptional differences in genes belonging to these biological processes suggests that the species likely use different mechanism to achieve similar overall hypoxia tolerance phenotype. Combined, this work demonstrates how phenotypic plasticity and adaptive evolution play a role in the variation of hypoxia tolerance among species of sculpin living in the nearshore environment.

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Hypoxia tolerance in fishes : cardiorespiratory performance and metabolism (2012)

Cardiac failure occurs in most vertebrates including humans following even short hypoxia exposure due to an inability to match cardiac energy demand to the limited energy supply. In contrast, hypoxia-tolerant ectothermic vertebrates show the remarkable ability to maintain cardiac energy balance and stable cardiac function during prolonged exposure to severe hypoxia (cardiac hypoxia tolerance, CHT). I investigated how CHT is achieved and its relationship to whole-animal hypoxia tolerance using measurements at multiple physiological levels in two study models: 1) tilapia, a hypoxia-tolerant teleost, and 2) a two-species comparison of elasmobranchs with different hypoxia tolerance. I tested the hypothesis that CHT depends upon the depression of cardiac power output (PO) (i.e., cardiac energy demand) to a level lower than the cardiac maximum glycolytic potential (MGP). All species showed a hypoxic PO depression via bradycardia and my work generally supports this hypothesis. However, in tilapia, hypoxic PO depression is not necessarily required to maintain cardiac energy balance, contrary to previous suggestions, because of an exceptionally high MGP. Thus, in certain species, PO depression may primarily benefit CHT by minimizing fuel use and waste production. I also tested the hypothesis that greater hypoxia tolerance is associated with enhanced hypoxic O₂ supply and consequently enhanced cardiovascular function (i.e., less PO depression and improved cardiac energy balance). My work on elasmobranchs supported this hypothesis and also suggested a role for strategic cardiac O₂ supply via O₂ sparing resulting from metabolic rate depression (MRD) in non-essential tissues. Finally, my work on elasmobranchs showed that critical oxygen tension (Pcrit) predicts hypoxic blood O₂ transport, supporting the use of Pcrit as an indicator of hypoxia tolerance. Next, I tested the hypothesis that hypoxic PO depression is associated with the depression of whole-animal O₂ consumption rate below Pcrit. I found that this occurred in all species, suggesting that modulation of peripheral demand for blood flow (e.g., via MRD) may influence CHT. Finally, my work on in vivo and in situ cardiac responses in tilapia provided little evidence for the hypothesis that hypoxic modulation of aerobic energy production pathways, including provision of aerobic fuels (specifically, fatty acids), contributes to CHT.

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Master's Student Supervision (2010 - 2018)
Establishing the optimal salinity for rearing salmon in recirculating aquaculture systems (2016)

Aquaculture of salmon worldwide is a 15.3 billion dollar industry and the majority of fish are produced in net-pen systems in coastal waters. Recently producers have begun investigating the feasibility of moving salmon production onto land and into recirculating aquaculture systems (RAS). The major downsides to RAS are the startup and operational costs; however the ability to optimize many environmental variables to enhance growth and feed conversion, something impossible to do in net-pen systems, may help defray these otherwise prohibitive costs. Salinity may be the most important of these variables due to the metabolic cost of osmoregulation, which has been estimated to account for 5-50% of routine metabolic rate. Decreased osmoregulatory costs could result in a greater allocation of energy toward growth, thus shortening production times and improving feed conversion efficiency. To establish an optimal salinity for growth in salmon, seven replicate, 15,000 liter RAS were constructed at the University of British Columbia’s InSEAS research facility. I conducted a preliminary study to validate that each system was able to control water quality parameters and yield similar levels of growth and feed conversion in coho salmon (Oncorhynchus kisutch). I then conducted salinity trials with Atlantic (Salmo salar) and coho salmon. Fish were grown in five salinities ranging from freshwater to seawater (0, 5, 10, 20, 30 ppt) for approximately five months. Growth rates and feed conversion ratios (FCR) were measured throughout the trial. The fastest growth rate and lowest FCR in coho salmon was at 10 ppt, which is approximately isosmotic to the blood. Growth rate of coho at intermediate salinities was almost double that at 0 or 30 ppt through the first growth period. This trend was not seen during the second coho growth period, possibly due to a size-dependent or density effect. Unexpectedly, salinity had no effect on growth rate and FCR in Atlantic salmon, although growth rates were consistent with those seen in industry. This research will help further move salmon production out of the oceans and onto land, alleviating some of the environmental costs associated with salmon grown in the oceans.

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Physiological basis of growth-performance trade-offs : insights from different strains of rainbow trout (2015)

Growth rate is a fundamental life history trait common to all taxa. Despite its importance, the underlying physiological mechanisms and constraints associated with growth remain poorly understood. The purpose of my research was to explore the physiological correlates and trade-offs associated with high growth. I examined a suite of physiological variables related to growth including metabolic rate, digestive capacity, and tissue energy content. Three strains of juvenile rainbow trout (Oncorhynchus mykiss) were chosen based on known differences in ecology and growth. Fish from each strain were assigned to one of three food ration treatments: (i) satiation over eight hours every day, (ii) fed 1% of body mass over eight hours every day, and (iii) complete deprivation of food. A wide range of growth rates were observed within and across all strains.I found that fast growing rainbow trout (hatchery strain) had higher standard metabolic rates and lower maximum metabolic rates and aerobic scopes, suggesting that high growth rate results in a reduced capacity to do metabolic work. I also found that trout with high growth rates, generally, had larger gastrointestinal tracts, higher maximum food consumption rate, and higher growth efficiency. Lipid content and water content had opposing correlations with growth rate; larger individuals with higher growth rates had a higher body lipid content, while water content was highest amongst smaller individuals.I show that there is a suite of physiological traits that correlate with growth rate, that these traits are affected by both genotype (strain) and environment (food ration), and that these traits appear to be consistently traded off against other correlates of performance.

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The effects of strain and ploidy on the physiological responses of rainbow trout (Oncorhynchus mykiss) to pH 9.5 exposure (2014)

British Columba (BC) has a well-established lake-stocking program that relies on hatchery-reared rainbow trout (Oncorhynchus mykiss) from multiple wild and domesticated strains. These strains are stocked into BC lakes as diploids and triploids and, in general, high mortality rates are common after lake stocking due to environmental conditions. Of particular concern is that some lakes in BC are approaching a pH of 9.5, and it is not known if strain and ploidy affects the physiological responses of trout to high pH exposure. The goal of this thesis is to understand the effects of pH exposure, in both soft and hard water, on wild and domestic strains of trout as diploids and triploids. In soft water, high pH exposure resulted in more than 40% loss of equilibrium in the wild strains of trout while the Fraser Valley domesticated strain had fewer than 10% of individuals lose equilibrium overall. There were no clear differences between ploidies in loss of equilibrium. High pH exposure caused significant increases in plasma and tissue ammonia, with no differences between strains or ploidies in ammonia accumulation. In the brain, glutamine increased in response to high pH exposure and glutamate decreased suggesting a protective mechanism of glutamine production in high pH. Plasma lactate accumulated in all groups, suggesting an increase in anaerobic metabolism as a result of high pH exposure. There were no physiological differences between high pH exposure in hard and soft water among the strains tested. However, triploid rainbow trout suffered a greater loss of equilibrium than diploid trout, occurring in conjunction with a significant elevation of brain ammonia in triploid rainbow trout when compared to diploid trout in high pH water. Overall, the results of this thesis demonstrate an effect of strain on high pH tolerance in trout, but the differences in tolerance appear to not be explained by differences in ion regulation and ammonia balance.

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Hypoxia tolerance and anaerobic capacity in Danio and Devario (2013)

It has been long suggested that hypoxia tolerant species should have a great capacity to generate energy through anaerobic pathways to maintain energy balance when oxygen is limited; however, this assertion has not been rigorously tested. In the present study, I characterized hypoxia tolerance in 12 groups representing 10 species from the genera Danio and Devario (with three strains of D. rerio) and examined whether there is a phylogenetically independent relationship between variation in hypoxia tolerance and anaerobic capacity as judged by enzyme activity and anaerobic substrate concentrations present in various tissues. Hypoxia tolerance was assessed using two measures: time to loss of equilibrium (LOE) and the oxygen tension that yields 50% LOE in a group of fish over 8 hr (TLE₅₀). Time to LOE to low oxygen was very sensitive to changes in water PO₂, with no LOE seen over 8 hr in some species at 16 torr (2.1 kPa) and complete LOE within 30 min at 8 torr (1.1 kPa). At 12 torr (1.6 kPa) however, there was significant variation in time to LOE among all the species investigated. In three species (Danio rerio, Danio albolineatus and Danio choprai) time to LOE at 12 torr showed the same pattern of hypoxia tolerance as TLE₅₀. Despite the variation in hypoxia tolerance seen among the species under study, there was very little variation in the critical oxygen tension (Pcrit), which is the environmental PO₂ at which fish transition from an oxyregulating strategy to an oxyconforming strategy. Routine Ṁ₀₂ varied between the species, but the variation was primarily explained by body size and not hypoxia tolerance. Anaerobic energy capacity was estimated by measuring maximal enzyme activities of pyruvate kinase (PK), lactate dehydrogenase (LDH) and creatine phosphokinase (CPK), and concentrations of glycogen and glucose in muscle, liver and brain, plus creatine phosphate (CrP) and ATP in muscle. Through comparative analysis, I showed that the variation in hypoxia tolerance seen among species was related to some aspects of anaerobic energy metabolism, but not in a consistent fashion, indicating that other factors contribute to describing the variation in hypoxia tolerance.

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Performance of wild and domestic strains of diploid and triploid rainbow trout (Oncorhynchus mykiss) in response to environmental challenges (2012)

To determine what may contribute to the poorer survival of triploid (3n) trout in lake stocking programs relative to their diploid (2n) counterparts, we compared whole animal performance in response to environmental challenges in juvenile 2n and 3n fish from four wild strains and one domestic strain of rainbow trout. Spanning four years (2008, 2009, 2010, and 2011), wild fish were caught from nature and spawned in-hatchery along with hatchery-reared domestic trout. Offspring from all strains were raised to eight months as both 2n and 3n and exposed to low oxygen, swimming, and high temperature challenges. The only measure of performance to show a consistent difference between 2n and 3n individuals across all strains was time to loss of equilibrium (LOE) as a result of hypoxia exposure (~10% air saturation, 16 torr). Triploid trout always showed a shorter time to LOE (by 15-86% depending on the strain) relative to their 2n counterparts, with the exception of lake reared trout which showed no significant differences between 2n and 3n time to LOE. Additionally, there were no consistent effects of ploidy on critical oxygen tension, ṀO2, critical swimming speed (Ucrit), critical thermal maxima (CTMax), or muscle enzyme activities. We observed significant effects of strain on all performance measures except for CTMax. In general, the Fraser Valley domestic strain had higher Ucrit, higher ṀO2, and greater muscle enzyme activities than did Blackwater, Tzenzaicut, and Pennask wild conspecifics, suggesting that domestication affects a variety of traits in addition to growth rates.

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The role of AMP-activated protein kinase in initiating metabolic rate suppression in goldfish hepatocytes (2010)

The ability to undergo metabolic rate suppression (MRS) markedly improves chances of survival during aquatic hypoxia. In this thesis, I specifically tested the hypothesis that AMP-activated protein kinase (AMPK) initiates MRS in hepatocytes from the common goldfish (Carassius auratus). My first goal was to investigate the responses of isolated hepatocytes to changes in O₂. Goldfish hepatocytes showed a gradual decrease in cellular oxygen consumption rate (MO₂) as O₂ was decreased from normoxia (~310 µM O₂) down to the apparent P₉₀ of 13 µM, below which there was a steep decline in MO₂. The apparent P₉₀ for hepatocyte respiration matched published measurements of venous [O₂], which suggests that hepatocyte MO₂ in vivo may be regulated by O₂. To address the relationship between AMPK and MRS, several drugs were used to manipulate AMPK activity. I was able to activate AMPK with 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) under normoxic conditions, which caused a reduction in MO₂; this decrease was mediated through a decrease in protein synthesis rate via eukaryotic elongation factor 2 (eEF2) phosphorylation. Specifically, a maximal 7.5-fold activation of AMPK resulted in a 24% reduction in MO2, thus supporting the notion that AMPK activation initiates MRS. We then used compound C, a general protein kinase inhibitor, in an attempt to reverse the AICAR effects on AMPK activation, but compound C did not reverse the effects of AICAR. A recently discovered specific AMPK activator, A769662, was also used to manipulate AMPK activity. However, at all doses, A769662 failed to activate AMPK. Nevertheless, whenever I was able to activate AMPK via AICAR incubation, there was a consistent lowering of metabolic rate. Thus I have provided evidence to support the hypothesis that AMPK is important in the initiation of MRS in goldfish hepatocytes.

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