M.Sc. / Please refer to full text to view abstract

The increasing incidence of infections due to antibiotic resistant bacteria has led to renewed interest in bacteriophages (= phages) and phage therapy. Although phage therapy has been applied to control bacterial diseases in plants, poultry, livestock and humans, its application in aquaculture is still relatively limited. The emergence of phage-resistant bacterial mutants has been considered to be one of the major limitations of phage therapy. This study aimed to (i) isolate and characterise phages; (ii) select phages and their bacterial hosts to set up in vivo phage therapy models with aquaculture animals, and estimate the efficiency of phage therapy; (iii) investigate the generation and characteristics of phage-resistant mutants, and thus estimate the consequence of applying phage therapy when phage-resistant mutants emerge; and (iv) discuss the prospects for application of phages in aquaculture. Two Vibrio isolates and their phages were isolated from a Scottish marine fish farm. Based on the results of conventional phenotype testing and 16S rRNA gene sequencing analysis, the two vibrios, V9 and V13, were identified as Vibrio splendidus and Vibrio cyclitrophicus, respectively. The bacterial characteristics including morphology, temperature and salinity range of growth, production of extracellular enzymes, and the possession of virulence genes were examined. According to the morphological characteristics observed using transmission electron microscopy by negative staining, phage PVS9 of V. splendidus V9 was identified as a myophage, while phage PVC13 of V. cyclitrophicus V13 was identified as a siphophage. The phages could only lyse one bacterial host strain and their genomic DNA was double stranded with a size of ~46 kb. The two Vibrio isolates were found to be non- or of low virulence to rainbow trout, goldsinny wrasse and Artemia in pathogenicity experiments. Thus an in vivo phage therapy model could not be set up using these Vibrio isolates and their phages. Two phages pAS-3 and pAS-6 were isolated using the Aeromonas salmonicida subsp. salmonicida Hooke strain as the host. Phages pAS-3 and pAS-6 had a similar genome size of ~50 kb, and the same relatively narrow host range within A. salmonicida subsp. salmonicida strains. The siphophage pAS-3 formed clear plaques and inhibited A. salmonicida Hooke growth in vitro completely for at least 18 hours when using MOI = 1,000, whereas the podophage pAS-6 formed turbid plaques and weakly inhibited Hooke growth. Rainbow trout exposed by intraperitoneal injection with 0.1 mL of the raw phage preparations at a concentration of 108 PUF mL-1 showed no adverse effects over 14 days. In the phage therapy trial, fish were firstly injected with 1 x 102 CFU fish-1 of A. salmonicida Hooke, then immediately injected with phage preparations of pAS-3 and pAS-6, respectively, using MOI = 10,000. Compared with the control group (which did not receive phage treatment), phage treated groups showed a delay in the time to death, and lower mortalities. However, the mortalities and time to death between phage treated and non-treated groups were not significantly different. Phage-resistant mutants of pathogenic A. salmonicida strain Hooke were induced by repeatedly challenging with phage pAS-3. One of the mutants, termed HM, was chosen to compare the characteristics with the parental wild-type strain Hooke. Test results including the formation of ‘smooth’ colonies on TSA, autoagglutination negative, the formation of creamy colonies on Coomassie Brilliant Blue agar, and the degradation of a thick/furry layered structure on the cell surface indicated a deficiency of the A-layer in the phage-resistant mutant HM. Therefore, it was deduced that the A-layer either directly acted as the receptor of A. salmonicida phage pAS-3, or was affected indirectly by the change of an unknown phage receptor. The greater wax moth larvae model was used to compare the virulence of the phage-resistant mutant HM and the parental wild-type strain Hooke, as it is an ethically acceptable animal model, which has the advantages of being low cost and convenient for injection, and is also a recognised alternative model for bacterial pathogens of fish. The results showed that virulence of the phage-resistant mutant HM did not decline in the greater wax moth larvae model compared with that of the parental wild-type strain Hooke. In conclusion, different approaches were used to isolate and characterise phages from different aquaculture environments for potential use in phage therapy. A rainbow trout model was set up using pathogenic A. salmonicida strain Hooke and two A. salmonicida phages pAS-3 and pAS-6. The use of phage treatment led to lower cumulative mortalities and delay to the time of death, although the differences between the groups were not significant, futher work is required to determine if these phages have potential in phage therapy. The consequence of applying phage therapy when phage-resistant mutants emerge was estimated based on their characteristics and virulence, and no decline in virulence of the phage-resistant mutant from this study indicates the importance of fully testing the virulence of phage-resistant mutants before carrying out large scale field trials of phage therapy. It appears feasible to use phage therapy as an alternative approach to control bacterial infections in aquaculture, but further studies are required to focus on improving effectiveness, and also to overcome the concrete limitations and hurdles in application and commercialisation. Moreover, a broader range of applications of phages in aquaculture should be explored.

Aquatic organisms in response to toxic insults from environmental pollutants activate defence systems including the aryl hydrocarbon receptor (AhR) in an attempt to metabolize and excrete these toxicants and their metabolites. These detoxification mechanisms however may come with certain energetic costs. I hypothesize that the activation of the AhR by β-Naphthoflavone (β-NF), a model AhR agonist, results in increased energetic costs requiring metabolic reorganization in rainbow trout hepatocytes. While the results obtained suggest that there are no significant energetic costs of AhR activation, analysis of enzyme activities suggests possible metabolic reorganization. This study also showed significant changes in cellular processes in hepatocytes over the incubation periods which previously were not reported. Furthermore, for the first time in fish hepatocytes, metabolic flux analysis (MFA) was used to examine intra-cellular metabolism, the applicability of which is discussed.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

Film is commonly used to estimate the fast-start performance of fish. An analysis of hypothetical, film-derived, and accelerometer-measured acceleration-time data of fish fast-starts indicates that the total error in film studies is the sum of the sampling frequency error (i.e., the error due to over-smoothing at low film speeds) and measurement error. The error in film based studies on the acceleration performance of fish is estimated to be about 33 to 100% of the maximum acceleration, suggesting that other methods of estimating acceleration should be employed. The escape performance of rainbow trout Oncorhynchus mykiss and northern pike Esox lucius (mean lengths 0.32 m and 0.38 m, respectively) were measured here with subcutaneously implanted accelerometers. Acceleration-time plots reveal two types of escape fast-starts for trout and three for pike. Simultaneous high-speed ciné films demonstrate a kinematic basis for these differences. Trout performing C-shaped fast-starts produce a unimodal acceleration-time plot (type I), while during S-shaped fast-starts a bimodal acceleration-time plot (type II) results. Pike also exhibit similar type I and II fast-starts, but also execute a second S-shaped fast-start that does not involve a net change of direction. This is characterized by a trimodal acceleration-time plot (type III). Intraspecific and interspecific comparisons of displacement, time, mean and maximum velocity, and mean and maximum acceleration rate indicate that fast-start performance is significantly higher for pike than for trout, for all performance parameters. This indicates that performance is related to body form. Overall mean maximum acceleration rates for pike were 120.2 ± 20 m s⁻² (x ± 2S.E.) and 59.7 ± 8.3 m s⁻² for trout. Performance values directly measured from the accelerometers exceed those previously reported. Maximum acceleration rates for single events reach 97.8 m s⁻² and 244.9 m s⁻² for trout and pike, respectively. Maximum final velocities of 7.06 m s⁻¹ (18.95 L s⁻¹, where L is body length) were observed for pike and 4.19 m s⁻¹ (13.09 L s⁻¹) for trout; overall mean maximum velocities were 2.77 m s⁻¹ for trout and 3.97 m s⁻¹ for pike. The fast-start performance of pike during prey capture was also measured with subcutaneously implanted accelerometers. Acceleration-time plots and simultaneous high-speed cin6 films reveal four behaviours with characteristic kinematics and mechanics. As for the escape data, fast-start types are identified by the number of large peaks that appear in the acceleration-time and velocity-time data. Comparisons of mean performance were made between each type of feeding fast-start. Type I fast-starts were of significantly (i.e., p < 0.05) shorter duration (0.084 s) and displacement (0.132 m) than type III (0.148 s and 0.235 m) and type IV (0.189 s and 0.307 m) behaviours, and higher mean and maximum acceleration (38.6 and 130.3 m s⁻², respectively) than the type II (26.6 and 95.8 m s⁻²), type III (22.0 and 91.2 m s⁻²), and type IV (18.0 and 66.6 m s⁻²) behaviours. The type II behaviours were also of shorter duration and displacement, and of higher mean acceleration than type IV fast-starts, and were of significantly shorter duration than the type LU behaviours. Prey capture performance was compared to escapes by the same individuals. When data are combined, regardless of mechanical type, mean acceleration (37.6 versus 25.5 m s⁻²), maximum acceleration (120.2 versus 95.9 m s⁻²), mean velocity (1.90 versus 1.57 m s⁻¹), and maximum velocity (3.97 versus 3.09 m s⁻¹) were larger, and duration shorter (0.108 versus 0.133 s) during escapes than during prey capture. No differences were found through independent comparisons of the performance of feeding and escape types II and III, but type I escapes had significantly higher mean velocity (2.27 versus 1.58 m s⁻¹), maximum velocity (4.70 versus 3.12 m s⁻¹), and mean acceleration (54.7 versus 38.6 m s⁻²) than the type I feeding behaviours. Prey capture performance was also related to prey size, apparent prey size (defined as the angular size of the prey on the pike's retina), and strike distance (the distance from the pike to the prey at the onset of the fast-start). Mean and maximum acceleration increased with apparent size and decreased with strike distance, while the duration of the event increased with strike distance and decreased with apparent size. No relation was found between the actual prey size and any performance parameter. Strike distance ranged from 0.087 to 0.439 m, and decreased as the apparent size increased from 2.6 to 9.9° (r² = 0.75). The type I behaviour was usually employed when the strike distance was small and the prey appeared large. As strike distance increased and apparent size decreased, there was a progressive selection of type II, then III, then IV behaviours. / Science, Faculty of / Zoology, Department of / Graduate

This research reports on the determination of the effects of fish size and feed pellet size on the settling characteristics of Rainbow trout (Salmo gairdneri) culture, tank cleaning wastes. Flocculant particle settling curves (Type II) were developed from settling column analysis of cleaning wastes from 11-311 gram Rainbow trout fed a moist pellet diet (Oregon Moist Pellet ®). Four feed pellet sizes were investigated: 3/32, 1/8, 5/32 and 3/16 inch. Overall non-filterable residue removal curves and individual particle settling velocity distribution curves, derived from the Type II settling curve of each fish size and feed pellet size group, were compared. Slopes and y-intercepts of the linearized overall non-filterable residue removal curves and individual particle settling velocity distribution curves were compared using the Equality of Slope Test (S:SLTEST). Results of the test for a common regression equation indicated there were no significant differences in the proportional distribution of particle sizes within the cleaning wastes. Variations observed in the initial rates of removal within the overall non-filterable residue removal curves were considered insignificant Settling trials were pooled in order to obtain single curves, characterizing the overall solids removal rate and the individual particle settling velocity distribution of the waste solids. / Applied Science, Faculty of / Chemical and Biological Engineering, Department of / Graduate

This study provides comparative data on the effects of temperature on zygote and alevin survival, rate of development to 50 percent hatching and emergence, and alevin and fry size for five species of Pacific salmon (Oncorhynchus) and for rainbow trout (Salmo gairdneri). Fertilized eggs from each species were incubated in controlled temperature baths at five constant temperatures (2°, 5°, 8°, 11° and 14°C). At 2°C, survival for coho salmon zygotes was high (85 percent), moderate for sockeye salmon zygotes (40 percent) and low for chinook salmon zygotes (4 percent). No pink and chum salmon or rainbow trout zygotes survived at 2°C. However, at 14°C survival for chum salmon and rainbow trout zygotes was high (67 and 85 percent), moderate for chinook and pink salmon zygotes (50 and 55 percent) and low for sockeye salmon zygotes (10 percent). No coho salmon zygotes survived at 14°C. The same general pattern for temperature and survival holds for alevins. These data suggest that coho and sockeye salmon are adapted to lower incubation temperatures than the other species. All six species showed an inverse relationship between temperature and incubation time to 50 percent hatching and emergence. The data were analysed using linear regression but, even after a series of transformations, the relationship between temperature and development time remained curvilinear. The only exceptions were for chum salmon at hatching and pink salmon at emergence. Incubation temperature also influences both alevin and fry size. In general, low incubation temperatures produce larger alevins and fry than high incubation temperatures. In addition to data on constant incubation temperatures, the effects of varying temperature regimes on the survival, rate of development and size of coho salmon and rainbow trout alevins and fry were also documented. Fertilized eggs from coho salmon and rainbow trout were incubated at two varying temperature regimes. The varying temperature regimes either gradually increased from 5° to 14°C (the spring regime) or gradually decreased from 14° to 5°C (the fall regime). The increasing temperature regime produces higher survival in rainbow trout zygotes and alevins than the decreasing temperature regime. However, in coho salmon there was no clear difference in zygote and alevin survival with either regime. The rate of development to hatching for zygotes incubated at either varying temperature regime was similar within a species because of similar mean incubation temperatures between regimes. But, the rate of development to emergence for alevins incubated at either varying temperature regime was different because of different mean temperatures between regimes. The linear regressions to hatching and emergence for coho salmon and rainbow trout were used to predict rates of development for zygotes and alevins incubated with each varying temperature regime. The actual and predicted rates of development to hatching and emergence are similar within a species. Varying temperature regimes also affect both alevin and fry size. The decreasing temperature regime produces larger alevins and fry in coho salmon and rainbow trout than the increasing temperature regime. / Science, Faculty of / Zoology, Department of / Graduate

Studies were undertaken to determine the cardiac and ventilatory responses of restrained and unrestrained rainbow trout (Salmo gairdneri) to changes in inspired oxygen and carbon dioxide tensions. The role of blood oxygen carrying capacity in the control of ventilation was investigated, as well as the location and innervation of oxygen receptors activated by hypoxia. Ventilation volume (Vg) was measured directly in restrained fish using a ventilation chamber that separated inspired from expired water, the latter being collected in a graduated cylinder. In receptor localization experiments a wooden tongue depressor held vertically in the buccal cavity in the median plane divided water flows to the gills on each side of the fish. Thus, one set of gills could be irrigated with hyperoxic water to maintain arterial oxygen tension, while hypoxic water was passed over the other set of gills. Blood samples were obtained from cannulae implanted in both the dorsal aorta and right common cardinal vein. Vg increased in hypercapnea (inspired CO₂ tension (PICO₂) 0.5-2.0 kPa) due to increased "stroke volume (frequency remained constant), with higher levels of Vg recorded at higher C0₂ tensions. In fish exposed to PICO₂ levels of 0.5 and 0.9 kPa, raising the inspired oxygen tension (PIO₂) to 60.4 kPa eliminated the ventilatory response to hypercapnea. Hyperoxia had little or no effect on ventilatory responses to (PICO₂) levels of 1.5 and 2.0 kPa. Ventilation volume was inversely related to blood oxygen content (CaO₂) in trout. CaO₂ decreased and Vg increased during hypercapnea (PICO₂ 0.8 kPa), hypoxia (PIO₂12.4 kPa) and anaemia (haematocrit reduced from 22.3% to 14.3%), while CaO₂ increased and Vg decreased during hyperoxic hypercapnea (PIO₂ 60.4 kPa, PICO₂ 0.8 kPa). Increased Vg during hypercapnea is attributed to hypoxaemia produced by Bohr and Root off-shifts which result from increased blood CO₂ tension and reduced blood pH. Oxygen uptake remained constant during all experimental trials, indicating that the manoeuvre of increasing Vg is effective in relieving adverse effects of hypoxaemia. The significance of elevated Vg as a short-term adaptation to hypoxaemia is discussed, Heart rate decreased and ventilation increased in unrestrained fish exposed to gradual hypoxia (PIO₂ decreased from 20 kPa to 4 kPa) at 7°C and 16°C. The initial heart rate of fish acclimated to 16°C was higher than that of the 7°C group, but at the lowest level of PIO₂, heart rates of both groups dropped to the same level. Thus, the cardiac chronotropic response to hypoxia in trout is temperature independent. Receptors causing hypoxic bradycardia are located in the dorsal region of the first gill arch. Hypoxic bradycardia was eliminated by removing the first gill arch, or by sectioning the branches of cranial nerves IX and X innervating the arch. Blood flow through the arch does not appear to be necessary for this response, since ligation of the arch at its ventral insertion on the body wall did not affect hypoxic bradycardia. The pseudobranch has no role in cardiac control since interrupting the flow of blood through, and deafferentation of, the pseudobranch had no effect on the cardiac response to hypoxia. The biological significance of hypoxic bradycardia, and ventilatory-circulatory interaction during hypoxia, are discussed. Ventilatory responses to hypercapnea and hypoxia were unchanged after bilateral section of the nerves to the first gill arch. Receptors in the first gill arch thus have no role in control of ventilation during either hypercapnea or hypoxia. Possible locations for receptors responsible for control of ventilation are discussed. / Science, Faculty of / Zoology, Department of / Graduate

This study was conducted to satisfy three objectives. The first was to provide a detailed description of the differences between the prey contents of rainbow trout (Salmo qairdneri) and kokanee (Oncorhynchus nerka) compared either to each other or to the prey contents of the natural environment. The second was to determine how elements of anatomy, physiology and behaviour interact to promote the acquisition of species specific diets by trout and kokanee. The third was to precisely identify the foraging strategies of the two predators by assessing how different anatomical and behavioural characteristics serve as interrelated adaptations that suit each species to effectively use a specific habitat-prey complex. Matched samples of trout and kokanee from Marion Lake exhibit only modest A dietary overlap (mean of C λ = .462, range.136 to .881). Although kokanee appear to track the environmental abundance of prey more closely than trout, both predators exhibit pronounced patterns of "density independent" acquisition of prey from the total complex of prey that is apparently available in the lake. To test hypotheses about the factors that control these dietary patterns, I conducted a series of studies concerning where trout and kokanee choose to forage, when they choose to forage, how they search for prey, how they attack prey, and how experience in encountering various prey alters the predator's foraging behaviour. Temporal segregation of trout and kokanee foraging activities is not well-developed under field conditions and appears unlikely to promote strong patterns of food-resource partitioning. By contrast, spatial segregation is well-developed and clearly plays a major role in promoting the acquisition of relatively large numbers of nearshore benthic prey ( eg. planorbid snails or odonates ) by trout and of relatively large numbers of offshore, water-column prey ( eg. chironomid pupae ) by kokanee. Close inspection of the details of predator and prey distributions indicates that many aspects of food-resource partitioning are not logical outcomes of spatial segregation. Differences in predator search-techniques do not determine the presence or absence of various prey types in predator diets, however, differences in predator search behaviours do bias them to obtain different quantities of particular classes of prey. Kokanee search procedures allow them to detect prey in both exposed and concealed locations while trout detect only exposed prey. While searching for benthic or lake-surface prey, kokanee maintain search positions which allow them to detect prey of smaller sizes than trout. This clearly favours the trend for kokanee to include greater quantities of small prey (eg. Hyalella sp.) in their diets than trout. Differences in search procedures do not explain why kokanee obtain a greater proportion of their diet than trout from small zooplankton (≤ 1 mm ) in late summer or why kokanee seldom exploit any of the relatively large ( > 4 mm ), armoured prey that are common in the diet of similar sized trout. Differences in both behavioural and morphological characteristics involved in the attack phase of foraging by trout and kokanee serve as the basis for explanations of a number of differences between the diets of free-ranging predators. These differences include: the greater utilization of aerial prey by trout, the inclusion of large numbers of copepods in the diet of kokanee but not of trout, the generally greater utilization of zooplankton by kokanee compared to trout, and the relative-scarcity of large ( > 4 mm ), armoured prey, in the diet of kokanee. A series of laboratory experiments was used to examine the extent to which short term experience might influence food-resource partitioning by trout and kokanee. These experiments offered convincing evidence that differential effects of experience will amplify the trends in resource partitioning already set in motion by differences in habitat selection, search procedures, and attack procedures. I argue that the morphological and behavioural traits that control food "selection" by trout and kokanee in Marion Lake are a consequence of the evolution of mutually exclusive foraging strategies. Trout are portrayed as D-strategists that concentrate on relatively large, dispersed prey for the bulk of their energy requirements. Adaptations which enable trout to differentially exploit large prey include: procedures for area-extensive search; a predisposition to attack relatively large, armoured-prey; large mouth-size; and persistent responses to opportunities to attack large prey. An inability to sustain high attack rates on small prey ( < 1 mm ) at high density ( 35 per liter ) and a tendency to ignore or reject such prey suggest that trout are not well-adapted to exploit relatively small, morphologically-uniform,prey. Kokanee are portrayed as C-strategists which concentrate on relatively small, contagiously-distributed prey for the bulk of their energy requirements. Adaptations which enable kokanee to differentially exploit small prey include: procedures for area-intensive search; a predisposition to attack relatively small, morphologically-uniform prey; small mouth-size; well-developed gill-rakers; and an ability to sustain high attack rates on small planktonic prey. Low ingestion success with a variety of large benthic prey and a tendency to ignore or reject such prey under laboratory conditions where they serve as the sole source of food are evidence that kokanee are not well adapted to exploit large, armoured-prey. Adaptations associated with the search, capture, manipulation and ingestion phases of the foraging cycle appear in each instance to be evolutionary responses to specific features of a given habitat-prey complex. C-selected or D-selected foraging strategies appear to be mutually exclusive evolutionary avenues down which trout and kokanee have been directed by the fundamental nature of a given habitat-prey complex. / Science, Faculty of / Zoology, Department of / Graduate

The effects of steady-state, aerobic swimming exercise upon blood volume and flow distribution in the rainbow trout {Salmo gairdneri) were examined. Isotopic Rubidium-86, and radiolabelled microspheres were injected into trout forced to swim against a current at 80% of their critical velocity (U[sub=crit]) in a Brett-type water tunnel respirometer. The results gathered from experiments using these radioactive tracers within the circulatory system of the trout indicated that blood flow during exercise was redistributed to favour working muscles, at the expense of diminished blood flow to those organs and tissues in the systemic circulation which could tolerate periods of transient hypoxia. Active hyperaemia in the skeletal muscle and vasoconstriction of the coeliacomesenteric artery, via adrenergic receptor mechanisms are proposed as the main sites of the control for blood volume and flow redistribution in the systemic circulation in trout during exercise. The gills of these fish must be able to maintain adequate gas transfer in order to keep pace with the increased metabolic demands of the working muscles during exercise. An isolated, saline-perfused trout head preparation and a spontaneously ventilating, blood-perfused whole trout preparation were developed in order to study how increases in the pulsatility of input and increases in the cardiac output through these gills; cardiovascular alterations known to occur during exercise in vivo in these fish, affect fluid flow distribution through, and within the branchial vasculature, and gas exchange across the gills. Data from these preparations indicated that pulsatility of flow increased venolymphatic fluid drainage from within gill tissues, as well increasing the fluid flux/reflux across the branchial microvasculature. However, these changes in fluid distribution associated with increased pulse pressure did not significantly change the rate of gas transfer across the gills. Although gill vascular resistances to flow were very sensitive to alterations in pulse pressure and flow rate, only perfusion flow rate through the gills could cause significant changes in the rate of mass transfer of gases across the gills. The gills of trout therefore were found to be perfusion and not diffusion limited for gas transfer, under conditions which simulated those found at rest and during exercise, in vivo. It also was shown that, given oxygen uptake and cardiac output data from the literature, combined with those for blood flow redistribution during exercise from the present study, the working muscles, which were operational during steady-state, aerobic swimming exercise in rainbow trout, could account for nearly all the measured increase in the total oxygen uptake at this level of exercise. The circulatory system of the rainbow trout, both branchial and systemic, was shown to be quite efficient in its ability to take up and distribute oxygen to the tissues during prolonged, aerobic swimming exercise. The numerous cardiorespiratory adjustments noted during exercise account for this animal's ability to maintain swimming activity in the face of increased oxygen demands put upon the circulation by enforced activity. / Science, Faculty of / Zoology, Department of / Graduate