Ecological interactions between zooplankton and photosynthetic bacteria in Crawford Lake, Ontario

The present study was carried out to test the hypothesis that photosynthetic bacteria contribute a large portion of the food of filter feeding zooplankton populations in Crawford Lake, Ontario. The temporal and spatial variations of both groups of organisms are strongly dependent on one another. 14 By using C-Iabelled photosynthetic bacteria. the ingestion and clearance rates of Daphnia pulex, ~. rosea, and Keratella spp were estimated during summer and fall of 1982. These quantitative estimations of zooplankton ingestion and clearence rates on photosynthetic bacteria comprised an original addition to the literature. Photosynthetic bacteria comprised a substantial portion of the diet of all four dominant zooplankton species. The evidence for this is based on the ingestion and clearance rates of the dominant zooplankton species. Ingestion rates of D. pulex and D. rosea ranged 5 5 -1 -1 - -- 5 - -- 5 from 8.3X10 -1 to 14.6XlO -1 cells.ind. hr and 8.1X10 to 13.9X10 cells.ind. hr • Their clearance rates ranged from 0.400 to 1.000 -1 -1 -1 -1 ml.ind. hr. and 0.380 to 0.930 ml.ind. hr • The ingestion and clearance -1 -1 -1 -1 rates of Keratella spp were 600 cell.ind. hr and 0.40 ul.ind. hr respectively. Clearance rates were inversely proportional to the concentration of food cells and directly proportional to the body size of the animals. It is believed that despite the very short reg~neration times of photosynthetic bacteria (3-8 hours) their population densities were controlled in part by the feeding rates of the dominant zooplankton in Crawford Lake. By considering the regeneration times of photosynthetic bacteria and the population clearance rates of zooplankton, it was estimated that between 16 to 52% and 11 to 35% of the PHotosynthetic bacteria were' consumed· by Daphnia· pulex. and Q.. rosea per day. The temporal and spatial distribution of Daphnia pulex, !.. rosea, Keratella quadrata, K. coChlearis and photosynthetic bacteria in Crawford Lake were also investigated during the period of October, 1981 to December, 1982. The photosynthetic bacteria in the lake, constituted a major food source for only those zooplankton Which tolerate anaerobic conditions. Changes in temperature and food appeared to correlate with the seasonal changes in zooplankton density. All four dominant species of zooplankton were abundant at the lake's surface (O-4m) during winter and spring and moved downwards with the thermocline as summer stratification proceeded. Photosynthetic bacteria formed a 2 m thick layer at the chemocline. The position of this photosynthetic bacterial J-ayer changed seasonally. In the summer, the bacterial plate moved upwards and following fall mixing it moved downwards. A vertical shift of O.8m (14.5 to 15.3m) was recorded during the period of June to December. The upper limit of the photosynthetic bacteria in the water column was controlled by dissolved oxygen, and sulfide concentrations While their lower limit was controlled by light intensity. A maximum bacterio- 1 chlorophyll concentration of 81 mg Bchl.l was recorded on August 9, 1981. The seasonal distribution of photosynthetic bacteria was controlledinpart' by ·theg.-"z1ai'_.Q;~.zoopl. ank:tCm;-.Qther -ciactors associated with zooplankton grazing were oxygen and sulfide concentrations.

Factors influencing the seasonal changes in primary productivity of the photosynthetic bacteria of Crawford Lake, Ontario

A naturally occurring population of photosynthetic bacteria, located in the meromictic Crawford Lake, was examined during two field seasons (1979-1981). Primary production, biomass, light intensity, lake transparency, pH and bicarbonate concentration were all monitored during this period at selected time intervals. Analysis of the data indicated that (l4C) bacterial photosynthesis was potentially limited by the ambient bicarbonate concentration. Once a threshold value (of 270 mg/l) was reached a dramatic (2 to 10 fold) increase in the primary productivity of the bacteria was observed. Light intensity appeared to have very little effect on the primary productivity of the bacteria, even at times when analyses by Parkin and Brock (1980a) suggested that light intensity could be limiting (i.e., 3.0-5.0 ft. candles). Shifts in the absorption maxima at 430 nrn of the .bacteriochlorophyll spectrum suggested that changes in the species or strain composition of the photosynthetic bacteria had occurred during the summer months. It was speculated that these changes might reflect seasonal variation in the wavelength of light reaching the bacteria. Chemocline erosion did not have the same effect on the population size (biomass) of the photosynthetic bacteria in Crawford Lake (this thesis) as it did in Pink Lake (Dickman, 1979). In Crawford Lake the depth of the chemocline was lowered with no apparent loss in biomass (according to bacteriochlorophyll data). A reverse current was. proposed to explain the observation. The photosynthetic bacteria contributed a significant proportion (10-60%) of the lake1s primary productivitya Direct evidence was obtained with (14C) labelling of the photosynthetic bacteria, indica.ting that the zooplankton were grazing the photosynthetic bacteria. This indicated that some of the photosynthetic bacterial productivity was assimilated into the food chain of the lake. Therefore, it was concluded that the photosynthetic bacteria made a significant contribution to the total productivity of Crawford Lake.

Transient and Pulsed Electron Paramagnetic Resonance Spectroscopy on Type I Photosynthetic Reaction Centers

Two time-resolved EPR techniques, have been used to study the light induced electron transfer(ET) in Type I photosynthetic reaction centers(RCs). First, pulsed EPR was used to compare PsaA-M688H and PsaB-M668H mutants of Chlamydomonas reinhardtii and Synechosystis sp. PCC 6803.The out-of-phase echo modulation curves combined with other EPR and optical data show that the effect of the mutations is species dependent. Second, transient and pulsed EPR data are presented which show that PsaA-A660N and PsaB-A640N mutations in C. reinhardtii alter the relative quantum yield of ET in the A- and B-branches of PS I. Third, transient EPR studies on RCs from Heliobacillus mobilis that have been exposed to oxygen show partial inhibition of ET. In the RCs in which ET still occurs, the ET kinetics and EPR spectra show evidence of oxidation of some but not all of the, BChl g and BChl g' to Chl a.

An investigation into the functional role of the D1:1 and D1:2 polypeptides in photosystem II in cyanobacteria : the effect of changing PSI/PSII ratio on photoinhibition in Synechococcus sp. PCC7942 /

The cyanobacterium Synechococcus sp. PCC 7942 (Anacystis nidulans R2) adjusts its photosynthetic function by changing one of the polypeptides of photosystem II. This polypeptide, called Dl, is found in two forms in Synechococcus sp. PCC 7942. Changing the growth light conditions by increasing the light intensity to higher levels results in replacement of the original form of D 1 polypeptide, D 1: 1, with another form, D 1 :2. We investigated the role of these two polypeptides in two mutant strains, R2S2C3 (only Dl:l present) and R2Kl (only Dl:2 present) In cells with either high or low PSI/PSII. R2S2C3 cells had a lower amplitude for 77 K fluorescence emission at 695 nm than R2Kl cells. Picosecond fluorescence decay kinetics showed that R2S2C3 cells had shorter lifetimes than R2Kl cells. The lower yields and shorter lifetimes observed in the D 1 and Dl:2 containing cells. containing cells suggest that the presence of D 1: 1 results in more photochemical or non-photochemical quenching of excitation energy In PSII. One of the most likely mechanisms for the increased quenching in R2S2C3 cells could be an increased efficiency in the transfer of excitation energy from PSII to PSI. However, photophysical studies including 77 K fluorescence measurements and picosecond time resolved decay kinetics comparing low and high PSI/PSII cells did not support the hypothesis that D 1: 1 facilitates the dissipation of excess energy by energy transfer from PSII to PSI. In addition physiological studies of oxygen evolution measurements after photoinhibition treatments showed that the two mutant cells had no difference in their susceptibility to photoinhibition with either high PSI/PSII ratio or low PSI/PSII ratio. Again suggesting that, the energy transfer efficiency from PSII to PSI is likely not a factor in the differences between Dl:l and Dl:2 containing cells.

Synechococcus sp. PCC 7002 CpcB lyase null mutations alter phycocyanin chromophore function and, consequently, affect the redistribution of excitation energy via the light state transition /

Phycobilisomes are the major light harvesting complexes for cyanobacteria and phycocyanin is the primary phycobiliprotein of the phycobilisome rod. The phycocyanobilin lyases responsible for chromophorylating the phycocyanin p subunit (CpcB) have been recently identified in the cyanobacterium Synechococcus sp. PCC 7002. Surprisingly, mutants missing the CpcB lyases were nevertheless capable of producing pigmented phycocyanin. 10K absorbance measurements revealed that the energy states of the p phycocyanin chromophores were only subtly shifted; however, 77K steady state fluorescence emission spectroscopy showed excitation energy transfer involving the targeted chromophores to be highly disrupted. Such evidence suggests that phycobilin orientation within the binding domain is specifically modified. We hypothesized that alternate, less specific lyases are able to act on the p binding sites. A phycocyanin linker-polypeptide deficient mutant was similarly characterized. The light state transition, a short term adaptation of the photosynthetic light harvesting apparatus resulting in the redistribution of excitation energy among the photo systems, was shown to be dominated by the reallocation of phycocyanin-absorbed excitation energy. Treatment with a high M phosphate buffer effectively prevented the redistribution of both chlorophyll a- and phycobilisome- absorbed excitation energy, suggesting that the two effects are not strictly independent. The mutant strains required a larger redistribution of excitation energy between light states, perhaps to compensate for their loss in phycobilisome antenna function.

Changes in the magnitude and pattern of translocation of photoassimilated ¹CO in soybean plants following an acute exposure to gamma radiation /

Young soybean plants (Glycine ~. L. cultivar Harosoy '63), grown under controlled conditions, were exposed to gamma radiation on a single occasion. One hour following exposure to 3,750 rads, the mature trifoliate leaf of the soybean plant was isolated in a closed system and permitted to photoassimilate approximately 1-5 pCi of 14C02 for 15 minutes. After an additional 45 minute-period, the plant was sacrificed and the magnitude of translocation and distribution pattern of 14C determined. In the non-irradiated plants 18~ of the total 14C recovered was outside the fed leaf blades and of this translocated 14c, 28~ was above the node of the fed leaf, 38~ in the stem below the node, 28~ in the roots and 7~ in the petiole. As well, in the irradiated plants, a smaller per cent (6~) of the total 14 C recovered was exported out of the source leaf blades. Of this translocated 14c , a smaller per cent (20~) was found in the apical region above the node of the source leaf and a higher per cent (45~) was recovered from the stem below the node and in the petiole (11~). The per cent of exported 14 C recovered from the root was unaffected by the radiation. Replacement of the shoot apex with 20 ppm IAA immediately following irradiation, only J partially increased the magnitude of translocation but did completely restore the pattern of distribution to that observed in the non-irradiated plants. From supplementary studies showing a radiationinduced reduction of photosynthetic rates in the source leaf and a reduction of the cumulative stem and leaf lengths in the apical sink region, the observed effects of radiation on the translocation process have been correlated to damage incurred by the source and sink regions. These data suggest that the reduction in the magnitude of translocation is the result of damage to both the source and sink regions rather than the phloem conducting tissue itself, whereas the change in the pattern of translocation is probably the result of a reduced rate of 14C-assimilate movement caused by a radiation-induced decrease of sink metabolism, especially the decrease in the metabolism of the apical sink.

A relationship between photosynthesis and translocation in plants stressed by ionizing radiation

Since previous investigations have shown that low levels of ionizing radiation can induce a reduction in the rates of apparent photosynthesis and in the magnitude of photoassimilated l4C exported out of a leaf, the present studies were designed and conducted to determine the relationship, if any, between the radiation effects on these two physiological processes. The experiments were particularly designed to determine if the radiation-induced reduction in export is the result of the reduction in photosynthesis and hence availability of materials for translocation or the result of a reduction in the amount of energy available for the vein loading process. This study has shown that the radiation-induced reduction in l4C export out of a leaf is likely related to a loss of energy available for the vein loading process rather than a reduction in the supply of materials available for export due to reduced C02 uptake. The process of photophosphorylation was shown to be reduced by exposure to radiation to an extent similar to the reduction in the export of l4C which was also observed. Both of these processes returned to their pre-irradiation rates 120 minutes following radiatruon exposure. The rate of photosynthetic C02 uptake was also reduced by radiation exposur~ howeve~ this process did not return to the control level nor was the extent of reduction as large as observed for photophosphorylation and photoassimilate export. The observed relationship between the reductions of export and photoph~sphorylation pointed to the utilization of photosynthetically produced ATP in the vein loading process. The radiation-induced reduction in the export of l4C was observed at the highest light intensity used in this study which would also imply the involvement of the photophosphorylation process as an energy seurce for vein loading. The lack of radiation-induced reduction in export at low light intensities was interpreted as being due to the utilization of respiratory derived ATP, a process known to be insensitive to radiation at the levels used in this study, as the energy source for the vein loading process. Studies using plants not stressed by radiation showed that there was an increase in export of 14C with higher light intensities. In summary, the data has been interpreted as showing that at high light intensities the ATP, produced by photophosphorylation, is available for use in the vein loading process. The site of ATP utilization could not be determined from the data obtained in this study but possible sites have been indicated from the work done by other physiologists and are discussed in the thesis.

Effect of diurnal cycling temperatures on cardiovascular- ventilatory function in statically-acclimated rainbow trout, Salmo gairdneri

Four groups of rainbow trout, Salmo gairdneri, were acclimated to 2°, 10°, and 18°e, and to a diurnal temperature cycle (100 ± 4°C). To evaluate the influence of cycling temperatures in terms of an immediate as opposed to acclimatory response various ventilatory-cardiovascular rate functions were observed for trout, either acclimated to cycling temperatures or acclimated to constant temperatures and exposed to a diurnal temperature cycle for the first time (10° ± 4°C for trout acclimated to 10°C; 18°+ 4°C for trout acclimated to l8°e). Gill resistance and the cardiac to ventilatory rate ratio were then calculated. Following a post preparatory recovery period of 36 hr, measurements were made over a 48 hour period with the first 24 hours being at constant temperature in the case of statically-acclimated fish followed by 24 hours under cyclic temperature conditions. Trout exhibited marked changes in oxygen consumption (Vo ) with temp- 2 erature both between acclimation groups, and in response to the diurnal temperature cycle. This increase in oxygen uptake appears to have been achieved by adjustment of ventilatory and, to some extent, cardiovascular activity. Trout exhibited significant changes in ventilatory rate (VR), stroke volume (Vsv), and flow (VG) in response to temperature. Marked changes in cardiac rate were also observed. These findings are discussed in relation to their importance in convective oxygen transport via water and blood at the gills and tissues. Trout also exhibited marked changes in pressure waveforms associated with the action of the resp; ratory pumps with temperature. Mean differenti a 1 pressure increased with temperature as did gill resistance and utilization. This data is discussed in relation to its importance in diffusive oxygen transport and the conditions for gas exchange at the gills. With one exception, rainbow trout were able to respond to changes in oxygen demand and availability associated with changes in temperature by means of adjustments in ventilation, and possibly pafusion, and the conditions for gas exchange at the gills. Trout acclimated to 18°C, however, and exposed to high cyclic temperatures, showed signs of the ventilatory and cardiovascular distress problems commonly associated with low circulating levels of oxygen in the blood. It appears these trout were unable to fully meet the oxygen requirements associated with c~ling temperatures above 18°C. These findings were discussed in relation to possible limitations in the cardiovascular-ventilatory response at high temperatures. The response of trout acclimated to cycling temperatures was generally similar to that for trout acclimated to constant temperatures and exposed to cycling temperatures for the first time. This result suggested that both groups of fish may have been acclimated to a similar thermal range, regardless of the acclimation regime employed. Such a phenomenon would allow trout of either acclimation group to respond equally well to the imposed temperature cycle. Rainbow trout showed no evidence of significant diurnal rhythm in any parameters observed at constant temperatures (2°, 10°, and 18° C), and under a 12/12 light-dark photoperiod regime. This was not taken to indicate an absence of circadian rhythms in these trout, but rather a deficiency in the recording methods used in the study.

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

Ionic regulation in goldfish, Carassius auratus, acclimated to constant and diurnally-cycling temperature conditions

The effects of a diurnal sine-wave temperature cycle (250 +- 5° C) on the wa terI-e etc r o1 yt est a t us 0 f gol df1' Sh , Carassius auratus, was assessed through determination of Na+, K+, Mg2+, Ca2+, Cl- and water content in plasma, Red blood cells and muscle tissue. Animals were also acclimated to o 0 0 static temperatures (20 C, 25 c, 30 C) corresponding to the high, low and mid-ooint temperatures of the cycle. All groups were sampled at 03:00, 09:00, 15:00 and 21:00 hr. Hemoglobin content and packed cell volume, as well as electrolyte and 'water levels were determined for each animal and red cell ion concentrations and ion : hemoglobin ratios estimated. Cycled animals were distinct from those at constant temperatures in several respects. Hematological parameters were elevated above those of animals at constant temperature and were, on a diurnal basis, more stable. Red blood cell electrolyte levels varied in an adaptively appropriate fashion to cycle temperatures. This was not the case in the constant temperature groups_ Under the cycling regime, plasma ion levels were more diurnally stable than those of constant temperature fish. Although muscle parameters in cycled fish exhibited more fluctuation than was observed in plasma, these also tended to be relatively more stable than was the caseErythrocytic data are discussed in terms of their effects on hemoglobin-oxygen affinity while plasma and muscle observations were considered from the standpoint of overall water-electrolyte balance. In general, cycled fish appeared to be capable of stabilizing overall body fluid composition, while simultaneously effecting adaptively-appropriate modifications in the erythrocytic ionic microenvironment of hemoglobin. The sometimes marked diurnal variability of water-electrolyte status in animals held at constant temperature as opposed to the conservation of cycled fish suggests that this species is, in some fashion, programmed for regulation in a thermally-fluctuating environment. If this interpretation is valid and a phenomenon of general occurrence, some earlier studies involving constant acclimation of eurythermal species normally occupying habitats which vary in temperature on a daily basis may require reconsideration. at constant temperature.

Palaeopigment accumulation and implcations for paleolimnology over the lost century for two Ontario lakes

Crawford Lake is a meromictic lake, which is 24 m deep and has an area of 2.5 ha, and has never been reported to have mixed below 16 m. Lady Evelyn Lake, which became a reservoir when a dam was built in 1916, is dimictic with a maximum depth of about 35 m. 1 My research proved that both native chlorophylls and the ratio of chlorophyll derivatives to total carotenoids were better preserved in the shallower lake (Crawford Lake) because it was meromictic. Thus the anaerobic conditions in Crawford Lake below 16 m (monimolimnion) provide excellent conditions for pigment preservation. Under such conditions, the preservation of both chlorophylls and carotenoids, including oscillaxanthin and myxoxanthophyll, are extremely good compared with those of Lady Evelyn Reservoir, in which anaerobic conditions are rarely encountered at the mud-water interface. During the period from 1500 to 1900 A. D. in Crawford Lake, the accumulation rates of oscillaxanthin and myxoxanthophyll were extremely high, but those of chlorophyll derivatives and total carotenoids were relatively low. This was correlated with the presence of a dense benthic mat of cyanobacteria near the lake's chemocline. Competition for light between the deep dwelling cyanobacteria and overlying phytoplankton in this meromictic lake would have been intensified as the lake became more and more eutrophic (1955-1991 A. D.). During the period from 1955 to 1991 A. D., the accumulation rates of chlorophyll derivatives and total carotenoids in the sediment core from Crawford Lake (0-7.5 cm, 1955-present) increased. During this same period, the accumulation rates of cyanobacterial pigments (Le. oscillaxanthin and myxoxanthophyll) declined as the lake became more eutrophic. Because the major cyanobacteria in Crawford Lake are benthic mat forming Lyngbya and Oscillatoria and not phytoplankton, eutrophication resulted in a decline of the mat forming algal pigments. This is important because in previous palaeolimnological studies the concentrations of oscillaxanthin and myxoxanthophyll have been used as correlates with lake trophic levels. The results of organic carbon a13c analysis on the Crawford Lake sediment core supported the conclusions from the pigment study as noted above. High values of a13c at the depth of 34-48 cm (1500-1760 A. D.) were related to a dense population of benthic Oscillatoria and Lyngbya living on the bottom of the lake during that period. The Oscillatoria and Lyngbya utilized the bicarbonate, which had a high a 13C value. Very low values were found at 0-7 cm in the Crawford sediment core. At this time phytoplankton was the main primary producer, which enriched 12C by photosynthetic assimilation.

Radiation-induced changes in the export of photoassimilated carbon /|nBarry Jess Shelp. -- 260 0 St. Catharines, [Ont. : s. n.],

Low levels of ionizing radiation induce two translocation responses in soybean: a reduction in photoassimilate export from leaves and a change in the distribution pattern of exported photoassimilate within the plant. In this investigation these responses have been further studied specifically to ascertain the site of radiation damage and to better understand the physiological responses observed. Experimentally the primary data was obtained from studies in which a mature trifoliate leaf of a young soybean plant (Glycine ~ L. cultivar Harosoy '63) is isolated in a closed transparent chamber and allowed to photoassimilate 14C02 for 15 minutes. This is followed by an additional 45 ~_il'1;ute period before the plant is sectl.o ne d an d 14 C-ra dl' oactl.v.l ty d eterml. ne d'l n a 11 parts. Such 14c data provides one with the magnitude and distribution pattern of translocation. Further analyses were conducted to determine the relative levels of the major photosynthetic products using the techniques of paper chromatography and autoradiography. Since differences between control and irradiated P 1 ants were not 0 b serve d l' n t h e par tl't"lo nlng 0 f 14 C between the 80% ethanol-soluble and -insoluble fractions 14 or in the relative amounts of C-products of photosynthesis, the reduction in export in irradiated plants is not likely due to reduced availability of translocatable materials. Data presented in this thesis shows that photoassimilate export was not affected by gamma radiation until a threshold dose between 2.0 and 3.0 krads was reached. It was also observed that radiation-induced damage to the export process was capable of recovery in a period of 1 to 2 hours provided high light intensity was supplied. In contrast, the distribution pattern was shown to be extremely radiosensitive with a low threshold dose between .25 and .49 krads. Although this process was also capable of recovery,lt" occurred much earlier and was followed by a secondary effect which lasted at least for the duration of the experiments. The data presented in this thesis is interpreted to suggest that the sites of radiation action for the two translocation responses are different. In regards to photoassimilate export, the site of action of ionizing radiation is the leaf, quite possibly the process of photophosphorylation which may provide energy directly for phloem loading and for membrane integrity of the phloem tissue* In regards to the pattern of distribution of exported photoassimilate, the site is likely the apical sink, possibly the result of changes of levels of endogenous hormones. By the selection of radiation exposure dose and time post-irradiation, it is possible to affect independently these two processes suggesting that each may be regulated independent of the other and involves a distinct site.

The effects of static and diurnally-cycling temperature acclimations on thermal tolerances of rainbow trout, Salmo gairdneri

:ofiedian lethal temperatures ( LT50' s ) were determined for rainbow trout, Salmo gairdnerii, acclimated for a minimum of 21 days at 5 c onstant temperatures between 4 and 20 0 C. and 2 diel temperature fluctuations ( sinewave curves of amplitudes ± 4 and ± 7 0 C. about a mean temperature of 12 0 C. ) . Twenty-four-, 48-, and 96-hour LT50 estimates were c alculated f ollowing standard flow-through aquatic bioassay techniques and probi t transformation of mortality data. The phenomenon of delayed thermal mortality was also investigated. Shifts in upper incipient lethal temperature occurred as a result of previous thermal conditioning. It was shown that increases in constant acclimation temperature result in proportional l inear increases in thermal tolerances. The increase i n estimated 96-hour LT50's was approximately 0.13 0 c. X 1 0 C:1 between 8 and 20 0 C. The effect of acclimation to both cyclic temperature regimes was an increase in LT50 to values between the mean and maximum constant equivalent daily temperatures of the cycles. Twenty-four-, 48-, and 96-hour LT50 estimates of both cycles corresponded approximately to the LT50 values of the 16 0 C. c onstant temperature equivalent . This increase i n thermal tolerance was further demonstrated by the delayed thermal mortality experiments . Cycle amplitudes appeared to i nfluence thermal resistance through alterations in initi al mortality since mortality patterns characteristic of base temperature acclimations re-appeared after approximately 68 hours exposure to test temperatures for the 12 + 4 0 C. group, whereas mortality patterns stabilized and remained constant for a period greater than 192 hours with the larger therma l cycle ( 12 + 7 0 C. ). NO s ignificant corre lations between s pecimen weight and time-to-death was apparent. Data are discussed in relation to the establishment of thermal criteria for important commercial and sport fishes , such as the salmonids , as is the question whether previously reported values on lethal temperature s may have been under estimated.

Thermoacclimatory variations in the activities of enzymes implicated in ion transport in the rainbow trout, salmo gairdneri

Two groups of rainbow trout were acclimated to 20 , 100 , and 18 o C. Plasma sodium, potassium, and chloride levels were determined for both. One group was employed in the estimation of branchial and renal (Na+-K+)-stimulated, (HC0 3-)-stimulated, and CMg++)-dependent ATPase activities, while the other was used in the measurement of carbonic anhydrase activity in the blood, gill and kidney. Assays were conducted using two incubation temperature schemes. One provided for incubation of all preparations at a common temperature of 2S oC, a value equivalent to the upper incipient lethal level for this species. In the other procedure the preparations were incubated at the appropriate acclimation temperature of the sampled fish. Trout were able to maintain plasma sodium and chloride levels essentially constant over the temperature range employed. The different incubation temperature protocols produced different levels of activity, and, in some cases, contrary trends with respect to acclimation temperature. This information was discussed in relation to previous work on gill and kidney. The standing-gradient flow hypothesis was discussed with reference to the structure of the chloride cell, known thermallyinduced changes in ion uptake, and the enzyme activities obtained in this study. Modifications of the model of gill lon uptake suggested by Maetz (1971) were proposed; high and low temperature models resulting. In short, ion transport at the gill at low temperatures appears to involve sodium and chloride 2 uptake by heteroionic exchange mechanisms working in association w.lth ca.rbonlc anhydrase. G.l ll ( Na + -K + ) -ATPase and erythrocyte carbonic anhydrase seem to provide the supplemental uptake required at higher temperatures. It appears that the kidney is prominent in ion transport at low temperatures while the gill is more important at high temperatures. 3 Linear regression analyses involving weight, plasma ion levels, and enzyme activities indicated several trends, the most significant being the interrelationship observed between plasma sodium and chloride. This, and other data obtained in the study was considered in light of the theory that a link exists between plasma sodium and chloride regulatory mechanisms.

Thermoaclimatory variations in the microenvironment of hemoglobin in the rainbow trout, Salmo gairdneri and the carp, Cyprinus carpio

Several inorganic substances (e.g., C£ , Mg , Ca , H ) are potent negative modulators of hemoglobin-oxygen affinity. To evaluate the possibility that potentially adaptive changes in the red cell ionic environment of hemoglobin may take place during acclimation of fishes to increased environmental temperature, hematological status (hemoglobin, hematocrit, red cell numbers, mean erythrocytic volume and hemoglobin content), plasma + + 2+ 2+ and packed red cell electrolyte levels (Na , K , Ca , Mg , C£ ) were evaluated in summer and winter populations of the stenothermal rainbow trout, Salmo gairdneri, following acclimation to 2°, 10°, 18°C, and in a spring population of eurythermal carp, Cyprinus carpio, held at 2°, 16° and 30°C. From these data cell ion concentrations and ion:hemoglobin ratios were estimated. In view of the role of red cell carbonic anhydrase in the reductions of blood C02 tensions and the recruitment of Na and C£~ lost by fishes, a preliminary investigation of thermoacclimatory changes in the activity of this system in rainbow trout erythrocytes was conducted. Few changes in hematological status were encountered following acclimation. There was, however, some evidence of weight-specific differential hematological response in carp. This lead to markedly greater increases in hemoglobin, hematocrit and red cell numbers in smaller rather than in larger specimens at higher temperatures; variations which were 2+ well correlated with changes in plasma Ca . Plasma composition in summer trout was not altered by acclimation. In winter trout plasma Na and K increased at higher temperatures. Carp were characterized by increases in plasma calcium, and reductions in sodium and magnesium under these conditions. Several significant seasonal differences in plasma ion levels were observed in the trout. (n) In trout, only erythrocytic K and K :Hb were altered by acclimation, rising at higher temperatures. In carp Na , Na :Hb, C£~ and C£~:Hb in- 2+ 2+ creased with temperature, while Mg and Mg :Hb declined. Changes in overall ionic composition in carp red cells were consistent with increases in H content. In both species significant reciprocal variations in C£~ 2+ - + and Mg were found. In mammalian systems increases in C£ and H reduce hemoglobin-oxygen affinity by interaction with hemoglobin. Reduction in 2+ 2+ Mg maximizes organophosphate modulator availability by decreasing ATP»Mg complex formation. Thus, the changes observed may be of adaptive value in reducing hemoglobin-oxygen affinity, and facilitating oxygen release to cells at higher temperatures. Trout appear to maintain a high chloridelow magnesium state over the entire thermal tolerance zone. Carp, however, achieved this state only at higher temperatures. In both species mean erythrocytic volume was decreased at higher temperatures and this may facilitate branchial oxygen loading. Since mean erythrocytic volume was inversely related to red cell ion content, it is hypothesized that reductions in cell volume are achieved by export of some unidentified solute or solutes. Variations in the carbonic anhydrase activity that could be attributed to the thermoacclimatory process were quite modest. On the other hand, assays performed at the temperature of acclimation showed a large temperature effect where under in vivo conditions of temperature fish acclimated to higher temperatures might be expected to have higher activities. Furthermore, since hematocrit increased with temperature in these fish, while carbonic anhydrase is present only in the erythrocyte, the whole blood levels of this enzyme are expected to increase and further augment the temperature effect. This, in turn, could aid in the reduction of C02 (111) tension and increase the production of H and HC0~~ used in the active uptake of Na and C£ at higher temperatures.