Lake (Coregonus clupeaformis) and round (Prosopium cylindraceum) whitefish are two species of cold-adapted freshwater fish that spawn in shallow (<10m) cobble beds in the Laurentian great lakes. Developing whitefish embryos are potentially exposed to various anthropogenic sources of warming, including climate change and thermal effluents discharged in the nearshore environment. Several studies have investigated the effects of elevated incubation temperatures on whitefish embryos (particularly lake whitefish) but little work has been done to examine post-hatch effects. Thermal preferenda (temperature preference) describe the range of temperatures an organism will occupy when given a choice and are traditionally thought to be species-specific. Temperature preference can be modulated by a variety of abiotic and biotic factors including environmental temperature and is typically correlated with optimal growth temperature for a species. Assays for thermal preferenda require at least 24 hours, which includes a long tank-acclimation period that limits throughput and thus impacts replication in the study. A shuttle box thermal preference assay was optimized from 24-hours to 4-hours; length of acclimation time and trial length had no significant impact on thermal preference. Whitefish were incubated at natural and elevated temperatures until hatching; all groups were moved to common garden conditions (15°C) during the post-hatching stage. Temperature preference was determined at 12 months of age; lake whitefish were also tested at 8 months. Round whitefish displayed a significant decrease in temperature preference when incubated at 2°C and 6°C compared to 0.5°C. Lake whitefish had similar temperature preferences regardless of age and incubation temperature. This suggests that there is a difference in thermal tolerance between these species, as round whitefish were more sensitive to elevated incubation treatments. This thesis identified a persistent effect of elevated incubation treatments on the thermal preference of juvenile round whitefish, lasting up to 12 months post-hatch, which highlights the importance of examining sub-lethal thermal effects and thermal plasticity of cold-adapted species. / Thesis / Master of Science (MSc) / Lake and round whitefish are cold-adapted freshwater species that spawn in shallow coastal water in the Laurentian great lakes. Anthropogenic warming from various sources (e.g. climate change, thermal effluent) has been shown to reduce survival of whitefish embryos, but few studies have investigated the effects of elevated embryonic incubation temperatures on surviving juveniles. Fish typically prefer a small range of temperatures (temperature preference) that they will occupy if given a choice, which can be affected by a variety of factors including early life thermal exposure. This thesis shows round whitefish incubated at elevated temperatures (2°C, 6°C) display decreased temperature preference up to 12 months post-hatch, while lake whitefish had similar temperature preference regardless of incubation temperature. Therefore, this thesis provides more evidence that round whitefish are a more thermally sensitive species.

How temperature affects physiological development in the chicken embryo is unknown. Embryos incubated at 38°C or 35°C showed no difference in growth or survival. The time to hatching was longer in 35°C than 38°C embryos (23.7 vs. 20.6 days), but unaffected was the relative timing of appearance of developmental landmarks (internal, external pipping). At stage 43-44, 38°C embryos maintained oxygen consumption around 1 mL/g/h despite acute temperature reduction (suggesting thermoregulatory maturation), unlike 35°C embryos. In 35°C embryos the lower oxygen-carrying capacity and temperature insensitive blood O2 affinity (P50 about 30 mmHg) may restrict O2 delivery to tissues, limiting metabolism during decreased ambient temperature. Reduced incubation temperature retards normal hematological and thermoregulatory development.

The small intestine and yolk sac (YS) are important organs for nutrient absorption and innate immunity in chickens during the post-hatch or prehatch periods. These organs share a similar structure of epithelial cell-lined villi with tight junctions between adjacent cells. Probiotics have been reported to improve chicken growth performance and gut health including promotion of intestinal morphology. However, there are few studies that show the effect of probiotics on ontogeny of intestinal epithelial cells and antimicrobial peptides, or intestinal integrity in young healthy chicks. Heat stress during incubation was shown to increase mortality and decrease hatchability of chicks, while no studies have investigated the effect of heat stress on the integrity of the YS, which might be related to hatching performance. There were four studies conducted in this research: 1) a comparison of the effect of two probiotics on the ontogeny of small intestinal epithelial cells in young chicks; 2) the effect of two probiotics on mRNA abundance of tight junction proteins in the small intestine of young chicks; 3) the effect of high incubation temperature on mRNA abundance of tight junction proteins in the YS of broiler embryos; and 4) comparison of avian defense peptide mRNA abundance in the YS of broilers and layers. In study 1, Probiotics transiently decreased body weight gain (BWG) from day 2 to day 4, but did not affect body weight (BW) from day 2 to day 8, and small intestinal weight and intestinal morphology from day 2 to day 6. Probiotics did not affect marker gene expression of intestinal stem cells (Olfm4) and goblet cells (Muc2) in all small intestinal segments, but did increase expression of a marker gene of proliferating cells (Ki67), and decreased an antimicrobial peptide (liver-enriched antimicrobial peptide 2, LEAP2) in the jejunum at day 4. Probiotic 1 decreased PepT1, a marker of enterocytes in the duodenum at day 4. These results suggest that probiotics did not improve growth performance and intestinal morphology in young healthy chicks, but temporarily promoted intestinal epithelial cell proliferation and decreased LEAP2 antimicrobial peptide expression in the jejunum. In situ hybridization (ISH) showed that Ki67+ proliferating cells were mainly located in the crypt region and the blood vessels of villi. In study 2, Probiotic supplementation to newly hatched chicks for less than one week did not affect mRNA abundance of the tight junction proteins in the small intestine. Occludin (OCLN) mRNA, which was detected by ISH to be expressed in intestinal epithelial cells in both the villus and crypt regions, was greater in the duodenum of female chicks than males. In study 3, high incubation temperature starting from embryonic day 12 (E12) affected mRNA abundance of the tight junction proteins in the YS, including increased zonula occluden 1 (ZO1) at E13, increased junctional adhesion molecule A (JAMA) and heat shock protein 90 (HSP90) at E17, but decreased tight junction protein JAMA at E19 and OCLN at day of hatch (DOH). These results showed that the YS tight junction proteins were increased by short term heat exposure but decreased by long term heat exposure. In study 4, the expression of avian β defensin 10 (AvBD10), CATHs and toll-like receptors in the YS was examined. Toll-like receptors were highly expressed in the YS at early incubation stages (E7), while CATHs showed a peak expression from E9 to E13, which was similar to the expression pattern of AvBD10. CATHs and AvBD10 mRNA temporal expression patterns were similar in broilers and layers, while their expression levels were different. Layers, especially brown layers, had greater mRNA abundance for antimicrobial peptides such as AvBD10, CATH1, and CATH2 in the YS. These results demonstrate that the antimicrobial peptide temporal expression patterns in the YS are not affected by breed, but their expression levels are affected by breed. In summary, the small intestine and the YS are essential for nutrient uptake, innate immunity, and maintenance of integrity. The ontogeny of intestinal epithelial cells, such as proliferating cells can be modulated by probiotic supplementation. Similar to the small intestine, the YS can also express tight junction proteins, which can be affected by high incubation temperature. Antimicrobial peptide expression in the intestine of healthy young chicks is also transiently decreased by probiotic supplements. Avian defensin and cathelicidin expression patterns in the YS were not affected by breed. / Doctor of Philosophy / The small intestine and yolk sac are important organs for nutrient absorption in hatched chicks or embryonic chicks. These organs also serve as a barrier to prevent pathogens from entering the blood circulation. Intestinal epithelial cells along the villi renew rapidly by proliferation and differentiation. In this research, probiotics which are also known as direct fed microbials temporarily increased expression of the proliferating cell marker Ki67 in the jejunum of healthy young chicks, which suggests that probiotics promote intestinal epithelial cell proliferation. However, probiotics transiently decreased expression of an antimicrobial peptide, which may reduce immune protection in the gut. The yolk sac can also express tight junction proteins. The expression of tight junction proteins was affected by elevated incubation temperature in broiler embryos, which might be related to low hatchability of eggs exposed to heat stress. Avian defense peptides and pathogen recognition receptors were expressed in the YS, which implied that the yolk sac contained an innate immune function. The expression pattern of avian defense peptides was affected by breed (broilers and layers), while the expression level of avian defense peptides was greater in layers than broilers. In summary, the small intestine and the yolk sac are multifunctional organs. Their cell composition, structural integrity, and secretion of antimicrobial peptides can be affected by environmental factors, such as probiotic supplementation or high incubation temperature.

The incubation period is crucial for development and overall quality of a chick. The selection for fast growing broilers has allowed the birds to reach market weight at a faster rate making the incubation period a larger portion of a broiler's life. A faster growth rate can lead to the release of more metabolic heat inside of the egg toward the second half of incubation because the embryo shifts to a homeothermic state. More heat being released into the incubator can cause the incubation temperature to rise if the incubator is not electronically regulated or cannot be ventilated properly due to malfunction. A high incubation temperature can impact the hatchability, growth, and development of the chick. This thesis provides a more in-depth analysis of the effects of high incubation temperature (37.5°C versus 39.5°C) on the developing small intestine and yolk sac, which provide the chick with nutrients posthatch and during embryogenesis. Studying these organs and mechanisms occurring during this time could potentially indicate why chicks from eggs subjected to a higher incubation temperature are not developing and growing properly. Chicks from eggs incubated at a higher temperature had lower body weights, lower hatchability and lower villus height in the duodenum, jejunum, and ileum. There were also differences seen in the depth of the crypt, which is the site for stem cells. Chicks from eggs incubated at a higher temperature had a lower crypt depth in the duodenum and jejunum. There was no difference in the expression of the intestinal stem cell marker olfactomedin 4 (Olfm4) and mucin 2, which is secreted by goblet cells and forms mucus. In the yolk sac, heat shock proteins (HSP) 70 and 90 were elevated at embryonic day 15, and HSP90 still remained elevated at embryonic day 17. Chicks from eggs incubated at a higher temperature showed greater expression of peptide transporter 1 and avian beta-defensin 10 mRNA at embryonic day 13. Even though small intestinal morphology was impacted early posthatch and expression of genes in the yolk sac were elevated at embryonic day 13, there does not seem to be a long-lasting effect on the development of the small intestine or the yolk sac. It is still important to study the impact of the incubation environment to understand the development and growth of the chicks and how different incubation factors can impact the overall hatchability and health of the chick. / Master of Science / The incubation period is crucial for development and overall quality of a chick. The selection for fast growing broilers has allowed the birds to reach market weight at a faster rate making the incubation period a larger portion of a broiler’s life. A faster growth rate can lead to the release of more metabolic heat inside of the egg toward the second half of incubation because the embryo shifts to a homeothermic state. More heat being released into the incubator can cause the incubation temperature to rise if the incubator is not electronically regulated or cannot be ventilated properly due to malfunction. A high incubation temperature can impact the hatchability, growth, and development of the chick. This thesis provides a more in-depth analysis of the effects of high incubation temperature (37.5°C versus 39.5°C) on the developing small intestine and yolk sac, which provide the chick with nutrients posthatch and during embryogenesis. Studying these organs and mechanisms occurring during this time could potentially indicate why chicks from eggs subjected to a higher incubation temperature are not developing and growing properly. Chicks from eggs incubated at a higher temperature had lower body weights, lower hatchability and lower villus height in the duodenum, jejunum, and ileum. There were also differences seen in the depth of the crypt, which is the site for stem cells. Chicks from eggs incubated at a higher temperature had a lower crypt depth in the duodenum and jejunum. There was no difference in the expression of the intestinal stem cell marker olfactomedin 4 (Olfm4) and mucin 2, which is secreted by goblet cells and forms mucus. In the yolk sac, heat shock proteins (HSP) 70 and 90 were elevated at embryonic day 15, and HSP90 still remained elevated at embryonic day 17. Chicks from eggs incubated at a higher temperature showed greater expression of peptide transporter 1 and avian beta-defensin 10 mRNA at embryonic day 13. Even though small intestinal morphology was impacted early posthatch and expression of genes in the yolk sac were elevated at embryonic day 13, there does not seem to be a long-lasting effect on the development of the small intestine or the yolk sac. It is still important to study the impact of the incubation environment to understand the development and growth of the chicks and how different incubation factors can impact the overall hatchability and health of the chick.

The effect of incubation temperature on different morphological, physiological, cognitive and behavioral characteristics in reptiles is a well-studied topic, although the underlying mechanism leading to the differences between individuals incubated at different temperatures remains largely unknown. In this thesis I studied the effect of incubation temperature on cognitive abilities and the number of neurons and non-neuronal cells in the gecko Paroedura picta incubated at two different temperatures, 24řC and 30řC. The geckos were tested in two cognitive tasks with simulated predatory attack. 14-day-old hatchlings were tested in a Y-maze, while 6-months-old geckos were tested in an arena with shelters of different colors. After testing, the number of neurons and non- neuronal cells in several parts of the brain were estimated using the isotropic fractionator in selected individuals. Although incubation temperature did not affect the success in the cognitive task in hatchlings, it did affect the total time needed to find the shelter. This difference remained significant also in adult geckos. The number of neurons, which was used as a proxy for the information processing capacity of the brain, did not affect success in the cognitive tasks. However, absolute brain size correlated with success in the...

Afin de mieux comprendre et évaluer l'impact du changement climatique sur les stocks de carbone organique du sol (COS) et les flux de carbone, et en particulier la respiration hétérotrophe du sol (RS), il est nécessaire d'étudier la sensibilité de la RS à la température. Plusieurs études sur les facteurs contrôlant la dépendance de la RS à la température ont été faites antérieurement et montrent que la sensibilité de la RS à la température diminue avec la température. Ces études ont suggéré que cette diminution de sensibilité de la RS à la température était liée à la modification de la disponibilité en substrat. D'autres études ont suggéré que cette diminution est liée à l'adaptation de la communauté microbienne aux températures élevées. La sensibilité de la RS à la température est plus particulièrement critique dans les régions semi-arides, comme le Nord Ouest de la Tunisie où le stock de COS est faible. Il est nécessaire de connaître l'effet de la disponibilité en substrat sur la sensibilité de la RS à la température. Dans cette étude, les échantillons de sol ont été incubés pendant 28 jours après une période de 28 jours de pré-incubation. Les pré-incubations et incubations ont été réalisées à 20, 30, 40 et 50°C. Pour tester l'effet de la disponibilité en substrat sur la sensibilité de la RS à la température, du glucose a été ajouté au sol au début de la période d'incubation. L'analyse des résultats a montré que les températures élevées de pré-incubation réduisent la sensibilité de la RS à la température d'incubation. L'addition de glucose réduit l'effet de températures élevées de pré-incubation sur la réponse de la RS. Il apparaît ainsi que la diminution de la sensibilité de la RS après un mois de pré-incubation aux fortes températures est due à la diminution de la disponibilité en substrat. Elle semble aussi liée à une diminution de la biomasse microbienne. Puisque, le sol utilisé pour cette étude est un Cambi sol Calco-magnésimorphe, une deuxième expérimentation a été réalisée afin de déterminer la part de CO2 provenant des carbonates et la part de CO2 provenant du COS. Cette étude a été réalisée par des mesures des signatures isotopiques (δ13C) du COS, des carbonates et du CO2 émis. Après 28 jours d'incubation, 23±9% de CO2 provient des carbonates. Cette contribution est faible par rapport à la teneur initiale élevée de C-CaCO3 dans le sol (4,3%) et est indépendante de la température d'incubation. Cette étude a montré que la réduction de la sensibilité de la RS aux fortes températures est surtout liée à une réduction de la disponibilité en substrat organique et de la biomasse microbienne aux fortes températures. / To better understand and assess the impact of climate change on the stocks of soil organic carbon (SOC) and carbon fluxes, and particularly heterotrophic soil respiration (SR), it is necessary to study the sensitivity of SR to temperature. Several studies have been achieved to improve the understanding of factors controlling the temperature dependence of SR and showed that the temperature sensitivity of SR decreases with temperature. These studies suggested that this decrease in temperature sensitivity of SR was related to change in substrate availability. Other studies presented microbial adaptation to warmed conditions. The temperature sensitivity of SR is especially critical in semi-arid regions, such as North West Tunisia, where the SOC stock is low. It is necessary to know the influence of substrate availability on the sensitivity of SR to temperature. In this study, soil samples were incubated for 28 days after a 28-day pre-incubation per iod. Pre-incubation and incubation were carried out at 20, 30, 40 and 50°C. To test the substrate availability effect on the temperature sensitivity of SR, glucose was added to soil at the beginning of the incubation period. Results showed that the highest pre-incubation temperature reduced the temperature sensitivity of SR during the subsequent incubation period. Glucose addition reduced the effect of high pre-incubation temperature on SR response. Thus, it appears that the observed decrease in SR sensitivity to temperature after one month pre-incubation at high temperature was due to a reduce in substrate availability and to a decrease in microbial biomass. Since the soil used in this study is a Calcari-Leptic Cambisol, a second experiment was also performed to determine the amount of CO2 from carbonates and the amount of CO2 from SOC. This study was carried out by measurements of the isotopic signatures (δ13C) of SOC, carbonates and emitted CO2. After 28 days of inc ubation, 23±9% of CO2 came from carbonates. This contribution was low compared to the high initial C-CaCO3 content in soil (4.3%), and it was independent to the incubation temperature. This study showed that reduce in the sensitivity of SR to high temperatures was probably due to a reduction in the substrate availability and to a decrease in microbial biomass.

The average global temperature is expected to rise by 3-5 °C at the end of the century, as a consequence of global warming. Negative effects are expected on poikilothermic animals, including fish, with changes in their physiology including metabolism. Metabolism has earlier been proven to have an association with propensities to migrate, which might be affected by raised temperatures as a consequence of global warming. The purpose of this study was to investigate if raised temperatures during incubation of eggs has an effect on metabolism in a partly migrating species of fish. The hypotheses for this study were: (i) ventilation rate (VR) will decrease as a consequence of warmer incubation temperatures, and (ii) offspring of anadromous parents are expected to have a higher VR compared to offspring of residential parents. VR, as a proxy for metabolic rate, was measured on young-of-the-year brown trout (Salmo trutta) during trials in respiration chambers. Fertilized eggs from four different crossings of parents (anadromous x anadromous, residential x residential, anadromous male x residential female, residential male x anadromous female) underwent incubation in normal and warm (normal + 3 °C) water temperatures respectively. A Two-way ANOVA showed a significant interaction between incubation temperature and crossing on VR. Warmer incubation temperatures in 3 out of 4 crossings resulted in a higher VR, except for individuals with two anadromous parents. Individuals with an anadromous father had a higher VR than individuals with a residential father. Evidence that partly supports both hypotheses was therefore found. Metabolism might have a strong genetic component, and other factors that might have an effect on metabolism and VR are epigenetics, stress, egg size etcetera. Studies of this kind will be of importance in getting a greater understanding of the effects of global warming on migrating species of fish, and on poikilothermic vertebrates in general.

The earth’s climate is rapidly changing. With climate change currently altering the environment, water temperature is an important environmental factor that will have various effects on fishes since they are ectotherms. As the changes are expected to be greatest during winter, when salmonid fishes incubate their eggs, one might expect substantial effects on egg development. In this experiment pairs of fish, matched by length, were compared. For each pair, one fish had experienced ambient cold temperatures (cold treatment) during embryogenesis and the other elevated temperatures (warm treatment, ca. 4-5 oC). The fish were tested in stream. Aggressive interactions were recorded and dominance was determined by scoring each fish’s position relative to where food was delivered from and the amount of food taken. Salmon from the cold treatment had a strong tendency to have a higher dominance score than salmon from the warm treatment. The individual with the highest dominance score was not consistently larger or smaller than the individual with the lower dominance score. The results support the hypothesis that salmonids exposed to a cold environment during embryogenesis will be more dominant. Due to climate change, it is expected that water temperature will continue to rise in the future, which in turn will alter the fish’s behaviour. / Jordens klimat förändras snabbt. Med klimatförändringar som för närvarande förändrar miljön är vattentemperaturen en viktig miljöfaktor som kommer att ha olika effekter på fiskar eftersom de är ektoterma. Eftersom förändringarna förväntas vara störst under vintern, när laxfiskar inkuberar sina ägg, kan man förvänta sig betydande effekter på äggutvecklingen. I det här experimentet jämfördes fisk par, matchade med längd. För varje par hade en fisk upplevt kalla vattentemperaturer (kall behandling) under embryogenesen och de andra förhöjda temperaturerna (varm behandling, ca 4-5 oC). Fisken testades i strömakvarie. Aggressiva interaktioner registrerades och dominans bestämdes genom att poängsätta varje fisks position i förhållande till var mat levererades från och mängden mat som tagits. Lax från den kalla behandlingen hade en stark tendens att ha högre dominanspoäng än lax från den varma behandlingen. Individen med högsta dominanspoäng var inte konsekvent större eller mindre än individen med lägre dominanspoäng. Resultaten stöder hypotesen att salmonider utsätts för en kall miljö under embryogenes kommer att vara den mer dominerande. På grund av klimatförändringen är det förväntat att vattentemperaturen fortsätter att stiga i framtiden, vilket i sin tur kommer att förändra fiskens beteende.

The influence of incubation temperature on bird neonate phenotype is widely studied. In waterfowl (Anatidae), maternal effect can be divided to two main parts: clutch formation and its incubation. Therefore, waterfowl are a good model for this type of study. The egg temperature can strongly influence embryo development and survival of the hatchlings. The goal of this work was to examine the effect of incubation temperature on hatchability, incubation length, sex ratio and phenotype of European Mallard ducklings immediately after hatching. Eggs were randomly placed in one of six incubation temperatures, which ranged from 35 to 39 řC. This range corresponds with temperature range in natural mallard nests in the Czech Republic. Eggs were then artificially incubated. Newborn hatchlings were weighted and their structural size was measured, then they were killed. Some hatchlings from incubation temperatures 35.5, 36 and 37 řC were used to examine residual yolk sac mass and basic chemical composition of the yolk-free body. Hatchability was markedly lower in both extreme temperatures (35 and 39 řC). Incubation length significantly decreased with increasing incubation temperature. I failed to prove the influence of incubation temperature on hatchling body mass, but I found temperature-dependent changes of...

Global warming, a consequence of the ongoing climate change, is expected to increase the mean global temperature by up to 5°C this century. This is predicted to have various impacts on the earth's ecosystems and especially on ectotherms which cannot regulate body temperature by endogenous heat production. There is an intensified interest in investigating the outcomes of climate change and the role of developmental phenotypic plasticity in relation to environmental conditions experienced early in life. Here I compare the body shape and fin size of juvenile Atlantic salmon that had been incubated at two different temperature regimes during embryogenesis. Morphometric methods were used to compare body size through a box-truss network of Euclidean distances and the data were then analyzed using discriminant analysis. Fin size was analyzed by comparing fin area using analysis of covariance with body size as the covariate. The fish incubated in ambient (cold) temperatures differed from the fish incubated in ca. 4°C warmer temperatures by having a deeper body shape and bigger pectoral fin area. There was no significant difference for dorsal fin area. My findings support the notion that Atlantic salmon are morphologically plastic when exposed to different temperature regimes during embryogenesis, but additional studies are needed to determine the ecological consequences of these changes. / Global uppvärmning, en konsekvens av pågående klimatförändringar, förväntas öka den globala medeltemperaturen med upp till 5°C det här århundradet. Det här förväntas ha stor påverkan på jordens ekosystem och speciellt ektotermer som inte kan reglera sin kroppstemperatur genom endogen värmeproduktion. Intresset har intensifierats för att undersöka klimatförändringarnas utfall och rollen som fenotypisk plasticitet har som svar på tidiga miljöförhållanden. Kroppsform och fenstorlek jämfördes hos juvenil atlantlax som inkuberats i två olika temperaturbehandlingar under embryogenesen. Morfometri användes för att undersöka kroppsformen genom ett box-truss nätverk av euklidiska avstånd som sedan analyserades med en diskriminant funktionsanalys. Fenstorleken analyserades genom att mäta fenornas area som sedan användes i en kovariansanalys med kroppsstorlek som kovariat. Fiskarna som inkuberades i normal (kall) temperatur skiljde sig från fiskarna som inkuberats i ca 4°C varmare temperatur genom att ha en djupare kroppsform och större area på bröstfenorna. Det fanns ingen signifikant skillnad mellan ryggfenornas area. Mina resultat stödjer idén att atlantlaxen är morfologiskt plastisk när den utsätts för olika temperaturbehandlingar under embryogenesen, men ytterligare studier behövs för att identifiera de ekologiska konsekvenserna av dessa förändringar.