Many organisms have geographical distributions extending from the tropics to near polar regions or can experience up to 30°C temperature variation within the lifespan of an individual. Two forms of evolutionary adaptation to such wide ranges in ambient temperatures are frequently discussed: local adaptation and phenotypic plasticity. The freshwater planktonic crustaceanDaphnia magna, whose range extends from South Africa to near arctic sites, shows strong phenotypic and genotypic variation in response to temperature. In this study, we use D. magna clones from 22 populations (one clone per population) ranging from latitude 0° (Kenya) to 66° North (White Sea) to explore the contributions of phenotypic plasticity and local adaptation to high temperature tolerance. Temperature tolerance was studied as knockout time (time until immobilization, Timm) at 37°C in clones acclimatized to either 20°C or 28°C. Acclimatization to 28°C strongly increased Timm, testifying to adaptive phenotypic plasticity. At the same time, Timm significantly correlated with average high temperature at the clones' sites of origin, suggesting local adaptation. As earlier studies have found that haemoglobin expression contributes to temperature tolerance, we also quantified haemoglobin concentration in experimental animals and found that both acclimatization temperature (AccT) and temperature at the site of origin are positively correlated with haemoglobin concentration. Furthermore, Daphnia from warmer climates upregulate haemoglobin much more strongly in response to AccT, suggesting local adaptation for plasticity in haemoglobin expression. Our results show that both local adaptation and phenotypic plasticity contribute to temperature tolerance, and elucidate a possible role of haemoglobin in mediating these effects that differs along a cold-warm gradient.

Daphnia is a widespread freshwater zooplankton species, which is both a classic and emerging new model for research in ecological physiology, ecotoxicology and evolutionary biology of adaptation to novel environments. Heat tolerance in Daphnia is known to depend both upon evolutionary history of a genotype and on individuals' acclimation to elevated temperature and to correlate with the level of haemoglobin expression. We demonstrate the existence of north-south gradient of heat tolerance in North American D. pulex, which is not associated with any parallel changes in haemoglobin expression. Geographically distinct clones differ in the way their haemoglobin expression changes due to acclimation to a sub-stressful (28°C) temperature, but these changes are not correlated with the latitude of clones' origin. Likewise, the effect of acclimation to sub-stressful temperature is independent from, and cannot be fully explained by, haemoglobin expression changes during acclimation. The degree of oxidative damage to haemoglobin, measured as the ratio of absorbance at 540:576 nm at the acclimation temperature, is a strong predictor of 28°C-acclimated Daphnia survival during an acute heat exposure. The comparison of haemoglobin expression in resistant and tolerant clones acclimated to different temperatures indicates that tolerant clones exhibit canalization of haemoglobin expression, possessing a high level of haemoglobin even at non-stressful temperatures. We discuss the evolutionary biology of adaptation and acclimation to elevated temperatures in an ecologically important component of freshwater ecosystems in the context of global climate change.

Geographic variation in thermal tolerance in Daphnia seems to represent genetic load at the loci specifically responsible for heat tolerance resulting from conditional neutrality. We see no evidence of trade-offs between fitness-related traits at 25 °C vs. 10 °C or between two algal diets across Daphnia magna clones from a variety of locations representing the opposite ends of the distribution of long-term heat tolerance. Likewise, we found no evidence of within-environment trade-offs between heat tolerance and fitness-related traits in any of the environments. Neither short-term and long-term heat tolerance shows any consistent relationship with lipid fluorescence polarization and lipid peroxidation across clones or environments. Pervasive positive correlations between fitness-related traits indicate differences in genetic load rather than trade-off based local adaptation or thermal specialization. For heat tolerance such differences may be caused by either relaxation of stabilizing selection due to lower exposure to high temperature extremes, i.e., conditional neutrality, or by small effective population size followed by the recent range expansion.

Examples of phenotypic plasticity—the ability of organisms of identical genotypes to produce different phenotypes in response to the environment—are abundant, but often lack data on the causative physiology and biochemistry. Phenotypes associated with increased protection against or reduced damage from harmful environments may, in fact, be downstream effects of hidden adaptive responses that remain elusive to experimental measurement or be obscured by homeostatic or over-compensatory effects. The freshwater zooplankton crustacean Daphnia drastically increases its heat tolerance as the result of acclimation to high temperatures, an effect often assumed to be based on plastic responses allowing better protection against oxidative stress. Using several geographically distant Daphnia magna genotypes, we demonstrate that the more heat tolerant individuals have a higher total antioxidant capacity (TAC) both in the comparison of heat-acclimated vs. non heat-acclimated females and in the comparison of females to age- and body size-matched males, which show lower heat tolerance than females. However, experimental manipulations of hypothesized antioxidant pathways by either glutathione addition or glutathione synthesis inhibition had no effect on heat tolerance. Lipid peroxidation (LPO), contrary to expectations, did not appear to be a predictive measure of susceptibility to thermal damage: LPO was higher, not lower, in more heat tolerant heat-acclimated individuals after exposure to a lethally high temperature. We hypothesize that LPO may be maintained in Daphnia at a constant level in the absence of acute exposure to elevated temperature and increase as a by-product of a possible protective antioxidant mechanism during such exposure. This conclusion is corroborated by the observed short-term and long-term changes in phospholipid composition that included an increase in fatty acid saturation at 28 °C and up-regulation of certain long-chain polyunsaturated fatty acids. Phospholipid composition was more strongly affected by recently experienced temperature (4-day transfer) than by long-term (2 generations) temperature acclimation. This is consistent with partial loss of thermal tolerance after a short-term switch to a reciprocal temperature. As predicted under the homeoviscous adaptation hypothesis, the more heat tolerant Daphnia showed lower membrane fluidity than their less heat tolerant counterparts, in comparison both between acclimation temperatures and among different genotypes. We conclude that thermal tolerance in Daphnia is influenced by total antioxidant capacity and membrane fluidity at high temperatures, with both effects possibly reflecting changes in phospholipid composition.

Ivy geranium (Pelargonium peltatum) is a heat susceptible species with its heat tolerance varying among varieties. Reactive oxygen species (ROS) and in-vivo defense systems are related to plant heat damage and heat tolerance. Application of chelated-iron has also been reported to enhance ivy geranium heat tolerance; however, the correlation of ROS, relative enzyme stability, and iron content to differences in heat tolerance in ivy geraniums is unknown. Here we show that the H2O2 content and ROS scavenging enzyme stability in ivy geranium varies with varieties and active iron is not related to heat tolerance in ivy geranium. H2O2 content in mature leaves in both heat tolerant 'Beach' and sensitive 'Butterfly' increased under heat stress, but 'Butterfly' had a relatively greater increase of this toxic compound. Catalase (CAT) activities in young leaves in both varieties decreased. In young leaves of 'Butterfly', CAT activities decreased to a level significantly lower than that in old leaves while this did not occur in 'Beach'. Superoxide dismutase (SOD) activities in 'Butterfly' young leaves were also decreased. All these phenomenon coincided with the heat tolerance differences of the two varieties. Active iron content only changed with leaf age and did not vary between varieties or treatments. Our results demonstrated that ROS scavenging ability and relative enzyme stability may indicate heat tolerance in ivy geranium and that iron deficiency was not the cause of heat damage. Cell Membrane Themostability (CMT) and Triphenyl Tetrazolium Chloride (TTC) cell viability tests are alternative, laboratory-based screening methods for screening for heat-tolerance. Both CMT and TTC tests can represent the variance in heat tolerance observed in ivy geraniums. The results of both CMT and TTC tests correlated well with plant width and growth indexes although their correlations to plant chlorosis were low. Unlike TTC, CMT strongly correlated with plant width. CMT and TTC tests are complementary laboratory-based methods that can be applied to cultivar screening for heat tolerance in ivy geraniums.

A set of 37 strains including 13 serotypes of Listeria monocytogenes (Lm) were analyzed for heat tolerance at 60°C for 10 min and further categorized into three groups; low (strains with <2 log survival), medium (2-4 log survival), and high (4-6 log survival) heat tolerant. When Lm strains representing each group were subjected to sub-lethal heatstress at 48°C prior to 60°C, the survivals of all strains were increased by at least 5 log CFU/ml when compared to controls. Sub-lethal heat-stress at 48°C for 30-60 min increased the heat-stress resistance of Lm strains by doubling D60°C values from 1.9-4.3 to 5.0-10.4 min. When Lm cells were cooled after sublethal heat-stress at 48°C prior to 60°C treatment, such acquired heat-stress adaptation was unstable at 22°C but was found to be highly stable for up to 24 h at 4°C. These results will have potential implications in food safety risk analysis for Lm.

Thermal adaptation in freshwater planktonic crustacean Daphnia pulex has been investigated by means of heatshock (35°C) experiments using individuals acclimated to different water temperatures (18, 24, and 28°C). Retention of mobility and survival during the heatshock experiments differed among clones and strongly depended on acclimation temperature: clones acclimated to substressful temperature (28°C) survived significantly longer than clones acclimated to lower temperatures. Both retention of mobility and survival correlated with geographic latitude of the clones' origin. There was, on the other hand, no significant correlation between clones' performance in heatshock experiments and hemoglobin content. Clones do not differ in the degree of the effect of acclimation on survival during heatshock but differ with respect to the effect of acclimation on mobility retention.

<p> As the effects of climate change become more evident, the development of effective strategies for improving livestock climatic adaptation and the long-term sustainability of animal food production have become key priorities around the world, including in the US. Together with nutrition, infrastructure, and management practices, genetically improving animals is an effective and lasting alternative to simultaneously improve productive efficiency and climatic adaptation of animals. Genetic improvement requires basic understanding of the genomic architecture of the indicator traits of interest and the availability of large-scale datasets. Understanding the role of evolution and selection (both natural and artificial) on shaping animal genomes is of paramount importance for the optimization of breeding programs and conservation of genetic resources. In addition, properly quantifying environmental stress and individual animal responses to thermal stress are still important challenges in breeding programs. Thus, the identification of optimal statistical methods and traits that better capture key biological mechanisms involved in the heat stress response has the potential to enable more accurate selection for thermal tolerant individuals. Therefore, this thesis aimed to investigate complementary topics related to thermal tolerance in livestock species based on genomic information. A total of 946 genotypes from 34 cattle breeds, as well as Datong yak (<em>Bos grunniens</em>) and Bali (<em>Bos javanicus</em>) populations, adapted to divergent climatic conditions, were used to investigate the genetic diversity and unravel genomic regions potentially under selection for thermal tolerance, with a focus on Chinese local cattle breeds and yak. Different signature of selection analyses and a comprehensive description of genetic diversity in 32 worldwide cattle and Datong yak populations was presented. Moderate genetic diversity was observed within each Chinese cattle population. However, these results highlighted the need to adopt strategies to avoid further reduction in the genetic diversity of these populations. Several candidate genes were identified as potentially under selection for thermal tolerance, and important biological pathways, molecular functions, and cellular components were identified, which contribute to our understanding of the genetic background of thermal tolerance in <em>Bos</em> species. Secondly, 8,992 genotyped individuals were used to provide a comprehensive description of genotype-by-environment interaction effects, defining optimal environmental variables based on public weather station data, and critical periods to evaluate heat tolerance for various reproduction, growth, and body composition traits in US Large White pigs. The period of 30 days before the measurement date was suggested to analyze genotype-by-environment interaction for off-test weight, muscle depth, and backfat thickness. While for number of piglets weaned and weaning weight, the suggested period ranged from the last trimester of gestation until weaning. This same population was used to access the genomic predictive ability of heat tolerance based on routinely-measured traits and explore candidate regions involved in the biological mechanisms that underlie heat stress response in pigs. Genotype-by-environment interaction was identified for most of the traits evaluated, and moderate (>0.36 ± 0.05) breeding values prediction accuracy were achieved using genomic information. Lastly, various behavioral, anatomical, and physiological indicators of heat stress were measured in a population of 1,645 multiparous Large White x Landrace lactating sows. This dataset was used to identify the best statistical models and estimate genomic-based genetic parameters for 23 indicators of heat stress, including automatically-measured vaginal temperature, skin surface temperatures, respiration efficiency, respiration rate, panting score, body condition scores, hair density, body size, and ear measurements. All the traits evaluated are heritable, with heritability estimates ranging from 0.04 ± 0.01 to 0.40 ± 0.09. The genetic correlations among these traits ranged from -0.49 (between repeated records of vaginal temperature measured at 0800 hours and caliper body condition score) to 1.0 (between repeated records of vaginal temperature measured at 0800 hours and single record of vaginal temperature measured at 0800 hours; and between repeated records of vaginal temperature measured at 1200 hours and single record of vaginal temperature measured at 1200 hours). These findings indicate that genetic progress for thermotolerance in pigs can be achieved through direct indicators of heat stress in selection schemes. However, special attention is needed due to complex relationship between these traits as evidenced by their genetic correlations. In conclusion, this thesis provides important information to be used when designing breeding strategies for improving thermal tolerance in cattle and pigs, important genomic regions and metabolic pathways that are important for understanding the biological mechanism regulating thermal tolerance, as well as future directions for investigations in the area of livestock climatic adaptation.</p>

O presente trabalho teve como objetivo avaliar o comportamento de pastejo de bovinos de corte com e sem acesso a recursos de sombreamento e determinar o Índice de Tolerância ao Calor Individual (ITCI) e uma possível relação com o uso da sombra. Foram utilizados 8 touros da raça Simental, com 80 meses. As observações de comportamento ocorreram do nascer ao pôr do sol, a cada 15 minutos, pelo método focal. Os comportamentos observados foram: Posição (ao sol, à sombra natural, à sombra artificial); Postura (em pé, deitado) e Atividade (em pastejo, em ruminação, em deslocamento, em ócio), em três tratamentos: Sombra Natural (SN), Sombra Artificial (SA) e sem sombra (SS). Numa segunda fase os animais foram submetidos ao Teste de Tolerância ao Calor e foi determinado o ITCI. O índice de globo negro e umidade médio foi de 88,5. O uso da sombra foi maior no SA em relação ao SN (43% e 32%) iniciando mais cedo e deixando-a mais tarde (10h00 e 16h00 no SA, 10h30 e 15h00 no SN). Os animais permaneceram mais tempo em pé no SS, seguido pelo SN e pelo SA (86,5%, 80,6%, 73,7%). No tratamento SN os animais pastejaram 65,4% do tempo total de observação, ruminaram 18,3%, permaneceram em ócio 15,9% e a atividade de deslocamento tomou 0,5% do tempo. No tratamento SA, as atividades foram divididas em 54,7% para o pastejo, 22,9% para ruminação, 21,2% para ócio e 1,2% para deslocamento. No tratamento SS, o pastejo ocorreu em 50,3% do tempo, 6,2% do tempo foi gasto com ruminação, 41,7% em ócio e 1,7% com deslocamento. Houve diferença estatística entre os três tratamentos (P<0,01) para todas as atividades analisadas. Verificou-se a interferência da disponibilidade de sombra e do tipo de sombra na distribuição das atividades ao longo do dia. Os animais que tiveram acesso à sombra natural sem restrição de espaço e com ótima ventilação devido às altas copas das árvores gastaram menos tempo em ruminação e ócio e mais tempo com o pastejo. O índice de tolerância ao calor médio foi de 9,33, com máximo de 9,57 e mínimo de 9,03. A relação entre o ITC e a busca pela sombra teve média-alta correlação (0,62) mas sem ser considerada significativa devido ao baixo número de repetições. / The objective of this study was to analyse the grazing behavior of beef cattle with and without shade and to determine the Individual Heat Tolerance Index (ITCI) and a possible relationship with the utilization of shade. The behavioral patterns of eight Simental bulls were observed during the day light, every 15 minutes, using a focal sampling method. The categories observed were the position (in the sun or under the shade), the posture (standing up or lying down) and the activities (grazing, ruminating, moving or resting). In three different treatments: natural shade (SN), artificial shade (SA) and no shade (SS). In a second stage the animals were submitted to the Heat Tolerance Test and the ITCI was calculated. The mean of black globe and humidity index was 88,5. The shade was more used in SA than in SN (43% e 32%), getting in there earlier and leaving it later (10h00 and 16h00 in SA, 10h30 and 15h00 in SN). The animals stayed more standing up in SS, followed by SN and SA (86,5%, 80,6%, 73,7%). In the SN treatment the animals grazed 65,4% of the total time of observation, ruminated 18,3%, rested 15,9% and moved 0,5%. In the SA treatment the animals grazed 54,7% of the total time of observation, ruminated 22,9%, rested 21,2% and moved 1,2%. In the SS treatment the animals grazed 50,3% of the total time of observation, ruminated 6,2%, rested 41,7% and moved 1,7%. There was statistic difference between the three treatments (P<0,01) for all the analyzed activities. There was interference of the availability of shade and kind of shade in the diary activities. The mean of the ITCI was 9,33, with maximum of 9,57 and minimum of 9,03. The relationship between ITCI and seek for shade had high correlation (0,62) but no statistic evidence because of the small repetition.

Trinta e oito vacas leiteiras, 19 da raça Holandês e 19 da raça Girolando (½ e ¾), foram submetidas à elevadas temperaturas por restrição à sombra durante o período entre a ordenha da manhã e da tarde. Mensurações de temperatura retal, frequências respiratória e cardíaca e escore de ofegação, além de coletas de sangue, foram realizadas nos períodos pré ordenha. Produção de leite e características físico-químicas do mesmo foram acessadas. Durante os períodos de coleta se avaliou características ambientais com vistas a calcular um índice de temperatura e umidade (ITU). Primeiramente se buscou estabelecer diferenças em parâmetros fisiológicos, de leite e sangue de vacas com diferentes porcentagens de alelos oriundos da raça Holandesa no genoma (100, 75 e 50%) em função de aumentos no ITU. Animais puros Holandês apresentaram características que condizem com menor tolerância ao calor do que animais Girolando. Em um segundo momento se analisou o efeito do estresse térmico sobre a permeabilidade das junções firmes das células epiteliais da glândula mamária de vacas Holandês. Além disso, buscou-se perceber alterações na estabilidade do leite ao teste do álcool decorrentes de um possível aumento na permeabilidade de tais estruturas celulares. O parâmetro utilizado para aferir tal efeito foi o nível sanguíneo de lactose no plasma. Alterações lácteas foram percebidas em função de aumentos de ITU e se devem principalmente à reduções em produção de leite. Aumento nos parâmetros fisiológicos não influenciou a estabilidade do leite. Leite instável apresentou maior teor de lactose. Maior número de dias em lactação pode ser o responsável pela redução na estabilidade do leite. Percebeu-se relação inversamente proporcional entre permeabilidade das junções firmes e estabilidade do leite, porém o estado de estresse térmico, ao contrário do esperado, não apresentou influência nas células da glândula mamária. / Thirty-eight dairy cows, 19 Holstein and 19 Girolando (½ e ¾), were submitted to elevated temperatures due to shade deprivation between morning and evening milkings. Rectal temperature, heart and respiratory rates and panting score measurements, besides blood samples collection, were performed before each milking. Milk production and its physical-chemical characteristics were studied. Climatic variables were measured during data collection to calculate a temperature-humidity index (THI). Firstly, the study evaluated changes in physiological, blood and milk parameters according to the percentage of alleles derived from the Holstein breed (100, 75 and 50%) due to increases in THI. Pure Holstein cows presented characteristics that indicate lower heat tolerance than Girolando cows. Secondly, heat stress effects on mammary gland cells tight junctions permeability were evaluated in Holstein cows. Alterations in milk stability due to changes in permeability were also assessed. Plasma lactose was the parameter used to detect changes in tight junctions permeability. Modifications in milk characteristics were probably due to reduction in milk production. Increase in physiological parameters did not affect milk stability. Unstable milk samples presented higher lactose levels. Higher days in milk might be the main responsible for reductions in milk stability. There was an inversely proportional relation between tight junctions permeability and milk stability to the ethanol test, although heat stress, contradicting the expected, did not influenced mammary gland cells.