Bumblebees are experiencing declines and range contractions globally that are, in some cases, independent of anthropogenic pesticide- or land-use change, leaving rising global temperatures as the primary driver of such loses. With ambient temperature (Ta) and thermal limitations being a crucial component in these observed declines, I sought to determine the physiological limitations that high Ta imposes on both individuals and colonies of a temperate bumblebee species, Bombus impatiens. Through Chapter 2, I first established the upper thermal tolerance (CTmax) of the species, testing both adults and larvae to determine which of these colony castes are most thermally sensitive to heat. Collective thermoregulation at the colony-level is then important to ensure that the most heat sensitive individuals are protected from changes in optimal nest temperature (Tn). I thus identified the energetic costs associated with colonial thermoregulation and whether large colonies could successfully achieve thermal homeostasis under various Ta. Chronic bouts of heat stress are also of concern as colonies invest time and energy into thermoregulation, especially given that heatwave events are becoming more frequent. In Chapter 3, I examined whether there exists a trade-off between thermoregulation and foraging effort for colonies under chronic heat stress and how various measures of colony success are impacted. Finally, foraging requires individuals to employ flight for the procurement of resources. In Chapter 4, I investigated if the temperate adaptation of an insulative pile layer would hinder flight performance under high Ta by assessing the metabolic rates of adult castes during flight. I found that larvae were more thermally sensitive compared to bumblebee adults, which emphasizes the importance of colonial thermoregulation – a task successful at low Ta. Under heat stress, however, Tn could not be maintained despite elevated energetic investments (Chapter 2). These findings suggest that Ta which exceeds optimal Tn may pose significant challenges to colonies; not only energetically but also to the health of thermally sensitive larvae within. A trade-off between thermoregulation and foraging effort did not emerge for colonies experiencing chronic exposure to high Ta. Instead, only high incidences of thermoregulation were observed which failed to prevent increases in Tn. Furthermore, a greater number of individuals were found to abandon the colony at high Ta, and fewer offspring were produced (Chapter 3). Here, findings suggest that chronic high Ta may pose the greatest risk to the production of thermally sensitive offspring by way of reduced worker population and failed thermoregulation. Finally, the metabolic output during flight at high Ta was not found to be affected by an insulative layer of pile (Chapter 4), indicating that either pile may play a role in limiting other measures of flight performance at high Ta, or that alternate physiological mechanisms may be responsible instead. Together the findings from this thesis broaden the understanding of how a temperate species of bumblebee responds physiologically to high Ta both at the individual and colonial level, providing further evidence on thermal limitations in a changing climate.

Doctor of Philosophy / Department of Agronomy / Allan K. Fritz / Heat stress is one of the major environmental constraints for wheat production worldwide. High temperature during grain filling in wheat leads to a significant reduction in yield. In this research, three different projects were completed. The first project was to study cytoplasmic effects on heat tolerance in wheat, where ten different alloplasmic lines of wheat were backcrossed with four different wheat varieties: „Karl 92‟, „Ventnor‟, „U1275‟ and „Jagger‟. The nuclear genome of the alloplasmic lines was substituted by backcrossing six times using the recurrent parents as males. During the fifth and/or fourth backcross, reciprocal crosses were made to develop NILs (Near Isogenic Lines) for cytoplasm. Sixty-eight NILs and their parents were evaluated in growth chambers for post-anthesis heat tolerance. Plants were grown in the greenhouse and placed under heat stress for 14 days starting at 10 days after anthesis. Growth chambers were maintained at 35°/30°C for heat stress and the greenhouse was maintained at 20°/15°C as the optimum temperature. Effects of high temperature on chlorophyll content and Fv/Fm (a chlorophyll fluorescence measuring parameter) were found to be significant. Cytoplasms 1, 4, 5, 8, 9 and10 provided greater tolerance in one or more nuclear backgrounds. These results indicated that cytoplasmic effects can contribute to heat tolerance of wheat. The second project focused on identification of quantitative trait loci (QTL) for thylakoid membrane damage (TMD), SPAD chlorophyll content (SCC) and plasma membrane damage (PMD), as these traits are found to be associated with resistance to heat stress and contributes to relatively stable yield under high temperature. A RIL (Recombinant Inbred line) population of a cross between winter wheat cultivars „Ventnor‟ and „Karl 92‟ was evaluated using two different temperature regimes (20°/15°C, 36°/30°C) imposed at ten days after anthesis. The aforementioned traits were evaluated and associated with various molecular markers (SSR, AFLP and SNP). The putative QTL associated are localized on chromosomes 6A, 7A, 1B, 2B and 1D and have the potential to be used in marker assisted selection for improving heat tolerance in wheat. In the third project, a transgenic approach to increase grain fill during high temperatures was investigated. Grain fill is reduced at temperatures above 25°C in wheat partly due to the inactivity of soluble starch synthase. We isolated a soluble starch synthase gene from rice that has the potential to overcome this deficiency during high temperatures and placed it behind both a constitutive promoter and an endosperm-specific promoter. Transgene expression and the effects of the transgene expression on grain yield-related traits for four generations (T0, T1, T2 and T3) were monitored. The results demonstrated that even after four generations, the transgene was still expressed at high levels, and transgenic plants produced grains of greater seed weight than Bobwhite control plants under the same environmental conditions. Thousand-seed weight under high temperatures increased 21-34% in T2 and T3 transgenic plants when compared to the non-transgenic control plants. In addition, the duration of photosynthesis was longer in transgenic wheat than in non-transgenic controls. Our study demonstrated that expression of rice soluble starch synthase gene in wheat can improve wheat yield under heat stress conditions.

Master of Science / Department of Agronomy / William T. Schapaugh / Heat and drought stress are common problems for crops grown in Kansas. Rarely do these problems occur separately, more often than not they occur in tandem if not simultaneously. The interaction of heat stress and pollen germination was investigated in order to determine if a physiological screen was a feasible method of determining heat tolerance in soybean [Glycine max (L.) Merr]. Ten soybean lines (Group A) from the 2006 Northern Region Uniform Soybean Tests were analyzed over two years in four locations consisting of irrigated and dryland field environments, with an additional twenty lines (Group B) analyzed in the second year. Pollen was collected from plants and incubated at either 28o, 34o, or 38o C to determine pollen germination for optimal and stress-inducing temperatures. A three-way interaction of entry x incubation temperature x environment was observed, as well as significant differences among entries, incubation temperatures and environments. Average pollen germination for soybean entries ranged from 25% to 38% across three incubation temperatures and four environments in Kansas during 2006 – 07. The average environment effect for pollen germination ranged from 29% (dryland, 2006) to 34% (irrigated, 2007), while the average incubation temperature effect on pollen germination ranged from 25% (38oC) to 44% (28oC). This experiment has shown that increasing incubation temperatures significantly decreases pollen germination in vitro. It has also shown that soybean genotypes differ in pollen germination and that an in vitro screen can be used to characterize these differences. Further studies are needed to establish the relationship between pollen germination, seed set and seed yield in soybean. Work also needs to be completed to determine the proper sample size to adequately characterize differences in pollen germination so that performance differences among genotypes can be used as selection criteria in a plant breeding program.

Este estudo teve como objetivo a compreensão dos efeitos desencadeados pelas temperaturas elevadas na expressão gênica de proteínas de choque térmico, nomeadamente da HSPA1A e HSP90AA1, em linfócitos de fêmeas bovinas de diferentes raças e origens. Parte do projeto foi desenvolvida em Portugal, onde foram utilizadas 20 novilhas da raça Limousine e 22 da raça Mertolenga. A parte desenvolvida no Brasil decorreu com 11 novilhas da raça Holstein Frísia e 20 da raça Brahman. Os animais da raça Holstein participaram do Experimento I no qual foram coletados 4 tubos de sangue para posterior choque térmico in vitro, onde cada tudo de sangue foi submetido a diferentes temperaturas: 40 °C e 42 °C (ambos através de banhos-maria), 23 °C (mantido a temperatura ambiente) e 10 °C (na geladeira), durante duas horas. Todos os animais de todas as raças participaram no Experimento II, onde foram sujeitos ao teste de tolerância ao calor, tendo sido aferidas a temperatura retal e a frequência respiratória, e coletado sangue (após os tratamentos sombra e sol). A todas as amostras de ambos os experimentos foi realizada a lise das hemácias de modo a obter o buffy-coat. O RNA foi isolado através do método do TRIzol e a RT-PCR realizada com SuperScript III após digestão com DNAse I. A PCR em tempo real decorreu no aparelho 7500 Fast Real Time, utilizando TaqMan Gene Expression Assays para os genes alvo HSPA1A e HSP90AA1, e ACTB e PPIA como genes endógenos. Foram calculados os ΔCt (Ctalvo - Ctendógeno) assim como a expressão gênica através do método 2-ΔΔCt. A análise estatística foi realizada através de modelos lineares mistos, recorrendo ao programa R Software Project (versão 3.0.1). No Experimento I foi atestada a qualidade da relação para ambos os endógenos nas relações que estabelecem com o mRNA-HSPA1A e o mRNA-HSP90AA1, através de uma análise de regressão pairwise. Tal como era esperado os valores de expressão gênica a uma temperatura de 23 °C foram os menores, seguidos 10 °C (estresse por frio), 40 °C e 42 °C para o gene HSPA1A. No caso da HSP90AA1, foi a 40 °C que se verificou uma maior expressão gênica. No Experimento II verificou-se uma variação intra-raça algo acentuada para valores de frequência respiratória, permitindo supor que o esforço termorregulatório dos animais de uma mesma raça possa ter sido diferente. Em relação à temperatura retal a raça Brahman apresentou valores significativamente diferentes das restantes raças para a situação sol. As diferenças observadas foram provavelmente consequência dos diferentes níveis de estresse aos quais os animais estiveram sujeitos. As diferenças observadas nos ΔCt não foram muito expressivas e apenas se observaram diferenças significativas na expressão gênica relativa na raça Mertolenga entre as situações sombra e sol. Podemos concluir que o aumento planificado da temperatura de linfócitos bovinos leva a diferentes expressões gênicas relativas de HSPA1A e HSP90AA1. A expressão de mRNA-HSPA1A é tanto maior quanto maior o estresse térmico, quer por frio quer por calor. As expressões gênicas relativas de HSPA1A e HSP90AA1 exibidas por animais com diferentes capacidades termolíticas são também elas diferentes, existindo uma variabilidade individual na expressão gênica relativa de proteínas de choque térmico. / The present study aimed to understand the effects triggered by high temperatures in heat shock proteins gene expression, HSPA1A and HSP90AA1, in cow lymphocytes from different breeds and origins. Part of the project was developed in Portugal, where 20 Limousin and 22 Mertolenga breed heifers were used and the second part was developed in Brazil with 11 Holstein Friesian and 20 Brahman heifers. The Holstein animals participated in Experiment I in which four blood samples were collected for subsequent in vitro heat shock, where each one of the blood samples was submitted to different temperatures: 40 ° C and 42 ° C (through water baths), 23 ° C (kept at room temperature) and 10 ° C (in refrigerator) for two hours. All animals from all breeds participated in Experiment II, where they were subjected to the heat tolerance test, where rectal temperature and respiratory rate were measured, and blood samples were collected (after shadow and after sun treatments). In all samples of both experiments was carried out the erythrocytes lysis so as to obtain the buffycoat. The RNA was isolated by the TRIzol method and RT-PCR performed with SuperScript III after digestion with DNase I. The real-time PCR apparatus took place in 7500 Real Fast Time, using TaqMan Gene Expression Assays for HSPA1A and HSP90AA1 target genes, ACTB and PPIA as endogenous genes. The ΔCt (Cttarget - Ctendogenous) were calculated as well as gene expression through the 2-ΔΔCt method. Statistical analysis was performed using linear mixed models, using the program R Project Software (version 3.0.1). In Experiment I it was attested the quality of the relationship for both endogenous with the mRNA-HSPA1A and mRNAHSP90AA1 through a pairwise regression analysis. As expected values, gene expression at a temperature of 23 °C were lower, followed by 10 °C (cold stress), 40 °C and 42 °C for the HSPA1A gene. In the case of HSP90AA1 the higher gene expression was found at 40 °C. In Experiment II, there was a slightly pronounced intra-race variation for respiratory rate values, allowing the assumption that the thermoregulatory effort of animals of the same breed may have been different. Regarding the rectal temperature, in Brahman breeds it was significantly different from the other breeds in the sun treatment. The differences observed were probably a result of different levels of stress to which the animals were subjected. The differences observed in ΔCt were not very expressive and significant differences were only observed in relative gene expression of Mertolenga breed between shade and sun treatments. We conclude that planned increasing temperature in bovine lymphocytes leads to different relative gene expression of HSPA1A and HSP90AA1. The mRNA-HSPA1A expression is greater in higher thermal stress, either by cold or by heat. The HSPA1A and HSP90AA1 relative gene expressions exhibited by animals with different thermolytic capabilities, are also different, existing an individual variability in relative gene expression of heat shock proteins.

Heat stress adversely affects wheat production in many regions of the world and is particularly detrimental during reproductive development. The objective of this study was to identify quantitative trait loci (QTL) associated with improved heat tolerance in hexaploid bread wheat (Triticum aestivum). To accomplish this objective, an analysis of both the phenotypic and genetic responses of two recombinant inbred line (RIL) populations was conducted. RIL populations Halberd x Cutter and Halberd x Karl 92 (H/K) both derive heat tolerance from Halberd and segregate in their response to heat stress. A heat susceptibility index (HSI) was calculated from the reduction of three yield components; kernel number, kernel weight, and single kernel weight, following a three-day 38 degrees C heat stress treatment during early grain-filling. The HSI, as well as temperature depression of the main spike and flag leaf were used as measurements of heat tolerance. Genetic linkage maps were constructed for both populations and were used in combination with phenotypic data and statistical software to detect QTL for heat tolerance. In a comparison across the two across populations, seven common QTL regions were identified for HSI, located on chromosomes 1B, 3B, 4A, 5A, 5B, and 6D. Subsequent analysis of temperature depression in the H/K population identified seven QTL that co-localized for both cooler organ temperature and improved HSI. Four of the beneficial alleles at these loci were contributed Halberd. The genetic effect of combining QTL, including QHkw.tam-1B, QHkwm.tam-5A.1, and QHskm.tam-6D showed the potential benefit of selection for multiple heat tolerant alleles simultaneously. Analysis of the H/K population in the field under abiotic stress detected QTL on chromosome 3B and 5A, which were in agreement with results from the greenhouse study. The locus QYld.tam-3B was pleiotropic for both temperature depression and HSI in both experiments and was associated with higher biomass and yield under field conditions. The results presented here represent a comprehensive analysis of both the phenotypic response of wheat to high temperature stress and the genetic loci associated with improved heat tolerance and will be valuable for future understanding and improvement of heat stress tolerance in wheat.

Due to concerns with synthetic neurotoxic insecticides used in insect pest management, alternative control methods are sought. Methoprene is a juvenile hormone analogue, which is well-known for its lethal effects on immature insects, and is registered as a stored-grain insecticide in the U.S.A. and Australia. However, less is known about its sub-lethal effects. Experiments were conducted to investigate the effects of methoprene (Diacon II) on heat tolerance, cold tolerance and progeny production of Tribolium castaneum (Herbst) when late-instar larvae or adults were exposed to wheat treated with methoprene. Methoprene at 3.33 ppm or higher, reduced heat tolerance of adults at 46ºC. In contrast, methoprene did not affect the heat tolerance of larvae. Cold tolerance and cold acclimation of both adults and larvae was not affected by methoprene. Exposure of larvae to 0.001 or 0.0165 ppm of methoprene on wheat had 37 and 72% reduction in adult emergence, respectively. The surviving adults had normal movement, but their progeny production was reduced by 71%. Males were affected more than females. Exposure of adults to methoprene (66.6 ppm) did not reduce progeny production. Methoprene is used as a surface treatment to control insects in empty grain bins, processing facilities and warehouses. Experiments were conducted to assess the effect of surface material, temperature and cleaning practices on methoprene residual efficacy. A bioassay with late-instar larvae at 30ºC until adult emergence was used to measure methoprene residual efficacy. Methoprene applied at the label rate (0.0003 mg/cm2) on varnished wood, held at 20, 30 or 35ºC for 24 weeks, prevented all development of larvae into adults. Conversely, concrete surfaces allowed 22% adult emergence after 8 weeks and 69% after 24 weeks. Temperatures, 20, 30 or 35ºC, that the concrete surfaces were held before the bioassay, did not affect this decline in efficacy. Presence of flour or repeated removal of flour (cleaning) reduced the efficacy of methoprene on concrete surfaces, but not on varnished wood. Maintaining concrete surfaces at 65ºC for 48 hours did not reduce efficacy. The implications of these results are discussed in the context of controlling stored-product insects, as well as insect physiology.

Due to concerns with synthetic neurotoxic insecticides used in insect pest management, alternative control methods are sought. Methoprene is a juvenile hormone analogue, which is well-known for its lethal effects on immature insects, and is registered as a stored-grain insecticide in the U.S.A. and Australia. However, less is known about its sub-lethal effects. Experiments were conducted to investigate the effects of methoprene (Diacon II) on heat tolerance, cold tolerance and progeny production of Tribolium castaneum (Herbst) when late-instar larvae or adults were exposed to wheat treated with methoprene. Methoprene at 3.33 ppm or higher, reduced heat tolerance of adults at 46ºC. In contrast, methoprene did not affect the heat tolerance of larvae. Cold tolerance and cold acclimation of both adults and larvae was not affected by methoprene. Exposure of larvae to 0.001 or 0.0165 ppm of methoprene on wheat had 37 and 72% reduction in adult emergence, respectively. The surviving adults had normal movement, but their progeny production was reduced by 71%. Males were affected more than females. Exposure of adults to methoprene (66.6 ppm) did not reduce progeny production. Methoprene is used as a surface treatment to control insects in empty grain bins, processing facilities and warehouses. Experiments were conducted to assess the effect of surface material, temperature and cleaning practices on methoprene residual efficacy. A bioassay with late-instar larvae at 30ºC until adult emergence was used to measure methoprene residual efficacy. Methoprene applied at the label rate (0.0003 mg/cm2) on varnished wood, held at 20, 30 or 35ºC for 24 weeks, prevented all development of larvae into adults. Conversely, concrete surfaces allowed 22% adult emergence after 8 weeks and 69% after 24 weeks. Temperatures, 20, 30 or 35ºC, that the concrete surfaces were held before the bioassay, did not affect this decline in efficacy. Presence of flour or repeated removal of flour (cleaning) reduced the efficacy of methoprene on concrete surfaces, but not on varnished wood. Maintaining concrete surfaces at 65ºC for 48 hours did not reduce efficacy. The implications of these results are discussed in the context of controlling stored-product insects, as well as insect physiology.

Made available in DSpace on 2015-03-26T13:40:01Z (GMT). No. of bitstreams: 1 texto completo.pdf: 716927 bytes, checksum: 2d7eff1a3130da5b28fc4573144a70c4 (MD5) Previous issue date: 2013-12-03 / Even though wheat is originated from cold climates, the use of adapted cultivars allows its production in regions with tropical climates, but high temperatures during the crop cycle may become one of the main factors that cause abiotic stress, limiting the well development of the plant and, consequently, a drastic decline in the productivity potential. The wheat production in the central region of Brazil is observed to have been affected because of the increases in temperature which are presented during the different phenological stages, becoming alike to the main limiting factor to its full development. The knowledge of methodologies that are easy to access and are of practical use, which are efficient for plant selection in field conditions is indispensable for the recognition and obtainment of cultivars with tolerance to heat. The objective of this work was to Evaluate canopy temperature depression through the use of infrared thermometry and agronomic traits in wheat cultivars, sown in pots with soil in through conventional cultivation with the intent to compare cultivar reaction to each environment, aiming to confirm which of these present tolerance to heat stress.The experiment was conducted at the Experimental Station Professor Diogo Alves de Melo - of the Federal University of Viçosa (UFV), Viçosa-MG. A completely randomized design was utilized. Six cultivars are utilized for use in the central region of Brazil: Aliança, Anahuac, Embrapa 22, BRS 254, BRS 264 and BRS 207. The cultivars were sown towards the end of July with varying temperatures throughout the development of the plant. There were no significant differences between cultivars when comparing the decline in the temperature of the evaluated plant organs. Only local effects were existent, which was attributed to the fact that, during the general culture development the average air temperature didn't reach values that are considered stressful, being found between 18 and 21° which is considered an C, ideal temperature range for wheat culture. The occurrence of winds may have reduced the canopy temperature of the plants sown in pots, which made these present higher temperature decline values when compared to plants cultivated in the environment's soil. Most of the agronomic traits evaluated present significant differences between environments and between cultivars, being that the pots' environment had the highest average values except for the harvesting index and the weight of one thousand grains, which were bigger in the conventional cultivation. / Mesmo o trigo sendo originário de climas frios, o uso de cultivares adaptadas possibilita sua produção em regiões de climas tropicais, mas as altas temperaturas durante seu ciclo podem tornar um dos principais estresses abióticos, limitando seu bom desenvolvimento e, consequentemente, levando a diminuição drástica no potencial produtivo. A produção de trigo na região central do Brasil é afetada devido aos incrementos da temperatura que se apresentam durante os diferentes estados fenológicos da planta. Deste modo, o conhecimento de metodologias de acesso fácil e de uso prático, que sejam eficientes para a seleção de plantas em condições de campo é imprescindível para a identificação e a obtenção de cultivares com tolerância ao calor. O objetivo deste trabalho foi Avaliar a depressão da temperatura do dossel por meio do método da termometria infravermelha e caracteres agronômicos em cultivares de trigo, semeadas em vasos com solo e em cultivo convencional a fim de comparar o comportamento das cultivares nos dois ambientes, visando qual destas apresentam tolerância ao estresse de calor. O experimento foi conduzido na Estação Experimental Prof. Diogo Alves de Melo da Universidade Federal de Viçosa (UFV), Viçosa-MG. Utilizou-se o delineamento experimental inteiramente casualizado. As cultivares avaliadas foram: Aliança, Anahuac, Embrapa 22, BRS 254, BRS 264 e BRS 207, sendo semeadas no final do mês junho com temperaturas variáveis ao longo do desenvolvimento das plantas. A depressão da temperatura no dossel não apresentou diferenças entre as cultivares. O efeito apresentado foi de local, o que foi atribuído ao fato de que, durante o desenvolvimento das plantas o ambiente não apresentou estresse acentuado pelo calor, considerando que a temperatura média do ar encontrava-se numa faixa entre 18 a 21° considerada adequada para o desenvolvimento da cultura de C, trigo. A ocorrência de ventos possivelmente reduziu a temperatura no dossel das plantas cultivadas em vasos, fazendo com que estas apresentassem maiores valores de depressão de temperatura em comparação com as plantas cultivadas no solo. Para a maioria dos caracteres agronômicos avaliados houve diferença significativa entre ambientes e entre cultivares, apresentando-se o ambiente nos vasos com maiores valores de médias, exceto o índice de colheita e massa de mil grãos que foram maiores no cultivo convencional.

Este estudo teve como objetivo a compreensão dos efeitos desencadeados pelas temperaturas elevadas na expressão gênica de proteínas de choque térmico, nomeadamente da HSPA1A e HSP90AA1, em linfócitos de fêmeas bovinas de diferentes raças e origens. Parte do projeto foi desenvolvida em Portugal, onde foram utilizadas 20 novilhas da raça Limousine e 22 da raça Mertolenga. A parte desenvolvida no Brasil decorreu com 11 novilhas da raça Holstein Frísia e 20 da raça Brahman. Os animais da raça Holstein participaram do Experimento I no qual foram coletados 4 tubos de sangue para posterior choque térmico in vitro, onde cada tudo de sangue foi submetido a diferentes temperaturas: 40 °C e 42 °C (ambos através de banhos-maria), 23 °C (mantido a temperatura ambiente) e 10 °C (na geladeira), durante duas horas. Todos os animais de todas as raças participaram no Experimento II, onde foram sujeitos ao teste de tolerância ao calor, tendo sido aferidas a temperatura retal e a frequência respiratória, e coletado sangue (após os tratamentos sombra e sol). A todas as amostras de ambos os experimentos foi realizada a lise das hemácias de modo a obter o buffy-coat. O RNA foi isolado através do método do TRIzol e a RT-PCR realizada com SuperScript III após digestão com DNAse I. A PCR em tempo real decorreu no aparelho 7500 Fast Real Time, utilizando TaqMan Gene Expression Assays para os genes alvo HSPA1A e HSP90AA1, e ACTB e PPIA como genes endógenos. Foram calculados os ΔCt (Ctalvo - Ctendógeno) assim como a expressão gênica através do método 2-ΔΔCt. A análise estatística foi realizada através de modelos lineares mistos, recorrendo ao programa R Software Project (versão 3.0.1). No Experimento I foi atestada a qualidade da relação para ambos os endógenos nas relações que estabelecem com o mRNA-HSPA1A e o mRNA-HSP90AA1, através de uma análise de regressão pairwise. Tal como era esperado os valores de expressão gênica a uma temperatura de 23 °C foram os menores, seguidos 10 °C (estresse por frio), 40 °C e 42 °C para o gene HSPA1A. No caso da HSP90AA1, foi a 40 °C que se verificou uma maior expressão gênica. No Experimento II verificou-se uma variação intra-raça algo acentuada para valores de frequência respiratória, permitindo supor que o esforço termorregulatório dos animais de uma mesma raça possa ter sido diferente. Em relação à temperatura retal a raça Brahman apresentou valores significativamente diferentes das restantes raças para a situação sol. As diferenças observadas foram provavelmente consequência dos diferentes níveis de estresse aos quais os animais estiveram sujeitos. As diferenças observadas nos ΔCt não foram muito expressivas e apenas se observaram diferenças significativas na expressão gênica relativa na raça Mertolenga entre as situações sombra e sol. Podemos concluir que o aumento planificado da temperatura de linfócitos bovinos leva a diferentes expressões gênicas relativas de HSPA1A e HSP90AA1. A expressão de mRNA-HSPA1A é tanto maior quanto maior o estresse térmico, quer por frio quer por calor. As expressões gênicas relativas de HSPA1A e HSP90AA1 exibidas por animais com diferentes capacidades termolíticas são também elas diferentes, existindo uma variabilidade individual na expressão gênica relativa de proteínas de choque térmico. / The present study aimed to understand the effects triggered by high temperatures in heat shock proteins gene expression, HSPA1A and HSP90AA1, in cow lymphocytes from different breeds and origins. Part of the project was developed in Portugal, where 20 Limousin and 22 Mertolenga breed heifers were used and the second part was developed in Brazil with 11 Holstein Friesian and 20 Brahman heifers. The Holstein animals participated in Experiment I in which four blood samples were collected for subsequent in vitro heat shock, where each one of the blood samples was submitted to different temperatures: 40 ° C and 42 ° C (through water baths), 23 ° C (kept at room temperature) and 10 ° C (in refrigerator) for two hours. All animals from all breeds participated in Experiment II, where they were subjected to the heat tolerance test, where rectal temperature and respiratory rate were measured, and blood samples were collected (after shadow and after sun treatments). In all samples of both experiments was carried out the erythrocytes lysis so as to obtain the buffycoat. The RNA was isolated by the TRIzol method and RT-PCR performed with SuperScript III after digestion with DNase I. The real-time PCR apparatus took place in 7500 Real Fast Time, using TaqMan Gene Expression Assays for HSPA1A and HSP90AA1 target genes, ACTB and PPIA as endogenous genes. The ΔCt (Cttarget - Ctendogenous) were calculated as well as gene expression through the 2-ΔΔCt method. Statistical analysis was performed using linear mixed models, using the program R Project Software (version 3.0.1). In Experiment I it was attested the quality of the relationship for both endogenous with the mRNA-HSPA1A and mRNAHSP90AA1 through a pairwise regression analysis. As expected values, gene expression at a temperature of 23 °C were lower, followed by 10 °C (cold stress), 40 °C and 42 °C for the HSPA1A gene. In the case of HSP90AA1 the higher gene expression was found at 40 °C. In Experiment II, there was a slightly pronounced intra-race variation for respiratory rate values, allowing the assumption that the thermoregulatory effort of animals of the same breed may have been different. Regarding the rectal temperature, in Brahman breeds it was significantly different from the other breeds in the sun treatment. The differences observed were probably a result of different levels of stress to which the animals were subjected. The differences observed in ΔCt were not very expressive and significant differences were only observed in relative gene expression of Mertolenga breed between shade and sun treatments. We conclude that planned increasing temperature in bovine lymphocytes leads to different relative gene expression of HSPA1A and HSP90AA1. The mRNA-HSPA1A expression is greater in higher thermal stress, either by cold or by heat. The HSPA1A and HSP90AA1 relative gene expressions exhibited by animals with different thermolytic capabilities, are also different, existing an individual variability in relative gene expression of heat shock proteins.

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.