High temperatures and low humidity promote the occurrence of microsporidians (Microsporidia) in mosquitoes (Culicidae).

BACKGROUND: In the context of climate change, a growing concern is that vector-pathogen or host-parasite interactions may be correlated with climatic factors, especially increasing temperatures. In the present study, we used a mosquito-microsporidian model to determine the impact of environmental factors such as temperature, humidity, wind and rainfall on the occurrence rates of opportunistic obligate microparasites (Microsporidia) in hosts from a family that includes important disease vectors (Culicidae). METHODS: In our study, 3000 adult mosquitoes collected from the field over 3 years were analysed. Mosquitoes and microsporidia were identified using PCR and sequencing of the hypervariable V5 region of the small subunit ribosomal RNA gene and a shortened fragment of the cytochrome c oxidase subunit I gene, respectively. RESULTS: DNA metabarcoding was used to identify nine mosquito species, all of which were hosts of 12 microsporidian species. The prevalence of microsporidian DNA across all mosquito samples was 34.6%. Microsporidian prevalence in mosquitoes was more frequent during warm months (> 19 °C; humidity < 65%), as was the co-occurrence of two or three microsporidian species in a single host individual. During warm months, microsporidian occurrence was noted 1.6-fold more often than during the cold periods. Among the microsporidians found in the mosquitoes, five (representing the genera Enterocytospora, Vairimorpha and Microsporidium) were positively correlated with an increase in temperature, whereas one (Hazardia sp.) was significantly correlated with a decrease in temperature. Threefold more microsporidian co-occurrences were recorded in the warm months than in the cold months. CONCLUSIONS: These results suggest that the susceptibility of mosquitoes to parasite occurrence is primarily determined by environmental conditions, such as, for example, temperatures > 19 °C and humidity not exceeding 62%. Collectively, our data provide a better understanding of the effects of the environment on microsporidian-mosquito interactions.

DNA liquid crystals with AIE effect toward humidity-indicating biomaterials.

In this study, by fabricating DNA doped with tetraphenylethene-containing ammonium surfactant, the resulting solvent-free DNA ionic complex could undergo a humidity-induced phase change that could be well tracked by the fluorescence signal of the surfactant. Taking advantage of the humidity-induced change in fluorescence, the reported ionic DNA complex could accurately indicate the humidity in real time.

High temperature and humidity in the environment disrupt bile acid metabolism, the gut microbiome, and GLP-1 secretion in mice.

High temperature and humidity in the environment are known to be associated with discomfort and disease, yet the underlying mechanisms remain unclear. We observed a decrease in plasma glucagon-like peptide-1 levels in response to high-temperature and humidity conditions. Through 16S rRNA gene sequencing, alterations in the gut microbiota composition were identified following exposure to high temperature and humidity conditions. Notably, changes in the gut microbiota have been implicated in bile acid synthesis. Further analysis revealed a decrease in lithocholic acid levels in high-temperature and humidity conditions. Subsequent in vitro experiments demonstrated that lithocholic acid increases glucagon-like peptide-1 secretion in NCI-H716 cells. Proteomic analysis indicated upregulation of farnesoid X receptor expression in the ileum. In vitro experiments revealed that the combination of lithocholic acid with farnesoid X receptor inhibitors resulted in a significant increase in GLP-1 levels compared to lithocholic acid alone. In this study, we elucidate the mechanism by which reduced lithocholic acid suppresses glucagon-like peptide 1 via farnesoid X receptor activation under high-temperature and humidity condition.

Relative Humidity-Dependent Phase Transitions in Submicron Respiratory Aerosols.

Respiratory viruses, such as influenza and severe acute respiratory syndrome coronavirus 2, represent a substantial public health burden and are largely transmitted through respiratory droplets and aerosols. Environmental factors such as relative humidity (RH) and temperature impact virus transmission rates, and a precise mechanistic understanding of the connection between these environmental factors and virus transmission would improve efforts to mitigate respiratory disease transmission. Previous studies on supermicrometer particles observed RH-dependent phase transitions and linked particle phase state to virus viability. Phase transitions in atmospheric aerosols are dependent on size in the submicrometer range, and actual respiratory particles are expelled over a large size range, including submicrometer aerosols that can transmit diseases over long distances. Here, we directly investigated the phase transitions of submicrometer model respiratory aerosols. A probe molecule, Nile red, was added to particle systems including multiple mucin/salt mixtures, a growth medium, and simulated lung fluid. For each system, the polarity-dependent fluorescence emission was measured following RH conditioning. Notably, the fluorescence measurements of mucin/NaCl and Dulbecco's modified Eagle's medium particles indicated that liquid-liquid phase separation (LLPS) also occurs in submicron particles, suggesting that LLPS can also impact the viability of viruses in submicron particles and thus affect aerosol virus transmission. Furthermore, the utility of fluorescence-based measurements to study submicrometer respiratory particle physicochemical properties in situ is demonstrated.

Variable angle of insulated shotcrete under different loading rates and temperature and humidity cycles- shear test and analysis.

The new thermal insulating shotcrete is of great significance for the management of thermal damage in deep mines, and its own strength has a greater impact on the roadway insulation and safe production, so it is very necessary to study the shear strength of the new thermal insulating shotcrete under the influence of the deep hot and humid environment and the stress of mining. For the heat-insulating shotcrete, firstly, we carried out the concrete variable angle shear test under different loading rates, which concluded that the shear rate and peak shear stress showed a trend of increasing and then decreasing; as the angle increases, the different rates have a greater impact on the peak shear stress of the specimen. Secondly, the concrete variable angle shear test was carried out under the temperature and humidity cycle, which revealed that the shear strength of thermal insulated shotcrete increased firstly and then decreased with the increase of temperature at the same number of cycles. Finally, the empirical equations between the cohesive force c, the angle of internal friction ϕ and the number of warm and wet cycles n and the temperature of warm and wet cycles T are fitted with the MATLAB software respectively, and the research results provide technical references for the management of geothermal temperature in deep well projects.

Non-destructive visualization of internal structural changes in humidified magnesium oxide tablets using X-ray computed tomography.

Detailed examinations of the internal structure of tablets are imperative for comprehending their formulation, physical attributes, and ensuring their safe utilization. While X-ray computed tomography (CT) is valuable for noninvasively analyzing internal structural changes, the influence of humidity on these structural changes remains unexplored. Accordingly, we aimed to assess the viability of X-ray CT in non-destructively evaluating the internal structure of humidified magnesium oxide (MgO) tablets. MgO tablets were subjected to conditions of 40 °C and 75% humidity for 7 days, weighed pre- and post-humidification, and subsequently stored at room temperature (22-27 °C) until day 90. Their internal structure was evaluated using X-ray CT. We observed a substantial increase in the weight of MgO tablets concomitant with moisture absorption, with minimal changes observed upon storage at room temperature. The skewness reduced immediately post-moisture absorption, remained almost the same post-storage at room temperature, and failed to revert to pre-humidification levels during the storage period. These findings highlight the utility of X-ray CT as an effective tool for non-destructive, three-dimensional, and detailed evaluation of internal structural transformations in MgO tablets.

Flexible Chitosan-Based Capacitive Humidity Sensors for Respiratory Monitoring.

As one of the most important human health indicators, respiratory status is an important basis for the diagnosis of many diseases. However, the high cost of respiratory monitoring makes its use uncommon. This study introduces a low-cost, wearable, flexible humidity sensor for respiratory monitoring. Solution-processed chitosan (CS) placed on a polyethylene terephthalate substrate was used as the sensing layer. An Arduino circuit board was used to read humidity-sensitive voltage changes. The CS-based sensor demonstrated capacitive humidity sensitivity, whereby the capacitance instantly increased from 10-2 to 30 nF when the environmental humidity changed from 43% to 97%. The capacitance logarithm sensitivity and response voltage change was 35.9 pF/%RH and 0.8 V in the RH range from 56% to 97%. And the voltage variation between inhalation and exhalation was ~0.5 V during normal breathing. A rapid response time of ~0.7 s and a recovery time of ~2 s were achieved during respiration testing. Breathing modes (i.e., normal breathing, rest breathing, deep breathing, and fast breathing) and tonal changes during speech could be clearly distinguished. Therefore, such sensors provide a means for economical and convenient wearable respiratory monitoring, and they have the potential to be used for daily health examinations and professional medical diagnoses.

Effect of temperature-humidity index on the evolution of trade-offs between fertility and production in dairy cattle.

BACKGROUND: In the current context of climate change, livestock production faces many challenges to improve the sustainability of systems. Dairy farming, in particular, must find ways to select animals that will be able to achieve sufficient overall production while maintaining their reproductive ability in environments with increasing temperatures. With future forecasted climate conditions in mind, this study used data from Holstein and Montbeliarde dairy cattle to: (1) estimate the genetic-by-temperature-humidity index (THI) interactions for female fertility, and (2) evaluate the production-fertility trade-off with increasing values of THI. RESULTS: Two-trait random regression models were fitted for conception rate (fertility) and test-day protein yield (production). For fertility, genetic correlations between different THI values were generally above 0.75, suggesting weak genotype-by-THI interactions for conception rate in both breeds. However, the genetic correlations between the conception rate breeding values at the current average THI (THI = 50, corresponding to a 24-h average temperature of 8 °C at 50% relative humidity) and their slopes (i.e., potential reranking) for heat stress scenarios (THI > 70), were different for each breed. For Montbeliarde, this correlation tended to be positive (i.e., overall the best reproducers are less affected by heat stress), whereas for Holstein it was approximately zero. Finally, our results indicated a weak antagonism between production and fertility, although for Montbeliarde this antagonism intensified with increasing THI. CONCLUSIONS: Within the range of weather conditions studied, increasing temperatures are not expected to exacerbate the fertility-production trade-off. However, our results indicated that the animals with the best breeding values for production today will be the most affected by temperature increases, both in terms of fertility and production. Nonetheless, these animals should remain among the most productive ones during heat waves. For Montbeliarde, the current selection program for fertility seems to be adequate for ensuring the adaptation of fertility traits to temperature increases, without adverse effects on production. Such a conclusion cannot be drawn for Holstein. In the future, the incorporation of a heat tolerance index into dairy cattle breeding programs would be valuable to promote the selection of animals adapted to future climate conditions.

Benchmarking the Humidity-Dependent Mechanical Response of (Nano)fibrillated Cellulose and Dissolved Polysaccharides as Sustainable Sand Amendments.

Soil quality is one of the main limiting factor in the development of the food sector in arid areas, mainly due to its poor mechanics and lack of water retention. Soil's organic carbon is nearly absent in arid soils, though it is important for water and nutrient transport, to soil mechanics, to prevent erosion, and as a long-term carbon sink. In this study, we evaluate the potential benefits that are brought to inert sand by the incorporation of a range of, mainly, cellulosic networks in their polymeric or structured (fiber) forms, analogously to those found in healthy soils. We explore the impact of a wide range of nonfood polysaccharide-based amendments, including pulp fibers, nanocellulose, cellulose derivatives, and other readily available polysaccharide structures derived from arthropods (chitosan) or fruit peels (pectin) residues. A practical methodology is presented to form sand-polymer composites, which are evaluated for their soil mechanics as a function of humidity and the dynamics of their response to water. The mechanics are correlated to the network of polymers formed within the pores of the sandy soil, as observed by electron microscopy. The response to water is correlated to both the features of the network and the individual polysaccharides' physicochemical features. We expect this work to provide a rapid and reproducible methodology to benchmark sustainable organic amendments for arid soils.

Multivariate Analysis of Light-Activated SMOX Gas Sensors.

Chemoresistive gas sensors made from SnO2, ZnO, WO3, and In2O3 have been prepared by flame spray pyrolysis. The sensors' response to CO and NO2 in darkness and under illumination at different wavelengths, using commercially available LEDs, was investigated. Operation at room temperature turned out to be impractical due to the condensation of water inside the porous sensing layers and the irreversible changes it caused. Accordingly, for sensors operated at 70 °C, a characterization procedure was developed and proven to deliver consistent data. The resulting data set was so complex that usual univariate data analysis was intricate and, consequently, was investigated by correlation and principal component analysis. The results show that light of different wavelengths affects not only the resistance of each material, both under exposure to the target gases in humidity and in its absence, but also the sensor response to humidity and the target gases. It was found that each of the materials behaves differently under light exposure, and it was possible to identify conditions that need further investigations.

Differential impact of environmental factors on airborne live bacteria and inorganic particles in an underground walkway.

We previously reported that variations in the number and type of bacteria found in public spaces are influenced by environmental factors. However, based on field survey data alone, whether the dynamics of bacteria in the air change as a result of a single environmental factor or multiple factors working together remains unclear. To address this, mathematical modeling may be applied. We therefore conducted a reanalysis of the previously acquired data using principal component analysis (PCA) in conjunction with a generalized linear model (Glm2) and a statistical analysis of variance (ANOVA) test employing the χ2 distribution. The data used for the analysis were reused from a previous public environmental survey conducted at 8:00-20:00 on May 2, June 1, and July 5, 2016 (regular sampling) and at 5:50-7:50 and 20:15-24:15 on July 17, 2017 (baseline sampling) in the Sapporo underground walking space, a 520-meter-long underground walkway. The dataset consisted of 60 samples (22 samples for "bacterial flora"), including variables such as "temperature (T)," "humidity (H)," "atmospheric pressure (A)," "traffic pedestrians (TP)," "number of inorganic particles (Δ5: 1-5 µm)," "number of live airborne bacteria," and "bacterial flora." Our PCA with these environmental factors (T, H, A, and TP) revealed that the 60 samples could be categorized into four groups (G1 to G4), primarily based on variations in PC1 [Loadings: T(-0.62), H(-0.647), TP(0.399), A(0.196)] and PC2 [Loadings: A(-0.825), TP(0.501), H(0.209), T(-0.155)]. Notably, the number of inorganic particles significantly increased from G4 to G1, but the count of live bacteria was highest in G2, with no other clear pattern. Further analysis with Glm2 indicated that changes in inorganic particles could largely be explained by two variables (H/TP), while live bacteria levels were influenced by all explanatory variables (TP/A/H/T). ANOVA tests confirmed that inorganic particles and live bacteria were influenced by different factors. Moreover, there were minimal changes in bacterial flora observed among the groups (G1-G4). In conclusion, our findings suggest that the dynamics of live bacteria in the underground walkway differ from those of inorganic particles and are regulated in a complex manner by multiple environmental factors. This discovery may contribute to improving public health in urban settings.

Antioxidant Defenses Against Air Humidity Stress in Fruit Bodies of Auricularia heimuer (Agaricomycetes).

Air humidity is an important environmental factor restricting the fruit body growth of Auricularia heimuer. Low air humidity causes the fruit body to desiccate and enter dormancy. However, the survival mechanisms to low air humidity for fruit bodies before dormancy remain poorly understood. In the present study, we cultivated A. heimuer in a greenhouse and collected the fruit bodies at different air humidities (90%, 80%, 70%, 60%, and 50%) to determine the contents of malondialdehyde (MDA) and non-enzymatic antioxidants such as ascorbic acid (AsA) and glutathione (GSH); and the activities of enzymatic antioxidants including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX) and glutathione reductase (GR). Results showed that the MDA contents tended to increase with decreasing relative air humidity. Relative air humidity below 90% caused membrane lipid peroxidation and oxidative stress (based on MDA contents) to the fruit body, which we named air humidity stress. In contrast to the control and with the degree of stress, the GSH contents and activities of SOD, CAT, GR, GPX, and APX tended to ascend, whereas AsA showed a declining trend; the POD activity only rose at 50%. The antioxidants favored the fruit body to alleviate oxidative damage and strengthened its tolerance to air humidity stress. The antioxidant defense system could be an important mechanism for the fruit body of A. heimuer in air humidity stress.

Predicting the impact of temperature and relative humidity on Salmonella growth and survival in sliced chard, broccoli and red cabbage.

This study assessed the fate of a Salmonella enterica cocktail (S. Typhimurium, S. Enteritidis, S. Newport, S. Agona and S. Anatum; initial counts 3.5 log CFU/g) in minimally processed sliced chard, broccoli and red cabbage at 16 conditions of different temperature (7, 14, 21 and 37 °C) and relative humidity (RH; 15, 35, 65 and 95%) over six days (144 h). Linear regression was used to estimate the rate change of Salmonella in cut vegetables as a function of temperature and relative humidity (RH). R2 value of 0.85, 0.87, and 0.78 were observed for the rates of change in chard, broccoli, and red cabbage, respectively. The interaction between temperature and RH was significant in all sliced vegetables. Higher temperatures and RH values favored Salmonella growth. As temperature or RH decreased, the rate of S. enterica change varied by vegetable. The models developed here can improve risk management of Salmonella in fresh cut vegetables.

Faecal cortisol metabolites, body temperature, and behaviour of beef cattle exposed to a heat load.

Feedlot cattle are at times exposed to high environmental temperatures. Faecal cortisol metabolites were related to possible indicators of heat stress that could be measured under field conditions: respiratory dynamics (respiration rate), body surface temperature and adaptive behaviours, such as water consumption, posture (standing, lying), and activity (eating, drinking and rumination). Twelve (12) yearling Black Angus steers were divided into two treatment groups: a hot treatment (HOT; n = 6) and a thermoneutral-treatment (TN; n = 6) and individually housed in a climate-controlled facility at The University of Queensland, Australia. In the TN treatment, all animals were exposed to an ambient temperature of 20.34 ± 0.25 °C, relative humidity 71.51 ± 3.26% and Temperature humidity index (THI) 66.91 ± 0.33 throughout. In the HOT treatment group, environmental conditions were exposed to different climatic phases from thermoneutral to hot conditions, where they remained for 7 d, and then returned to TN conditions in the recovery period. The dry bulb ambient temperature (TA) and relative humidity (RH) in the pens of cattle in the HOT treatment were increased from 28 °C (daily maximum ambient temperature) and 45% RH at 0700 h to a daily maximum TA and RH of 35 °C (daily maximum ambient temperature) and 50% (THI 77) at 1100 h, which was maintained until 1600 h, after which it declined until it reached the baseline at 2000 h. In both treatments, there was a significant decrease in faecal cortisol metabolite concentration from the start to the end of the experiments they adapted to the experimental facility. The concentration of faecal cortisol metabolites was greater in the HOT treatment, compared to the TN treatment during the heat exposure period, but there was no difference in the transition or recovery periods. Respiration rate was greater in the HOT treatment during heat exposure, and it increased with ambient dry bulb temperature above 26 °C, the latter being the upper critical temperature. Although positive correlations were detected between faecal cortisol metabolites and body surface temperature measurements, particularly the shoulder and rump, as well as standing time, panting score and drinking, a stepwise regression found that faecal cortisol metabolites were only significantly correlated with one variable, respiration rate. It is concluded that respiration rate is the best indicator of the stress induced by hot conditions for cattle.

High-resolution projections of outdoor thermal stress in the twenty-first century: a Tasmanian case study.

To adapt to Earth's rapidly changing climate, detailed modelling of thermal stress is needed. Dangerous stress levels are becoming more frequent, longer, and more severe. While traditional measurements of thermal stress have focused on air temperature and humidity, modern measures including radiation and wind speed are becoming widespread. However, projecting such indices has presented a challenging problem, due to the need for appropriate bias correction of multiple variables that vary on hourly timescales. In this paper, we aim to provide a detailed understanding of changing thermal stress patterns incorporating modern measurements, bias correction techniques, and hourly projections to assess the impact of climate change on thermal stress at human scales. To achieve these aims, we conduct a case study of projected thermal stress in central Hobart, Australia for 2040-2059, compared to the historical period 1990-2005. We present the first hourly metre-scale projections of thermal stress driven by multivariate bias-corrected data. We bias correct four variables from six dynamically downscaled General Circulation Models. These outputs drive the Solar and LongWave Environmental Irradiance Geometry model at metre scale, calculating mean radiant temperature and the Universal Thermal Climate Index. We demonstrate that multivariate bias correction can correct means on multiple time scales while accurately preserving mean seasonal trends. Changes in mean air temperature and UTCI by hour of the day and month of the year reveal diurnal and annual patterns in both temporal trends and model agreement. We present plots of future median stress values in the context of historical percentiles, revealing trends and patterns not evident in mean data. Our modelling illustrates a future Hobart that experiences higher and more consistent numbers of hours of heat stress arriving earlier in the year and extending further throughout the day.

Hybrid chiral nanocellulose-cyanidin composite with pH and humidity response for visual inspection and real-time tracking of shrimp quality and freshness.

Biobased multi-stimulation materials have received considerable attention for intelligent packaging and anti-counterfeiting applications. Cellulose nanocrystals (CNCs) and cyanidins are good material candidates for monitoring food freshness as they are eco-friendly natural substances. This work incorporated cyanidin with a CNC-hosting substrate to develop a simple, environment-friendly colorimetric device to visualize food freshness. Across the pH range of 2-13, the indicator exhibited noticeable color changes ranging from red to gray and eventually to orange. The CNC-cyanidin (CC) film exhibited a dramatic color change from blue to dark red and high sensitivity at a relative humidity of 30 %-100 %. In corresponding to the total volatile elemental nitrogen (TVB-N) level of shrimp, the indicator showed distinguishable colors at different stages of shrimp. The findings imply that the samples have substantial potential for use as an intelligent indicator for tracking shrimp freshness.

Encapsulation of 4-terpineol with ß-cyclodextrin: Inclusion mechanism, characterization and relative humidity-triggered release.

4-Terpineol (4-TA), a typical monocyclic monoterpene essential oil compound with important biological activities, poor stability and solubility severely hamper its biological activities. To date, ß-cyclodextrin (ß-CD) encapsulating essential oil to form inclusion complexes (ICs) is considered as a satisfactory treatment. Nevertheless, the detailed inclusion mechanism of ß-CD for 4-TA especially the behavior of 4-TA during inclusion formation have not available yet. Herein, 4-TA/ß-CD ICs were successfully synthesized by the co-precipitation method, and hydrogen bonds and hydrophobic interactions played a key role in the formation of ICs, and the isopropyl of 4-TA entered the cavity through the wide rim of ß-CD. Moreover, the release profile demonstrated that high RH (85 % and 99 %) triggered the release of TA from ICs. This study suggests the great potential of cyclodextrin inclusion strategy for improving the stability and sustained release of 4-TA in food preservation application.

The role of air relative humidity on the wettability of Pseudomonas fluorescens AR11 biofilms.

Biofilms are complex porous materials formed by microorganisms, polysaccharides, proteins, eDNA, inorganic matter, and water. They are ubiquitous in various environmental niches and are known to grow at solid-liquid, solid-air and air-liquid interfaces, often causing problems in several industrial and sanitary fields. Their removal is a challenge in many applications and numerous studies have been conducted to identify promising chemical species as cleaning agents. While these substances target specific components of biofilm structure, the role of water content in biofilm, and how it can influence wettability and detergent absorption have been quite neglected in the literature. Estimating water content in biofilm is a challenging task due to its heterogeneity in morphology and chemical composition. In this study, we controlled water content in Pseudomonas fluorescens AR 11 biofilms grown on submerged glass slides by regulating environmental relative humidity after drying. Interfacial properties of biofilm were investigated by measuring wetting of water and soybean oil. The morphology of biofilm structure was evaluated using Confocal Laser Scanning Microscopy and Scanning Electron Microscopy. The results showed that biofilm water content has a significant and measurable effect on its wettability, leading to the hypothesis that a preliminary control of water content can play a crucial role in biofilm removal process.

Enhanced homogeneity and flexibility in a humidity sensor using cellulose nanocrystal-based composite film with circular shear flow.

Cellulose nanocrystal (CNC) film is known to be one kind of dynamic color-sensing material, capable of reversible color changes in response to varying humidity levels. However, the brittleness, low hygroscopicity and poor homogeneity of these films have hindered their development. To address this limitation, we present a novel approach where we combine natural deep eutectic solvents (NADES) with sorbitol under the influence of circular shear flow to craft a CNC humidity-sensitive film with enhanced flexibility, hygroscopicity and homogeneity. The inclusion of sorbitol and NADES enhances hygroscopicity and improves the flexibility. Surprisingly, the introduction of circular shear flow was found not only to improve homogeneity, macroscopically and microscopically, but also to further enhance flexibility, toughness, and water absorption capability. The resulting composite films demonstrated highly reversible color changes across the whole visible spectrum depending on the relative humidity, showing their capability to be reliable humidity-sensing materials. Thanks to the improved homogeneity and flexibility, the obtained humidity-sensing composite film can be employed in its entirety without the need for cutting, making it a promising candidate for various applications.

Behavioural responses related to increasing core body temperature of grazing dairy cows experiencing moderate heat stress.

Exposure to direct solar radiation, high ambient temperature, lack of wind movement, coupled with own metabolic heat production, makes grazing dairy cows vulnerable to heat stress. In pastures, it would be beneficial to monitor heat stress by observable changes in behaviour. We hypothesised that grazing dairy cows exhibit behavioural changes due to increasing heat load in temperate climate. Over two consecutive summers, 38 full-time grazing Holstein dairy cows were investigated in 12 experimental periods of up to 3 consecutive days where the cows were repeatedly exposed to various levels of moderate heat load determined by the comprehensive climate index (CCI). The CCI defines the ambient climate conditions, combining air temperature, relative humidity, solar radiation and wind speed. Vaginal temperature (VT) was automatically measured as an indicator of heat stress. In addition, as a less invasive method, we investigated if reticular temperature (RET) can be indicative of heat stress on pastures. Walking activity, lying-, feeding, and ruminating durations were recorded continuously with sensors. Respiration rate (RR), proximity to and competition at the water trough, social licking, self-licking, inter-individual distance, and fly intensity were directly observed. Data were analysed in the morning (0900-1100 h) and during the hottest time of day when cows were on pasture (1230-1430 h). The VT and RET showed similar patterns in relation to the CCI, suggesting that RET can be suitable for continuous monitoring of heat stress on pastures. In the morning, the cow's VT and RET did not relevantly react to the CCI. During the period 1230-1430 h, the cow's mean VT (mean vaginal temperature (VTMEAN); range: 37.7-40.3 °C) and mean RET (mean reticular temperature; range: 37.0-41.1 °C) were positively related to the mean CCI (mean comprehensive climate index) in this period (mean ± SD: 25.9 ± 5.71 °C). For cows with greater VTMEAN, an increased mean RR and decreased durations of walking, lying, feeding, and ruminating were found. These cows were also more likely to be in proximity to the water trough and to have small inter-individual distances. Changes in these traits seem to reflect behavioural adaptations to heat stress in a temperate climate and could be used to detect the heat stress in individual dairy cows on pastures.