Honeybee larvae and pupae are extremely stenothermic, i.e. they strongly depend on accurate regulation of brood nest temperature for proper development (33–36°C). Here we study the mechanisms of social thermoregulation of honeybee colonies under changing environmental temperatures concerning the contribution of individuals to colony temperature homeostasis. Beside migration activity within the nest, the main active process is “endothermy on demand” of adults. An increase of cold stress (cooling of the colony) increases the intensity of heat production with thoracic flight muscles and the number of endothermic individuals, especially in the brood nest. As endothermy means hard work for bees, this eases much burden of nestmates which can stay ectothermic. Concerning the active reaction to cold stress by endothermy, age polyethism is reduced to only two physiologically predetermined task divisions, 0 to ∼2 days and older. Endothermic heat production is the job of bees older than about two days. They are all similarly engaged in active heat production both in intensity and frequency. Their active heat production has an important reinforcement effect on passive heat production of the many ectothermic bees and of the brood. Ectothermy is most frequent in young bees (<∼2 days) both outside and inside of brood nest cells. We suggest young bees visit warm brood nest cells not only to clean them but also to speed up flight muscle development for proper endothermy and foraging later in their life. Young bees inside brood nest cells mostly receive heat from the surrounding cell wall during cold stress, whereas older bees predominantly transfer heat from the thorax to the cell wall. Endothermic bees regulate brood comb temperature more accurately than local air temperature. They apply the heat as close to the brood as possible: workers heating cells from within have a higher probability of endothermy than those on the comb surface. The findings show that thermal homeostasis of honeybee colonies is achieved by a combination of active and passive processes. The differential individual endothermic and behavioral reactions sum up to an integrated action of the honeybee colony as a superorganism.

SUMMARYIn order to survive cold northern winters, honeybees crowd tightly together in a winter cluster. Present models of winter cluster thermoregulation consider the insulation by the tightly packed mantle bees as the decisive factor for survival at low temperatures, mostly ignoring the possibility of endothermic heat production. We provide here direct evidence of endothermic heat production by `shivering' thermogenesis. The abundance of endothermic bees is highest in the core and decreases towards the surface. This shows that core bees play an active role in thermal control of winter clusters. We conclude that regulation of both the insulation by the mantle bees and endothermic heat production by the inner bees is necessary to achieve thermal stability in a winter cluster.

The relation between the respiratory activity of resting honeybees and ambient temperature (T a ) was investigated in the range of 5-40 °C. Bees were kept in a temperature controlled flow through respirometer chamber where their locomotor and endothermic activity, as well as abdominal ventilatory movements was recorded by infrared thermography.Surprisingly, true resting bees were often weakly endothermic (thorax surface up to 2.8 °C warmer than abdomen) at a T a of 14-30 °C. Above 33 °C many bees cooled their body via evaporation from their mouthparts. A novel mathematical model allows description of the relationship of resting (standard) metabolic rate and temperature across the entire functional temperature range of bees. In chill coma (<11 °C) bees were ectothermic and CO 2 release was mostly continuous. CO 2 release rate (nl s −1 ) decreased from 9.3 at 9.7 °C to 5.4 at 5 °C. At a T a of >11 °C CO 2 was released discontinuously. In the bees' active temperature range mean CO 2 production rate (nl s −1 ) increased sigmoidally (10.6 at 14.1 °C, 24.1 at 26.5 °C, and 55.2 at 38

Graphical abstract.Research highlights▶ Thorax temperature was regulated from 37.0–45.3 °C (ambient temperature: 3–39 °C). ▶ Solar heat gain was used to increase thorax temperature by about 1–3 °C. ▶ High thorax temperature allowed regulation of an optimal head temperature. ▶ Flexible thermal strategy enabled foraging in a broad ambient temperature range.

Graphical abstract.Highlights► We demonstrate the benefits of a combined use of infrared thermography with respiratory measurements in insect ecophysiological research. ► Infrared thermography enables repeated investigation of behaviour and thermoregulation without behavioural impairment. ► Comparison with respirometry brings new insights into the mechanisms of energetic optimisation of bee and wasp foraging. ► Combination of methods improves interpretation of respiratory traces in determinations of insect critical thermal limits.

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SummaryDespite their tremendous economic importance, and apart from certain topics in the field of neurophysiology such as vision, olfaction, learning and memory, honey bees are not a typical model system for studying general questions of insect physiology. The reason is their social lifestyle, which sets them apart from a "typical insect" and, during social evolution, has resulted in the restructuring of certain physiological pathways and biochemical characteristics in this insect. Not surprisingly, the questions that have attracted most attention by researchers working on honey bee physiology and biochemistry in general are core topics specifically related to social organization, such as caste development, reproductive division of labour and polyethism within the worker caste. With certain proteins playing key roles in these processes, such as the major royal jelly proteins (MRJPs), including royalactin and hexamerins in caste development, and vitellogenin in reproductive division of labour and age polyethism, a major section herein will present and discuss basic laboratory protocols for protein analyses established and standardized to address such questions in bees. A second major topic concerns endocrine mechanisms underlying processes of queen and worker development, as well as reproduction and polyethism, especially the roles of juvenile hormone and ecdysteroids. Sensitive techniques for the quantification of juvenile hormone levels circulating in haemolymph, as well as its synthesis by the corpora allata are described. Although these require certain instrumentation and a considerable degree of sophistication in the analysis procedures, we considered that presenting these techniques would be of interest to laboratories planning to specialize in such analyses. Since biogenic amines are both neurotransmitters and regulators of endocrine glands, we also present a standard method for the detection and analysis of certain biogenic amines of interest. Further questions that cross borders between individual and social physiology are related to energy metabolism and thermoregulation. Thus a further three sections are dedicated to protocols on carbohydrate quantification in body fluid, body temperature measurement and respirometry. Métodos estándar para la investigación de la fisiología y bioquímica de Apis mellifera ResumenA pesar de su enorme importancia económica, y aparte de ciertos temas en el campo de la neurofisiología, tales como la visión, el olfato, el aprendizaje y la memoria, las abejas no son un sistema modelo típico para el estudio de cuestiones generales sobre la fisiología de los insectos. La razón de ello es su forma de vida social, lo que las diferencia de un "insecto típico" y que durante la evolución social, se ha traducido en la reestructuración de ciertas vías fisiológicas y bioquímicas propias de este insecto. Como era de esperar, las preguntas que han atraído mayor atención por parte de los investigadores que trabajan en la fisiología y la bioquímica de la abeja melífera, son en general tema...

Heterothermic insects like honeybees, foraging in a variable environment, face the challenge of keeping their body temperature high to enable immediate flight and to promote fast exploitation of resources. Because of their small size they have to cope with an enormous heat loss and, therefore, high costs of thermoregulation. This calls for energetic optimisation which may be achieved by different strategies. An ‘economizing’ strategy would be to reduce energetic investment whenever possible, for example by using external heat from the sun for thermoregulation. An ‘investment-guided’ strategy, by contrast, would be to invest additional heat production or external heat gain to optimize physiological parameters like body temperature which promise increased energetic returns. Here we show how honeybees balance these strategies in response to changes of their local microclimate. In a novel approach of simultaneous measurement of respiration and body temperature foragers displayed a flexible strategy of thermoregulatory and energetic management. While foraging in shade on an artificial flower they did not save energy with increasing ambient temperature as expected but acted according to an ‘investment-guided’ strategy, keeping the energy turnover at a high level (∼56–69 mW). This increased thorax temperature and speeded up foraging as ambient temperature increased. Solar heat was invested to increase thorax temperature at low ambient temperature (‘investment-guided’ strategy) but to save energy at high temperature (‘economizing’ strategy), leading to energy savings per stay of ∼18–76% in sunshine. This flexible economic strategy minimized costs of foraging, and optimized energetic efficiency in response to broad variation of environmental conditions.