A versatile recording device for the analysis of continuous daily external activity in colonies of highly eusocial bees

A versatile recording device for the analysis of continuous daily external activity in colonies of highly eusocial bees | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A versatile recording device for the analysis of continuous daily external activity in colonies of highly eusocial bees Arthur Roque Justino, Klaus Hartfelder This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4201960/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jun, 2024 Read the published version in Journal of Comparative Physiology A → Version 1 posted 9 You are reading this latest preprint version Abstract As pollinators, bees are key to maintaining the biodiversity of angiosperm plants, and for agriculture they provide a billion-dollar ecosystem service. But they also compete for resources (nectar and pollen), especially the highly social bees that live in perennial colonies. So, how do they organize their daily foraging activity? Here, we present a versatile, low-cost device for the continuous, automatic recording and data analysis of the locomotor activity in the colony-entrance tube of highly eusocial bees. Consisting of an in-house built block containing an infrared detector, the passage of bees in the colony entrance tunnel is registered and automatically recorded in an Arduino environment, together with concomitant recordings of temperature and relative humidity. With a focus on the highly diverse Neotropical stingless bees (Meliponini), we obtained 10-day consecutive recordings for two colonies each of the species Melipona quadrifasciata and Frieseomelitta varia , and also for the honey bee. The data were converted into CSV files, followed by the generation of actograms and Lomb-Scargle periodograms. We found a predominant circadian rhythmicity for all three species, but also indications of ultradian rhythms. For M. quadrifasciata , which is comparable in size to the honey bee, we found evidence for an anticipatory activity already before sunrise, followed by an early morning peak of activity. The cost and versatility of the device and the open-source options for data analysis make this an attractive system for conducting studies on circadian rhythms in social bees under natural conditions, complementing studies on flower visits by these important pollinators. Activity recording circadian rhythm foraging Meliponini Apis mellifera Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The high energetic cost of flights and the importance of collecting resources for colony maintenance are critical factors in the optimization of foraging behaviors in social bees (Stabentheiner and Kovacs 2014 ). As most of the bees’ foraging behavior is directed towards collecting nectar and pollen, this requires the adjustment of the flight activity with the timing when nectar and pollen becomes available in flowering plants. In the co-evolution between angiosperm plants and bees (Anthophila) since the Cretaceous, this created an advantageous relationship for both parts. In the Western honey bee, Apis mellifera , the adjustment of foraging flights with flowering time has been conceptually formulated as the Zeitgedächtnis (time memory) of the bees (Beling 1929 ; Kleber 1935 ; Renner 1957 ). Particularly, the capacity of the bees’ endogenous clock of approximately 24 h running time permits them to predict the best time for obtaining the desired plant resources, and for the plants to successfully achieve pollination (Bloch et al. 2001 ; Bloch et al. 2017 ). Since the length of the light/dark cycles varies across the year, this requires that the bees can shift and synchronize their endogenous clock to the environmental cycles. The factors for this adjustment are known as Zeitgebers , and the main ones are photoperiod and temperature (Kefuss and Nye 1970 ; Moore and Rankin 1993 ; Moritz and Kryger 1994 ; Bloch 2010 ). In highly eusocial insects, the colony context, primarily the contact with nestmates, also plays a role as a Zeitgeber , and this can even be stronger than the photoperiod (Moritz and Sakofski 1991 ; Frisch and Koeniger 1994 ; Beer et al. 2016 ; Shpigler et al., 2020 ). In the honey bee for instance, Fuchikama et al. (2016) demonstrated that workers experiencing conflicting photic and social synchronizations choose to follow the social signals, thus calling attention to the fact the social context needs to be taken into account when studying biological rhythms. In this sense, the honey bee is certainly one of the best characterized insect species concerning circadian rhythms and their relation with environmental and social Zeitgebers (Moore 2001 ; Abou-Shaara 2014 ; Beer and Helfrich-Förster 2020 ). Furthermore, the availability of a fully sequenced honey bee genome (The Honey Bee Genome Sequencing Consortium, 2006 ) and a plethora of information on the molecular underpinnings of the Drosophila circadian clock (Hardin 2005 ) has led to deep insights into the molecular nature of the honey bee circadian clock (Rubin et al. 2006 ; Bloch 2010 ). The only other bees that match the honey bees (Apini) in terms of social organization are the stingless bees (Meliponini), and their species richness and variation in biological characters (Grüter, 2020 ) provides an excellent opportunity to study factors and possible adaptations that shaped the biological rhythms in their foraging patterns. So far, most studies on foraging activity in stingless bees have set the focus on the diel rhythms of food collection, i.e., rhythmicity controlled by day-light cycles (Hilário et al. 2000 , 2001 ; Pierrot and Schlindwein 2003 ; Texeira and Campos, 2005). While the patterns of foraging along the day showed interspecific variation, a common trait in the stingless bees is a temporal separation with respect to resource collection, with pollen being collected mainly in the early morning, while nectar is collected over a longer period during the day (Sommeijer et al. 1983 ; Hilário et al. 2000 ; Pierrot and Schlindwein 2003 ; Nascimento and Nascimento 2012 ). This can potentially be explained by an increase in the nectar sugar content as temperatures rise (Roubik and Buchmann 1984 ; Roubik et al. 1995 ). In contrast, only few studies on stingless bees addressed the association of foraging activity with endogenous circadian rhythms. A study by Bellusci and Marques ( 2001 ) on Scaptotrigona aff. depilis , showed that the workers are capable of keeping a normal circadian rhythm of external activity even under constant light, and the same authors also showed that bees were active even during the night. There is also clear evidence for the presence of rhythmicity inside the nest (Giannini 1998 ; Bellusci and Marques 2001 ; Oda et al. 2007 ) and also in terms of physiological functions (Teixeira et al. 2011 ). Addressing questions of rhythmicity in colony behaviors, especially related to foraging activity, can be done with different methodological approaches. The simplest is the direct observation and counting of the ins-and-outs at the colony entrance. But this is very labor intensive, especially for large colonies and for several colonies at the same time, and it also cannot easily be done around the clock. An alternative is the constant videorecording of these activities at the colony entrance, but this requires good quality cameras and several hours of work for the analysis of the recordings. In contrast, radiofrequency identification (RFID) presents considerable advantages and has found several applications in studies of bees (Streit et al. 2003; Nunes-Silva et al. 2010 ; Nunes-Silva et al. 2020 ; Alburaki et al. 2021 ), including studies on circadian rhythms (Stelzer et al. 2010 ). Nonetheless, a limitation for RFID studies on bees is the size of the tag that the bees must carry, and for small bees this can severely hamper their flight performance. Furthermore, a complete RFID setup is rather costly, especially for simultaneous studies on multiple colonies. Another alternative is the system developed by Beer et al. ( 2016 ) that allows the monitoring of individual honey bees in a social context with respect to circadian locomotor activity. But this system is, so far, primarily designed for the use with miniature colonies kept under controlled conditions. Considering the size differences among stingless bee species and how these may relate to the foraging activity of colonies under natural environmental conditions, we set forth to develop an automated continuous recording system that can easily be inserted into the colony entry tube. We designed it to be versatile in terms of tube diameter, so that it can be used on stingless bees of a wide size range. Furthermore, it can simultaneously record external temperature and humidity. We report here on the continuous recordings of the external activity of a relatively large stingless bees, Melipona quadrifasciata , a smaller, slender species, Frieseomelitta varia , and for comparison, we also recorded the activity of honey bee observation colonies kept in the same apiary. Materials and methods The activity detection and recording device The registration device is composed of two parts, an Arduino hardware system with software written for the registration of the bees’ activity, and a sensor block containing an infrared (IR) emitter and an IR receiver to detect the passage of the bees, an adaptable tube holder and a case that protects the entire unit. The parts composing the sensor are illustrated in Fig. 1 A, and technical designs for the construction of these parts are available as supplementary material File S1. These parts were produced by the machining service of the university’s Precision Manufacturing Workshop from a black polyacetal block (Polyoxymethylene, 50 mm diameter) purchased from a local supplier of machining materials. Figure 1 B shows the registration device inserted into the exit tube of a F. varia colony kept in an observation room with free exit to the outside, and Fig. 1 C shows a M. quadrifasciata worker crossing the monitoring device. For the use with bees of different size we designed and built a series of adaptable tubes, which can hold glass tubes of different diameters. The internal diameter of the glass tube placed in the exit path of a colony has to be in accordance with the size of the bees, to ensure that only one bee at a time can cross the IR detector. Nonetheless, it is important that the bees can comfortably cross the tube, even when carrying pollen, resin, or mud on the corbicula. In our design, we used glass tubes of a 5 mm diameter for F. varia , 8 mm for M. quadrifasciata , and 10 mm for A. mellifera. We had good experience with the use of laboratory glass pipettes, as they come in different diameters and should be available from nowadays unused stocks in biochemistry laboratories. The registration system consists of an Arduino board and the open-source Arduino software ( https://www.arduino.cc/en/Guide/Introduction/ , Smart Projects, Italy) (Fig. 2 ). Detailed information on the Arduino system and the Sketch program written for the registration of the bees’ activity are available in the supplementary material File S2. For external temperature and relative humidity detection we used the low-power and high-accuracy module HDC1080 (Texas Instruments, Dallas, TX) with accuracies of ± 2%RH and 0.2 ºC, and 14 bits resolution ( https://www.ti.com/product/HDC1080 ). It is connected to the Arduino board by wires and measures the environmental variables at the end of the desired time intervals. The Arduino board, the IR diode and IR detector, and the HDC1080 module were purchased from Eletrogate (Belo Horizonte, MG, Brazil). The Arduino board was connected to a commercial notebook for storage of the data. An important point is the definition of critical parameters in the Sketch program for the registration process itself. To avoid false positive or false negative counts, it is important to establish an upper and a lower time threshold for an event setting. We based this on the time that it normally takes for a bee to cross a 1 cm distance in the glass tube. This time will depend on the biological characteristics of the species and needs to be empirically established. If there is a voltage drop within the given time thresholds, each such event is then added as a count, and this process is automatically repeated over the entire recording period of interest. Setting up the colonies for activity recording We used field colonies of similar size and health status for each of the two stingless bee species. For the recordings on honey bees, we prepared single frame observation hives from two field colonies of Africanized honey bee hybrids, containing about 1000 workers and a queen. All these colonies were obtained from the Experimental Apiary of the Ribeirão Preto Campus of the University of São Paulo and were transferred into an observation room in the apiary. The entries of the colonies were connected to the outside by plastic tubes that passed the wall. Sugar syrup 50% was provided twice a week. After a period of acclimatization of one week, the recording device containing the IR emitter and IR receiver was placed in the middle of the respective entrance tubes (Fig. 1 B,C). The HDC1080 module for temperature and humidity recording was passed over the tube to the outside, and its wires were connected to the Arduino board. The recordings were performed for 10 consecutive days. This was during the southern summer for the A. mellifera (December 20–29 2022) and the M. quadrifasciata colonies (January 16–25, 2023). For the F. varia colonies, the recordings were done slightly later, at the beginning of the fall (March 24 to April 03, 2023). This time difference entails certain environmental differences, not so much in terms of pluviosity, but day lengths are already slightly decreasing at the beginning of the fall. Data analysis In the Arduino program (Sketch, File S2), the time interval for each recording period was empirically set at 10 minutes. The software PuTTy ( https://www.putty.org/ , v. 0.78) was used to transform the values for the bees’ passages in the entrance tube and the temperature and humidity recordings as input into a CSV table. For generating double-plotted actograms and to assess the mean activity of each colony we used the plugin ActogramJ tool for Fiji ImageJ [© Benjamin Schmid and Taishi Yoshii, University of Würzburg, Department of Neurobiology and Genetics; Fiji ImageJ (Version 1.53, © Wayne Rasband, National Institutes of Health, USA)] (Schmid et al. 2011). In the plots, the environmental bar represents the mean length of the day and night time for the respective recording period. The acrophase (center of gravity in activity recordings) was calculated using the acrophase tool, and the time of activity onset and offset was calculated using the Activity on- and offset tool [Smoothing Gaussian std. dev. = 5.0000 min, Threshold = Median (with zero activity)]. Furthermore, to detect the rhythms of the colonies, a Lomb-Scargle periodogram analysis was performed using the package lomb (version 2.1.0) implemented in RStudio (v. 2023.06.2 + 561), with α < 0.01. The package ggplot2 (3.4.1) was used to generate the plots of the periodograms. Finally, the R package dygraphs (v. 2.2.1) was used to plot the recorded temperature and humidity values. The function cor.test() implemented in RStudio software was also used to perform a Spearman rank correlation between external activity and environmental factors. Results Confection and setup of the recording device Important aspects for the choice of a monitoring device are (a) the easiness of obtaining data and transforming these into tables or graphs, (b) the adaptability for more than one purpose, and (c) the cost per device. With our device, the passage of a bee crossing the IR emitter/detector in a given time interval generates a discrete electrical signal as a voltage drop. The integration of these signals via an Arduino board and corresponding software is then used as a direct input into a CSV table. In terms of adaptability, social bees of different size can be monitored via the choice of an appropriate adaptor and glass tube. And concerning costs, the electronic parts (Arduino board, IR emitter and detector, and the HDC180 module for registration of ambient conditions), as well as the raw material for the device structure, a polyacetal block, are low-cost items. We acquired them for less than US $ 60.00. The main cost factor will be the machining service requiring about 10 hours of a turner’s work, which in our case was provided free of charge by the university workshop. Actograms and temperature and humidity recordings for two stingless bee species The recordings of foraging activity and environmental conditions (temperature and relative humidity) were taken for 10 consecutive days for the two colonies of each of the two species. With respect to the bees’ external activity patterns, the double plotted actograms for the two M. quadrifasciata colonies (Fig. 3 A) revealed high activity levels always in the early morning, which then became reduced during the rest of the day. Interestingly, we noted that for the Melipona colonies, the activity inside the entrance tube showed an increase already before sunrise. For the two F. varia colonies (Fig. 3 B) we noted differences in their foraging activity patterns. Colony 1 showed a bimodal distribution of foraging activity, one in the morning and one in the afternoon, while for Colony 2, foraging activity was relatively constant during daytime. In addition, Colony 2 presented a notable and continuous transit of bees in the colony entrance tube even during the night (Fig. 3 B). The analysis of the acrophase of the bees’ activity rhythms (Fig. 3 , blue line) and of the timing of activity on- and offset (Table 1 ) also put in evidence the clear difference in the temporal structure of foraging activity among the two stingless bee species. The acrophase of activity in the M. quadrifasciata colonies occurs in the early morning hours, and as mentioned, the activity onset actually is already before sunrise. In contrast, for F. varia , the acrophase of activity is positioned in the middle of the day, and the onset of activity only starts with sunrise. Table 1 – Time of activity onset, offset, and the acrophase for the two stingless bee species and Apis mellifera . Species Onset Offset Acrophase M. quadrifasciata Colony 1 04.25 ± 0.41 h 16.53 ± 2.11 h 09.29 ± 0.61 h M. quadrifasciata Colony 2 04.26 ± 0.36 h 16.90 ± 1.26 h 09.36 ± 0.76 h F. varia Colony 1 06.43 ± 0.21 h 16.01 ± 0.21 h 12.36 ± 0.30 h F. varia Colony 2 06.20 ± 0.18 h 17.86 ± 0.31 h 12.06 ± 0.35 h A. mellifera Colony 1 06.25 ± 0.81 h 18.21 ± 0.85 h 12.52 ± 1.40 h A. mellifera Colony 2 04.33 ± 23.0 h 18.91 ± 1.02 h 10.66 ± 0.93 h The recordings of the environmental variables showed strong daily oscillations in both temperature and humidity (lower graphs in Fig. 3 A and B). There were no major differences in the daily temperature ranges for the two time periods, but the humidity levels were higher at the end of March, when the recordings were done on the F. varia colonies. Actograms and temperature and humidity recordings for Apis mellifera The recordings of foraging activity and environmental temperature and humidity for the two A. mellifera observation hives were taken at the end of December 2022. Strikingly, even though they were monitored simultaneously, the two colonies showed quite divergent patterns of locomotor activity (Fig. 4 , Table 1 ). While the activity pattern for Colony 1 showed considerable variation over the consecutive days, including some activity in the entrance tube during night time, Colony 2 showed two clear activity peaks, one in the early morning and one in the late afternoon. For both colonies, the activity patterns in the last three days of recording were rather unstructured. Yet, as can be seen from the concomitant temperature and humidity recordings, these were days of considerable and prolonged pluviosity, which was also accompanied by a drop in temperature, especially on day 8. Periodogram analysis To reveal generalized daily activity patterns, we calculated the mean from the 10-day consecutive recordings, and further analyzed the temporal organization of activity by performing a Lomb-Scargle periodogram analysis. The results for M. quadrifasciata and F. varia are shown in Fig. 5 and those for A. mellifera in Fig. 6 . As already indicated from the actograms, the two M. quadrifasciata colonies (Fig. 3 A) were highly similar in their mean activity (Fig. 5 A). The maximum flow in the entrance tube was seen concentrated between 5 and 9 am, and the Lomb-Scargle analysis identified the presence of a strong circadian rhythm (close to 24 h) in the foraging activity, as well as two minor peaks of ultradian rhythms (8 h and 12 h). In contrast, the two F. varia colonies (Fig. 5 B) showed a broad spread in their flow rates in the colony entrance tube along the day, between 7 am and 6 pm. With respect to the Lomb-Scargle analysis, both F. varia colonies showed a clear peak of circadian rhythmicity, but the slightly bimodal distribution of activity seen for Colony 1 was also reflected in a small peak of ultradian activity at 8 h. The difference already denoted in the actograms of the two honey bee colonies (Fig. 4 ) came out even stronger in their respective periodograms (Fig. 6 ). While Colony 2 showed a broad spread of activity over the entire day, from 7 am to 5 pm, similar to F. varia , the activity pattern for Colony 1 is strongly bimodal, with a high flow of activity between 7 to 9 am and another between 3:30 to 4:30 pm. Consequently, the Lomb-Scargle analysis for Colony 1 showed a strong and exclusive circadian activity pattern, while for Colony 2 it indicated an almost similar division between a circadian rhythm (24 h) and two strong ultradian rhythms (8 h and 12 h). Notably, as the two honey bee colonies were larger in population size than the stingless bee colonies, their flow rates in the exit tubes were much higher than in the stingless bees. Correlation between environmental factors and activity So as to reveal how the environmental conditions may affect the bees’ activity rhythm we performed a correlation analysis between the colony activity patterns and the external factors (temperature and humidity). As shown in Table 2 , the colonies of F . varia responded strongly to changes in environmental factors, with both colonies presenting a positive correlation coefficient (rho) with temperature and a negative one with humidity. In contrast, for the M. quadrifasciata colonies the correlation coefficients were much lower for both factors, which indicates that they are less susceptible to external factors. In fact, on other occasions, we have seen foragers of these same colonies flying even during rainy days (personal observation), and, as mentioned above, their preferential activity is during the early morning hours, when temperatures are still moderately low. For the two honey bee colonies, the differences seen in the activity patterns (Fig. 4 and Table 1 ) are also reflects in the respective correlation coefficients with the environmental conditions. While Colony 1 showed coefficients that were more similar to F. varia , those of Colony 2 were more similar to M. quadrifasciata. Table 2 Correlation analysis between external activity and environmental factors. Colony Temperature Humidity rho p-value rho p-value M. quadrifasciata Colony 1 0.10 0.00016 − 0.12 3.8e-06 M. quadrifasciata Colony 2 0.13 5.2e-07 − 0.09 0.00049 F. varia Colony 1 0.62 p < 2.2e-16 − 0.55 p < 2.2e-16 F. varia Colony 2 0.50 p < 2.2e-16 − 0.46 p < 2.2e-16 A. mellifera Colony 1 0.55 p < 2 .2e-16 − 0.46 p < 2.2e-16 A. mellifera Colony 2 0.24 p < 2.2e-16 − 0.12 4.5e-06 Discussion There are different ways and solutions to record the external activity of bees, from labor-intensive and time-consuming direct observations or video recordings of the transit at the colony entrance, to high-technology RFID monitoring of individually tagged bees. The latter has been applied to honey bees, including studies on circadian rhythms (Stelzer et al. 2010 ). An in-depth description of the hardware and software required for the setup of a RFID recording system of honey bees (de Souza et al. 2018 ) provides important information on the advantages and limits, as well as the costs of RFID recording. One of its limits clearly is the size and weight of the tag that needs to be applied onto a bee’s thorax. Furthermore, the constant presence of an RFID tag has been reported as be a stress factor for the bees (de Souza et al. 2018 ). A complete RFID setup is also rather costly, especially when the intention is to simultaneously study multiple colonies. Another alternative is the system developed by Beer et al. ( 2016 ) that allows the monitoring of the circadian locomotor activity of individual honey bees, but this requires minicolonies that are set up under controlled conditions. An automated recording device that is similar to ours was developed by Hilario et al. (2012), consisting of a photocell coupled to a programmable logic controller (PLC) system. They used it to analyze the relationship between climatic factors and the foraging activity of the stingless bee Plebeia remota . Here we describe in detail the confection and use of a non-invasive, low-cost device for the continuous monitoring of colony activity rhythms of highly eusocial bees of very different sizes. On two colonies each of two stingless bee species and on two colonies of the honey bee we tested the suitability of this device for the automated recording during 10 consecutive days of the locomotory activity in the entrance tube of the bees. The device permits to simultaneously record the environmental variables temperature and humidity that can affect the bees’ foraging activity. The 10-day period that we used was arbitrarily defined for the test period of the device, but there should be no system-inherent time limit, other than the computational capacity. Advantages provided by the device The system is based on an open-source Arduino technology coupled with a small detector for the environmental variables. The program (Sketch) is made available for public use in Supplementary File S2. The detection itself is based on the voltage interruption when a bee crosses the path of the IR-emitter/sensor in a glass tube of variable size that can be easily inserted into the colony entrance tube. The detector itself was produced by machining from a black polyacetal block. As the first step in the data analysis, we used the open-source software PuTTY to convert the recorded activity events into CSV format for representation in spreadsheets. In the testing phase of the device, we checked the stability of the recordings over 10-day periods and the easiness of switching the setup between different colonies and different bee species, especially species of different worker size. Stingless bees are perfectly suitable for such tests, as they greatly vary in size, ranging from very small, Drosophila -size bees, like the genera Plebeia and Leurotrigona , to bees of housefly size, like the genera Frieseomellitta, Tetragona, Scaptotrigona, Trigona and others, to bees of almost honey bee size (genus Melipona ). While our primary interest is to understand the nature of circadian rhythms in highly eusocial bees, especially in the context of the variable natural histories of the stingless bees, we envision several other utilities for this device. For instance, one application that immediately comes to mind is the analysis of the effects of sublethal doses of pesticides under field-realistic conditions on the foraging behavior of entire colonies. Currently most of these tests are done on worker bees kept in small cages (Lourenço et al. 2012 ; Farder-Gomes et al. 2024 ). Despite its advantages, there are also limitations concerning the device and the design of the current study. With respect to the device, a limitation is that the recording is done in the entrance tube, and hence, it cannot distinguish between general locomotory activity (outgoing, incoming, or just back and forth movement) and actual foraging (foragers entering with pollen and/or nectar). Separating these activities would require dual registration, e.g., in a tube that permits only exits and in a separate one reserved for entries. This could eventually be possible, but it will require a training period, especially for the returning bees to use the correct entrance. The current registration, however, allowed us to see activity that would have passed undetected when observing only actual exit and/or entry flights. This was the nocturnal activity in the colony entrance tube, which we detected for all colonies that we monitored, and this observation could be of interest for studies on sleep in social bees (Kaiser and Steiner-Kaiser 1983 ; Sauer et al. 2003 ). Furthermore, the before-sunrise activity in the M. quadrifasciata colonies is indicative of an anticipatory activity, driven by the bees’ circadian clock. Another important aspect to consider is colony size, as this will be reflected in the mean flow of bees in the entrance tube, as seen in the mean activity graphs for the three species that we studied (Figs. 5 and 6 ). The analysis of the data in the form of Lombs-Scargle periodograms, however, essentially eliminates this issue and permits deeper insights into circadian and eventually ultradian organization of external activity. Another limitation of our study could be perceived as the restriction of the recordings to the southern summer season and the relatively small number of colonies (replicates) per species. But covering these aspects was not our intention. Rather, our aim was to develop a versatile and low-cost device for automated, continued activity recordings in species of highly eusocial bees with workers of very different body size. External activity rhythms of colonies differ across species The comparative analysis of the recordings on the three species generated interesting data on the bees’ circadian locomotory activity in the entrance tube of the colonies. For M. quadrifasciata , the results indicate that the workers become active already before sunrise, and then, in the early morning hours they forage massively. In other words, the bees apparently anticipate the timing of their activity relative to sunrise based on their circadian clock. In both colonies, as indicated by the acrophase (Fig. 3 A, Table 1 ), the major activity peak was in the early morning hours when temperatures are still low to moderate. We and others (Roubik 1989 ) had already observed groups of Melipona bees sitting at colony entrance early in the morning, beating their wings and getting ready to forage. A second, minor level of activity in the entrance tube was then seen in the afternoon (Fig. 3 A). In the Lomb-Scargle periodograms of the M. quadrifasciata colonies (Fig. 5 A), this temporal division in external activity became apparent as a circadian rhythm of approximately 24 hours and two ultradian ones of around 12 hours and 8 hours, respectively. In contrast to M. quadrifasciata , the F. varia workers showed a much larger distribution of their external activity across daytime (Fig. 3 B), with an acrophase at around noon (Table 1 ). Furthermore, while the two Melipona colonies were very similar in their activity patterns, the Lomb-Scargle periodogram analysis indicated some degree of colony specificity in the activity patterns of F. varia . Colony 1 presented a slightly bimodal activity pattern, indicating an ultradian rhythm of around 8 hours in addition to the strong 24-hours circadian rhythm (Fig. 5 B). Interestingly, in the actograms of F. varia Colony 2, a notable and continuous activity of bees in the entrance tube was seen even during the night (Fig. 5 B). As there is a similar report for another stingless bee, Scaptotrigona aff. depilis (Bellusci and Marques 2001 ), of slightly larger size than F. varia , such activity at the colony entrance during the night could represent a more general activity pattern in stingless bee colonies. This activity is relatively constant throughout the night and, thus, different from the anticipatory and increasing activity seen in M. quadrifasciata colonies before sunrise. We interpret the latter as a preparation of the foraging activity that can start already during the twilight phase, but will further depend on the seasonal temperature conditions. In a high-altitude reserve of the Atlantic Rainforest in the State of São Paulo, Oliveira-Abreu et al. ( 2014 ) reported a peak of nectar and then pollen collecting activity between 08:30 to 09:50, when temperatures were between 20 and 23°C. Considering our location and the season when the measurements were taken, the temperatures in the early morning (06:30; Fig. 3 A) were certainly already adequate for foraging. An advantage of the early foraging in this species is likely the profit of high floral rewards and low competition at these early hours for the relatively large Melipona workers, compared to the generally much smaller other genera of stingless bees (Roubik 1989 ; Pereboom and Biesmeijer 2003 ; Teixeira and Campos 2005 ; Streinzer et al. 2016 ). Another important factor to consider is that large bees need to avoid problems, such as overheating, and by doing so, the activity during high sunlight intensity is decreased (Linsley 1958 ). For example, the preferred time of foraging of the carpenter bee Xylocopa (Proxylocopa) olivieri is before sunrise and after sunset, which allow these solitary bees to occupy a unique niche with much less competition, as well as to avoid direct exposition to sunlight (Gottlieb et al. 2005 ). Other important aspects to consider are whether a species is mass-recruiting or not, and whether the foragers show aggressive behavior against competitors at the foraging sites. These questions have been addressed in several studies (for reviews see Roubik 1989 ; Grüter 2020 ) and are specifically highlighted by I’Anson Price et al. (2021). These authors showed that mass-recruiting species of stingless bees do not necessarily have higher foraging returns and/or shorter foraging flights than non-recruiting ones. Rather, mass recruiting may serve to exploit resources against aggressive competitors and does not necessarily favor high-quality food sources. With respect to the colony activity recordings for the Africanized A. mellifera hybrids we saw clear differences in the activity patterns between the two colonies (Fig. 4 and Table 1 ). This is surprising, as the two colonies have been kept for a long time already in the same apiary, together with the stingless bee colonies used in the study. Furthermore, the workers of M. quadrifasciata and A. mellifera are similar in size, while the F. varia workers are much smaller and rather slender. Hence, while body size is likely an important factor in determining foraging activity patterns in the stingless bees, honey bee colonies apparently present more plasticity in the timing of their foraging patterns. This, however, is an issue that certainly needs a closer look, especially in the view of a recent study (Giannoni-Guzmán et al. 2024 ) reporting an effect of colony temperature during a sensitive period in early adult life on the entrainment of circadian rhythms in honey bee workers. Conclusions We describe here a versatile, low-cost device for the continuous automatic recording and integrated data analysis of locomotor activity in the entrance tubes of colonies of highly eusocial bees. We compared the activity of two colonies each for two species of stingless bees and compared these to two colonies of the honey bee, all kept at the same location and recordings done during a single southern summer to early fall season. The analysis of the data in terms of actograms and Lomb-Scargle periodograms showed a predominant circadian rhythm for all three species, but also indications of ultradian rhythms. For Melipona quadrifasciata , which is comparable in size to the honey bee, we found evidence for an anticipatory locomotor activity in the entrance tube already before sunrise, followed by an early morning peak of foraging, indicating an anticipatory activity driven by their endogenous clock. Hence, we believe that the low cost and the versatility of the device, as well as the open-source options for data analysis should be an attractive option for conducting studies on circadian rhythms in social bees under natural conditions, complementing studies on flower visits by these ecologically and economically important pollinators. Declarations Acknowledgments We thank Prof. Dr. Eduardo Brandt de Oliveira (USP) for the help with the Arduino technology and Dr. Katharina Beer (Univ. Würzburg) for helpful suggestions in the analysis of circadian rhythms. We also thank Anderson Roberto de Souza and the staff of the Precision Manufacturing Workshop of the USP-Ribeirão Preto campus for the design and manufacturing of the registration device. Jairo de Souza and Luis Roberto Aguiar provided assistance in maintaining the stingless bee and honey bee colonies. Financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process 302209/2022-0) and scholarships to the first author by CNPq (PIBIC) and the Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP, Process 2020/05723-0) are acknowledged. Author contributions ARJ and KH conceived and designed the experiments. ARJ performed the experiments and collected and analyzed the data. ARJ and KH jointly wrote and edited the manuscript and approved the final version. Data availability The data used to support the findings of this study are available from the corresponding author upon request. Conflict of Interest The authors declare no conflict of interest. References Abou-Shaara HF (2014) The foraging behaviour of honey bees, Apis mellifera : A review. Vet Med-Czech 9:1–10. https://doi.org/10.17221/7240-VETMED Alburaki M, Madella S, Corona M (2021) RFID technology serving honey bee research: A comprehensive description of a 32-antenna system to study honey bee and queen behavior. Appl Syst Innov 4:88. https://doi.org/10.3390/asi4040088 Beer K, Helfrich-Förster C (2020) Model and non-model insects in chronobiology. Front Behav Neurosci 14:601676. https://doi.org/10.1007/s00359-016-1103-2 Beer K, Steffan-Dewenter I, Härtel S, Helfrich-Förster C (2016) A new device for monitoring individual activity rhythms of honey bees reveals critical effects of the social environment on behavior. 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Sociobiology 61(4):441–448. https://doi.org/10.13102/sociobiology.v61i4.441-448 Pereboom JJM, Biesmeijer JC (2003) Thermal constraints for stingless bee foragers: The importance of body size and coloration. Oecologia 137:42–50. https://doi.org/10.1007/s00442-003-1324-2 Pierrot LM, Schlindwein C (2003) Variation in daily flight activity and foraging patterns in colonies of uruçu – Melipona scutellaris Latreille (Apidae, Meliponini). Rev Bras Zool 20:565–571. https://doi.org/10.1590/S0101-81752003000400001 Renner M (1957) Neue Versuche über den Zeitsinn der Honigbiene. Z Vergl Physiol 40:85–118. https://doi.org/10.1007/BF00298152 Roubik DW (1989) Ecology and Natural History of Tropical Bees. Cambridge University Press, New York Roubik DW, Buchmann SL (1984) Nectar selection by Melipona and Apis mellifera (Hymenoptera: Apidae) and the ecology of nectar intake by bee colonies in a tropical forest. Oecologia 61:1–10. https://doi.org/10.1007/BF00379082 Roubik DW, Yanega D, MA S, Buchmann SL, Inouye DW (1995) On optimal nectar foraging by some tropical bees (Hymenoptera: Apidae). Apidologie 26:197–211. https://doi.org/10.1051/apido:19950303 Rubin EB, Shemesh Y, Cohen M, Elgavish S, Robertson HM, Bloch G (2006) Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee ( Apis mellifera ) and shed new light on the molecular evolution of the circadian clock. Genome Res 16:1352–1365. https://doi.org/10.1101/gr.5094806 Sauer S, Kinkelin M, Herrmann E, Kaiser W (2003) The dynamics of sleep-like behaviour in honey bees. J Comp Physiol - Neuroethol Sens Neural Behav Physiol 189:599–607. https://doi.org/10.1007/s00359-003-0436-9 Shpigler HY, Yaniv A, Gernat T, Robinson GE, Bloch G (2020) The influences of illumination regime on egg-laying rhythms of honey bee queens. J Biol Rhythm 37:609–619. https://doi.org10.1177/07487304221126782 Sommeijer MJ, de Rooy GA, Punt W, de Bruijn LLM (1983) A comparative study of foraging behaviour and pollen resources of various stingless bees (Hym., Meliponinae) and honeybees (Hym., Apidae) in Trinidad, West-Indies. Apidologie 14:205–224. https://doi.org/10.1051/apido:19830306 Stabentheiner A, Kovac H (2014) Energetic optimisation of foraging honeybees: Flexible change of strategies in response to environmental challenges. PLoS ONE 9:e105432. https://doi.org/10.1371/journal.pone.0105432 Stelzer RJ, Stanewsky R, Chittka L (2010) Circadian foraging rhythms of bumblebees monitored by radio-frequency identification. J Biol Rhythms 25:257–267. https://doi.org/10.1177/0748730410371750 Streinzer M, Huber W, Spaethe J (2016) Body size limits dim-light foraging activity in stingless bees (Apidae: Meliponini). J Comp Physiol A -. Neuroethol Sens Neural Behav Physiol 202:643–655. https://doi.org/10.1007/s00359-016-1118-8 Teixeira LV, Campos FNM (2005) Início da atividade de vôo em abelhas sem ferrão (Hymenoptera, Apidae): influência do tamanho da abelha e da temperatura ambiente. Zoociências 7:195–202 Teixeira LV, Waterhouse JM, Marques MD (2011) Respiratory rhythms in stingless bee workers: Circadian and ultradian components throughout adult development. J Comp Physiol - Neuroethol Sens Neural Behav Physiol 197:361–372. https://doi.org/10.1007/s00359-010-0620-7 The Honey Bee Genome Sequencing Consortium (2006) Insights into social insects from the genome of the honeybee Apis mellifera . Nature 443:931–949. https://doi.org/10.1038/nature05260 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFileS1.docx SupplementaryFileS2.docx Cite Share Download PDF Status: Published Journal Publication published 20 Jun, 2024 Read the published version in Journal of Comparative Physiology A → Version 1 posted Editorial decision: Revision requested 06 May, 2024 Reviews received at journal 03 May, 2024 Reviews received at journal 17 Apr, 2024 Reviewers agreed at journal 12 Apr, 2024 Reviewers agreed at journal 03 Apr, 2024 Reviewers invited by journal 03 Apr, 2024 Submission checks completed at journal 02 Apr, 2024 Editor assigned by journal 02 Apr, 2024 First submitted to journal 01 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4201960","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":287168600,"identity":"7fa568a2-4835-4444-9c84-03db89324a64","order_by":0,"name":"Arthur Roque Justino","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Arthur","middleName":"Roque","lastName":"Justino","suffix":""},{"id":287168602,"identity":"4c741fdb-db99-406e-b021-d81a1d74c9b0","order_by":1,"name":"Klaus Hartfelder","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYBACCXYQWSEHF0ggrIUZRJ4xJlULYxspWiSbmZ9J/pxnEG0+u4FN8ucOhjzzBgJapJnZzKR5txnkzrlzgE2a9wxDscwBAlrkmBnMpBm3/cmdIZHAJs3YxpA4g5DD5JjZv0n+nGMA1iL5kxgt0sw8ZhK8DRAtErzEaJFs5im25jkG1CJzsNma94xEsQQhLRLH2zfe/FED1CLdfPDmzx02eQS1IGlmbGBgbCBBA1ALEAN1jYJRMApGwSjAAAD0iDVUXMuSogAAAABJRU5ErkJggg==","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":true,"prefix":"","firstName":"Klaus","middleName":"","lastName":"Hartfelder","suffix":""}],"badges":[],"createdAt":"2024-04-01 16:59:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4201960/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4201960/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00359-024-01709-2","type":"published","date":"2024-06-20T15:56:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54154633,"identity":"dfe11d1a-e26f-47af-9b96-80aaaaf378fe","added_by":"auto","created_at":"2024-04-05 11:41:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41468,"visible":true,"origin":"","legend":"\u003cp\u003eStructure and function of the registration device for bees moving in the colony exit tube. A) The different components of the device, consisting of a polyethylene block (left) containing the IR emitting diode and the IR sensor on opposite sides; the adaptor tube (middle) for glass tubes of different diameters; and the polyethylene case (right) that protects the entire setup. The adaptor tube has a hole that fits into the IR light path. B) Photo of the actual position of the registration device (red circle) in the entrance tube of a Frieseomelitta varia colony. The colony is set up in an observation room, where the Arduino plate and the computer is located. The exit tube and the wires for the temperature and humidity sensors are passed through the wall to the outside. C) Photo of a M. quadrifasciata worker crossing the detector block (red circle).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4201960/v1/1f7788bfa0bc2d7525f21318.jpg"},{"id":54154632,"identity":"342558e5-5dd7-4e0b-84fb-2e90837154b7","added_by":"auto","created_at":"2024-04-05 11:41:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24370,"visible":true,"origin":"","legend":"\u003cp\u003eElectronic circuitry of the device for foraging activity and external condition measurements. The figure shows the Arduino board with a sketch showing the entry for the IR detector. Each interruption caused by the passage of a bee is counted as a new event. A module HDC1080 that measures the temperature and relative humidity with high accuracy is connected to other entries. The Arduino board is connected to a computer, and the values can be assessed through the Arduino IDE (Integrated Development Environment). Electric components are shown as standardized symbols.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4201960/v1/8786b23953d67079a0b233b6.jpg"},{"id":54154636,"identity":"39ce95ce-9e7d-4f51-8530-2b39605088f4","added_by":"auto","created_at":"2024-04-05 11:41:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":204632,"visible":true,"origin":"","legend":"\u003cp\u003eLocomotor activity in the entrance tube and environmental temperature and humidity measurements for two stingless bee species during 10 days of recording. \u0026nbsp;Double-plotted actograms show the foraging rhythms for two colonies each for A) \u003cem\u003eMelipona quadrifasciata \u003c/em\u003eand B) \u003cem\u003eFrieseomelitta varia\u003c/em\u003e. The acrophase is shown as a blue line (mean) and for each day (blue triangle). The environmental bars represent the average length of day and night during the recording periods. The lower graphs in A and B show the respective temperature and humidity curves that were registered simultaneously with the activity records.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4201960/v1/ab7d69146933885edbfc57f9.jpg"},{"id":54154639,"identity":"5c3b8d0c-61c1-4fde-bfdb-638e180adb38","added_by":"auto","created_at":"2024-04-05 11:41:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":123308,"visible":true,"origin":"","legend":"\u003cp\u003eLocomotor activity in the entrance tube and environmental temperature and humidity measurements taken during 10 days for two colonies of \u003cem\u003eApis mellifera\u003c/em\u003e. Double-plotted actograms show the foraging rhythms, as well as the presence of some nocturnal activity in the exit tube. The acrophase is shown as a blue line (mean) and for each day (blue triangles). Environmental bars represent the average length of daylight and night during the periods recorded. The lower graph shows the respective temperature and humidity curves that were registered simultaneously with the activity records.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4201960/v1/80cb13da645df99acdc9cbb0.jpg"},{"id":54154637,"identity":"e9c8563c-3df5-41bb-ab48-da4b0884a01d","added_by":"auto","created_at":"2024-04-05 11:41:48","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":183651,"visible":true,"origin":"","legend":"\u003cp\u003eMean flow of activity in the entrance tube and Lomb-Scargle Periodogram analysis for each each of the two colonies of A) \u003cem\u003eMelipona quadrifasciata\u003c/em\u003e and B) \u003cem\u003eFriesomelitta varia\u003c/em\u003e. The mean activity graphs show means + SE. Environmental bars represent the average length of daylight and night during the recording periods. While all four colonies showed a strong circadian rhythm (peak at 1440 min), the \u003cem\u003eM. quadrifasciata\u003c/em\u003e colonies also have a prominent ultradian component of approximately 12 h in their foraging rhythm. The dashed blue line indicates statistical significance, P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4201960/v1/d3627c63362dc7d5a4e94b44.jpg"},{"id":54154635,"identity":"dba25e04-3df7-4a36-8820-d8b0b6c649d1","added_by":"auto","created_at":"2024-04-05 11:41:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":52391,"visible":true,"origin":"","legend":"\u003cp\u003eMean flow of activity in the entrance tube and Lomb-Scargle Periodogram analysis for the two\u003cem\u003e Apis mellifera \u003c/em\u003ecolonies. The mean activity graphs show means + SE. Environmental bars represent the average length of daylight and night during the recording period. Both colonies show a clear circadian rhythm (peak at 1440 min), but Colony 2 also shows strong peaks at 8 h and 12 h, indicating the presence of ultradian rhythms. The dashed blue line indicates statistical significance, P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4201960/v1/a7bf4cf98bf663c8116dc86f.jpg"},{"id":58823824,"identity":"bbb78ffa-6cde-4db2-9f51-a56f38e66a20","added_by":"auto","created_at":"2024-06-21 17:07:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1243389,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4201960/v1/5614d18a-c58f-40c1-a3ce-34948d85e41d.pdf"},{"id":54154634,"identity":"8e6ec05e-fe7c-43a6-bbb8-ec1f6c9eeaec","added_by":"auto","created_at":"2024-04-05 11:41:47","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":176950,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4201960/v1/d9dd6b312590550dbe8ca07d.docx"},{"id":54154638,"identity":"c8313261-8b1e-4310-aef3-ee9c10e855a4","added_by":"auto","created_at":"2024-04-05 11:41:48","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1443199,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4201960/v1/4c948a5f95cb6e1a960f8fc9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A versatile recording device for the analysis of continuous daily external activity in colonies of highly eusocial bees","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe high energetic cost of flights and the importance of collecting resources for colony maintenance are critical factors in the optimization of foraging behaviors in social bees (Stabentheiner and Kovacs \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). As most of the bees\u0026rsquo; foraging behavior is directed towards collecting nectar and pollen, this requires the adjustment of the flight activity with the timing when nectar and pollen becomes available in flowering plants. In the co-evolution between angiosperm plants and bees (Anthophila) since the Cretaceous, this created an advantageous relationship for both parts. In the Western honey bee, \u003cem\u003eApis mellifera\u003c/em\u003e, the adjustment of foraging flights with flowering time has been conceptually formulated as the \u003cem\u003eZeitged\u0026auml;chtnis\u003c/em\u003e (time memory) of the bees (Beling \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1929\u003c/span\u003e; Kleber \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1935\u003c/span\u003e; Renner \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1957\u003c/span\u003e). Particularly, the capacity of the bees\u0026rsquo; endogenous clock of approximately 24 h running time permits them to predict the best time for obtaining the desired plant resources, and for the plants to successfully achieve pollination (Bloch et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Bloch et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince the length of the light/dark cycles varies across the year, this requires that the bees can shift and synchronize their endogenous clock to the environmental cycles. The factors for this adjustment are known as \u003cem\u003eZeitgebers\u003c/em\u003e, and the main ones are photoperiod and temperature (Kefuss and Nye \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Moore and Rankin \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Moritz and Kryger \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Bloch \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In highly eusocial insects, the colony context, primarily the contact with nestmates, also plays a role as a \u003cem\u003eZeitgeber\u003c/em\u003e, and this can even be stronger than the photoperiod (Moritz and Sakofski \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Frisch and Koeniger \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Beer et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shpigler et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the honey bee for instance, Fuchikama et al. (2016) demonstrated that workers experiencing conflicting photic and social synchronizations choose to follow the social signals, thus calling attention to the fact the social context needs to be taken into account when studying biological rhythms. In this sense, the honey bee is certainly one of the best characterized insect species concerning circadian rhythms and their relation with environmental and social \u003cem\u003eZeitgebers\u003c/em\u003e (Moore \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Abou-Shaara \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Beer and Helfrich-F\u0026ouml;rster \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, the availability of a fully sequenced honey bee genome (The Honey Bee Genome Sequencing Consortium, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and a plethora of information on the molecular underpinnings of the \u003cem\u003eDrosophila\u003c/em\u003e circadian clock (Hardin \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) has led to deep insights into the molecular nature of the honey bee circadian clock (Rubin et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Bloch \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe only other bees that match the honey bees (Apini) in terms of social organization are the stingless bees (Meliponini), and their species richness and variation in biological characters (Gr\u0026uuml;ter, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) provides an excellent opportunity to study factors and possible adaptations that shaped the biological rhythms in their foraging patterns. So far, most studies on foraging activity in stingless bees have set the focus on the diel rhythms of food collection, i.e., rhythmicity controlled by day-light cycles (Hil\u0026aacute;rio et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Pierrot and Schlindwein \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Texeira and Campos, 2005). While the patterns of foraging along the day showed interspecific variation, a common trait in the stingless bees is a temporal separation with respect to resource collection, with pollen being collected mainly in the early morning, while nectar is collected over a longer period during the day (Sommeijer et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Hil\u0026aacute;rio et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Pierrot and Schlindwein \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Nascimento and Nascimento \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This can potentially be explained by an increase in the nectar sugar content as temperatures rise (Roubik and Buchmann \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Roubik et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, only few studies on stingless bees addressed the association of foraging activity with endogenous circadian rhythms. A study by Bellusci and Marques (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) on \u003cem\u003eScaptotrigona\u003c/em\u003e aff. \u003cem\u003edepilis\u003c/em\u003e, showed that the workers are capable of keeping a normal circadian rhythm of external activity even under constant light, and the same authors also showed that bees were active even during the night. There is also clear evidence for the presence of rhythmicity inside the nest (Giannini \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Bellusci and Marques \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Oda et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and also in terms of physiological functions (Teixeira et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAddressing questions of rhythmicity in colony behaviors, especially related to foraging activity, can be done with different methodological approaches. The simplest is the direct observation and counting of the ins-and-outs at the colony entrance. But this is very labor intensive, especially for large colonies and for several colonies at the same time, and it also cannot easily be done around the clock. An alternative is the constant videorecording of these activities at the colony entrance, but this requires good quality cameras and several hours of work for the analysis of the recordings. In contrast, radiofrequency identification (RFID) presents considerable advantages and has found several applications in studies of bees (Streit et al. 2003; Nunes-Silva et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Nunes-Silva et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Alburaki et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), including studies on circadian rhythms (Stelzer et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Nonetheless, a limitation for RFID studies on bees is the size of the tag that the bees must carry, and for small bees this can severely hamper their flight performance. Furthermore, a complete RFID setup is rather costly, especially for simultaneous studies on multiple colonies. Another alternative is the system developed by Beer et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) that allows the monitoring of individual honey bees in a social context with respect to circadian locomotor activity. But this system is, so far, primarily designed for the use with miniature colonies kept under controlled conditions.\u003c/p\u003e \u003cp\u003eConsidering the size differences among stingless bee species and how these may relate to the foraging activity of colonies under natural environmental conditions, we set forth to develop an automated continuous recording system that can easily be inserted into the colony entry tube. We designed it to be versatile in terms of tube diameter, so that it can be used on stingless bees of a wide size range. Furthermore, it can simultaneously record external temperature and humidity. We report here on the continuous recordings of the external activity of a relatively large stingless bees, \u003cem\u003eMelipona quadrifasciata\u003c/em\u003e, a smaller, slender species, \u003cem\u003eFrieseomelitta varia\u003c/em\u003e, and for comparison, we also recorded the activity of honey bee observation colonies kept in the same apiary.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe activity detection and recording device\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe registration device is composed of two parts, an Arduino hardware system with software written for the registration of the bees\u0026rsquo; activity, and a sensor block containing an infrared (IR) emitter and an IR receiver to detect the passage of the bees, an adaptable tube holder and a case that protects the entire unit. The parts composing the sensor are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, and technical designs for the construction of these parts are available as supplementary material File S1. These parts were produced by the machining service of the university\u0026rsquo;s Precision Manufacturing Workshop from a black polyacetal block (Polyoxymethylene, 50 mm diameter) purchased from a local supplier of machining materials. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows the registration device inserted into the exit tube of a \u003cem\u003eF. varia\u003c/em\u003e colony kept in an observation room with free exit to the outside, and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC shows a \u003cem\u003eM. quadrifasciata\u003c/em\u003e worker crossing the monitoring device.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eFor the use with bees of different size we designed and built a series of adaptable tubes, which can hold glass tubes of different diameters. The internal diameter of the glass tube placed in the exit path of a colony has to be in accordance with the size of the bees, to ensure that only one bee at a time can cross the IR detector. Nonetheless, it is important that the bees can comfortably cross the tube, even when carrying pollen, resin, or mud on the corbicula. In our design, we used glass tubes of a 5 mm diameter for \u003cem\u003eF. varia\u003c/em\u003e, 8 mm for \u003cem\u003eM. quadrifasciata\u003c/em\u003e, and 10 mm for \u003cem\u003eA. mellifera.\u003c/em\u003e We had good experience with the use of laboratory glass pipettes, as they come in different diameters and should be available from nowadays unused stocks in biochemistry laboratories.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe registration system consists of an Arduino board and the open-source Arduino software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arduino.cc/en/Guide/Introduction/\u003c/span\u003e\u003cspan address=\"https://www.arduino.cc/en/Guide/Introduction/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, Smart Projects, Italy) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Detailed information on the Arduino system and the Sketch program written for the registration of the bees\u0026rsquo; activity are available in the supplementary material File S2. For external temperature and relative humidity detection we used the low-power and high-accuracy module HDC1080 (Texas Instruments, Dallas, TX) with accuracies of \u0026plusmn;\u0026thinsp;2%RH and 0.2 \u0026ordm;C, and 14 bits resolution (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ti.com/product/HDC1080\u003c/span\u003e\u003cspan address=\"https://www.ti.com/product/HDC1080\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). It is connected to the Arduino board by wires and measures the environmental variables at the end of the desired time intervals. The Arduino board, the IR diode and IR detector, and the HDC1080 module were purchased from Eletrogate (Belo Horizonte, MG, Brazil). The Arduino board was connected to a commercial notebook for storage of the data.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn important point is the definition of critical parameters in the Sketch program for the registration process itself. To avoid false positive or false negative counts, it is important to establish an upper and a lower time threshold for an event setting. We based this on the time that it normally takes for a bee to cross a 1 cm distance in the glass tube. This time will depend on the biological characteristics of the species and needs to be empirically established. If there is a voltage drop within the given time thresholds, each such event is then added as a count, and this process is automatically repeated over the entire recording period of interest.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSetting up the colonies for activity recording\u003c/h2\u003e \u003cp\u003eWe used field colonies of similar size and health status for each of the two stingless bee species. For the recordings on honey bees, we prepared single frame observation hives from two field colonies of Africanized honey bee hybrids, containing about 1000 workers and a queen. All these colonies were obtained from the Experimental Apiary of the Ribeir\u0026atilde;o Preto Campus of the University of S\u0026atilde;o Paulo and were transferred into an observation room in the apiary. The entries of the colonies were connected to the outside by plastic tubes that passed the wall. Sugar syrup 50% was provided twice a week. After a period of acclimatization of one week, the recording device containing the IR emitter and IR receiver was placed in the middle of the respective entrance tubes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB,C). The HDC1080 module for temperature and humidity recording was passed over the tube to the outside, and its wires were connected to the Arduino board.\u003c/p\u003e \u003cp\u003eThe recordings were performed for 10 consecutive days. This was during the southern summer for the \u003cem\u003eA. mellifera\u003c/em\u003e (December 20\u0026ndash;29 2022) and the \u003cem\u003eM. quadrifasciata\u003c/em\u003e colonies (January 16\u0026ndash;25, 2023). For the \u003cem\u003eF. varia\u003c/em\u003e colonies, the recordings were done slightly later, at the beginning of the fall (March 24 to April 03, 2023). This time difference entails certain environmental differences, not so much in terms of pluviosity, but day lengths are already slightly decreasing at the beginning of the fall.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eIn the Arduino program (Sketch, File S2), the time interval for each recording period was empirically set at 10 minutes. The software PuTTy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.putty.org/\u003c/span\u003e\u003cspan address=\"https://www.putty.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, v. 0.78) was used to transform the values for the bees\u0026rsquo; passages in the entrance tube and the temperature and humidity recordings as input into a CSV table. For generating double-plotted actograms and to assess the mean activity of each colony we used the plugin ActogramJ tool for Fiji ImageJ [\u0026copy; Benjamin Schmid and Taishi Yoshii, University of W\u0026uuml;rzburg, Department of Neurobiology and Genetics; Fiji ImageJ (Version 1.53, \u0026copy; Wayne Rasband, National Institutes of Health, USA)] (Schmid et al. 2011). In the plots, the environmental bar represents the mean length of the day and night time for the respective recording period. The acrophase (center of gravity in activity recordings) was calculated using the acrophase tool, and the time of activity onset and offset was calculated using the Activity on- and offset tool [Smoothing Gaussian std. dev. = 5.0000 min, Threshold\u0026thinsp;=\u0026thinsp;Median (with zero activity)]. Furthermore, to detect the rhythms of the colonies, a Lomb-Scargle periodogram analysis was performed using the package \u003cem\u003elomb\u003c/em\u003e (version 2.1.0) implemented in RStudio (v. 2023.06.2\u0026thinsp;+\u0026thinsp;561), with α\u0026thinsp;\u0026lt;\u0026thinsp;0.01. The package \u003cem\u003eggplot2\u003c/em\u003e (3.4.1) was used to generate the plots of the periodograms. Finally, the R package \u003cem\u003edygraphs\u003c/em\u003e (v. 2.2.1) was used to plot the recorded temperature and humidity values. The function cor.test() implemented in RStudio software was also used to perform a Spearman rank correlation between external activity and environmental factors.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eConfection and setup of the recording device\u003c/h2\u003e \u003cp\u003eImportant aspects for the choice of a monitoring device are (a) the easiness of obtaining data and transforming these into tables or graphs, (b) the adaptability for more than one purpose, and (c) the cost per device. With our device, the passage of a bee crossing the IR emitter/detector in a given time interval generates a discrete electrical signal as a voltage drop. The integration of these signals via an Arduino board and corresponding software is then used as a direct input into a CSV table. In terms of adaptability, social bees of different size can be monitored via the choice of an appropriate adaptor and glass tube. And concerning costs, the electronic parts (Arduino board, IR emitter and detector, and the HDC180 module for registration of ambient conditions), as well as the raw material for the device structure, a polyacetal block, are low-cost items. We acquired them for less than US\u003cspan\u003e$\u003c/span\u003e 60.00. The main cost factor will be the machining service requiring about 10 hours of a turner\u0026rsquo;s work, which in our case was provided free of charge by the university workshop.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eActograms and temperature and humidity recordings for two stingless bee species\u003c/h2\u003e \u003cp\u003eThe recordings of foraging activity and environmental conditions (temperature and relative humidity) were taken for 10 consecutive days for the two colonies of each of the two species. With respect to the bees\u0026rsquo; external activity patterns, the double plotted actograms for the two \u003cem\u003eM. quadrifasciata\u003c/em\u003e colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) revealed high activity levels always in the early morning, which then became reduced during the rest of the day. Interestingly, we noted that for the \u003cem\u003eMelipona\u003c/em\u003e colonies, the activity inside the entrance tube showed an increase already before sunrise.\u003c/p\u003e \u003cp\u003eFor the two \u003cem\u003eF. varia\u003c/em\u003e colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) we noted differences in their foraging activity patterns. Colony 1 showed a bimodal distribution of foraging activity, one in the morning and one in the afternoon, while for Colony 2, foraging activity was relatively constant during daytime. In addition, Colony 2 presented a notable and continuous transit of bees in the colony entrance tube even during the night (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe analysis of the acrophase of the bees\u0026rsquo; activity rhythms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, blue line) and of the timing of activity on- and offset (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) also put in evidence the clear difference in the temporal structure of foraging activity among the two stingless bee species. The acrophase of activity in the \u003cem\u003eM. quadrifasciata\u003c/em\u003e colonies occurs in the early morning hours, and as mentioned, the activity onset actually is already before sunrise. In contrast, for \u003cem\u003eF. varia\u003c/em\u003e, the acrophase of activity is positioned in the middle of the day, and the onset of activity only starts with sunrise.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e Time of activity onset, offset, and the acrophase for the two stingless bee species and \u003cem\u003eApis mellifera\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOnset\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOffset\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcrophase\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. quadrifasciata\u003c/em\u003e Colony 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e04.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e09.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. quadrifasciata\u003c/em\u003e Colony 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e04.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e09.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eF. varia\u003c/em\u003e Colony 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e06.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eF. varia\u003c/em\u003e Colony 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e06.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.06 \u0026plusmn; 0.35 h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. mellifera\u003c/em\u003e Colony 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e06.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40 h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. mellifera\u003c/em\u003e Colony 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e04.33\u0026thinsp;\u0026plusmn;\u0026thinsp;23.0 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.66 \u0026plusmn; 0.93 h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe recordings of the environmental variables showed strong daily oscillations in both temperature and humidity (lower graphs in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). There were no major differences in the daily temperature ranges for the two time periods, but the humidity levels were higher at the end of March, when the recordings were done on the \u003cem\u003eF. varia\u003c/em\u003e colonies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eActograms and temperature and humidity recordings for\u003c/b\u003e \u003cb\u003eApis mellifera\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe recordings of foraging activity and environmental temperature and humidity for the two \u003cem\u003eA. mellifera\u003c/em\u003e observation hives were taken at the end of December 2022. Strikingly, even though they were monitored simultaneously, the two colonies showed quite divergent patterns of locomotor activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While the activity pattern for Colony 1 showed considerable variation over the consecutive days, including some activity in the entrance tube during night time, Colony 2 showed two clear activity peaks, one in the early morning and one in the late afternoon. For both colonies, the activity patterns in the last three days of recording were rather unstructured. Yet, as can be seen from the concomitant temperature and humidity recordings, these were days of considerable and prolonged pluviosity, which was also accompanied by a drop in temperature, especially on day 8.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePeriodogram analysis\u003c/h2\u003e \u003cp\u003eTo reveal generalized daily activity patterns, we calculated the mean from the 10-day consecutive recordings, and further analyzed the temporal organization of activity by performing a Lomb-Scargle periodogram analysis. The results for \u003cem\u003eM. quadrifasciata\u003c/em\u003e and \u003cem\u003eF. varia\u003c/em\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and those for \u003cem\u003eA. mellifera\u003c/em\u003e in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. As already indicated from the actograms, the two \u003cem\u003eM. quadrifasciata\u003c/em\u003e colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) were highly similar in their mean activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The maximum flow in the entrance tube was seen concentrated between 5 and 9 am, and the Lomb-Scargle analysis identified the presence of a strong circadian rhythm (close to 24 h) in the foraging activity, as well as two minor peaks of ultradian rhythms (8 h and 12 h). In contrast, the two \u003cem\u003eF. varia\u003c/em\u003e colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) showed a broad spread in their flow rates in the colony entrance tube along the day, between 7 am and 6 pm. With respect to the Lomb-Scargle analysis, both \u003cem\u003eF. varia\u003c/em\u003e colonies showed a clear peak of circadian rhythmicity, but the slightly bimodal distribution of activity seen for Colony 1 was also reflected in a small peak of ultradian activity at 8 h.\u003c/p\u003e \u003cp\u003eThe difference already denoted in the actograms of the two honey bee colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) came out even stronger in their respective periodograms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). While Colony 2 showed a broad spread of activity over the entire day, from 7 am to 5 pm, similar to \u003cem\u003eF. varia\u003c/em\u003e, the activity pattern for Colony 1 is strongly bimodal, with a high flow of activity between 7 to 9 am and another between 3:30 to 4:30 pm. Consequently, the Lomb-Scargle analysis for Colony 1 showed a strong and exclusive circadian activity pattern, while for Colony 2 it indicated an almost similar division between a circadian rhythm (24 h) and two strong ultradian rhythms (8 h and 12 h). Notably, as the two honey bee colonies were larger in population size than the stingless bee colonies, their flow rates in the exit tubes were much higher than in the stingless bees.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation between environmental factors and activity\u003c/h2\u003e \u003cp\u003eSo as to reveal how the environmental conditions may affect the bees\u0026rsquo; activity rhythm we performed a correlation analysis between the colony activity patterns and the external factors (temperature and humidity). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the colonies of \u003cem\u003eF\u003c/em\u003e. \u003cem\u003evaria\u003c/em\u003e responded strongly to changes in environmental factors, with both colonies presenting a positive correlation coefficient (rho) with temperature and a negative one with humidity. In contrast, for the \u003cem\u003eM. quadrifasciata\u003c/em\u003e colonies the correlation coefficients were much lower for both factors, which indicates that they are less susceptible to external factors. In fact, on other occasions, we have seen foragers of these same colonies flying even during rainy days (personal observation), and, as mentioned above, their preferential activity is during the early morning hours, when temperatures are still moderately low. For the two honey bee colonies, the differences seen in the activity patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) are also reflects in the respective correlation coefficients with the environmental conditions. While Colony 1 showed coefficients that were more similar to \u003cem\u003eF. varia\u003c/em\u003e, those of Colony 2 were more similar to \u003cem\u003eM. quadrifasciata.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation analysis between external activity and environmental factors.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eColony\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eHumidity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003erho\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003erho\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. quadrifasciata\u003c/em\u003e Colony 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.8e-06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. quadrifasciata\u003c/em\u003e Colony 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.2e-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00049\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eF. varia\u003c/em\u003e Colony 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eF. varia\u003c/em\u003e Colony 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. mellifera\u003c/em\u003e Colony 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;2 .2e-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. mellifera\u003c/em\u003e Colony 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.5e-06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere are different ways and solutions to record the external activity of bees, from labor-intensive and time-consuming direct observations or video recordings of the transit at the colony entrance, to high-technology RFID monitoring of individually tagged bees. The latter has been applied to honey bees, including studies on circadian rhythms (Stelzer et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). An in-depth description of the hardware and software required for the setup of a RFID recording system of honey bees (de Souza et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) provides important information on the advantages and limits, as well as the costs of RFID recording. One of its limits clearly is the size and weight of the tag that needs to be applied onto a bee\u0026rsquo;s thorax. Furthermore, the constant presence of an RFID tag has been reported as be a stress factor for the bees (de Souza et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A complete RFID setup is also rather costly, especially when the intention is to simultaneously study multiple colonies. Another alternative is the system developed by Beer et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) that allows the monitoring of the circadian locomotor activity of individual honey bees, but this requires minicolonies that are set up under controlled conditions. An automated recording device that is similar to ours was developed by Hilario et al. (2012), consisting of a photocell coupled to a programmable logic controller (PLC) system. They used it to analyze the relationship between climatic factors and the foraging activity of the stingless bee \u003cem\u003ePlebeia remota\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eHere we describe in detail the confection and use of a non-invasive, low-cost device for the continuous monitoring of colony activity rhythms of highly eusocial bees of very different sizes. On two colonies each of two stingless bee species and on two colonies of the honey bee we tested the suitability of this device for the automated recording during 10 consecutive days of the locomotory activity in the entrance tube of the bees. The device permits to simultaneously record the environmental variables temperature and humidity that can affect the bees\u0026rsquo; foraging activity. The 10-day period that we used was arbitrarily defined for the test period of the device, but there should be no system-inherent time limit, other than the computational capacity.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAdvantages provided by the device\u003c/h2\u003e \u003cp\u003eThe system is based on an open-source Arduino technology coupled with a small detector for the environmental variables. The program (Sketch) is made available for public use in Supplementary File S2. The detection itself is based on the voltage interruption when a bee crosses the path of the IR-emitter/sensor in a glass tube of variable size that can be easily inserted into the colony entrance tube. The detector itself was produced by machining from a black polyacetal block. As the first step in the data analysis, we used the open-source software PuTTY to convert the recorded activity events into CSV format for representation in spreadsheets.\u003c/p\u003e \u003cp\u003eIn the testing phase of the device, we checked the stability of the recordings over 10-day periods and the easiness of switching the setup between different colonies and different bee species, especially species of different worker size. Stingless bees are perfectly suitable for such tests, as they greatly vary in size, ranging from very small, \u003cem\u003eDrosophila\u003c/em\u003e-size bees, like the genera \u003cem\u003ePlebeia\u003c/em\u003e and \u003cem\u003eLeurotrigona\u003c/em\u003e, to bees of housefly size, like the genera \u003cem\u003eFrieseomellitta, Tetragona, Scaptotrigona, Trigona\u003c/em\u003e and others, to bees of almost honey bee size (genus \u003cem\u003eMelipona\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eWhile our primary interest is to understand the nature of circadian rhythms in highly eusocial bees, especially in the context of the variable natural histories of the stingless bees, we envision several other utilities for this device. For instance, one application that immediately comes to mind is the analysis of the effects of sublethal doses of pesticides under field-realistic conditions on the foraging behavior of entire colonies. Currently most of these tests are done on worker bees kept in small cages (Louren\u0026ccedil;o et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Farder-Gomes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite its advantages, there are also limitations concerning the device and the design of the current study. With respect to the device, a limitation is that the recording is done in the entrance tube, and hence, it cannot distinguish between general locomotory activity (outgoing, incoming, or just back and forth movement) and actual foraging (foragers entering with pollen and/or nectar). Separating these activities would require dual registration, e.g., in a tube that permits only exits and in a separate one reserved for entries. This could eventually be possible, but it will require a training period, especially for the returning bees to use the correct entrance. The current registration, however, allowed us to see activity that would have passed undetected when observing only actual exit and/or entry flights. This was the nocturnal activity in the colony entrance tube, which we detected for all colonies that we monitored, and this observation could be of interest for studies on sleep in social bees (Kaiser and Steiner-Kaiser \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Sauer et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Furthermore, the before-sunrise activity in the \u003cem\u003eM. quadrifasciata\u003c/em\u003e colonies is indicative of an anticipatory activity, driven by the bees\u0026rsquo; circadian clock.\u003c/p\u003e \u003cp\u003eAnother important aspect to consider is colony size, as this will be reflected in the mean flow of bees in the entrance tube, as seen in the mean activity graphs for the three species that we studied (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The analysis of the data in the form of Lombs-Scargle periodograms, however, essentially eliminates this issue and permits deeper insights into circadian and eventually ultradian organization of external activity.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAnother limitation of our study could be perceived as the restriction of the recordings to the southern summer season and the relatively small number of colonies (replicates) per species. But covering these aspects was not our intention. Rather, our aim was to develop a versatile and low-cost device for automated, continued activity recordings in species of highly eusocial bees with workers of very different body size.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExternal activity rhythms of colonies differ across species\u003c/h2\u003e \u003cp\u003eThe comparative analysis of the recordings on the three species generated interesting data on the bees\u0026rsquo; circadian locomotory activity in the entrance tube of the colonies. For \u003cem\u003eM. quadrifasciata\u003c/em\u003e, the results indicate that the workers become active already before sunrise, and then, in the early morning hours they forage massively. In other words, the bees apparently anticipate the timing of their activity relative to sunrise based on their circadian clock. In both colonies, as indicated by the acrophase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the major activity peak was in the early morning hours when temperatures are still low to moderate. We and others (Roubik \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) had already observed groups of \u003cem\u003eMelipona\u003c/em\u003e bees sitting at colony entrance early in the morning, beating their wings and getting ready to forage. A second, minor level of activity in the entrance tube was then seen in the afternoon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the Lomb-Scargle periodograms of the \u003cem\u003eM. quadrifasciata\u003c/em\u003e colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), this temporal division in external activity became apparent as a circadian rhythm of approximately 24 hours and two ultradian ones of around 12 hours and 8 hours, respectively.\u003c/p\u003e \u003cp\u003eIn contrast to \u003cem\u003eM. quadrifasciata\u003c/em\u003e, the \u003cem\u003eF. varia\u003c/em\u003e workers showed a much larger distribution of their external activity across daytime (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), with an acrophase at around noon (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, while the two \u003cem\u003eMelipona\u003c/em\u003e colonies were very similar in their activity patterns, the Lomb-Scargle periodogram analysis indicated some degree of colony specificity in the activity patterns of \u003cem\u003eF. varia\u003c/em\u003e. Colony 1 presented a slightly bimodal activity pattern, indicating an ultradian rhythm of around 8 hours in addition to the strong 24-hours circadian rhythm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Interestingly, in the actograms of \u003cem\u003eF. varia\u003c/em\u003e Colony 2, a notable and continuous activity of bees in the entrance tube was seen even during the night (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). As there is a similar report for another stingless bee, \u003cem\u003eScaptotrigona\u003c/em\u003e aff. \u003cem\u003edepilis\u003c/em\u003e (Bellusci and Marques \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), of slightly larger size than \u003cem\u003eF. varia\u003c/em\u003e, such activity at the colony entrance during the night could represent a more general activity pattern in stingless bee colonies. This activity is relatively constant throughout the night and, thus, different from the anticipatory and increasing activity seen in \u003cem\u003eM. quadrifasciata\u003c/em\u003e colonies before sunrise.\u003c/p\u003e \u003cp\u003eWe interpret the latter as a preparation of the foraging activity that can start already during the twilight phase, but will further depend on the seasonal temperature conditions. In a high-altitude reserve of the Atlantic Rainforest in the State of S\u0026atilde;o Paulo, Oliveira-Abreu et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) reported a peak of nectar and then pollen collecting activity between 08:30 to 09:50, when temperatures were between 20 and 23\u0026deg;C. Considering our location and the season when the measurements were taken, the temperatures in the early morning (06:30; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) were certainly already adequate for foraging. An advantage of the early foraging in this species is likely the profit of high floral rewards and low competition at these early hours for the relatively large \u003cem\u003eMelipona\u003c/em\u003e workers, compared to the generally much smaller other genera of stingless bees (Roubik \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Pereboom and Biesmeijer \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Teixeira and Campos \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Streinzer et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Another important factor to consider is that large bees need to avoid problems, such as overheating, and by doing so, the activity during high sunlight intensity is decreased (Linsley \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1958\u003c/span\u003e). For example, the preferred time of foraging of the carpenter bee \u003cem\u003eXylocopa\u003c/em\u003e (Proxylocopa) \u003cem\u003eolivieri\u003c/em\u003e is before sunrise and after sunset, which allow these solitary bees to occupy a unique niche with much less competition, as well as to avoid direct exposition to sunlight (Gottlieb et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Other important aspects to consider are whether a species is mass-recruiting or not, and whether the foragers show aggressive behavior against competitors at the foraging sites. These questions have been addressed in several studies (for reviews see Roubik \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Gr\u0026uuml;ter \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and are specifically highlighted by I\u0026rsquo;Anson Price et al. (2021). These authors showed that mass-recruiting species of stingless bees do not necessarily have higher foraging returns and/or shorter foraging flights than non-recruiting ones. Rather, mass recruiting may serve to exploit resources against aggressive competitors and does not necessarily favor high-quality food sources.\u003c/p\u003e \u003cp\u003eWith respect to the colony activity recordings for the Africanized \u003cem\u003eA. mellifera\u003c/em\u003e hybrids we saw clear differences in the activity patterns between the two colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is surprising, as the two colonies have been kept for a long time already in the same apiary, together with the stingless bee colonies used in the study. Furthermore, the workers of \u003cem\u003eM. quadrifasciata\u003c/em\u003e and \u003cem\u003eA. mellifera\u003c/em\u003e are similar in size, while the \u003cem\u003eF. varia\u003c/em\u003e workers are much smaller and rather slender. Hence, while body size is likely an important factor in determining foraging activity patterns in the stingless bees, honey bee colonies apparently present more plasticity in the timing of their foraging patterns. This, however, is an issue that certainly needs a closer look, especially in the view of a recent study (Giannoni-Guzm\u0026aacute;n et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reporting an effect of colony temperature during a sensitive period in early adult life on the entrainment of circadian rhythms in honey bee workers.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe describe here a versatile, low-cost device for the continuous automatic recording and integrated data analysis of locomotor activity in the entrance tubes of colonies of highly eusocial bees. We compared the activity of two colonies each for two species of stingless bees and compared these to two colonies of the honey bee, all kept at the same location and recordings done during a single southern summer to early fall season. The analysis of the data in terms of actograms and Lomb-Scargle periodograms showed a predominant circadian rhythm for all three species, but also indications of ultradian rhythms. For \u003cem\u003eMelipona quadrifasciata\u003c/em\u003e, which is comparable in size to the honey bee, we found evidence for an anticipatory locomotor activity in the entrance tube already before sunrise, followed by an early morning peak of foraging, indicating an anticipatory activity driven by their endogenous clock. Hence, we believe that the low cost and the versatility of the device, as well as the open-source options for data analysis should be an attractive option for conducting studies on circadian rhythms in social bees under natural conditions, complementing studies on flower visits by these ecologically and economically important pollinators.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Dr. Eduardo Brandt de Oliveira (USP) for the help with the Arduino technology and Dr. Katharina Beer (Univ. W\u0026uuml;rzburg) for helpful suggestions in the analysis of circadian rhythms. We also thank Anderson Roberto de Souza and the staff of the Precision Manufacturing Workshop of the USP-Ribeir\u0026atilde;o Preto campus for the design and manufacturing of the registration device. Jairo de Souza and Luis Roberto Aguiar provided assistance in maintaining the stingless bee and honey bee colonies. Financial support from Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq, process 302209/2022-0) and scholarships to the first author by CNPq (PIBIC) and the Funda\u0026ccedil;\u0026atilde;o de Apoio \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP, Process 2020/05723-0) are acknowledged.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eARJ and KH conceived and designed the experiments. ARJ performed the experiments and collected and analyzed the data. ARJ and KH jointly wrote and edited the manuscript and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbou-Shaara HF (2014) The foraging behaviour of honey bees, \u003cem\u003eApis mellifera\u003c/em\u003e: A review. 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Nature 443:931\u0026ndash;949. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature05260\u003c/span\u003e\u003cspan address=\"10.1038/nature05260\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-comparative-physiology-a","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcpa","sideBox":"Learn more about [Journal of Comparative Physiology A](http://link.springer.com/journal/359)","snPcode":"359","submissionUrl":"https://submission.nature.com/new-submission/359/3","title":"Journal of Comparative Physiology A","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Activity recording, circadian rhythm, foraging, Meliponini, Apis mellifera","lastPublishedDoi":"10.21203/rs.3.rs-4201960/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4201960/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs pollinators, bees are key to maintaining the biodiversity of angiosperm plants, and for agriculture they provide a billion-dollar ecosystem service. But they also compete for resources (nectar and pollen), especially the highly social bees that live in perennial colonies. So, how do they organize their daily foraging activity? Here, we present a versatile, low-cost device for the continuous, automatic recording and data analysis of the locomotor activity in the colony-entrance tube of highly eusocial bees. Consisting of an in-house built block containing an infrared detector, the passage of bees in the colony entrance tunnel is registered and automatically recorded in an Arduino environment, together with concomitant recordings of temperature and relative humidity. With a focus on the highly diverse Neotropical stingless bees (Meliponini), we obtained 10-day consecutive recordings for two colonies each of the species \u003cem\u003eMelipona quadrifasciata\u003c/em\u003e and \u003cem\u003eFrieseomelitta varia\u003c/em\u003e, and also for the honey bee. The data were converted into CSV files, followed by the generation of actograms and Lomb-Scargle periodograms. We found a predominant circadian rhythmicity for all three species, but also indications of ultradian rhythms. For \u003cem\u003eM. quadrifasciata\u003c/em\u003e, which is comparable in size to the honey bee, we found evidence for an anticipatory activity already before sunrise, followed by an early morning peak of activity. The cost and versatility of the device and the open-source options for data analysis make this an attractive system for conducting studies on circadian rhythms in social bees under natural conditions, complementing studies on flower visits by these important pollinators.\u003c/p\u003e","manuscriptTitle":"A versatile recording device for the analysis of continuous daily external activity in colonies of highly eusocial bees","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-05 11:41:43","doi":"10.21203/rs.3.rs-4201960/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-06T08:57:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-03T08:21:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-17T17:31:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"dad44c28-9b82-41f6-90e4-78a6dad9a0a0","date":"2024-04-12T10:03:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39818635-e35a-4d4f-82ff-c4df02a3e570","date":"2024-04-03T14:51:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-03T12:28:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-02T04:09:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-02T04:09:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Comparative Physiology A","date":"2024-04-01T16:50:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-comparative-physiology-a","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcpa","sideBox":"Learn more about [Journal of Comparative Physiology A](http://link.springer.com/journal/359)","snPcode":"359","submissionUrl":"https://submission.nature.com/new-submission/359/3","title":"Journal of Comparative Physiology A","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fd428af1-6e89-41f6-ac80-b2021a93079e","owner":[],"postedDate":"April 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-21T15:56:17+00:00","versionOfRecord":{"articleIdentity":"rs-4201960","link":"https://doi.org/10.1007/s00359-024-01709-2","journal":{"identity":"journal-of-comparative-physiology-a","isVorOnly":false,"title":"Journal of Comparative Physiology A"},"publishedOn":"2024-06-20 15:56:17","publishedOnDateReadable":"June 20th, 2024"},"versionCreatedAt":"2024-04-05 11:41:43","video":"","vorDoi":"10.1007/s00359-024-01709-2","vorDoiUrl":"https://doi.org/10.1007/s00359-024-01709-2","workflowStages":[]},"version":"v1","identity":"rs-4201960","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4201960","identity":"rs-4201960","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}