Journal of Economic Entomology, XX(XX), 2019, 1–4

doi: 10.1093/jee/toy420
Short Communication Short Communication

Scientific Note: Warming Nurses, a New Worker Role

Recorded for the First Time in Stingless Bees

Downloaded from https://academic.oup.com/jee/advance-article-abstract/doi/10.1093/jee/toy420/5304344 by guest on 31 January 2019

Yara S. Roldão-Sbordoni,1,3, Guilherme Gomes,2 Sidnei Mateus,1 and
Fábio S. Nascimento1

1
Laboratório de Comportamento e Ecologia de Insetos Sociais, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto. Av.
Bandeirantes, 3900. Universidade de São Paulo, Ribeirão Preto, Brasil, 2Laboratório de Neurobiofísica, Instituto de Física de São
Carlos. Av. Trabalhador São-carlense, 400. Universidade de São Paulo, São Carlos, Brasil And

Corresponding author, e-mail: yara.roldao@hotmail.com

3

Subject Editor: John Trumble

Received 6 August 2018; Editorial decision 27 October 2018

Abstract
Nest temperature is a crucial variable that determines colony survival in social insects. The successful pro-
duction and development of a new brood, therefore, depends on stable thermal conditions and limited tem-
perature oscillations. Thermoregulatory processes are useful in controlling both individual activity and for the
maintenance of colony temperature. We measured heat production generated by nurse bees working on brood
combs of the stingless bee Melipona scutellaris (Hymenoptera, Apidae, Meliponini) in this study; our results
enabled us to identify the existence of a new task performed by nurse bees, referred to here as ‘hot bees’
because of their higher thorax surface temperature (4°C above that of other bees within the brood area). This
additional heat has been little studied in stingless bees but is likely the result of thorax muscle contractions or,
indeed, the development of this musculature as these are recently emerged individuals. We hypothesize that
these ‘hot bees’ contribute to the maintenance of warmth within the nest brood area.

Key words: stingless bees, thermoregulation, hot bees, thermography

The developmental success of immature insects under natural condi-
tions is largely controlled by temperature (Liu et al. 1995). The hon-
eybee, Apis mellifera Linnaeus (Hymenoptera: Apidae), is probably
the best-studied of all insect species in this regard because colonies
are able to rigidly maintain their brooding area temperature at levels
between 34 and 36°C because of active heat production or cooling
mechanisms, respectively (Lindauer 1954; Kronenberg and Heller
1982; Pacheco and Kerr 1989; Heinrich 1993; Stabentheiner et al.
2003, 2010). In contrast with endothermic animals, honeybee colo-
nies are able to do this even at ambient temperatures far below freez-
ing, or well above 40°C. Previous research studies have shown that
the temperature of stingless bee nests is maintained by specific struc-
tures, including the involucrum that involves the brood area and
may be a structural heat retention adaptation (Zucchi and Sakagami
1972, Roubik and Peralta 1983, Jones and Oldroyd 2007).
In their earlier work, Jones and Oldroyd (2007) noted that the
most important factor that enables bee colonies to maintain constant
brood area temperatures is an appropriate nest location choice. The
thermal insulation provided by trees or the ground, microhabitats
usually chosen by stingless bees to build their nests, usually helps
to decrease environmental temperature oscillations and results
in a more stable environment inside cavities (Wille and Michener
1973, Sedgeley 2001, Wiebe 2001, Willis and Brigham 2007). Torres
et al. (2007) recorded a series of stingless bee (Tetragonisca angus-
tula) nest temperatures and noted a mean value of 30.3°C within
the brood area (i.e., inside the involucrum) compared with 19.6°C
within pollen and nectar storage areas (i.e., outside the involucrum).
These results suggest that the brood area comprises the thermal core
within stingless bee nests and heat loss is mitigated by concentric
involucrum cerumen layers. It has also long been speculated that
these layers surrounding the brood retain heat within this region and
act passively as thermoregulatory surfaces (Nogueira-Neto 1948,
Kerr et al. 1967, Jones and Oldroyd 2007, Torres et al. 2007).
Honeybees are also characterized by the use of physiological
thermoregulation, a phenomenon that is usually associated with high
metabolic activities such as flight. In this process, the thorax tends
to reach greater temperatures during flight than other body parts,
up to around 1.6°C warmer than either the head or the abdomen
(Harrison 1987, Roberts and Harrison 1998, Roberts et al. 1998,
Kovac et al. 2009). Increasing heat production rates at decreasing

© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. 1
All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
2 Journal of Economic Entomology, 2019, Vol. XX, No. XX
temperatures inside the nest emphasize both nonectothermic behav-
ior and active thermoregulation within this biological system (Torres
et al. 2009).
In another earlier study, Contrera and Nieh (2007) related ambi-
ent temperature effects with the thoracic temperature and forager
sound production to show that these bees have to reach a minimum
flight muscle temperature before take-off; this result means that for-
ager sounds can be linked to muscle warm-up. Prior to this, Nieh
and Sánchez (2005) had utilized infrared thermography to report
that Melipona panamica (Hymenoptera, Apidae) foragers elevate
their thoracic temperature when they reach useful food sources with
higher sucrose concentrations closer to the nest.
Body surface temperature has also been used to understand ther-
moregulatory mechanisms inside honeybee nests (Kleinhenz et al.
2003, Stabentheiner et al. 2010). No previous studies have so far
used stingless bees as a model, however, even though these insects
employ different mechanisms to control nest temperatures. Thoracic
surface temperatures measured in A. mellifera carnica (Hymenoptera,
Apidae) revealed values between 30.7 and 43.0°C, a mean varia-
tion across a 6°C range (Kleinhenz et al. 2003). Stabentheiner et al.
(2010) then subsequently related thoracic surface temperature to the
age and location of bees within a colony; these workers suggested
that endothermic bees are able to regulate brood comb temperature
more accurately than local air temperature. These bees apply heat
as close to the brood as possible; thus, workers heating cells from
within have a higher probability of endothermy than those on the
comb surface. Our hypothesis is, therefore, that hot nurse bees are
responsible for heat production within the brood area in stingless
species. So, the aim of the present study was to report heat produc-
tion by nurse bees of the stingless bee Melipona scutellaris working
on the brood combs.
Materials and Methods
The experiments reported here were performed at the Laboratório
de Comportamento e Ecologia de Insetos Sociais, Campus of the
University of São Paulo at Ribeirão Preto, Brazil. We used a M.
scutellaris colony that was connected to the outside world via plastic
tubes through which individuals could freely exit and enter the
nest. These bees collected pollen and nectar freely as they received
no artificial feeding. The M. scutellaris nest contained an estimated
population of between approximately 600 and 800 individuals
(Nogueira-Neto 1997).
Stingless bees are known to perform various tasks throughout
their lives, one of which is to nurse within the brood. Newly emerged
workers were, therefore, marked in this study with Acrilex paint in
order to record their activities, especially when they became nurses
(generally nurse bees assume this function between 14 and 20 d after
emergence). As we marked 115 individuals every day over an 11 d
period, age was known in each case. We also later observed both the
nest and marked bees; we used an infrared camera (SC-640, FLIR
Systems) to record thermal images within the brood area when the
latter began to exhibit care activities. We recorded thorax surface
temperatures for bees on the top comb only; temperatures from
combs below were not recorded so as to not disturb the structure
of the brood area and interfere with experiments even though bees
were present within these structures.
We used mean thorax temperature recorded over 2 d for bees
in general (i.e., cold bees) and observed and recorded nurse bees
with higher temperatures. Images were recorded at room tempera-
ture (i.e., an ambient mean temperature of 27.5°C) where bees were
located, and all data were analyzed using the software Thermacam
2.9 (Flir Systems). The infrared camera used in this experiment is
calibrated every 3 yr in an accredited laboratory at Flir Brasil in the
city of Sorocaba; this is the only accredited calibration laboratory in
Latin America. An inspection and calibration test involves 14 points
(see: https://www.flir.com/support-center/Instruments/service/infra-
red-camera-calibration/); during the test process, real surface tem-
perature is correlated with different black bodies to obtain a curve
that associates radiation intensity with temperature and, therefore,
Downloaded from https://academic.oup.com/jee/advance-article-abstract/doi/10.1093/jee/toy420/5304344 by guest on 31 January 2019
calibrating equipment according to required ISO 9001: 2008 stand-
ards (November 2013) for nondestructive tests.
The data reported in this study were also obtained using ther-
mocouples associated to known emissivity areas (i.e., insulation
tape, e = 0.95) close to individual images. These thermocouples
were inserted into individuals to obtain emissivity values as well as
to compare with the calibration performed in the FLIR accredited
laboratory. These calibrations, alongside correlations with data
obtained from areas with known emissivity, were then associated to
the thermocouple; this enabled us to obtain real object temperatures
and also mitigate future problems with experimental replication and
variance. Differences between individual images highlight variation
in thermogenesis between nurses and the other individuals observed
under the same conditions. This allowed us to observe differences in
radiation intensity and meant that we could also use the laboratory
calibration to obtain real individual temperatures.
Hot nurse bees (marked bees) were then captured and dissected;
this was done to verify whether, or not, these bees had developed
ovaries characteristic of nurses. We used a Stereo microscope (Wild
M5A) for our dissection protocol, following the method outlined by
Noll et al. (1997), and used a Canon EOS 7D camera to take pic-
tures. All experiments were carried out between 12 December 2013
and 13 November 2014.
Results and Discussion
The thermal images collected here reveal that some bees working in
the brood area had higher thoracic surface temperatures (i.e., 35°C,
a ‘hot bee’) than their counterparts performing other tasks nearby
(i.e., 31°C, a ‘cold bee’). The brood area mean temperature was also
between 28 and 30°C; our thermal images, therefore, indicate that
‘cold bees’ maintained their individual body temperatures at the lev-
els necessary for their activities (Fig. 1 and Supp Video 1 [online
only]).
On the basis of the division of labor previously observed in some
Meliponini bee species (Sakagami 1982, Cepeda 2006, Ferreira-
Caliman et al. 2010, dos Santos et al. 2010), bees between 13 and
18 d old are usually involved in reproductive and brood care tasks,
both nursing functions. In other words, these individuals perform
activities that are directly related with the brood area. In this case,
such nurse bees are referred to as ‘hot bees’ because we have been
able to show that they increase their thorax surface temperatures to
up to 4°C higher than their counterparts performing another task in
the brood area or nearby. We counted a total of 49 ‘hot bees’ over a
period of 6 h (counting was performed every 15 min) on the comb;
the mean proportion of ‘hot bees’ compared with the total under the
comb was 11.2 ± 5.8% (Fig. 2).
We observed nurse bees working within the brood area at ages
between 14 and 16 d. Indeed, of the 16 marked nurse bees observed
with thermal images with thorax temperatures greater than their
counterparts, 14 also had ovaries developed (Fig. 3). According to
the previous research of Sakagami (1982), Giannini (1997), Cepeda
Journal of Economic Entomology, 2019, Vol. XX, No. XX 3

Downloaded from https://academic.oup.com/jee/advance-article-abstract/doi/10.1093/jee/toy420/5304344 by guest on 31 January 2019

Fig. 1. Thermal image showing bees within the brood area with thorax surface temperature greater (around 35°C—hot bees) than others (31°C—cold bees).
These bees were 15 d old and showed a specialized function with activities directly related with the brood area.

30.0
Percentage of hot bees (%)

25.0
20.0
15.0
10.0
5.0
0.0
9:45 10:15 10:45 11:15 11:45 12:15 12:45 13:15 13:45 14:15 14:45 15:15 15:45 16:15
Time of day (hour:min)

Fig. 2. Proportion of hot nurse bees working on the brood area (on the top comb) in a 6 h period with respect to cold bees. It may be noted that the proportion
reached 25% at 11:00 a.m., a considerable amount of the total of bees that worked in the comb at that moment.

Fig. 3. Dissected nurse specialized bees showing ovary activation (dissection of the abdomen). Cold nurse bee (thorax surface temperature between 28 and
31°C) (A) and hot bee (thorax surface temperature around 35°C) with 15 d old that performed the function of nurses (brood care) (B).

(2006), dos Santos et al. (2010), and Corrêa-Gomes et al. (2015),
bees with developed ovaries that also work within the comb area
have been classified within the task age called ‘nurse bees’ and fre-
quently lay eggs during cell treatment and queen oviposition pro-
cesses. Cepeda (2006) also noted that heavy workers (i.e., those with
more fat tissue) retain activated ovaries and participate in functions
related to the brood area. It is also the case that stingless bees live for
60 d on average and so nurse bees aged between 13 and 18 d tend
to be very young. These individuals possess well-developed ovaries
as well as glands that regurgitate food within cells and produce wax
(Justino et al. 2018).
In earlier work, Adade and Cruz-Landim (2004) investigated
flight muscle aging in newly emerged, nurse, and forager bees within
the genus Apis. These workers found that the muscle fibers of nurse
bees are larger in diameter than those of newly emerged and for-
ager bees; at the same time, nurse bees also possess larger amounts
of stored glycogen when compared with the other two categories.
These results indicate that nurse bees have the highest individual cap-
acity for thermogenesis and contribute most to thermal homeostasis
within the colony. Adade and Cruz-Landim (2004) also showed that
these bees exhibit higher activity levels and have a greater capacity to
generate metabolic heat by contracting their thorax muscles.
4 Journal of Economic Entomology, 2019, Vol. XX, No. XX
The results of this study show for the first time that a group of
bees are responsible for heat production within the brood area in
a stingless species; the fact that such ‘hot bees’ were also seen over
consecutive days further indicates that they performed this func-
tion while nursing. We, therefore, suggest that bees of this age that
perform this task (hot bees) are able to maintain higher thoracic
temperatures. This heat production, combined with the fact that the
involucrum surrounds brood combs, may help to maintain elevated
temperatures in the brood area and thereby assists in incubation and
the development of immature individuals. The results of this study
are also important to our understanding of bee colony survival as the
brood area is the principal component of the nest and the develop-
ment of immature individuals depends on temperature.
Acknowledgments
We appreciate the loan of the infrared camera to us by Prof. Dr. Denis Otávio
Vieira de Andrade of the Laboratório de Fisiologia Comparada of UNESP
(Rio Claro - Centro de Aquicultura - Jacarezário). We also acknowledge an
English review performed by Prof. Dr. Wilfried Klein and Sam Morris as well
as financial support from Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP).
References Cited
Adade, C. M., and C. Cruz-Landim. 2004. Diferenciação e envelhecimento
do músculo do vôo em operárias de Scaptotrigona postica Latreille
(Hymenoptera, Apidae). Revista Brasileira de Zoologia. 21: 379–384.
Cepeda, O. I. 2006. Division of labor during brood production in stingless
bees with special reference to individual participation. Apidologie. 37:
175–190.
Contrera, F. A. L., and J. C. Nieh. 2007. The effect of ambient temperature on
forager sound production and thoracic temperature in the stingless bee,
Melipona panamica. Behav. Ecol. Sociobiol. 61: 887–897.
Corrêa-Gomes, R. L., C. Menezes, and F. A. L. Contrera. 2015. Worker lon-
gevity in an Amazonian Melipona (Apidae, Meliponini) species: effects of
season and age at foraging onset. Apidologie. 46: 133–143.
Ferreira-Caliman, M. J., F. S. Nascimento, I. C. Turatti, S. Mateus, N. P. Lopes,
and R. Zucchi. 2010. The cuticular hydrocarbons profiles in the stingless
bee Melipona marginata reflect task-related differences. J. Insect Physiol.
56: 800–804.
Giannini, K. M. 1997. Labor division in Melipona compressipes fasciculata
Smith (Hymenoptera: Apidae: Meliponinae). An. Soc. Entomol. Brasil. 26:
153–162.
Harrison, J. M. 1987. Roles of individual honeybee workers and drones in
colonial thermogenesis. J. Exp. Biol. 129: 53–61.
Heinrich, B. 1993. The hot-blooded insects: strategies and mechanisms of
thermoregulation. Harvard University Press, Cambridge, MA.
Jones, J. C., and B. P. Oldroyd. 2007. Nest thermoregulation in social insects.
Adv. Insect Physiol. 33: 153–191.
Justino, C. E. L., F. B. Noll, S. Mateus, and J. Billen. 2018. Wax gland size
according to worker age in Friesella schrottkyi. Apidologie. 49: 359.
Kerr, W. E., S. F. Sakagami, R. Zucchi, V. P. De Araujo, and J. M. F. Camargo.
1967. Observações sobre a arquitetura dos ninhos e comportamento
de algumas espécies de abelhas sem ferrão das vizinhanças de Manaus,
Amazonas (Hymenoptera: Apoidea). Atas. Simp. Biota Amazônica. 5:
255–309.
Kleinhenz, M., B. Bujok, S. Fuchs, and J. Tautz. 2003. Hot bees in empty
broodnest cells: heating from within. J. Exp. Biol. 206: 4217–4231.
Kovac, H., A. Stabentheiner, and R. Brodschneider. 2009. Contribution of
honeybee drones of different age to colonial thermoregulation. Apidologie.
40: 82–95.
Kronenberg, F., and H. C. Heller. 1982. Colonial thermoregulation in honey
bees (Apis mellifera). J. Comp. Physiol. 148: 65–76.
Lindauer, M. 1954. Temperaturregulierung und wasserhaushalt im bienen-
staat. Zeitschrift für vergieichende. Physiologie. 36: 391–432.
Liu, S. S., G. M. Zhang, and J. Zhu. 1995. Influence of temperature variations
on rate of development in insects: analyses of case studies from entomo-
logical literature. An. Entomol. Soc. Am. 88: 107–119.
Downloaded from https://academic.oup.com/jee/advance-article-abstract/doi/10.1093/jee/toy420/5304344 by guest on 31 January 2019
Nieh, J. C., and D. Sánchez. 2005. Effect of food quality, distance and height
on thoracic temperature in the stingless bee Melipona panamica. J. Exp.
Biol. 208: 3933–3943.
Nogueira-Neto, P. 1948. Notas bionômicas sobre meliponíneos I sobre a ven-
tilação dos ninhos e as construções com ela relacionadas. Rev. Bras. Biol.
8: 465–488.
Nogueira-Neto, P. 1997. Vida e criação de abelhas indígenas sem ferrão, pp.
445. Nogueirapis, São Paulo.
Noll, F. B., R. Zucchi, and S. Mateus. 1997. Morphological caste differences in
the neotropical swarm-founding and polygynous polistine wasp, Polybia
scutellaris. Stud. Neotrop. Fauna Environ. 32: 76–80.
Pacheco, R. L. F., and W. E. Kerr. 1989. Temperaturas em abelhas da espécie
Melipona compressipes fasciculata. Ciênc. Cult. 41: 490–495.
Roberts, S. P., and J. F. Harrison. 1998. Mechanisms of thermoregulation in
flying bees. Amer. Zool. 38: 492–502.
Roberts, S. P., J. F. Harrison, and N. F. Hadley. 1998. Mechanisms of thermal
balance in flying Centris pallida (Hymenoptera: Anthophoridae). J. Exp.
Biol. 201: 2321–2331.
Roubik, D. W., and F. J. A. Peralta. 1983. Thermodynamics in nests of two
Melipona species in Brazil. ACTA Amazôn. 13: 453–466.
Sakagami, S. F. 1982. Stingless bees, pp. 361–423. In H. R. Herman (ed.),
Social insects, vol. III. Academic Press, London.
dos Santos, C. G., B. Blochtein, F. L. Megiolaro, and V. L. Imperatriz-Fonseca.
2010. Age polyethism in Plebeia emerina (Friese) (Hymenoptera: Apidae)
colonies related to propolis handling. Neotrop. Entomol. 39: 691–696.
Sedgeley, J. A. 2001. Quality of cavity microclimate as a factor influencing
selection of maternity roosts by a tree-dwelling bat, Chalinolobus tubercu-
latus, in New Zealand. J. Appl. Ecol. 38: 425–438.
Stabentheiner, A., H. Pressl, T. Papst, N. Hrassnigg, and K. Crailsheim. 2003.
Endothermic heat production in honeybee winter clusters. J. Exp. Biol.
206: 353–358.
Stabentheiner, A., H. Kovac, and R. Brodschneider. 2010. Honeybee colony
thermoregulation—regulatory mechanisms and contribution of individu-
als in dependence on age, location and thermal stress. PLoS One. 5: 1–13.
Torres, A., W. Hoffmann, and I. Lamprecht. 2007. Thermal investigations
of a nest of the stingless bee Tetragonisca angustula Illiger in Colombia.
Thermochimica Acta. 458: 118–123.
Torres, A., W. Hoffmann, and I. Lamprecht. 2009. Thermal investigations of a
nest of the stingless bee Trigona (frieseomelitta) nigra paupera provancher
in Colombia. J. Therm. Anal. Calorim. 95: 737–741.
Wiebe, K. L. 2001. Microclimate of tree cavity nests: is it important for repro-
ductive success in northern flickers? Auk. 118: 412–421.
Wille, A., and C. D. Michener. 1973. The nest architecture of stingless bees
with special reference to those of Costa Rica (Hymenoptera: Apidae). Rev.
Biol. Trop. 21: 1–278.
Willis, C. K. R., and R. M. Brigham. 2007. Social thermoregulation exerts
more influence than microclimate on forest roost preferences by a cavity-
dwelling bat. Behav. Ecol. Sociobio. 62: 97–108.
Zucchi, R., and S. F. Sakagami. 1972. Capacidade termo-reguladora em Trigona
spinipes e em algumas espécies de abelhas sem ferrão (Hymenoptera:
Apidae: Meliponinae), pp. 301–309. In R. Zucchi and S. F. Sakagami
(eds.), Homenagem a Warwick E. Kerr. Rio Claro, São Paulo, Brazil.