Behavioural and developmental responses of a stingless ( depilis) to nest overheating Ayrton Vollet-Neto, Cristiano Menezes, Vera Lucia Imperatriz-Fonseca

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Ayrton Vollet-Neto, Cristiano Menezes, Vera Lucia Imperatriz-Fonseca. Behavioural and develop- mental responses of a (Scaptotrigona depilis) to nest overheating. Apidologie, Springer Verlag, 2015, 46 (4), pp.455-464. ￿10.1007/s13592-014-0338-6￿. ￿hal-01284459￿

HAL Id: hal-01284459 https://hal.archives-ouvertes.fr/hal-01284459 Submitted on 7 Mar 2016

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2015) 46:455–464 Original article * INRA, DIB and Springer-Verlag France, 2014 DOI: 10.1007/s13592-014-0338-6

Behavioural and developmental responses of a stingless bee (Scaptotrigona depilis ) to nest overheating

1 2 3 Ayrton VOLLET-NETO , Cristiano MENEZES , Vera Lucia IMPERATRIZ-FONSECA

1Departamento de Biologia da Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo 14040-900, 2Embrapa Amazônia Oriental, Tv. Dr. Enéas Pinheiro, s/n., Belém, Pará, Brazil 3Universidade Federal Rural do Semi-Árido, km 470-BR110, Mossoró, Rio Grande do Norte, Brazil

Received 15 April 2014 – Revised 16 November 2014 – Accepted 21 November 2014

Abstract – In this study, we investigate the behavioural response of the highly eusocial stingless bee Scaptotrigona depilis to high temperatures. We found that nest temperature in the brood area can be up to 2.5 °C lower relative to a control empty box during hot periods. We showed that high temperatures increase both water collection and wing fanning behaviour at the colony level, suggesting that these behaviours are adaptations to cooling the nest in this species. Additionally, we measured survival and development time of pupae incubated at five different constant temperatures (22, 26, 30, 34 and 38 °C). We found that the development time of S. depilis brood significantly varied between treatments and had a strong negative correlation with temperature. Brood survived over a large range of incubation temperatures; however, extreme temperatures (22 and 38 °C) were almost 100 % lethal, highlighting the importance of thermoregulation, at least in extreme conditions. social thermoregulation / nest cooling / water foraging / Scaptotrigona depilis

1. INTRODUCTION adult especially to foraging activities are af- fected even by 2 °C of difference between temper- Social bees are able to control the nest environ- atures they were raised (Tautz et al. 2003). More- ment to allow optimal performance of the adults as over, mortality and malformation increase when well as optimal development of the brood (Wilson brood is reared outside of a very narrow range of 1971; Jones and Oldroyd 2007). In particular, so- optimal temperatures (Himmer 1927; Heinrich cial bees are sensitive to, and invest considerable 1993; Mardan and Kevan 2002) effort in controlling, the temperature within the Nest temperature can be increased through nest. Honeybees (Apis mellifera ) are very effective metabolic heating generated by the shivering of at controlling their internal nest temperature. The flight muscles in bumblebees (, workers can perform quite well over a wide range , Bombini) and honeybees (Hymenoptera, of temperatures, but the brood is very sensitive to Apidae, Apini) (Jones and Oldroyd 2007). These temperature fluctuations. The cognition ability of groups also have the ability to cool their nests. The honeybee is able to maintain the internal nest temperature within a narrow range of 33 to Electronic supplementary material The online version of this article (doi:10.1007/s13592-014-0338-6) contains 36 °C, even when the external temperatures are supplementary material, which is available to authorized higher than 40 °C (Heinrich 1993). To accomplish users. this, workers fan their wings to create an air flow Corresponding author: A. Vollet-Neto, between the inside and the outside of the nest. [email protected] This behaviour is combined with water collection, Manuscript editor: Peter Rosenkranz where small droplets of water are deposited on 456 A. Vollet-Neto et al. surfaces on the inside of the nest. This reduces the that it plays a role in the cooling of the nest temperature of the nest through evaporative because the deposition of water droplets was nev- cooling (Lindauer 1954). In Bombus , only fan- er observed inside colonies and because cavity ning behaviour has been reported and is correlated nesting is considered to provide a thermally stable with an increase in nest temperature (Jones and environment (Engels et al. 1995). On the other Oldroyd 2007). hand, Macías-Macías et al. (2011), working with Stingless bees (Hymenoptera, Apidae, individual workers of colimana ,found Meliponini) are the largest and most diverse group that high temperatures led to an increase in water of highly eusocial bees (Michener 2000). They are consumption, and that workers regurgitated the restricted to tropical and subtropical habitats and water on the bottom of the boxes. The authors are, therefore, often exposed to high temperatures. suggested that this individual behaviour could be However, there is no evidence that they perform used at the colony level to decrease the tempera- behaviours to actively cool the nest (Jones and ture of the nest. Oldroyd 2007). Research shows that the internal In preliminary observations, we noted that nest temperature differs from ambient tempera- Scaptotrigona depilis can maintain relatively sta- ture, but most authors attribute this to the behav- ble temperature inside the nest during periods of iour of nesting in insulated cavities and assume extreme heat. This is in line with the observations that this is the main way by which stingless bees of Engels et al. (1995), showing that colonies of avoid nest temperature rising to damaging levels , a closely-related species, during hot periods (Engels et al. 1995; Nogueira- are able to maintain the temperature of the brood Neto 1997; Jones and Oldroyd 2007). On the area around 40 °C when exposed to high temper- other hand, wing fanning has been described in atures (close to 44 °C). Given the scattered, but several species in this group, leading some re- suggestive, evidence for active cooling of the nest searchers to suggest that this might be a cooling environment in stingless bees, we performed an behaviour (Nogueira-Neto 1948; Macías-Macías experimental study to test the hypothesis that et al. 2011). However, this hypothesis has yet to S. depilis has the ability to actively cool the nest be tested. Engels et al. (1995), working with environment. We addressed two main questions: Scaptotrigona postica , observed that colonies First, to what extent is S. depilis able to cool the experiencing high temperatures had elevated nest when exposed to high temperatures? Second, numbers of bees performing wing fanning. are there behavioural mechanisms for nest Macías-Macías et al. (2011) demonstrated that cooling? To answer the second question, we workers of Melipona colimana kept individually analysed two candidate behaviours, wing fanning in boxes inside incubators perform wing fanning and water collection. We also investigated the behaviour when exposed to temperatures of effects of temperature on the pupae survival and 40 °C. However, Moritz and Crewe (1988)point- development time. ed out that wing fanning plays a role in air circu- lation inside the nest, regulating the CO2 and O2 2. MATERIAL AND METHODS levels inside the nest cavities of the ground nesting species denotii . Nogueira-Neto 2.1. Study species and study site (1948) also reports the occurrence of fanning be- haviour in stingless bee colonies during cold win- The study was performed on the Ribeirão Preto ter days, suggesting that this behaviour is related campus of São Paulo University, SP, Brazil, during to gas exchange, rather than temperature summer time (from December 2011 to March regulation. 2012—maximum temperature of 40 °C), where There are a few reports of water collection in Scaptotrigona depilis is a common stingless bee spe- stingless bee species (Nogueira-Neto 1997; cies. This species was chosen because many aspects of Macías-Macías et al. 2011). However, the authors its biology have been described in the literature, includ- associate this behaviour with individual ingestion ing some responses to high temperature, such as wing of water and minerals and reject the hypothesis fanning behaviour (Engels et al. 1995). Colonies were Response of a stingless bee to nest overheating 457 kept outdoors, unshaded, in white wooden boxes cov- abdomens, their crop content was collected with ered by tiles under ambient conditions of light and a graduated capillary tube (Fig. S1D). The sugar temperature (Fig. S1A). Colonies usually contain sev- content of their crop content was then eral thousand workers and one, singly mated queen analysed using a digital refractometer (Krüss (Paxton et al. 2003). Optronic—Alemanha—DR201-95). Only the bees that contained at least 7 μLofliquidwere analysed. This is the minimum sample volume 2.2. Experimental setup required by our refractometer for accurate mea- surement of the solution’s concentration. Concen- a) Ability to cool the nest trations with less than 2 % of sugar were consid- To investigate the bees’ ability to cool the nest ered to be “water” (Leonhardt et al. 2007). during high temperature conditions, temperature c) Fanning behaviour probes (15 length×5 diameter mm; SENSIRION, To determine whether wing fanning behaviour oc- model SHT75; precision: 0.3 °C) were introduced curs as temperature increases, we monitored three into the hives through lateral holes (15-mm diam- colonies as described above. A transparent plastic eter). In each colony, the temperature of the brood tube was attached to the entrance (12 cm area (T BROOD) was monitored (Fig. S1A). The data length×2.5 cm diameter) to extend the entrance were collected and stored at intervals of 5 min and tube (where the workers position themselves to 24 h a day. Five colonies were monitored. Another perform fanning behaviours) and to allow counting empty wooden box with the same characteristics as of individual workers fanning their wings. This the one used for keeping the colonies was moni- behaviour is very characteristic, with the workers tored by a temperature probe (held by a piece of standing on the substrate (in this case, the plastic wood that prevented contact with the floor of the tube), forming a queue, with the posterior legs box) and placed in the same conditions as the stretched, and fanning their wings (Fig. S1B and experimental hives. This box, lacking bees but S1E). under the same environmental conditions, served Six bioassays were conducted in which the tem- as a control for the conditions the bee colonies perature of the boxes was gradually increased, were submitted to (T CONTROL). The ambient tem- using one 15 Watts incandescent lamp positioned perature was also recorded using a sensor placed in under the hives (2 cm below the box). After the shade, close to the hives (T AMB). All boxes 45 min, the lamps were turned off. After that, the were exposed to the sunlight, which produced high number of workers fanning in the plastic tube was

temperature conditions (>40 °C in the T CONTROL recorded every 5 min, until there were no more during the hottest time of the day), and conditions bees fanning. The bioassays were performed from were similar in all hives. The data used in the 11:00 to 15:00 hours. Temperature data were sam- analysis comprised 3 days in which the tempera- pled as described earlier. ture conditions were hot enough. d) Effect of temperature on pupae mortality and de- b) Water collection behaviour velopment time To test whether water collection behaviour is per- The bioassay consisted of incubating brood formed when the temperature increases, we per- combs containing pupae at five different tem- formed further observations on two of the colonies peratures: 22, 26, 30, 34 and 38 °C (BOD mentioned above. The colonies had a transparent incubator). Towards the end of normal pupal plastic tube attached to the entrance (15-cm developmental time, we observed the brood length×2.5-cm diameter), which had a small win- combs daily to check for emergence of dow (3×2 cm) that allowed the capture of foragers adults. We noted individual mortality (pupae as they returned to the nest (Fig. S1C). Every hour, were considered dead if they failed to emerge (from 08:00 to 17:00 hours) three foragers with or other characteristics of death, such as fun- neither nor resin in their corbiculae were gal growth, were observed) and, for the sur- collected from each colony. These foragers were viving pupae, the time until spontaneous

anaesthetized with CO2, and by pressing their emergence of the bees. We used 50 pupae 458 A. Vollet-Neto et al.

from five different colonies in each treatment. temperatures.. The relationship between temperature To ascertain the age of the pupae, we and development time was analysed using a Kruskal- uncapped 60 brood cells that contained larvae Wallis one-way ANOVA on Ranks test (Zuur 2009). in pre-pupae stages, which were adjacent to the pupae. As in this species, the brood comb is built as a disc, with new cells being added 3. RESULTS from the centre to the margins, and the colo- nies produce more than 200 cells per day, a) Nest cooling “ ” almost all the pre-pupae which neighbour pu- Cooling periods (when T BROOD was pae pupated the next day. This day was con- lower than T CONTROL) lasted about 5 h sidered day zero (Fig. S2). As we opened 60 (306.6±83.9 min), from approximately – cells, we removed the necessary amount of 10:00 14:00 hours. This was the time of individuals to reach 50 female pupae in each highest environmental temperature comb. The removal was performed when the (Figure 1). pupae reached the stage of black eyes, which These results suggest that there is a mech- allowed us to remove male pupae. The brood anism for cooling the nest during warm combs were placed inside a petri dish con- periods, while during the rest of the day, taining saturated solution of water and salt the brood was maintained at a higher tem- (NaCl) to maintain the humidity close to perature than the control (Figure 1; 75 %, which has previously been showed to Table S1). The peaks of maximum differ- be the ideal condition for in vitro queen ences between T CONTROL and T BROOD rearing on this species (Menezes et al. 2013). reached 1.6(±0.2)°C (averaged over all the colonies studied and all sampling days). The average maximum cooling 2.3. Data analysis achieved by the colonies was 2.5 (±0.28)°C for only one of the days sam- All analyses were performed in R 2.15.0 (R Devel- pled. The T AMB was considerably lower opment Core Team 2011). Cooling of the nest was than the T CONTROL during the cooling pe- considered to occur if the brood temperature riods (Figure 1).

(T BROOD) was lower than the control temperature b) Water collection (T CONTROL). The temperatures in this category About 20 % of the foragers (47 of 231) in (T BROOD

35

30

25

T. ambient T. control 20 T. brood

0120120120 Time (hours)

Figure 1. Temperature of the brood area (T BROOD—open dots showing mean and bars showing standard deviation) from the five colonies monitored, the control box (T CONTROL thick line ), and ambient (TAMB thin line ) during three non-consecutive days. The cooling periods (when T CONTROL is higher than average T BROOD) are highlighted (grey areas ).

extreme temperatures, 22 and 38 °C, showed Development time showed significant varia- high levels of mortality, 100 % and 96.6 %, tion among treatments (H =675.1; P <0.001). respectively (Figure 4; Table S4) (one-way Non-significantly different values of devel- ANOVA: F =519.43; P <0.001–Holm-Sidak: opment time were found only between 34 P <0.05). and 38 °C (Figure 4; Table S4). There was a Water collection probability Water 0.0 0.2 0.4 0.6 0.8 1.0

25 30 35 40 Brood temperature (°C)

Figure 2. The relationship between water collection and brood temperature. The fitted values (solid line ) represent the probability of a forager collecting water relative to the brood temperature (T BROOD) obtained by the binomial GLM analysis (slope=0.55±0.09, P <0.001). The dots are the observed values (1 represent water collection). 460 A. Vollet-Neto et al.

Slope = 3.9739 Adj R² = 0.48, p < 0.001 10 15 20 25 30 35 Number of fanning bees Number of fanning 5 0

30 32 34 36 Brood temperature (°C)

Figure 3. Scatterplot showing the effect of brood temperature on the number of fanning bees. The line represents the best-fit line and was drawn based on the parameter estimates obtained from the linear regressions (adjusted R 2=0.48, F =158.8, P <0.001).

40 a 35 30 25 b 20 c c 15 Time (days)

abbba 100

75

50

25 Survival (%)

0 22 26 30 34 38

Figure 4. Survival rate (bars ) and development time (boxplots )ofS. depilis pupae incubated at five different temperature. Bars , grey sections represent the percentage of survival, while the black sections represent percentage mortality. Boxplots , median (thick line ), upper and lower quartiles (upper and lower limits of the boxes), 95 % of data distribution (stems ) and the values out of the 95 % data distribution (ouliers ) are represented. Different superscribed letters indicate groups statistically different (ANOVA and Kruskal-Wallis associate with Dunn’stest,P <0.001). Response of a stingless bee to nest overheating 461

strong negative correlation between incuba- Our results suggest that S. depilis exhibit at tion temperature and development time least two active mechanisms which may play a (spearman rank correlation=−0.95). The role in the cooling of the nest: (i) fanning behav- mean time of development halved from 26 iour and (ii) water collection. Both behaviours are to 34 °C. present in the behavioural repertoire of the re- sponse of bees to high temperatures de- scribed by Lindauer (1954). 4. DISCUSSION Water collection for cooling purposes has up until now only been described in the Apini tribe Our results show that S. depilis brood nest and in several species of social (Jones and temperature is colder than the empty control box Oldroyd 2007). Here, we provide the first evi- during the hottest part of the day. Although the dence of water collection for the purpose of nest difference between T BROOD and T CONTROL might cooling in stingless bees. In Apini and wasps, the seem to show only moderate cooling, it has been mechanism of cooling seems to be similar: the suggested that even small increases in temperature water is deposited in small droplets on the sur- during very hot periods can be highly damaging to facesofthenest,orthesurfaceislicked,andasthe brood (Undurraga and Stephen 1980; Mardan and water evaporates, it lowers the temperature of the Kevan 2002; Tautz et al. 2003), as our data also nest (Lindauer 1954; Simpson 1961; Nicolson showed for extreme temperatures (“Results” (d)). 2009). Despite previous observations of water This damage is thought to mostly occur due to collection in Meliponini, this has not been linked disruption of cellular physiological processes. Bees to the cooling of the nest, but to nutritional needs and most other seem to be much more (hydration and minerals). Such a link was proba- robust to cold temperatures (Angilletta 2009). bly not previously made because the deposition of Although the data we collected indicate that water droplets or licking behaviour has not been active mechanisms of fanning and water collec- observed before (Jones and Oldroyd 2007). As in tion could be related to nest temperature decrease, honeybees, our results show that high ambient we cannot rule out the possibility that nest struc- temperatures are linked to water collection tures and simple presence of the bees are not (Lindauer 1954), but the mechanism underlying influencing the nest temperature oscillations, the process of cooling in stingless bees is still since they were not present in the empty control unknown. It is likely that the water is deposited box. The brood, adult bees, wax structures, honey on internal nest surfaces or evaporated through and pollen pots could result in thermal inertia, licking by the workers, but this awaits investiga- decreasing the rate of temperature rise in nests. tion. There is the possibility of the adults using However, both the adult workers and the brood this water to decrease their own body temperature, produce metabolic heat (Roubik and Peralta 1983; as has been described in honeybees (Heinrich Jones and Oldroyd 2007), which could have the 1979;Cooperetal.1985). This would nonetheless opposite effect, increasing the nest temperature be a way of decreasing the nest temperature. Our and make active cooling more difficult. Therefore, results suggest a causal link between nest temper- while further investigations are necessary to fully ature and water collection behaviour, rather than a understand the mechanisms that decrease nest coincidental occurrence of water collection at the temperature in stingless bees, our data strongly time of highest ambient temperature, because suggest that active behavioural efforts play a role stingless bee foraging activity for other resources in nest thermoregulation. It is noteworthy that (nectar, pollen or resin) normally decreases during T AMB is lower than T CONTROL during the hottest extremely high temperatures (Silva et al. 2011; period of the day, highlighting the necessity of a Hilário et al. 2012; Figueiredo-Mecca et al. control box when studying the thermoregulation 2013). The same phenomenon was observed in of social that nest in cavities. Such a con- large bees, such as Bombus terrestris (Kwon and trol is absent in all other studies on nest cooling by Saeed 2003). Water foragers may be able to toler- social insects. ate foraging at higher temperatures by using 462 A. Vollet-Neto et al. evaporative cooling from their bodies (Prange temperature treatments which showed the fastest 1996; Pereboom and Biesmeijer 2003). brood development and highest survival (30 and We also confirm previous suggestions (Engels 34 °C). Extreme temperatures were found to be et al. 1995; Nogueira-Neto 1997; Jones and very damaging to the brood, highlighting the im- Oldroyd 2007) that the wing fanning behaviour portance of temperature control. A change of even is indeed a colony-level response to an increase in 1 °C could make the difference between survival nest temperature and, therefore, is likely to be or death. However, it is important to note that performed to cool the nest. The higher the tem- these temperatures were held constant in our de- perature inside the nest, the more bees start to fan velopment bioassay, but temperatures are not con- their wings in the entrance tube. We also observed stant in nature. The impact of short episodes of that this behaviour occurs inside the nest, in re- extremes temperatures on brood development gions not connected to the entrance. It is important would benefit from investigation. Even 1 h of to note that this behaviour is also a response to exposure to temperatures of 50 °C caused a mor- high levels of CO2 inside the nest in other social tality of 100 % in pupae and pre-pupae of the bees, and is used to improve gas exchange in the solitary bee Megachile rotundata (Hymenoptera, nest (Simpson 1961; Moritz and Crewe 1988; Megachilidae), while an exposure of several hours Weidenmuller et al. 2002). Indeed, high CO2 to 45 °C did not significantly increased mortality levels could also be the ultimate cause of the (Undurraga and Stephen 1980). The development ventilation, since increased metabolic rates during time of Osmia bicornis (Hymenoptera, high temperature periods could increase CO2 Megachilidae) brood was also decreased when levels. This variable should be included in further the temperatures fluctuated, as compared to con- studies to conclusively determine whether fanning ditions of constant temperatures based on the av- is a response to overheating per se, or to an in- erage of the fluctuations (Radmacher and Strohm crease in CO2 concentrations caused by 2011). overheating. Lastly, another possibility is that the In honeybees, a variation of 5 to 6 °C from the fanning behaviour could have a role of decreasing 33 °C optimal temperature for brood development humidity inside the nest by facilitating the air greatly increases brood mortality and exchange between the brood area and the outside. malformations (Himmer 1927; Mardan and Such an integration between ventilation and evap- Kevan 2002), while in S. depilis , mortality does orative cooling would be particularly important not change over a range of at least 8 °C. However, when the ambient air temperature exceeds the the development of S. depilis is considerably upper critical temperature. When this limit is slower than that of A. mellifera . The minimal reached, non-evaporative heat loss is progressive- development time from pupae to emergence of ly reduced because the air temperature is higher S. depilis is around 15.7 and 16.2 days, at 38 than the temperature inside the nest, causing sim- and 34 °C, respectively. In honey bees, the closed ple ventilation itself to be ineffective. This hy- cell phase (prepupal and pupal) development time pothesis also remains to be tested. is around 9.5 to 12 days (Michener 1974). We The effects of temperature on brood develop- suggest that the precise temperature control ment time varied greatly between intermediate achieved by honeybees when compared to temperature treatments (26, 30 and 34 °C; Fig- S. depilis has led to adaptation to a narrow range ure 4). However, while we found a small positive of temperatures. The stingless bees species which correlation between an increase in temperature are poorer thermoregulators would be better and pupae survival, this correlation was not sig- adapted to a wider range of temperatures, leading nificant. However, the extreme temperatures (22 to slower development, but one that is more robust and 38 °C) increased brood mortality (Figure 4). to temperature fluctuations (Angilletta 2009). This Our data showed that the T BROOD was on average still remains to be thoroughly tested, especially 30.5±2.9 °C (mean±s.d.) over the whole day and considering the difference in individual sizes and 34±1.4 °C (mean±s.d.) during the cooling periods. weight between S. depilis and A. mellifera .Sting- These temperatures are most similar to the two less bee species are good candidates to test this Response of a stingless bee to nest overheating 463 hypothesis, since many different thermoregulato- REFERENCES ry strategies are present among closely related species. 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