Behav Ecol Sociobiol (2002) 51:415–425 DOI 10.1007/s00265-002-0457-3
O R I G I N A L A RT I C L E
T. Schmickl · K. Crailsheim
How honeybees (Apis mellifera L.) change their broodcare behaviour in response to non-foraging conditions and poor pollen conditions
Received: 28 June 2001 / Revised: 18 December 2001 / Accepted: 15 January 2002 / Published online: 14 March 2002 © Springer-Verlag 2002
Abstract We observed the nursing of larvae during all 5 days of larval development. We caged a queen in a specific area of empty combs inside the broodnest and filmed nursing episodes within this area. We created 5-day observation periods with and without artificial rain, as well as periods with and without manual reduction of pollen stores and reduction of pollen income. In rain periods, there were significantly fewer nursing episodes for young larvae (1–3 days old) than in no-rain periods. The nursing frequency was significantly correlated with the amount of pollen in the hive, as well as with the total amount of unsealed larvae. The ratio of available pollen to larvae had the strongest influence on the nursing frequency: the more pollen available per larva, the higher the nursing frequency of young larvae. Higher nursing frequency, as well as a longer total duration of nursing episodes, resulted in a higher protein content of the larvae. In contrast, the frequency of nursing of older larvae (4 days old) did not depend on the amount of pollen or on the ratio of pollen to larvae, even after some days of severe pollen reduction. The amount of honey stores and the weight of the hive were not correlated with the nursing frequency of any larval age group. When pollen becomes scarce, older larvae receive preferential treatment. They represent a considerable investment for the colony. From an economic point of view, it is important for the colony that they reach the “safe” final capping stage. Keywords Honeybees · Broodcare · Nutrition · Weather · Pollen supply
Communicated by T. Seeley/T. Czeschlik T. Schmickl · K. Crailsheim (✉) Department of Zoology, Karl-Franzens-University, Universitätsplatz 2, 8010 Graz, Austria e-mail:
[email protected] Tel.: +43-316-3805616, Fax: +43-316-3809875
Introduction Compared to the offspring of most other insect species – which can vary markedly in developmental speed and time due to changes in temperature and nutrition (Ratte 1985; Tsukada 1994; Gilbert and Raworth 1996; Shibata 1998) – the offspring of most eusocial insects are much more dependent on stable nutrition and care at a certain temperature. This is especially true for the larvae of honeybees, which have very inflexible environmental and nutritional demands. One important factor that allows eusocial insects to achieve such stable conditions for the large amount of brood they usually raise was the development of division of labour and, sometimes, task partitioning (Ratnieks and Anderson 1999). Colony members are separated into castes of reproductives and workers, and the workers are separated into temporal castes such as foragers, food storers, nurses, etc. by age polyethism, lifetime polyethism or polymorphism (Wheeler and Wheeler 1979; Sudd 1982; Buschinger 1990; Calderone 1998; Page and Peng 2001). But the need for intracolonial homoeostasis confronts colonies with a further problem: foraging intensity has to be adjusted to the current storage situation and brood demand, while reproduction and nursing are determined by nutritional supply and seasonal population dynamics. Therefore honeybees have developed several feedback mechanisms that regulate recruiting, foraging, food storage, comb construction, brood production and nursing (Fewell and Winston 1992; Ratnieks and Anderson 1999). This allows the colony to react very flexibly and sensitively to changes in demand (Robinson et al. 1992; Huang and Robinson 1996). Although a single larva is not very flexible in its developmental needs, the whole brood is controlled by mechanisms such as adjustment of the oviposition rate (Bodenheimer 1937) or cannibalism (Woyke 1977; Weiss 1984), according to seasonal and ecological parameters. Honeybees collect and store honey, which provides the main carbohydrate source, and pollen, which offers mainly proteins. For adjusting foraging intensity according
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to the colony’s actual demands (Al-Tikrity et al. 1972; Winston and Fergusson 1986), information about the food storage state and the brood state can be shared via interadult feedings (Camazine et al. 1998), pheromones (Pankiw et al. 1998) or individual inspections (Dreller and Tarpy 2000). The state of the brood plays an important role in the honeybees’ pollen foraging and storage strategy. Honeybee larvae are especially dependent on proteins, because they gain mass very quickly (Wang 1965). The nursing of brood is usually carried out by a group of young adult “nurse bees” (Haydak 1963) which feed the larvae with a product of the hypopharyngeal and mandibular glands called jelly (Haydak 1970; Crailsheim 1990b) and sometimes feed them honey and pollen directly (cf. Crailsheim 1998). Both the behavioural patterns of these feeding episodes (Beetsma 1985; Brouwers et al. 1987) and the composition of the larval food (Haydak 1970; Brouwers 1984) change as a larva ages. Additional factors determining the nursing a larva receives are the total number of nurses, their level of activity, the age and the sex of the larva (Lindauer 1952) and the current hunger status of the larva (Huang and Otis 1991). Many studies have revealed the dependence of the brood on the pollen supply. Pollen stored near the broodnest has a higher attraction to nurse bees than pollen stored further away (Doull 1974) and the rate of pollen consumption by young and middle-aged adult bees correlates with brood levels (Hrassnigg and Crailsheim 1998). Barker (1971) showed that when colonies are deprived of pollen to a variable extent, there is a correlation between pollen collection and the amount of reared brood afterwards. Also, the total amount of brood in the hive is known to have an impact on the rate of raised progeny per adult bee in later brood cycles, and on the physiological fitness of the progeny and the nurses (Eischen et al. 1983). While the adjustment of foraging to colony needs has been studied intensively in recent decades, little is known about the regulation of nursing according to the colony’s needs and restrictions. Foraging behaviour has often been studied in connection with brood and foodstore manipulations, but there have been no previous studies of nursing behaviour in response to restrictions on foraging. Bad weather or bad pollen supply in the foraging area can soon bring a colony into a situation where there is not enough pollen available for all pollen consumers. One reason for this is the usually small amount of stored pollen (compared to the amount of honey stores) in a honeybee colony. If such periods of pollen shortage occur repeatedly, the effects can still be measured some generations later (Dustmann and Ohe 1988). But even under conditions of very poor pollen supply, the bees somehow manage to continue nursing brood at a level that (in most cases) enables the colony to survive (Wille 1984), whereas without any available pollen, bees can raise brood only for a very short time (Haydak 1935). Dietz and Stephenson (1975) showed that only colonies
that have access to a certain amount of fresh pollen are able to perform successful brood rearing. The main objective of our study was to investigate the impact of weather and pollen shortage on the treatment of larvae of different ages.
Methods General methods The experiments were performed in the summers of 1998 and 1999 in Graz, Austria. We observed the nursing episodes of larvae at five different stages (days 1–5 after hatching of the egg) by filming them. We generated cohorts of eggs of known and almost similar age in the broodnests of observation hives. To test the effect of weather and resource supply on nursing behaviour, we produced artificial rain (experiment 1, done with one colony in one hive) and created a poor pollen supply through manual manipulations (experiment 2, done with another colony in another hive). To obtain cohorts of similarly aged eggs, we confined a queen in a 5 cm×5 cm area inside the broodnest for 2 h. Before the caging of the queen, all eggs and larvae were removed from this area and the worker bees – but not the queen – were allowed to enter the space to clean and prepare the brood cells. We could thus generate larvae that were almost exactly (±2 h) 1, 2, 3, 4 or 5 days old, designated as ages “L1d” to “L5d”. After the 2-h confinement, the queen was placed outside the cage, but the cage remained in place and excluded the queen for three additional days. Workers could still enter the area and (possibly) lay eggs there, but worker-laid eggs are normally removed by “worker policing” (Ratnieks 1993) and they develop only into drones. We only collected data from female worker larvae that survived until the final capping event; therefore we can disregard the problem of replacing queen-laid eggs by worker-laid ones. Successive cohorts of eggs were produced on different combs, by alternating the side of the observation hive on which the queen was caged. To decrease disturbance of the hive, we used several small glass windows instead of one large one, so that we had access to a small part of the comb without opening the whole hive. These manipulations were always done after video-recording (which ended around 1400 hours) and under red-light conditions. We counted a larva as being 1 day old (L1d) on the 4th day after the laying of the egg, because the egg stage lasts 3 days. We timed the caging of the queen so that we always had L1d larvae when observation started on the 1st day of our observation turns. This way the ages of larvae were always the same as the day numbers in our observation turns. Each day we recorded the survival of larvae and eggs by drawing a “map of larvae” of our observation area on transparency media through the glass windows of the observation hives. For recording the larval nursing episodes, we filmed the observation area with a video camera under red-light conditions each day for 2 h starting around 12 noon. We call one (2 h) observation session of one larva on the video an “observation”. The whole development period of one cohort (5 days), we call an “observation turn”. After each observation turn we chose six larvae that were successfully capped. Viewing the video, we recorded all episodes of brood nursing that these particular larvae had received. From these data we calculated the nursing frequency, the total nursing period per hour and the mean duration of nursing episodes within each single observation. An adult bee was classified as nursing a larva when she was sticking her head and at least part of her thorax into a larva’s cell for more than 2 s and less than 3 min. According to Lindauer (1952), short cell inspections (without intensive contact with the larva) usually last 2–3 s whereas a normal feeding lasts for up to 3 min. He also noticed “nurses resting in larval cells” which lasted much longer than 3 min. We did not classify either of these behaviours as nursing activities. While “cell inspections” were observed very frequently, “resting in cell” was observed very rarely and only in cells containing young
417 larvae and mostly during rain periods (data not shown). Also, Brouwers et al. (1987) reported that most feeding episodes are shorter than 180 s. Temperature was recorded by thermoelements inside and outside the hive, and the natural weather conditions outside were also recorded. We prevented thermoregulation problems by heating the room that contained the observation hives throughout the nights. Experiment 1: artificial rain In this experiment we used an 8-frame observation hive housing a colony of about 13,000 bees. In front of the hive’s entrance, we installed a “rain-machine” (Riessberger and Crailsheim 1997; Blaschon et al. 1999) which simulated continuous heavy rainfall by showering the area in front of the entrance, which prevented all bees from leaving the hive. The cooled water from the rain machine additionally caused a decrease in temperature in the entire entrance area (to 9–11°C), and we also shaded the hive entrance, so that the bees accepted the artificial rain as “real” and did not try to leave the hive during the artificial-rain periods. The “rain machine” does not affect barometric pressure. We made each artificial rain period last for 5 days, followed by 6 days without artificial rain. Thus, we experimentally prevented any pollen or nectar foraging for five continuous days (=“rain period”), and then gave the bees a 6-day period (=“no-rain period”) to restore the pollen and nectar levels. Although the no-rain period lasted for 6 days (to allow the bees to refill the pollen stores before the next rain period started), we only filmed the first 5 days of larval development in both types of periods. To satisfy the bees’ daily need to defecate outside the hive, during rain periods, we turned off the rain for 1 h in the evening, when the sun was already down and the bees could not collect significant amounts of pollen or nectar. To obtain information about the changes in pollen stores, we mapped the pollen cells in the whole observation hive on transparent sheets each evening. We calculated the amount of pollen in the hive at 12 noon as the mean value of the totals from the hive maps made on the observation day and the day before the observation. We also estimated the levels of honey stores. We repeated this rain/no-rain cycle several times. We present here the results only from those periods that finally led to at least six capped larvae in our observation area. We noticed a loss of larvae due to cannibalism in all of our observation periods (Schmickl and Crailsheim 2001). There were two rain periods where at least six larvae survived to the capping stage. We made 120 observations in these 2 rain/no-rain cycles. Each video observation of a larva lasted 2 h. Experiment 2: manual pollen reduction In a second experiment, we concentrated on how changes in the amounts of pollen stores and pollen income affect brood nursing behaviour. Therefore we manipulated these two parameters without any artificially produced changes in weather and without preventing the bees from leaving the hive. We used a 3-frame observation hive housing a colony of about 7,000 bees and used a “queen excluder” to keep the queen off the lowest frame and ensure that it contained no brood. A queen excluder is a barrier that only the worker bees (which are smaller than the queen) can pass through. It has the side effect that bees that return from foraging trips do not like to carry their pollen loads through it, so almost all pollen stores were on the lowest frame of the hive. Therefore, by taking out the lowest frame and replacing it with a pollenless one, we greatly reduced the amount of available pollen on day 1 of each observation period. During the following days, the pollen stores increased again. To prevent the bees from restoring the pollen stores too quickly, we placed a pollen trap in front of the hive entrance. We had three observation periods with and three periods without the pollen reduction and the pollen trap. As in experiment 1, each observation period with pollen reduction lasted for 5 days, and the periods without reduction lasted 6 days
each. In this experiment, the time between the hatching of the egg and the capping of the larval cell varied markedly. In some of our test periods the larvae were already capped or were being prepared for capping at observation time on day 5. This pre-capping time is known to be associated with a change in nursing behaviour (Lindauer 1952), so we decided to consider only the nursing on days 1–4 of larval development in our results. We used a hive balance that recorded the total weight of the hive every 2 h automatically throughout each observation turn. We counted the number of bees in our observation area five times per observation period to monitor changes in the number of adult bees in the broodnest. The broodnest temperature was recorded three times a day: in the morning, at 12 noon and in the evening. We also mapped all cells in the hive on transparent sheets. We classified each cell by its contents: pollen, unsealed honey, sealed honey, egg, young larva (=1–3 days old), old larva (more than 3 days old), capped brood or empty. In these “whole hive maps”, we estimated the age of each larva by its size, as the size of a larva is crucial in estimating its pollen needs. In total we observed 36 larvae, each for 4 stages (days) of larval development, for a total of 144 two-hour observations. We defined the following indices to quantify the pollen situation and the brood situation in the hive. The “pollen index” (PI) This index quantifies the current pollen situation, taking into consideration the pollen level from the previous day, because the nurse bees need time to process pollen into the jelly they feed to larvae (Crailsheim 1990a, b). (1) Equation 1. The “pollen index” (PI) is the mean number of pollen cells on the observation day and on the previous day. The “pollen-change index” (PCI) This index describes how the pollen level changed between the day before the observation and the observation day, to test for an effect of the rate of change of pollen income on the nurses’ behaviour. A negative value indicates a reduction in pollen stores, while a positive value means that the amount of pollen stores was increasing. (2) Equation 2. The “pollen-change index” (PCI) expresses the pollen gain or pollen loss on the observation day compared to the day before the observation. The “larvae index” (LI) This index is a measure of the larval pollen demand, taking into account the numbers of young larvae (L1d–L3d) and old larvae (L4d–L5d). Because old larvae have higher protein (pollen) needs than young larvae, the number of old larvae is multiplied by a weighting factor, k. We used k=20, because 20 is the mean ratio between the weight of older larvae and the weight of young larvae (Wang 1965). Recalculating Wang’s data, we found almost the same relative mass gain (in percent) per 24 h for young and for old larvae. Our assumption of an approximately linear relation between larval mass and nutritional needs is strengthened by the findings of Lindauer (1952), who showed that the gain of mass through larval development is proportional to the feeding time received by the larvae. Also, the presence of older larvae leads to a greater increase in pollen-foraging frequency (Hellmich and Rothenbuhler 1986), and pollen is consumed more frequently near old larvae than near younger ones (Taber 1973), which points to a higher protein need of older larvae.
418 (3) Equation 3. The “larvae index” (LI) quantifies the total larval demand for pollen, taking into account that old larvae have a higher need for protein than young larvae. Based on data of Wang (1965), we used 20 as the value for k. The “pollen-larvae index” (PLI) This index describes how well the hive’s current pollen supply meets its current pollen demand. A value of PLI=1 means that there are 20 filled pollen cells available for each older larva, and 1 filled pollen cell for each young larva, throughout the preceding 24 h. (4) Equation 4. The “pollen-larvae index” (PLI) expresses how well the current pollen situation meets the pollen demands of the current amount of brood.
Fig. 1 Mean areas of filled pollen cells in the hive during rain and no-rain periods (n–2 rain periods and 2 no-rain periods). The amount of pollen stores decreased in rain periods until the 1st day of the no-rain period, and then increased again. The 1st day of a rain period still has large areas filled with pollen
Physical condition of larvae Starting on the 5th day of larval development during different weather conditions, we filmed the brood area to obtain the exact time of all cell-capping events. We collected each larva 24 h after cell capping, weighed it, and analysed its protein content using the Lowry method (Lowry et al. 1951). A total of 97 larvae were collected and analysed in this way. Statistics We used two-tailed Mann-Whitney U-tests to test for significant differences between two groups of data. We used the Spearman rank correlation when one of the parameters was ordinal (as for most of the indices described above); otherwise we used Pearson correlations. In testing the dependence of nursing frequencies on the indices defined above, we adjusted α to 0.01 (multiple test correction).
Results Experiment 1: the influence of rain on brood nursing In rain periods, the pollen stores steadily decreased, and in no-rain periods the pollen stores increased again, as shown in Fig. 1. We found significant differences between rain and no-rain periods in the nursing frequency of larvae aged 1–3 days (Fig. 2a shows all five observed ages). One-day-old larvae were nursed more often (n1=12, n2=12, z=–3.71, P
