Original article

Do bicolor (, Meliponinae) workers distinguish relatedness among different physogastric queens?

Wladimir J. Alonso Tarcila Lucena Carla M. Fracasso Hayo H.W. Velthuis Vera Lucia Imperatriz-Fonseca

Departamento de Ecologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Travessa 14, no. 321 CEP: 05508-900, Cidade Universitária, São Paulo,

(Received 2 January 1998; accepted 8 July 1998)

Abstract - The prediction that the polygynous workers should dis- tinguish physogastric queens to whom they have different degrees of relatedness was tested. One colony with two physogastric queens was monitored for 5 consecutive days for the occurrence of the provision and oviposition processes (POP) of individually marked workers. We recorded cell provisioning, egg laying, and cell sealing. The presence of one or both of the queens during each POP was recorded. After that, one of the queens from the colony used in the first experiment was removed from the colony and substituted by two other non-related physogastric queens, from other colonies. In both cases preferences by workers towards their mother or aunt or to the unrelated queens were not detected. In a third experiment we demonstrated that guard bees at entrance of a colony do not reject a physogastric foreign queen. Evolutionary and husbandry implications are discussed. © Inra/DIB/AGIB/Elsevier, Paris kin recognition / polygyny / provision and oviposition processes / Melipona bicolor

1. INTRODUCTION cial , nestmates with different degrees of relatedness are often found; this is due After Hamilton’s publications [10] it is to polyandry, to polygyny and the meeting now widely accepted that an individual of different generations (sisters, nieces, etc.) increases its overall fitness by helping to in the same colony. rear the offspring of a relative. It is expected that this help should be preferentially In honeybees, polyandry brings about directed toward individuals that share a great important varieties of intra-colonial relat- degree of relatedness with the actor. In euso- edness. In experiments where some evidence

* Correspondence and reprints E-mail: [email protected] of worker discrimination based on degrees important component of their diet [29, 32], of relatedness was found the results have and are dependent on the cell provisioning been questioned because: A) the preference by the workers. These actions happen in the for full sisters may be an artefact of the use provisioning and oviposition process (POP) of heritable phenotypic markers to distin- in a highly stereotyped fashion [28, 29, 38]. sub-families; task guish B) specialisation In the present study we tested the ten- as observations on the same (such repeated dency of workers to behave differently was not controlled; bee) among lineages C) towards physogastric queens to which they statistical and no blind obser- faulty analyses have different degrees of relatedness, one vations were used; and/or D) the genetic of them being their mother, the other being of the was low diversity colony artificially possibly an aunt (exp. 1) or being a foreign because were insem- queens experimentally queen (exp. 2). Additionally it was tested inated with the of two or three sperm only whether the system of foreign queen recog- mate with males, although they usually nition could occur at the entrance of the seven to 17 males [1, 4, 6, 15, 24, 25, 34, colony (exp. 3). Evolutionary and husbandry 35]. We can add that, even so, the discrim- implications are discussed. ination found could be merely a by-product of nestmate recognition [5, 6, 8, 9, 31] To add empirical data to this discussion 2. METHODS into account these criticisms, we taking per- 2.1. Experiment 1 formed experiments that did not involve artificial lineages, used marked bees (if dis- For this experiment, a polygynous colony crimination occurs), and chose a stingless originating from Cunha (São Paulo State, Brazil) bee that has a low level of naturally was chosen and maintained in the Bee Laboratory relatedness variability. Furthermore, the crit- of the Ecology Department of the São Paulo Uni- icism that the discrimination could be a by- versity. It was kept in an observation hive (mod- product or a kind of nestmate recognition ified from [27]) at a constant temperature (27-29 °C). Bees were allowed to out- can be partially overcome. This, if we deal forage side, but sometimes the was also with situations of discrimination that colony pro- hardly vided syrup (sugar with water 1:1) and dehy- could be directed to individuals of other drated . Observations were made using red colonies instead of nestmates (e.g. brood light. This colony had two physogastric queens care), but involve individuals with differ- (Q1, Q2) who were seen in the colony for the ent degrees of relatedness into the colony. first time in October 1996 - and who were prob- ably sisters. The queens were marked with Melipona bicolor Lepeletier is a facul- coloured dots of paint on their thorax. tatively polygynous species [2, 16, 30] For 1 month before the beginning of the which is abundant in the Brazilian Atlantic behavioural observations, we removed the lower Rain forest [26, 36]. Since queens are taught combs (which are, for this species, the older ones) and them in a hive extension to mate only once [17-19, 30], the polygy- placed (15 x 10 x 6 cm) connected to the main nest nous condition allows us to assess the inter- by a short tube (6 cm). In this hive extension, nurse actions individuals with different among bees could enter and help new bees to emerge. levels of relatedness. The highly eusocial These young bees, which could be identified by condition (as defined by Michener [22]) pro- their pale integument, were collected daily (also vides us with a repertoire of behaviours that 505during the days of behavioural observations), with numbered seem unlikely to be confounded with those provided individually paper tags onto the thorax and returned to the behaviours that can be directed towards indi- pasted colony. For 5 consecutive and (15-20 viduals of other colonies. In days nights January fact, stingless 1997) all POPs were monitored, and the indi- bees have massive provisioning, and queens viduals that had taken part in the following cat- often eat worker-laid eggs, which are an egories (or states) were recorded. Workers: cell provisioning prior to oviposi- introduced, which quickly began to participate. tion; egg laying prior and after queen oviposi- For this reason the first day of our recording is not tion; cell sealing after queen oviposition. considered for the present data analysis. The introduced and are neither Queens: presence in front of the cell during queens Q3 Q4 daugh- ters nor sisters of All the other methods for the POP process; egg laying. Q2. the monitoring session (31 June-5 July) and data The behaviours the workers performed by analysis are as described for experiment 1. were scored in relation to the queen(s) present, but cell closure behaviour was scored in relation to the queen that oviposited. In order to assess 2.3. Experiment 3 a possible preference toward one queen or the other, the total number of behaviours performed Between 21 and 25 July, eight physo- by each worker to each queen was recorded. A July were removed from their score of one was attributed when the behaviour gastric queens colony, in a tube 3 cm, diameter was to one queen. In cases where transported (length performed only 1.3 and at the entrance both were in the POP the score cm), immediately placed queens present of another chosen was divided in two halves, one half for each randomly queenright colony. In order to enter the colony, the queens had to queen. Only those workers that had been workers a tube of 40-50 cm observed in four or more POPs were pass along transparent participating in and 1.3 in diameter that connected the chosen for Repetitions of the cell pro- length analysis. brood nest with the exterior. The tube is behaviour the same worker in the long visioning by to allow the and bees same POP were not considered. We assumed enough guards foraging (the most aggressive ones) to meet and reject an that each one of these behavioural categories has intruder. Furthermore, this artificial tube is con- the same ’altruistic value’, and therefore are sidered the workers as an internal equally computed. by being part of the nest. This is indicated by the fact that the The possibility that workers stimulated by the guards bees always stay defending its exterior presence of one queen were not inhibited by the extremity (which is closed by the bees with of the second was also considered. presence with the exception of a small hole which allows in a second those POPs Therefore, analysis, only the passage of one bee at a time). in which only one queen was present (and again with workers that had been observed participat- A successful introduction was considered when, at least 3 h after the introduction, the for- ing in four or more POPs) were considered, and was observed on the comb, in again it was tested whether the workers had any eign queen being preference. apparent good condition and without experienc- ing any aggression from the workers. After this To test worker towards preference any queen period each queen was reintroduced into the we used the log-likelihood ratio statistics (G test), colony of origin. which may be used instead of chi-square when n is not large [37]. In order to test the null hypoth- esis (no preference of workers toward any queen), each worker was supposed to perform behaviours 3. RESULTS to each queen in a ratio similar to the ratio of the number of the POPs in which each queen was 3.1. Experiment 1 present. A total of 84 POP sequences were recorded the 5 of the 2.2. Experiment 2 during days experi- ment. Of these, 20 with Q1, 37 with Q2 and 31 with both in the POP From the beginning of June 1997, we again queens present (the some of the POPs marked the emerged bees, for a period of over sum is not 88 because 1 month. Both queens (Q1 and Q2) were taken had changes regarding the queen present). from the colony just before starting POP moni- A total of 154 marked workers were toring. Queen Q3 from a different colony which, recorded participating in the POPs. Of these, with was intro- together Q2 (control treatment), 104 workers participated in at least four duced as a substitute for one of the queens (Q1). POPs and thus could be used for Q3 was immediately accepted, but had a low studying participation in the POPs. Therefore, after 1 day the degree to which preference for a partic- of observations, another queen Q4, was also ular queen occurs (figure 1). If the colony were to consist of two sep- result is the same (figure 2). Again prefer- arate groups of workers, each group sup- ence toward anyone of the queens was not porting mainly their own mother queen, then detected (log-likelihood ratio G-test = 41.5, this figure should have shown a distinct sep- df = 39, P = 0.36). aration of the workers. This is not the case. Instead, there is a gradual change from 3.2. Experiment 2 workers having a high ratio of performing behaviour in favour of queen 1 into a high A total of 79 POP sequences were ratio in favour of queen 2. This gradual recorded during the 4 days of the experi- change reflects random differences rather ment. Of these, 24 with Q1, 25 with Q3 or than a distinct choice by individual bees Q4, and 43 with two or three queens pre- (Log likelihood ratio G-test = 42.2, df = 52, sent in the POP (the sum is not 79 because p = 0.83). some of the POPs had changes regarding the queen present). A total of 131 marked If we restrict the analysis to those POPs workers were recorded participating in the in which only one queen was present, the POPs. Of these, 74 participated in at least four POPs and thus could be used for the court, but the workers did not attack her, analysis of preference (figure 3). and the queen was able to reach the brood nest and remained there without the distribution of the POP activ- being Again, harassed. ities of these bees does not show a popula- tional segregation. Instead, it agrees well 4. DISCUSSION with a random distribution of participation in the POPs, irrespective of whether the In both situations, exp. 1, when two sis- familiar or the foreign queen oviposited (log- ter queens were present, and exp. 2, when likelihood ratio G-test = 21.8, df = 36, one of the queens had been replaced by an P = 0.97). If we restrict the analysis to those unrelated queen, we were unable to detect POPs where only one queen was present any kind of preference from the workers (figure 4), we arrive at the same conclusion toward one of the queens - at least regarding ratio G-test = 24.0, df = 26, (log-likelihood the behavioural and P = 0.57). categories frequencies observed. These results have implications both for evolutionary interpretations and 3.3. Experiment 3 with respect to the husbandry field.

All the queens introduced at the entrance 4.1. Evolutionary implications of the colony were accepted in the colony. Each introduced queen was surrounded by If there is a possibility to discriminate several workers in the tube making a kind of queens on the basis of genetic cues, it is not being used in an absolute sense by the work- ination during the POP in polygynous ers present in the colony. We can then colonies, or that they lack the possibilities to deduce that workers either do not enhance do so, in the sense that: "The social behaviour their inclusive fitness through kin discrim- of a species evolves in such a way that in each distinct behaviour-evoking situation nomic management of this species. Other the individual will seem to value his neigh- unpublished data confirm it. In fact, till now bours’ fitness against his own according to this has been a current method of colony the coefficients of relationship appropriate to management - e.g. to strengthen weak ones that situation" [10]. In a stingless bee, (see also [33]). Our results also agree with M. compressipes [23] (and for other bees other experiments that revealed the easiness see references therein), it was also verified of exchanging queens in Meliponinae that workers do not interfere in the fight colonies [20, 23] - and even among colonies between a related resident queen and an of different species (reviewed by [23]). introduced unrelated one, accepting the win- ner.

If we consider queen-worker recogni- ACKNOWLEDGEMENTS tion not as a mechanism to discriminate relatedness among the same colony mem- We thank Cynthia Schuck-Paim, Euphly bers, but as a mechanism to avoid the enter- Jalles-Filho, Jorge R. Lima, Dick Koedam and ing of foreign queens in order to parasite the anonymous referees for their helpful com- the colony, we, in fact, should not expect ments. CAPES (33021163) and CNPq workers to discriminate among physogas- (521776/96-1 and 521178/95-9) supported this work. tric queens, because in Meliponinae these queens are unable to fly and thus unable to a but see parasite foreign colony ([22] [13]). Résumé - Les ouvrières de bico- a is Melipona Entering foreign colony only possible lor faire la distinction entre for young queens before or after the mat- peuvent-elles le de de diverses reines However, this aspect of queen degré parenté ing flight. La théorie de la sélection behaviour has not been studied yet. physogastriques ? de la parentèle prédit qu’un individu peut We also showed (exp. 3) that when a parvenir à l’adéquation adaptative (fitness) is introduced at the physogastric queen au moyen de la reproduction indirecte en entrance of a foreign colony, there is no aidant l’élevage des descendants d’un parent the workers contrast with aggression by (in et l’on s’attend à ce que cette aide soit diri- the work- strong rejection experienced by gée de préférence vers les individus qui par- ers of the same when Melipona species they tagent un fort degré de parenté avec l’acteur. are introduced into a strange colony [3, 12, M. bicolor est la seule espèce fortement At this it does not seem to be a 14]). stage eusociale connue qui présente une polygy- mechanism which workers discriminate by nie facultative de longue durée. Nous avons among their own and foreign queens. Prob- testé la tendance éventuelle des ouvrières à ably the recognition and acceptance of a faire la distinction entre des reines physo- a role when fecun- queen only plays newly gastriques qui leur sont plus ou moins appa- dated queens are involved. However this rentées. Une colonie possédant deux reines acceptance is a long process that already physogastriques, qui s’étaient accouplées begins before the virgin queen has taken quatre mois plus tôt et étaient probablement flight [7, 11, 21, 30]. soeurs, a été suivie pendant cinq jours et cinq nuits de suite du point de vue des proces- sus d’approvisionnement et de ponte (POPs). 4.2. Beekeping Nous avons enregistré les types de com- portement suivants d’ouvrières marquées Because the interchange of physogastric individuellement : aprovisionnement de la queens between colonies is not affected by cellule avant la ponte, ponte avant et après la discriminative behaviours of workers, this ponte de la reine, obturation de la cellule provides us with a useful tool for the eco- après la ponte de la reine. Afin d’évaluer l’éventuelle préférence de ces ouvrières mar- les ouvrières présentes dans la colonie. Nous quées pour l’une ou l’autre reine, nous avons pouvons en déduire soit que les ouvrières enregistré le nombre total de comportement n’améliorent pas leur adéquation adaptative effectués par chaque ouvrière vis-à-vis de globale par la discrimination de parentèle chaque reine. La note 1 a été attribuée à la au cours des POPs dans les colonies poly- reine lorsque le comportement n’était effec- gynes, soit que les possibilités de le faire tué que vis-à-vis d’une reine, et la note un leur font défaut. Avantages pour l’apicul- demi à chaque reine, lorsque les deux reines ture : puisque les reines physogastriques étaient présentes au cours du POP. Seules peuvent être interchangées entre colonies les ouvrières que nous avons vu participer à sans que cela ne perturbe les comportements quatre POPs au moins ont été retenues pour discriminatifs des ouvrières, nous disposons l’analyse (figure 1). La possibilité que les là d’un outil utile pour gérer cette espèce ouvrières aient pu être stimulées par la pré- sur le plan économique. Nos résultats sont en sence d’une reine et n’aient pas été inhibées accord avec d’autres expériences qui ont par la présence de l’autre a été prise en montré la facilité d’échanger les reines entre considération. Aussi dans une autre analyse, colonies de Meliponinae - même entre colo- seuls les POPs au cours desquels une seule nies d’espèces différentes. © Inra/DIB/ reine était présente, ont été retenus et nous AGIB/Elsevier, Paris avons à nouveau testé l’éventuelle préfé- rence des ouvrières (figure 2). Dans les deux Melipona bicolor / reconnaissance cas, les ouvrières ayant réparti leurs activi- parentèle / polygynie / processus tés au hasard sur les cellules fréquentées par d’approvisionnement et de ponte les reines, nous en concluons qu’elles n’ont l’une ou l’autre des pas de préférence pour Zusammenfassung - Erkennen Arbeite- reines. Par ailleurs, nous avons retiré de la rinnen von Melipona bicolor ihren Ver- colonie utilisée 1 l’une pour l’expérience wandtschaftsgrad zu verschiedenen phy- des reines et l’avons remplacé par deux sogastrischen Königinnen ? Nach der Kin reines physogastriques non apparentées, pré- selection’ Theorie kann ein Individuum auch levées dans d’autres colonies. Nous avons durch nicht-eigene Vermehrung einen Fit- le suivi des et refait la répété comportements neßgewinn erreichen, indem es bei der Auf- de là encore nous même analyse données ; zucht von Nachkommen eines Verwandten n’avons trouvé aucune des préférence hilft. Hierbei ist zu erwarten, daß diese Hilfe ouvrières, que la reine leur soit apparentée vorzugsweise den in hohem Maße mit dem ou non (fïgures 3 et 4). Dans une troisième Helfer verwandten Tieren zugute kommt. expérience nous avons recherché si les gar- Melipona bicolor ist die einzige bekannte diennes à l’entrée de la colonie rejetaient hoch eusoziale Bienenart, bei der für lange les reines physogastriques étrangères. Pour Zeit mehrere Königinnen zusammen in cela huit reines physogastriques ont été reti- einem Nest vorkommen. Es wurde unter- rées de leur colonie et immédiatement pla- sucht, ob eine Tendenz der Arbeiterinnen cées à l’entrée d’une colonie choisie au besteht, auf Grund eines unterschiedlichen hasard et possédant une reine. L’introduction Verwandtschaftsgrades zu den physogas- a été considérée comme réussie lorsqu’au trischen (eierlegenden) Königinnen zwi- moins trois heures plus tard la reine pou- schen diesen zu differenzieren. Ein Volk vait être observée sur le rayon apparemment mit zwei physogastrischen Königinnen, die en bonne santé. Toutes les reines introduites wahrscheinlich Schwestern waren und die ont été acceptées. Implications sur le plan sich beide 4 Monate zuvor gepaart hatten, de l’évolution : si la possibilité existe de wurde an 5 einander folgenden Tagen und discriminer les reines d’après des informa- Nächten in Bezug auf Zellvorräte und Eiab- tions génétiques, elle n’est pas utilisée par lage überwacht (POP). Wir notierten fol- gende Verhaltenskategorien von individu- eine Königin auf der Wabe mindestens 3 ell markierten Arbeiterinnen: Anlegen der Stunden nach dem Zusetzen auf einer Wabe Zellvorräte vor der Eilage, Eilage vor und beobachtet wurde. Alle Königinnen wur- nach dem Eierlegen durch die Königin und den angenommen. Evolutionäre Schluß- Zellverdeckelung nach der Eilage durch die folgerungen: Wenn es die Möglichkeit einer Königin. Um eine mögliche Bevorzugung Unterscheidung der Königinnen auf Grund dieser markierten Arbeiterinnen für die eine von genetischen Merkmalen gibt, so würde oder andere Königin zu bestimmen, wurde dies jedenfalls nicht in einer absoluten Weise die Gesamtzahl von Verhaltensakten jeder von den Arbeiterinnen eines Volkes genutzt. Arbeiterin zu jeder Königin registriert. Hier- Wir können danach folgern, daß Arbeite- bei wurde jeweils ein Punkt vergeben, wenn rinnen entweder ihre inklusive Fitneß nicht durch ’Kin während der sich eine Arbeiterin nur einer Königin oder Diskriminierung’ POP in Völkern oder wenn sie sich bei gleichzeitiger POP beider polygynen steigern, ihnen die zu einer Diskri- Königinnen jeweils zur Hälfte jeder Königin daß Möglichkeit zuwendete. Es wurden nur Arbeiterinnen minierung fehlt. Vorteile bei der Bienen- Da der Austausch von zur Auswertung herangezogen, die bei 4 oder haltung: physoga- strischen zwischen den Völkern mehr POPs aktiv beteiligt waren (Abb. 1). Königinnen Zusätzlich wurden die Möglichkeit in nicht durch das Verhalten von Arbeiterin- nen wird, wird uns eine nützli- Betracht gezogen, daß eine Arbeiterin durch beeinflußt die Anwesenheit der einen Königin stimu- che Methode zur ökonomischen Haltung liert wurde, aber nicht durch die Anwesen- dieser Art ermöglicht. Unsere Ergebnisse heit der anderen Königin gehemmt wurde. stimmen mit anderen Versuchen überein, Deshalb wurden in einer weiteren Analyse die ebenfalls den mühelosen Austausch von in Völkern der nur solche POPs berücksichtigt, in der nur Königinnen Meliponen - zwischen Völkern von verschiedenen eine war. Auch hier wurde sogar Königin beteiligt Arten - © Inra/DIB/AGIB/Else- getestet, ob die Arbeiterinnen eine Bevor- aufzeigten. vier, Paris zugung zeigten (Abb. 2). Aus den Beob- achtungen, daß in beiden Fällen die Arbei- Kin recognition / Polygynie / Prozesse des terinnen in einer zufälligen Weise bei Zellen Zellvorrats und Eilage / Melipona bicolor aktiv waren, die von jeder der Königinnen besucht wurde, wurde geschlossen, daß sie keine der beiden Königinnen bevorzugten. REFERENCES Weiterhin wurde eine Königin aus dem im [1] Alexander R.D., and kin ersten Versuch benutzten Volk entfernt und Social learning recog- nition, Ethol. 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