Ovarian activation in quadrifasciata queens triggered by mating plug stimulation (, ) Gabriel Melo, Maria Luisa T. Buschini, Lucio Campos

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Gabriel Melo, Maria Luisa T. Buschini, Lucio Campos. Ovarian activation in Melipona quadrifasciata queens triggered by mating plug stimulation (Hymenoptera, Apidae). Apidologie, Springer Verlag, 2001, 32 (4), pp.355-361. ￿10.1051/apido:2001135￿. ￿hal-00891887￿

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Original article

Ovarian activation in Melipona quadrifasciata queens triggered by mating plug stimulation (Hymenoptera, Apidae)

Gabriel A.R. MELOa*, Maria Luisa T. BUSCHINIb, Lucio A.O. CAMPOSc

a Departamento de Zoologia, Universidade Federal do Paraná, Cx. Postal 19020, 81531-990, Curitiba, PR, Brazil b Departamento de Biologia, Universidade Estadual do Centro-Oeste, Rua Presidente Zacarias 875, 85015-430, Guarapuava, PR, Brazil c Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-001, Viçosa, MG, Brazil

(Received 20 April 2000; revised 22 March 2001; accepted 9 April 2001)

Abstract – The effect of the detached male genital capsule on ovarian activation in queens of the stin- gless bee Melipona quadrifasciata, and the amount of time the genital capsule remains attached to the queen genital chamber after mating, were investigated. Twenty-four controlled matings were carried out and the male capsules were manually removed at preset time intervals. The results indicate that the experimental removal of the mating plug on the first three days after mating inhibits ovarian activation or strongly decreases the chances that it will happen. After this period it would not affect ovarian activation, despite continuing to be attached to the queen for a few more days. It is sug- gested that the male genital capsule provides mechanical stimulation only.

Hymenoptera / Melipona quadrifasciata / stingless bees / ovarian development

1. INTRODUCTION oogenesis, to being the sole factor respon- sible for the onset of egg maturation in these In , several internal and external (Engelmann, 1970; Wheeler, 1996). factors influence the total egg production by females. Among such factors, mating can Nothing is known about the factors that vary from causing a slight acceleration in trigger ovarian activation in stingless bees.

* Correspondence and reprints E-mail: [email protected] 356 G.A.R. Melo et al.

In their close relatives of the genus Apis, 2. MATERIALS AND METHODS unmated queens mature their ovaries very slowly compared to mated queens; how- The M. quadrifasciata bees used in our ever, virgin queens introduced into orphaned experiments were obtained from colonies colonies after double CO2 narcosis develop maintained in hives at the Federal University their ovaries as fast as mated queens (Engels of Viçosa, Minas Gerais State, Brazil. The et al., 1976). Queens in the controlled matings and the establishment of Melipona quadrifasciata are born with the new colonies (newly mated queen plus small, inactive ovaries. Virgin queens kept workers) were carried out accordingly to with food and workers do not mature their the technique described by Camargo (1972a, ovaries, and eventually die or are killed by 1976). We took combs with brood close to the workers in a few weeks; also, differently emergence to obtain virgin queens and from Apis, they do not respond to CO2 treat- workers, each comb being kept separately in ment (Melo, unpublished data). a Petri dish. Mature males were collected inside the hives and were also kept in Petri In M. quadrifasciata, the queens mate dishes until mating. All the Petri dishes had only once and during mating the male loses a circular filter paper covering the bottom his genitalia, leaving it attached to the and a small container with diluted honey. queen’s genital chamber (Kerr and Krause, 1950; Kerr et al., 1962; Silva et al., 1972). The virgin queens were 1–3 days old and According to Silva et al. (1972), the detached the matings took place in a small wooden × × male genitalia are removed by the queen her- box (11 8 3 cm) covered with a trans- self soon after returning from the mating parent plastic lid. The bees were mated one flight. Single mating seems to be the rule pair at a time. After mating, the queens were in stingless bees (Peters et al., 1999). [Evi- observed under a dissecting microscope to dence for multiple mating found by Paxton check the position of the male genitalia in et al. (1999) might be due to mating with their genital chamber and were marked with physogastric queens (see Campos and Melo, a paint spot in the mesoscutum. Each mated 1990) during artificial disturbance of queen was transferred to a Petri dish with colonies]. Also, judging from the relatively 20 newly born workers plus food (honey and pollen from M. quadrifasciata colonies or uniform morphology of the male genitalia in honeybee pollen fermented with M. quadri- stingless bees (Michener, 1990), loss of the fasciata pollen seed). Every day, 15 newly male genitalia during mating is probably born workers were added to the new universal among them. colonies. Eight days after mating, the queens The role of the detached male genitalia in and workers were transferred to a wood box × × stimulating ovarian activation in recently (20 20 6 cm) supplied with food and mated queens of M. quadrifasciata was first soft cerumen. All the experimental material noticed in an earlier unintentional experi- (combs, males and new colonies) was kept ment when controlled matings were being in a incubator under 28 °C. carried out for a genetic study: two out of The different treatments consisted of four queens had the male genital capsule removing the mating plug (detached male manually removed one day after mating; genital capsule) from the queen genital these two queens showed no ovarian acti- chamber at different intervals after the mat- vation 30 days after mating, while the two ing. The time intervals used are shown in unmanipulated queens had already matured Table I (treatments 1 to 7). In treatments 6 their ovaries and started oviposition. This and 7, the queens were manipulated 10 and unexpected result gave us the impetus to 13 days, respectively, after mating. The carry out further investigations. experimental queens were taken from the Ovarian activation in Melipona bees 357

Table I. Influence of the experimental removal of the mating plug on ovarian activation in queens of Melipona quadrifasciata. Treatment: time interval between mating and removal of male genitalia; n: number of queens submitted to each treatment.

Treatment Time between n Number of queens Number of queens Number of queens mating and removal that did not show that showed that died before of male genitalia ovarian activation ovarian activation the mean period for the onset of oviposition

1 5 hours 12 2 1– – 2 1 day 12 – 1– 2 3 3 days 15 4 11 – 4 5 days 14 – 13 1 5 6 days 131 11 1 6 unknown† 12– 12 – 7 unknown† 12 – 12– 8 Control* 14 – 14 –

Total 24 7 13 4

† Queens in treatments 6 and 7 had no attached male genitalia when examined 10 and 13 days, respectively, after mating; * non-manipulated queens.

Petri dish (or from the wood box for treat- 3. RESULTS ments 6 and 7) and had the male genitalia removed, if present, with the aid of a very All the queens from treatments 1 to 5 had fine forceps under a dissecting microscope. the male genitalia attached to their genital The queens were returned to their respec- chamber when they were manipulated. tive colonies immediately after the manip- Queens from treatments 6 and 7 had no male genitalia when examined, indicating that the ulation. Queens in treatments 3 to 7 were male genitalia was lost or removed between held with the help of a cotton ball to pre- six and 10 days after mating, under our vent direct contact with fingers, since work- experimental conditions. ers older than 2 to 3 days can discriminate and kill non-physogastric queens that have The results of the different treatments are been touched with bare fingers (Melo, summarized in Table I. In treatment 1, the two queens were observed for a total period unpublished data). The control treatment of, respectively, 25 and 29 days, and did not consisted of queens not manipulated after exhibit any activation of their ovaries. mating. The queens were followed for at Queens in treatment 2 died three and eight least 25 days to observe their ovarian mat- days after the mating, without any sign of uration and to determine when they initi- ovarian activation. In treatment 3, only one ated egg laying. Ovarian maturation was queen fully developed her ovaries, while evaluated through increase in the volume the four remaining queens still had unde- of the metasoma. After this period, some of veloped ovaries 21 (n = 2), 24 and 30 days the queens had the number of spermatozoa after mating, respectively. One out of three in their spermatheca counted, using the tech- queens in treatment 5 showed ovarian devel- nique described by Camargo (1972b). opment; the other two died eight and 18 days 358 G.A.R. Melo et al.

Table II. Time, in days, elapsed between mat- the frequency in which the queens had their ing and onset of oviposition in queens of ovaries activated differs significantly Melipona quadrifasciata. between these two categories when apply- Treatment n Time (in days) ing a contingency test (G = 9.027, p < 0.01; cases involving premature death excluded). 3 1 15 The negative result obtained in treatment 4 3 15, 16 and 20 5 might be related to another experimental 5 1 15 effect: an increase in mortality rate among 6 2 16 and 21 the manipulated queens. Although it was 7 2 15 and 16 considered a negative result, since the queen 8 4 12, 15, 15 and 19 died 18 days after mating without showing Total 13 mean = 16.2; sd = 2.44 any significant ovarian activation, her rela- tively early death might have been caused by experimental manipulation. The two queens of treatment 2, one in treatment 4, and one in treatment 5 died before the average inter- after mating, respectively. Finally, all the val taken by the control queens to mature queens in treatment 6, 7 and 8 (control) fully their ovaries and start oviposition. They died developed their ovaries and began oviposi- between 3 and 8 days after mating and had tion 16.2 days, on average, after mating no signs of ovarian activation. Melo and (Tab. II). Santana (unpublished data) also found a The sperm counts revealed that two con- higher mortality rate in an experiment where trol queens had 682500 and 885000 sper- the queens had the mating plug removed a matozoa, respectively, in their spermatheca. few hours after mating and were put to They were physogastric and had just begun remate after that. As the tip of the genitalia oviposition. Two other queens, one from valves are pulled deeply into the membrane treatment 1 and one from treatment 3, had of the queen’s genital chamber, the forced 990000 and 1027500 spermatozoa, respec- removal of the genitalia may cause the acci- tively. Both queens had small, undeveloped dental perforation of the membrane and indi- ovaries. rectly death of the queen. Membrane per- forations caused by the valves seem to be involved in the death of physogastric queens of M. quadrifasciata when mated experi- 4. DISCUSSION mentally with several males (Campos and Melo, 1990). Although the sample size is small for most treatments, the results indicate that the The genital capsule of M. quadrifasciata experimental removal of the mating plug males seems to provide only mechanical on the first days after mating inhibits ovar- stimulation. The pressure exerted by the gen- ian activation or strongly decreases the italia valves onto the queen’s genital cham- chances that it will happen. The mating plug ber walls could provide the necessary stim- seems to exert a strong influence on the first ulus, via nervous system, to activate days (from one to three days) and after this production of juvenile hormone by the cor- period it does not affect ovarian activation, pora allata, as has been demonstrated in despite continuing to be attached to the queen other insects. In Diploptera cockroaches, for for a few more days. If the data presented example, egg maturation is mechanically in Table I is collapsed into only two treat- stimulated through copulation and virgin ment categories – (1) capsule removed before females develop their ovaries when artifi- day 4 and (2) capsule removed after day 4 – cial spermatophores are inserted into their Ovarian activation in Melipona bees 359 bursa copulatrix; ovarian activation, how- artificially inseminate them after they ever, does not occur when the ventral nerve become physogastric. Matings with radia- chord is severed prior to mating (Engelmann, tion-sterile males, followed by normal ovar- 1959). ian maturation and oviposition, have been successfully carried out in M. quadrifasciata Seminal products transferred during mat- (Melo, unpublished data). Also, Campos ing have been implicated in triggering and Melo (1990) have shown that egg-lay- ovarian maturation in a number of insects ing queens in M. quadrifasciata remain (Eberhard, 1996). We do not believe, how- physiologically capable of receiving and ever, that such substances are involved in storing more spermatozoa. ovarian activation in queens of M. quadri- fasciata. Although the seminal vesicles are transferred together with the male genital capsule during mating (Kerr and Krause, Résumé – L’activation ovarienne des 1950; Camargo, 1972a), complete transfer of reines de Melipona quadrifasciata (Hyme- male products to the female reproductive noptera, Apidae) déclenchée par la sti- tract apparently takes less than one hour (the mulation du bouchon d’accouplement. results from the sperm counts indicate that On ne connaît rien des facteurs qui déclen- the experimental treatments did not inter- chent l’activation ovarienne chez les abeilles fere with sperm transfer). Also, the soft tis- sans aiguillon. Lors d’une expérience pré- sues associated with the male capsule dry cédente, dans laquelle on pratiquait des out in a few hours and it seems unlikely that accouplements contrôlés pour une étude de the capsule would continue to release any génétique, on a pour la première fois remar- significant amount of a substance capable qué le rôle du bouchon d’accouplement of interfering with the queen’s physiology. (capsule génitale mâle qui se détache) dans la stimulation de l’activation ovarienne chez Silva et al. (1972) and Camargo (1972a) des reines de M. quadrifasciata récemment reported that queens of Melipona quadri- fécondées : on a retiré manuellement la cap- fasciata and M. quinquefasciata get rid of sule génitale mâle à deux des quatre reines the male genitalia soon after returning from un jour après l’accouplement ; ces reines the mating flight. Our results do not sup- n’ont présenté aucune activation ovarienne, port their observations, since under our alors que les ovaires des deux reines non experimental conditions the male genital manipulées avaient mûri et que celles-ci capsule remained attached to the queen for at s’étaient mises à pondre. Ce résultat inat- least six days. Judging from the description tendu nous a décidé à faire d’autres expé- given in Silva et al. (1972), these authors riences. Dans l’étude présentée ici, 24 accou- might have taken the elimination of the plements contrôlés ont été effectués selon whitish soft tissues attached to the male gen- la technique décrite par Camargo (1972a, ital capsule as evidence for removal of the 1976) et les différents traitements ont capsule itself. consisté à retirer le bouchon d’accouple- The findings reported here have impli- ment de la chambre génitale de la reine à cations for the development of techniques divers intervalles de temps après l’accou- of artificial insemination in Melipona. plement (traitements 1 à 7, cf. Tab. I). Les Although a way of triggering ovarian acti- résultats montrent que le retrait manuel du vation in Melipona queens without involv- bouchon d’accouplement durant les trois ing mating might be found in the future, one premiers jours après l’accouplement inhibe alternative way to carry out artificial insem- l’activation ovarienne ou diminue fortement inations in these bees would be to mate les chances qu’elle ait lieu (Tab. I). Après queens with males made sterile by high cette période, le bouchon n’affecterait pas dosages of radiation (60 to 80 krad) and then l’activation ovarienne même s’il reste encore 360 G.A.R. Melo et al. attaché quelques jours. Les produits sémi- die nicht manipulierten Königinnen ent- naux transférés au cours de l’accouplement wickelte Ovarien hatten und bereits Eier sont impliqués dans le déclenchement de legten. Diese unerwartete Beobachtung ver- l’activation ovarienne chez un certain anlasste uns weitere Versuche zu machen. nombre d’insectes (Eberhard, 1996). Nous Es wurden 24 kontrollierte Paarungen nach ne pensons pas que de telles substances der Methode von Camargo (1972a, 1976) soient impliquées dans l’activation ova- durchgeführt. In verschiedenen zeitlichen rienne des reines de M. quadrifasciata. La Abständen nach der Paarung wurde der capsule génitale des mâles de M. quadri- Begattungspfropf (abgetrennte Genitalkap- fasciata semble ne fournir qu’une stimula- sel der Männchen) aus den Genitalkammern tion mécanique. La pression exercée par les der Königinnen entfernt. Die Zeitabstände valves des génitalia sur les parois de la sind in Tabelle I (Versuch 1–7) angegeben. chambre génitale de la reine pourrait fournir Nach diesen Ergebnissen verhindert die Ent- le stimulus nécessaire, via le système ner- fernung des Begattungspfropfs innerhalb veux, pour activer la production d’hormone der ersten 3 Tage die Reifung der Eierstöcke juvénile par les corpora allata, comme cela oder mindert zumindest die Chance, dass l’a été montré chez d’autres insectes. Nos die Reifung einsetzt (Tab. I). Nach dieser résultats ne confortent pas les observations Zeitperiode scheint er keinen Einfluss mehr de Silva et al. (1972) et de Camargo (1972a), zu haben, obwohl normalerweise der Pfropf selon lesquelles les reines de M. quadrifas- noch mehrere Tage in der Königin verbleibt. ciata et M. quinquefasciata se débarrassent Substanzen aus dem Ejakulat sollen bei vie- des génitalia mâles peu de temps après être len Insekten die Reifung der Ovarien aus- rentrées de leur vol de fécondation, puisque lösen (Eberhard, 1966). Wir glauben nicht, dans nos conditions expérimentales la cap- dass solche Substanzen bei der Ovarakti- sule génitale mâle est restée attachée à la vierung von M. quadrifasciata Königinnen reine pendant au moins six jours. eine Rolle spielen. Von der Genitalkapsel der M. quadrifasciata Männchen scheint Melipona quadrifasciata / abeille sans nur eine mechanische Stimulation auszuge- aiguillon / développement ovarien hen. Der Druck, der von den Genitalklap- pen auf die Wände der Geschlechtskammer der Königinnen ausgeht, könnte den not- Zusammenfassung – Reizung durch den wendigen Stimulus bilden, der über das Ner- Begattungspfropf bewirkt eine Aktivie- vensystem die Produktion von Juvenilhor- rung der Ovarien bei Melipona quadrifa- mon bei den Corpora allata aktiviert, wie sciata Königinnen (Hymenoptera, Api- bei anderen Insekten gezeigt wurde. Unsere dae). Über Faktoren, die eine Reifung der Ergebnisse unterstützen die Beobachtungen Eierstöcke stimulieren, ist bei Stachellosen von Silva et al. (1972) und Camargo (1972a) Bienen nichts bekannt. Die Funktion von nicht, nach denen Königinnen von M. qua- Begattungspfropf bei der Stimulation kurz drifasciata und M. quinquefasciata die nach der Paarung von Melipona quadrifa- Genitalkapsel der Männchen kurz nach der sciata Königinnen wurde zufällig bei ande- Rückkehr vom Hochzeitsflug abstreifen, ren Experimenten beobachtet, die auf Grund weil unter unseren Bedingungen dieser genetischer Studien gemacht wurden: Bei Pfropf für mindestens 6 Tage in der Königin 2 von 4 Königinnen wurde die männliche verblieb. Genitalkapsel einen Tag nach der Paarung mit der Hand entfernt. Bei diesen beiden Königinnen hatten sich die Ovarien 30 Tage Hymenoptera / Melipona quadrifasciata / nach der Paarung nicht entwickelt, während Stachellose Bienen / Ovarreifung Ovarian activation in Melipona bees 361

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