150 Reproduccion Abejas Eusociales

Reproduction in eusocial bees
(Apidae: Apini, Meliponini)
TONG XUAN CHINH REPRODUCTION IN EUSOCIAL BEES Department of Behavioural Biology Faculty of Biology, Utrecht University Bee Research and Development Centre Hanoi, Vietnam ISBN: 90-393-3661-X Printed by: Central Reproduction FSB, Utrecht University Cover photos: Nest of Apis cerana (front) and orientation flights of Trigona ventralis flavibasis Layout and photos by Tong Xuan Chinh

Voortplanting bij eusociale bijen
(met een samenvatting in het Nederlands)
Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de Rector Magnificus, Prof. Dr. W.H. Gispen, ingevolge het besluit van het College voor Promoties in het openbaar te verdedigen op maandag 17 mei 2004 des namiddags te 12:45 uur
door
Tong Xuan Chinh geboren op 16 mei 1965, te Thai Binh, Vietnam
Promotor: Co-promotor:
Prof. Dr. Jan A.R.A.M. van Hooff Dr. Marinus J. Sommeijer Department of Behavioural Biology Faculty of Biology, Utrecht University
Co-promotor:
Dr. Willem J. Boot Laboratory of Entomology, Wageningen University
The investigations were financially supported by the Royal Netherlands Embassy, Hanoi, Vietnam; the Netherlands Universities Foundation for International Cooperation (NUFFIC); Utrecht University, the Netherlands and the United Nations University.
Contents
Chapter 1 General introduction and summary Chapter 2 General methodology: the bees and study site Chapter 3 Nest-architecture and colony-characteristics of three stingless bees in north Vietnam with the first description of the nest of Lisotrigona carpenteri (Hymenoptera: Apidae, Meliponini) With Marinus J. Sommeijer, Willem J. Boot & Charles D. Michener Submitted to Journal of the Kansas Entomological Society Chapter 4 Patterns of male production in the stingless bee Melipona favosa (Apidae, Meliponini) With Gijs B.J. Grob; Franciscus J.A.J Meeuwsen & Marinus J. Sommeijer Adapted from a publication in Apidologie 42 (2003): 161-170 Chapter 5 Seasonal production of sexuals in Trigona (Lepidotrigona) ventralis flavibasis Cockerell (Apidae: Meliponini) in North Vietnam With Marinus J. Sommeijer Accepted for publication in Apidologie Chapter 6 Production of sexuals in the honeybee species Apis cerana Fabricius 1793 (Apidae: Apini) in North Vietnam 10 16
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With Willem J. Boot & Marinus J. Sommeijer Accepted for publication in Journal of Apicultural Research Chapter 7 Reproductive strategies in sympatric species of eusocial bees With Willem J. Boot & Marinus J. Sommeijer Samenvatting 90
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References Acknowledgements Curriculum vitae Publications
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CHAPTER 1
Introduction and summary: reproduction in eusocial bees (Apidae: Apini, Meliponini)
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1. Reproduction at the individual level
Both honey bees (Apini) and stingless bees (Meliponini) are highly eusocial bees that live in perennial colonies. Colonies of these bees are characterized by morphologically distinct female castes (gynes or queens, and workers), division of reproductive labour between the castes and generation overlap (Michener, 1974). At individual level, most reproduction aims to increase the number of worker bees in the colony, which is needed to produce new colonies. Colony growth by production of workers also increases the number of gynes and males that can be reared by the colony. These sexuals transmit the genes of all individuals of the colony to the next generation, since bees in the same colony are usually related. After mating, a gyne may become a queen, either because she replaces the old queen, or she serves a new colony in relation to the swarming process. The production of sexuals and colony multiplication are complex processes that are triggered by a combination of extranidal and intranidal factors.
2. Colony multiplication
Colonies of stingless bees and honey bees are founded through swarming because single individuals of a colony are unable to survive and queens of these bee species cannot forage. Swarming involves the departure of a cohort of workers with a queen that leaves the original colony for a new nest site. In honeybees, the old queen leaves the original nest with a large group of workers. Subsequent swarms may depart from the colony in the company of one or a number of the young queens that are produced during the swarming process. In the original colony, one of the new queens takes over reproduction. The number of swarms that a colony can issue is not regulated by the number of gynes produced, because survival of colonies depends largely on population size. This implies a reluctance to divide in too many parts. Surplus gynes get killed in mutual fights or are killed by the workers after the acceptation of one of the young queens by the colony. In stingless bees, the number of swarms issued is even less reflected by the number of gynes produced in the colony. In general, stingless bees have a low swarming frequency and besides population size it appears that also the amount of food reserves and the supply of nest building materials are related to the swarming capacity of a colony. Typically, the swarming process involves the transfer of these materials by the workers from the mother colony to the daughter colony. The old queen always stays in the original colony and a young queen will start reproduction in the new colony (Michener, 1974).
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Chapter 1. Introduction and summary
3. The research of this thesis
In this thesis, some key aspects of the regulation and the mechanisms of colony reproduction in honeybees and stingless bees are presented. Special attention is paid to key questions about how the production of males, gynes and swarms takes place, and what intranidal and extranidal factors are related to these processes. The natural pattern of male production in Melipona favosa was investigated in Trinidad and Tobago (W.I.) and subsequently the relationship between emerging males and haploid eggs laid by laying workers was studied in the laboratory at Utrecht University. Secondly, the regulation of reproductive processes such as the production of males, gynes and swarms were studies in the sympatric eusocial bee species Trigona (Lepidotrigona) ventralis and Apis cerana in North Vietnam. Thirdly, similarities and differences in reproductive strategies between the tribes Apini and Meliponini in the same habitat are compared and discussed to generalize collective evolutionary and adaptive significance in highly eusocial bees. 3.1. Three sympatric stingless bee species and their nest architecture Nest architecture and colony population was studied in three sympatric stingless bee species (chapter 3). Trigona (Lepidotrigona) ventralis and Trigona (Heterotrigona) laeviceps usually nested in trees with trunk diameters of more than 35 cm. In contrast, Lisotrigona carpenteri built its nests commonly in small diverse cavities such as occurring in man-made objects. The structure of the nest entrance among the three species is different and appears a species-specific feature. The nest entrance is thin soft funnel-shaped for T. ventralis, small brittle cylindrically shaped for T. carpenteri and black round-ringed for Trigona laeviceps. In T. ventralis, the dimension of the nest entrance tube appears to be positively correlated to the nest cavity and the population of the colony. Brood cells are arranged in combs in T. ventralis, and in clusters in the other two species. 3.2. Periodic emergence of males in M. favosa In stingless bees (Meliponini), males are produced throughout the year, although their number is variable, depending on the season (van Veen et al., 1999; Moo-Valle et al., 2001; Sommeijer et al., 2003). To gain insight into how males are produced, six colonies of the stingless bee Melipona favosa were observed in the field. The results showed that the production of males is periodic in this species and males emerge in a clumped pattern in distinct periods (chapter 4). Neighbouring colonies do not produce males synchronously. When certain colonies are producing males, other colonies are producing all females. On the scale of the population, however, males are always produced. 12
Reproduction in eusocial bees 3.3. Periodic laying of reproductive worker eggs resulting in clumped male production in M. favosa. From the results on male emergence in colonies obtained from the field, we hypothesized that the periodic pattern of male production is the consequence of cyclic periods of workers laying reproductive eggs. In the laboratory in Utrecht, detailed observations were carried out on egg laying behaviour by means of permanent video recordings. All cells in which eggs were laid were mapped and followed until the emergence of workers, males and queens. This was to reveal whether the queen or the workers were responsible for the production of males. Our results (chapter 4) supported the above hypothesis. We clearly recorded that the majority of males were produced as sons of workers. The negative correlation between reproductive and trophic worker eggs suggests that the number of laying workers in a colony is limited, so the more workers lay reproductive eggs the fewer workers can produce trophic eggs for feeding the queen. The importance of trophic eggs to the queens productivity was established. The background for the periodic pattern of the male production in M. favosa is still poorly understood. Several hypotheses are presented as an explanation (chapter 4). 3.4. Seasonal production of sexuals in T. ventralis So far, our information on the production of sexuals and colony reproduction for stingless bees is based on species that live in the neo-tropical South American Region. The reproductive behaviour of stingless bees from other regions such as Asia or Africa has until now completely been ignored. To obtain information about the reproduction of stingless bees from the Asian Region we carried out observations in Vietnam. Ten colonies of Trigona (Lepidotrigona) ventralis were investigated to study the production of sexuals and worker bees over the year. The results show that at population level both the males and gynes were produced almost year round although the number of sexuals was variable, depending on the availability of flowers to forage on (chapter 5). At colony level, male production was periodic and occurred in distinct bouts over the season. Different from the production of males, gynes were produced in low numbers. The production of sexuals was positively correlated with the amount of food reserves and the number of bees in the colony. It is surprising that the same periodical production of males in a single colony without synchronization between colonies was found both in the old world species T. ventralis and in the new world-species Melipona favosa. This suggests that the mechanisms underlying allocation of resources over sexes may be general for many stingless bee species.
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Chapter 1. Introduction and summary
3.5. Behaviour of newly emerged gynes in T. ventralis The behavioural repertoire of newly emerged gynes was described, with emphasis on the behavioural interactions between gynes and workers (chapter 5). Workers showed aggressive behaviour to gynes, and gynes tried to hide somewhere in the brood nest to escape the attacks. We observed dead adult gynes contained in their royal cells, which has never been reported before. We did not only observe this phenomenon in the observation-hives, but also in natural nests at the moment that these were opened in the forest for transfer to hives. These dead queens may result from differences in the provisioning of brood cells. In honeybee colonies, nursing workers feed larvae progressively in open cells arranged in combs. The cells are capped when the larvae stop feeding in order to pupate. In this system, workers have the possibility to rear emergency gynes from larvae that were earlier destined to become workers. In Stingless bees, cells are provisioned with the total amount of food needed and after egg laying the cell is immediately capped. This excludes rearing of emergency gynes in case the queen dies and therefore a number of gynes have to be reared continuously as a safeguard. Gynes may be stored for some time inside their cells but eventually they will die. Such gynes were probably observed in nests of T. ventralis. 3.6. The regulation of male and gyne production in A. cerana In 20 colonies of Apis cerana, in northern Vietnam, observations on colony growth, on production of drones and queens, and on swarming and supersedure were related to available flowers to forage on and climatic data. Despite the tropical setting of the study area with forage available year-round, production of males and gynes was restricted to two periods from March to July and from September to December. Positive correlations between available forage, colony growth and production of sexuals suggest that the synchronized production of males and gynes is defined by the forage flow into the colony. If this flow is high, the colony starts growing; when the colony is large enough males and gynes are produced, and eventually the colony swarms. Production of sexuals is synchronized because foraging conditions are only part of the year sufficient to allow growth. Patterns in male and gyne rearing by A. cerana are similar to patterns found in Apis mellifera. Variation probably reflects differences in environment rather than differences between species. 3.7. Colony multiplication in A. cerana A total of 35 swarms and 8 supersedures of the queen were investigated in the A. cerana population (chapter 6). The colonies prepared for swarming when their worker population had reached to about twenty-one thousand bees. On 14
Reproduction in eusocial bees average, the colonies swarmed once per year, mostly during May when forage was most abundant.
4. Reproductive strategies of honeybees and stingless bees
In tropical areas many species of eusocial bees are found. In the last chapter (chapter 7) our new knowledge of reproductive behaviour of eusocial bees (see above) is integrated to explore general reproductive strategies. We reviewed differences in provisioning of the brood, in the general swarming process, in which caste (worker or queen) produces the males, in how caste is determined and in seasonal production of sexuals. These differences are related to general strategies for reproduction and swarming. We focus on three eusocial bee species occurring in the same forest in Vietnam. The honeybee Apis cerana is well adapted to exploit seasonal or temporal resources, because it responds quickly to changing environmental conditions and swarms can travel far from the original nest. Stingless bees respond much slower and new nests are started as satellites in the vicinity of the old nest. Hence, it is expected that stingless bees will in general flourish most in stable environments. However, some differences can be observed among sympatric stingless bee species. Species with large colonies like Trigona ventralis focus more on colony defence and growth of the colony itself. Species with small colonies like Lisotrigona carpenteri probably focus more on colonizing many new nest sites. Together with resource partitioning and specialization on various nest sites, the different reproductive strategies help to explain why so many eusocial bee species are living sympatrically.
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CHAPTER 2
General methods: the bees and the study sites
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1. The bees
1.1. Taxonomic position of the Apini and the Meliponini within the Apinae Worldwide about 16,000 bee species have been recognized. These species have placed in 425 genera, 22 subfamilies, 6 families (Michener, 2000). They are divided into two main groups: the more primitive short-tongued bees and the more advanced long-tongued bees. Both honeybees and stingless bees belong to the latter group. The corbiculate Apidae, characterized by the presence of a corbicula or pollen basket on the outer surface of the hind tibia, consists of three subfamilies: Cyclopinae, Nomadinae and Apinae. The tribes Bombini, Meliponini, Euglossini, and Apini are placed in one subfamily, the Apinae (Winston, 1991). Honeybees (Apini) and stingless bees (Meliponini) have highly developed social ("eusocial") behaviour; all species live in complex colonies. As typical characteristics of eusociality they have large perennial colonies, morphological distinctions between female castes, reproductive division of labour, generation overlap, and a complicated communication and recruitment related to foraging. Although their eusociality has evolved independently, the Apini and the Meliponini are sister groups (Michener, 1990). 1.2. Some distinctive differences between the Apini and Meliponini The honeybees are discriminated from the stingless bees and the other Apinae by a few minor morphological criteria, but by essential behavioural characteristics as follows: males of honeybees (drones) have a membranous endophallus, whereas this characteristic is lacking in males of stingless bees. The pattern of wing venation stretches in the former, but is reduced in the latter group. The tribe Meliponini far exceeds the Apini in diversity and original native distribution. Stingless bees have recently been reported to consist of about 374 species (Michener, 2000) versus about nine species of honeybees (Otis, 1997); the former naturally distribute over the pan- tropics whilst the latter are indigenous in the Old World, both in tropical and in temperate areas (Roubik, 1989; Camago & Pedro, 1992). Temperature regulation inside bee colonies needs to be taken into consideration to explain differences in the distribution between the sister tribes. The Apini lack a thermo-humidity insulating cover that potentially could be of very high adaptive value in cool climates, which is found in the Meliponini. In compensation, the Apini have the property of three effective characteristics of thermo-regulation: clustering, heating generated by muscle wing vibration, and water carrying to the nest for reducing nest temperature. This results in a constant temperature in the centre 17
Chapter 2. General methods
of the nest. Lacking these characteristics may be a barrier for stingless bees to spread to temperate regions (Ruttner, 1988). Temperature regulation is not completely absent in stingless bees, however. A few of the several hundred meliponid species have the ability to regulate nest temperature within certain limits, and normally they are species with large colonies in voluminous nests (Roubik, 1989). The involucrum surrounding the brood helps stingless bee colonies to maintain nest temperature above ambient temperature; however, no temperature homeostasis has been found. The geographic distribution of the Meliponini seems to indicate a centre of speciation in South America (Ruttner, 1988). However, Wille (1979) strongly suggests that in spite of a more recent distribution of species in South America, the centre of origin of the Meliponini is in Africa. The honeybee species probably originated in southeastern Asia (Cornuet, 1986). 1.3. The honey bee species Apis cerana 1.3.1. Species diversity of Apis in Southeast Asia Based on essentially morphological and behavioural analyses and the aid of different genetic techniques, the classification systematic of the true honeybees has obtained great achievements in the last two decades of the twentieth century. The number of recognized honeybee species has been reduced since the descriptions of Maa (Maa, 1953), because many types are now seen as subspecies (Otis, 1997). The tribe, Apini, consists of only one small monophyletic genus, Apis that comprises nine honeybee species: Apis mellifera, A. cerana, A. koschevnikovi, A. nigrocincta, A. nuluensis, A. dorsata, A. laboriosa, A. florea and A. andreniformis (Otis, 1997; Tingek et al., 1996). Out of these nine species the five initial species nest in cavities have a number of combs. The last four are nest in the open and have a single comb. Apis species are divided into three lineages: the cavity-nesting bees, Apis mellifera, A. cerana, A. koschevnikovi, A. nigrocincta and A. nuluensis; the dwarf bees, A. florea and A. andreniformis; the giant bees, A. dorsata and A. laboriosa. Of the nine species, only A. mellifera and A. cerana have been domesticated for a long time (Koeniger, 1976). A. mellifera is the most studied and economically exploited species. All Apis species, except for A. mellifera, are native to Southeast Asia. This region is a centre of Apis diversity and makes scientists pay great attention to the recently recognized species such as A. nigrocincta and A. nuluensis. In Vietnam, five native honey bee species are found, namely A. cerana Fabricius 1793, A. dorsata Fabricius 1793, A. florea Fabricius 1787, A. andreniformis F Smith 1958 (Ha & Lap, 1992) and A. laboriosa F Smith 1871 (Trung et al., 1996). They distribute in different areas of the country from north to south, especially in mountainforest areas. This rich species-diversity promotes prospective sources for 18
basic research on biology, ecology and behaviour in honeybees. Furthermore, this diversity is of interest to applied researchers because of the great importance of pollination in Vietnamese agricultural and in the forestry ecosystem. 1.3.2. The distribution of A. cerana A. cerana is the Asiatic honeybee or the oriental honeybee because they are only found in Asia, from Iran in the east to Pakistan in the west, and from Japan in the north to the Philippines in the south. Thus, A. cerana does not live only in tropical and subtropical areas of Asia, but also in colder areas as Siberia, Northern China and the high mountain area of the Himalayan region (Koeniger, 1976). A. cerana occurs at altitudes from 0 to 3333 m above the sea level (Rahman, 1945). For many social insects a tendency has been found that the higher the altitude and latitude the bees inhabit, the bigger the body size and the colony population is (Paspari & Vargo, 1995). This relationship is the rule for A. mellifera (Ruttner, 1988; Ruttner, 2000; Hepburn et al., 2000), for A. cerana (Verma et al., 1994), for A. florea (Ruttner et al., 1995), and for some stingless bee species (Pereboom & Biesmeijer, 2003). A high degree of variation in size and coloration probably reflect the ecological diversity of A. cerana. The influence of latitude and altitude on the size of worker bees was also found for A. cerana in Vietnam (Niem et al., 1992). A. cerana colonies occur in all provinces of Vietnam (except Uminh forest) but their natural types are commonly found in mountain-forested areas such as Viet Bac, Hoang Lien Son, Truong Son; in coconut-grown provinces as such Ben Tre, Tien Giang, etc., and island-districts as Cat Ba, Phu Quoc, Con Dao (Chinh, 1996). 1.3.3. Studies on the taxonomy of A. cerana in the World A. mellifera is the best-studied species in honeybees in particular and in social bees in general. Twenty-six subspecies and ecotypes are discriminated and have been studied in detail. Compared to A. mellifera there is very little research on the morphology of A. cerana (Verma, 1990). Based on the analysis of 34 morphological criteria of 68 samples collected from different areas of Asia, Ruttner (1988) divided A. cerana into four subspecies: Apis cerana indica This is the subspecies with the smallest body size. It lives in the south of India, in the south of Thailand, Cambodia and Vietnam, in Malaysia, in Indonesia and in The Philippines. The length of proboscis and forewing is 4.58-4.78 mm and 7.42-7.78 mm respectively (Ruttner, 1988).
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Apis cerana cerana This subspecies with the biggest body size of A. cerana occurs in northern parts of China, in the northwest of India, in the north of Pakistan and Afghanistan, and in the north of Vietnam. On average, the proboscis and forewing length measure 5.25 mm and 8.63 mm respectively. Apis cerana himalayana The body size of this subspecies is intermediate between A. c. cerana and A. c. indica. It occurs in the east of the Himalayas from Nepal to northern Thailand. On average, the proboscis and forewing length measure 5.14 and 8.03 mm respectively. Apis cerana japonica This subspecies is endemic in Japanese temperate climates except the island of Hokkaido. This subspecies is divided into two separate ecotypes: Honshi and Tsushima. The body size of Apis cerana japonica is relatively big, with an average proboscis length of 5.18 mm and an average forewing length of 8.69 mm. A. c. japonica gradually has been replaced by introduced A. mellifera (Okada, 1986). The infraspecific classification of the Asiatic honeybee species, A. cerana is in a state of flux and uncertainty (Hepburn et al., 2001). Next to the four subspecies distinguished by Ruttner (Ruttner, 1988), four other subspecies have been proposed: A. c. abaensis, A. c. philippina, A. c. skorikovi and A. c. hainanensis (reviewed by Hepburn et al., 2001). Five of these subspecies occur in China: A. c. indica, A. c. cerana, A. c. skorikovi, A. c. hainanensis and A. c. abaensis. A. c. cerana is divided further into five ecotypes known as Quangdong-Quangxi, Hainan, Yunnan north, and Changbei-Shan (Zhen-Ming et al., 1992). Based on multivariate morphometric analyses of 557 colonies of A. cerana from all of the southern mainland of Asia, Hepburn et al. (in press) have recently established that A. cerana is placed in three separable groups that are not entirely distinct morphoclusters of bees: 1. bees from the Hindu Kush, Kashmir, northern Myanmar, northern Vietnam, and southern China; 2. bees from northern India, Nepal, central Myanmar and Thailand, Cambodia, southern Vietnam and southern China; 3. bees from central and southern India, southern Myanmar, southern Thailand and peninsular Malaysia. However, the nomenclature of these infraspecific taxa of A. cerana remains unadjusted in this paper. Deviations in the infraspecific classification of A. cerana probably reflect differences in sampling and methodology.
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1.3.4. Studying the taxonomy of A. cerana in Vietnam The traditional beekeeping with A. cerana in Vietnam was recorded in ancient literature by Pham Le in the eighth century and Le Qui Don in the eighteenth century. Information on the traditional beekeeping with A. cerana in Vietnam can be found in different books and articles (reviewed by Chinh, 1996). Little information is available on the biological and ecological aspects of this native species, but research on the morphology of native A. cerana in different places in Vietnam has been reported fragmentarily (Niem et al., 1992). These authors established that the worker size of A. cerana is reduced in correlation with the latitude, e.g. workers in the south are smaller than those in the north; bees of Thanhhoa province are smaller than those of Hoabinh. The authors concluded that A. cerana in northern Vietnam belongs to the subspecies of A. c. cerana (Niem et al., 1992). However, their morphological studies are based on only 14 criteria of workers instead of 35-55 criteria that are regarded as international standard for morphological studies (Verma, 1990; Ruttner, 1988). Therefore, the question of which subspecies of A. cerana occurs in Vietnam has not yet been clarified. Based on four samples of worker bees of A. cerana in northern Vietnam, Ruttner (1988) reported that A. cerana of this area is A. c. cerana and A. cerana in the south is probably A. c. indica. At present, based on multivariate morphometric analyses of workers from 61 colonies collected in various localities over the country, A. cerana in Vietnam is established to belong to two different groups. However, the name of the subspecies is not yet designated (Hepburn et al., in press). 1.4. Stingless bee species: Melipona favosa and Trigona ventralis The classification of stingless bees has been presented very differently by various authors (see review by Sakagami, 1982), which makes the taxonomic study of stingless bees complex. Stingless bees are grouped in two principal genera Trigona and Melipona (Schwarz, 1948), which are divided into 23 genera (no subgenera) (Moure, 1961). Detailed summaries on the classification have been given by Wille (1979), Sakagami (1975; 1982), Michener (1990) and Camago & Pedro (1992). In the most recent classification of stingless bees, the Meliponini comprise 23 genera and 18 subgeneric names, which consist of 374 recognized species (Michener, 2000). Stingless bees occur mostly in tropical parts of the world such as Africa, Asia and South America. They are most abundant in the neotropics (Sakagami, 1982; Wille, 1983). The genus Melipona has about 40 species, in which Melipona favosa is common in some countries in South America such as The Guyanas, Trinidad and Tobago (W.I.), Venezuela, Colombia and Panama. M. favosa colonies have several hundreds of workers that are medium-sized and rather timid in 21
behaviour when disturbed. Brood is reared in identical-sized cells, and is arranged in vertical combs from which all castes and sexes emerge (Roubik, 1982). The genus Trigona consists of 10 subgenera in which about 120 species have been recognized. This is the largest and most widely distributed genus of the Meliponini. Trigona stingless bees are found in the tropics from Mexico to Argentina, and in the Indo-Australian region from India and Sri Lanka to Taiwan, east to the Caroline Islands, the Solomon Islands, and south throughout Indonesia and New Guinea to about 34o latitude in Australia. However, the majority of Trigona species are found in the Americas (Michener, 2000). The subgenus Lepidotrigona, one of Trigonas subgenera, is widely distributed in Indo-Australia and has four species, one of which is Trigona (Lepidotrigona) ventralis. This species consists of four subspecies: Trigona (L.) v. ventralis, T. (L.) v. hoozana, T. (L.) v. flavibasis and T. (L.) v. doipaensis. This species is found in northeast India, Burma, Thailand, Sumatra, Java, Borneo, and rather isolated in Taiwan. Sakagami (1975) recognized that the worker body size is the smallest in the mountainous areas of central Vietnam, larger in Malaysia and the largest in northern Thailand. Colonies of T. ventralis construct nests in tree hollows; the brood nest is covered by a multi-layered involucrum and brood cells are arranged in combs (Sakagami et al., 1983).
2. The study sites
All observations and experiments described in this study were carried out in three places: Tobago an island of Trinidad & Tobago (W.I.); Laboratory at Utrecht University, The Netherlands and National Cuc Phuong Forest, northern Vietnam. 2.1. Tobago (West Indies) This island is part of the republic of Trinidad and Tobago. This country, geological part of South America, is situated near the coast of Venezuela in the mouth of the Orinoco river. The climate in Tobago (11o 20' 11o 9' North Latitude, maximum altitude 549 m) (Fig. 1) is characterized by a rainy season (June to December) and a dry season (January to April). The annually average temperature and rainfall is 29oC (83o F) and 3750 mm respectively. The topography of Tobago comprises a tropical forest and a mountainous midsection. Several stingless bee species are found here of which Trigona nigra is the most common.
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Figure 1. Map of Tobago (W.I.) 2.2. Cuc Phuong National Park, Vietnam The Cuc Phuong National Forest comprises an area of 22,220 ha ((20o14-20o 24 N and 105o29-105o44 E) (Fig. 2) in Cuc Phuong commune, Nho Quan district, Ninh Binh province. This area is covered by a primary tropical rain forest and is situated on two parallel limestone mountain ranges, running from northwest to southeast direction. The average altitude of the Park is 300-400 m above sea level with the highest mountain peak of 650m, gradually decreasing from northwest to southeast. The Buoi river runs via the northwest part of the Park, along the southwest border for about 4-5 km. The climate of Cuc Phuong is characterized by a rainy season (MayNovember) and a dry season (December-April). Maximum daily temperature during the rainy season is about 37oC. In the dry season, maximum daily temperature is about 35oC, although sometimes it is reduced to 3oC. Annual rainfall is about 224 and 1923 mm in the dry and rainy season, respectively. Cuc Phuong National Park has an area of 20,473 ha forest, which accounts for 92,2 % of the total area. The vegetation of this area belongs to the evergreen tropical rainforest type, and is divided into 3 categories as follows. The forest in the valley and piedmont areas contains 5 canopy layers, three of which are woody, one layer with shrubs, and one with underground. The forest on the mountain slopes consists of just 3 layers; and the mountain 23
top forest consists of small and short trees, divided into 2 layers. The rich flora of the park harbours 1944 species, 908 genera, and 229 families. The plant species, genera, and families account for 24.6%, 43.6%, and 68.9%, respectively, of the totals for Vietnam.
Figure 2. Map of Vietnam The angiosperms group is represented by a considerable number of species. In Cuc Phuong forest there are 64 orders and 164 families found, accounting for 71 %, and 38%, respectively, of the 89 orders and 433 families of the world (Qui et al., 1996). Hanh (2000) investigated the most profitable plants for bees in both Cuc Phuong forest and its bufferzones. The study was based on direct observations of flowers visited by the native honey species, A. cerana and through morphological analysis of pollen grains from samples of pollen loads collected from returning forager bees. His study showed that out of 41 24
families, 85 species are of importance to these bees, 20 species only for nectar, 13 only for pollen, and 53 for both. The fauna of Cuc Phuong is very rich with 71 mammal species, 319 bird species, 33 species of reptiles, and 16 species of amphibians. There are more than 50 large and medium sized mammal species. Some rare and endangered species such as Neofelis nebulosa, Felis temmimck, Selenarctos thibetanus, and Hylobates concolor live here, although they are difficult to be seen as they live deep in the forest. The beautiful langur, Trachipythecus francoisi delacouri, is endemic sub-species and is considered as the symbol of the park. The insect population is even more abundant. There are approximately 1800 species of 200 families from 30 orders. Solitary and social bees are also rich in species. Three of the five species of native honeybees in Vietnam are found: A. cerana, A. dorsata, and A. florea. Three different species of stingless bees occur in the forest. They have been preliminarily classified as belonging to the genus Trigona (Qui et al., 1996).
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CHAPTER 3
Nest architecture and colony characteristics of three stingless bees in North Vietnam with the first description of the nest of Lisotrigona carpenteri Engel (Hymenoptera: Apidae, Meliponini)
Tong X. Chinh1, Marinus J. Sommeijer2, Willem J. Boot3 & Charles D. Michener4
Submitted to Journal of the Kansas Entomological Society
Bee Research and Development Centre, 68 Nguyenhong, Langha, Dongda, Hanoi, Vietnam
Department of Social Insects, Utrecht University, P.O.Box 80.086, NL- 3508 TB Utrecht, The Netherlands Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EH Wageningen, The Netherlands
4
Division of Entomology, University of Kansas, 1460 Jayhawk Boulevard (Snow Hall), Lawrence, Kansas 66045-7523, USA.
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Summary. In the tropical primary forest of Cuc Phuong National
Conservative Biosphere, North Vietnam and its bufferzones, we studied three sympatric stingless bee species: Lisotrigona carpenteri Engel, Trigona (Tetragonula) laeviceps Smith and Trigona (Lepidotrigona) ventralis Smith. Nest-architecture, adult population, the number of brood cells, the presence of adult reproductives, the proportion of males in brood, the number of queen cells, honey and pollen pots, and other features were recorded from 35 wild nests. Some behavioural observations were made. L. carpenteri and T. laeviceps arrange brood cells in clusters. In contrast, brood cells in T. ventralis are arranged in horizontal combs in a brood compartment that is surrounded by an involucrum of multiple lamellae. L. carpenteri constructed its nests mainly in small crevices of man-made structures while colonies of T. laeviceps and T. ventralis were generally found to nest in live trees. The flexibility for using nest substrates other than living trees appears in these species related to colony size. T. ventralis has large colonies (up to around 10,000 adults), T. laeviceps is intermediate with colonies of up to 1,200 adult bees, and L. carpenteri has by far the smallest colonies of only up to about 400 adult bees. The construction of the external nest entrance was different in three species. L. carpenteri builds a hard brittle cylindrical tube. The nest entrance of T. laeviceps is a hole decorated with a batumen rim. T. ventralis constructs a long medium-soft tube with a funnel-extension at the end. In this paper we present the first description of the nest of the recently described species L. carpenteri.
1. Introduction
Southeast Asia is well-know for its diversity of the social honey bees, the Apini and it is considered to be the origin of the only genus recognized, Apis (Cornuet, 1986). All Apis species are found in Southeast Asia (Otis, 1997), even Apis mellifera that naturally occurs in Africa and Europe (Ruttner, 1988) but has been introduced during the last few hundred years. Despite the sympatric occurrence of all Apis species, the general bee fauna in tropical Southeast Asia is rather poor in species compared with the Neotropics and tropical Africa (Michener, 1979), although this region has a rich angiosperm flora (Withmore, 1984). This may be related to humid weather conditions throughout the year, since bees are most diverse in the warm temperate dry regions (Michener, 1979). It is full of bees, albeit with fewer species. Both honey bees and stingless bees (Meliponini) are highly social. Like Apini and unlike all other bees, Meliponini live in perennial colonies and have distinct female castes: queen (or gyne if not yet mated) and worker. The distribution of the Meliponini is confined to the tropical and southern subtropical areas throughout the world. Colony size is diverse and stingless 27
Chapter 3. Nest architecture and colony characteristics of three stingless bees in north Vietnam
bees live in colonies ranging from a few dozen to 100,000 or more individuals (Michener, 2000). Stingless bees are particularly common in large areas in tropical America and in Southeast Asia. They are considered among the major pollinators in the tropics (Michener, 1974). All stingless bees build elaborate nests with structures that are often characteristic for the species or for higher taxa (Michener, 1974; Sakagami, 1982). Taxonomically, the stingless bees of Southeast Asia are relatively well known (Schwarz, 1937, 1939; Moure, 1961; Sakagami, 1975, 1978), but bionomical data are still scarce. Even data on nest architecture, information that is basic to any biological and ecological study, are only recorded fragmentarily in a few species as reviewed by Wille and Michener (1973). For neotropical species this problem is partly alleviated by the beautifully illustrated works of Camargo and his associates; for a recent example with references to others, see Camargo and Pedro (2003); for Australian species, see Michener (1961). Worldwide stingless bees are classified in 23 genera and 18 subgenera, comprising about 374 described species. Of which 43 recognized species belonging to two genera: Lisotrigona and Trigona occur in Asia (Michener, 2000). Until a few years ago, only two species were recognized in the genus Lisotrigona: Lisotrigona cacciae and Lisotrigona scintillans. Both species have been found in the region extending from Sri Lanka and Madhya Pradesh in India to Vietnam, Borneo, and Sumatra (Michener, 2000). Recently, two new species have been described: Lisotrigona carpenteri from northern Vietnam and Lisotrigona furva from Thailand (Engel, 2000). Information on biology, ecology and nests of the genus Lisotrigona has not been described previously. The genus Trigona encompasses worldwide about 120 species, placed in 10 subgenera, of which Homotrigona, Lepidotrigona and Heterotrigona are endemic subgenera for tropical and subtropical Asia (Michener, 1990, 2000). The subgenus Homotrigona has only one species, Trigona (Homotrigona) fimbriata, and is found from western Malaysia to Vietnam and south to Sumatra and Borneo (Michener, 2000). According to the classification system by Schwarz (1939), Trigona (Lepidotrigona) ventralis is divided into four subspecies: T. v. ventralis, T. v. flavibasis, T. v. doipaensis, and T. v. hoozana. Based on results of a comparison of worker specimens from various localities in Southeast Asia, Sakagami (1975) could not find any superspecific segregation among ventralis, flavibasis and hoozana. He also showed that the body size is smallest in the mountainous areas of central Vietnam, larger in Malaysia and Laos, and largest in northern Thailand. 28
The subgenus Heterotrigona of the genus Trigona consists of about 36 species (Michener, 2000), of which six Australian species have been redescribed (Dollin et al., 1997). According to the classification of Michener, this group includes the subgenus (or genus) Tetragonula (Michener, 2000). Heterotrigona in this classification is the largest and most widespread subgenus in the Indo-Pacific area and includes the numerous and abundant species of Tetragonula (Sakagami and Inoue, 1985). However, in the classification system by Wille (1979), which was later modified by Sakagami and Inoue (1985), Heterotrigona and Tetragonula are considered separate subgenera of the genus Trigona, and we recognize Tetragonula as a subgenus in the present work. The taxonomic characteristics of Tetragonula as a separate subgenus have been described by Moure (1961), revised by Sakagami (1978) and further modified by Sakagami & Inoue (1985). Tetragonula species are distinguished from Heterotrigona species by the conspicuously projecting mesoscutellum of workers and males (not in queens) that is easily recognized by the raised vertex and medially setore propodeum (Sakagami, 1978; Sakagami & Inoue, 1985). In addition, all studied members of Tetragonula show the same simple oviposition behaviour (Sakagami & Inoue, 1990). Tetragonula is the most successful group of the Indo-pacific stingless bees in terms of the number of species and of their relative abundance in most areas (Dollin et al., 1997; Michener, 2000). In Central Vietnam four of the 13 Tetragonula species described have been found: Trigona laeviceps, Trigona gressitti, Trigona pagdeni and Trigona fuscobalteata (Sakagami, 1978). 1.1. The objective of this study Vietnam is situated in the tropical monsoon climatic zone in Southeast Asia. The country stretches from 23o22 North to 8 o30 South (1650 km) with a coastline of 3260 km (from 21o28 North to 8 o22 South). Stingless bees are distributed over the country and form an important group of pollinators in agricultural and natural ecosystems. However, hardly any information exists on the biology and ecology of the stingless bees in Vietnam. For this reason we studied the characteristics of three species of stingless bees that are common in the primary forest of Cuc Phuong. In this paper we describe their nest architecture and share our observations on their biology and behaviour.
2. Methods
The study was carried out from March 1999 to December 1999 in the buffer zone around Cuc Phuong National Forest, which is a section of the primary forest that remains on the limestone mountains, located near the Red River Delta. The area is characterized by flora typical for a tropical rain forest. The 29
o forest comprises an area of 22,220 ha (20o14' o24' N and 105 - 1 05o44' -20 29' E, 300-400 m above sea level). In total 35 wild nests of three stingless bee species were collected from live tree trunks and walls in different places at the Cuc Phuong National Forest. After opening the cavities, we made estimations of the volume of the nest, population size, size of brood and storage area and the presence of gynes and drones. The volume of the nest cavity was estimated from the length and the average diameter of the cavity in the tree trunk. If cavities were not cylindrical in shape the average height, width and depth were used to estimate the volume. One of the species constructed round combs with an average of 20.5 cells per square cm. The radius of all combs was measured after which the total comb area was calculated. The total number of brood cells per colony was estimated after correction of the comb area for empty cells from which the brood had emerged. The other two species arranged their brood cells in clusters. Here the number of brood cells was estimated by measuring the volume of the brood masses. Storage pots (for pollen and honey) were normally found clumped in more than one compact cluster, which did in general not allow for a direct count of their numbers. The amount of food reserves was therefore recorded by estimating the total volumes of these cell masses. In some nests, colony stores were assessed by counting and measuring individual pots, however. Here measurements were taken by measuring the height and the width of a number of the smallest and largest pots in each colony and their mean dimensions were used in calculating the volume of food stores by different formulas (either sphere or ellipsoid) depending on the dominant shape (Roubik, 1979). The thickness of storage pot walls was usually less than one mm, and was assumed negligible in the estimation of the storage pots volume. To estimate the numbers of adult bees in each colony as precise as possible, each nest was blocked the entrance tube at night for keeping all the foragers at home. In the next day, the tree log with the nest was cut down and opened in a tent of mosquito netting. Nests constructed in a stone wall were directly opened under a covering net. This facilitates the measurements of the numbers of a swarm of flying bees. Flying bees mostly return to an observation hive to which the nest was transferred at sunset. Estimates of total numbers of adult bees therefore represent the order of magnitude after judging the approximate numbers of bees in flight and on the comb. The number of virgin queens (gynes) present in the nest was directly counted, and the number of males was estimated by taking a sample of about 100 adult bees that was preserved in 70% alcohol to be sexed later. The number of queen
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cells was directly counted by inspecting the brood combs or the brood cluster. A sample of mature brood of about one hundred cells was taken to determine the percentage of males in the brood. After the colony and nest measurements had been conducted, nests were removed to observation hives and, transported to the Cuc Phuong Commune. They were kept in a dark room of our bee research field station for further behavioural observations. Bee samples were taken and preserved in ethanol 70 % and later identified by C.D.M. as belonging to three different species: Lisotrigona carpenteri, Trigona laeviceps and Trigona ventralis. .
3. Results
3.1. Lisotrigona carpenteri Engel Workers are pale-coloured, of which body length is 3-3.4 mm. Seventeen nests were opened, of which two were hived for further observations. In describing Lisotrigona carpenteri, Engel (2000) showed (by shading in the illustration) yellow in the lower paraocular areas next to the eye margins. This yellow is absent in the 20 specimens identified by C.D. Michener, in specimens from Cambodia (see below), and in Engel's paratype in the American Museum of Natural History, New York City. Presumably it was variable in the series studied by Engel. Another character that caused some confusion is the shape of the hind basitarsus, which is not parallel-sided as illustrated by Engel but is widest in its distal half. An additional record for L. carpenteri, hitherto known only from Vietnam, is the following: Cambodia, O Rang District, Mandulkari, km 170, 27 November, 2002 (D. Roubik), specimens in the Entomology Division, Natural History Museum, University of Kansas. 3.1.1. Nest architecture and colony characteristics Unlike the two following species, nests of L. carpenteri were found in all kinds of substrates, such as tree trunks, brick walls, rock crevices, and other man-made objects. The inner surface of the nest cavity is usually decorated with a black cerumen (1.5-2 mm). The detailed results concerning the nest architecture and colony characteristics are presented in Table 1. Nest entrances were found at an average height of 90 cm the above ground level. Sometimes two to four nests were found together in crevices of a brick wall. L. carpenteri seemed to be the commonest stingless bee species in Cuc Phuong forest. The bees used relatively small cavities for their colonies with volumes ranging from less than 200 cm3 to1000 cm3. The external entrance tube ranges from 10 to 50 mm long, brownish, precisely cylindrical, thin and brittle. The average outer of the tube was 6.5 mm. Sometimes in winter, the entrance tube was temporarily closed at night, possibly helping the very small 31
colonies to defend the nest or regulate temperature. One colony constructed its nest with two external entrances in a fissure of a main doorframe. Brood cells were positioned in amorphous clusters, of which up to three could occur in a single nest. Brood cells were spherical, pale-coloured, 3.0-3.2 mm long and 2.0-2.2 mm diameter. The numbers of brood cells varied considerably, with an average of 57 cells (SD = 63, range: 0-206, n = 17). Of 17 nests opened, ten nests contained a mated queen, six had no queens and brood and one had only a virgin queen. Queen cells (5.0-5.5 mm long, 3.2-3.4 mm ) were randomly found among worker and male brood, and their number ranged from none to fourteen. Table 1. Characteristics of nest-architecture and colony measurements in 17 wild nests of L. carpenteri Measurement criteria Range Means SD Length of external entrance tube (mm) of external entrance tube (mm) Number of stored honey pots Number of stored pollen pots Number of brood clusters Number of queen cells Total number of brood cells Field estimate of total adult bees 31.6 11.7 6.5 1.5 60.0 42.0 18.0 16.1 1.1 1.1 4.5 5.4 57.0 63.0 144.2 96.0 10.0 50.0 4.8 11.0 4.0 145.0 2.0 56.0 0.0 3.0 0.0 14.0 0.0 206 50.0 375.0
Queen cells are also pale, connected with brood cells by from one to four short pillars. All physogastric queens had intact wings, a swollen abdomen and their body length was 4.9-5.2 mm. Of one hundred bees sexed, only two males were found. The external morphology of the males is rather similar to that of the workers and the lacking corbiculae are the convenient feature for discrimination. The population size of this species was small, with an average of 144 (SD = 96, range: 50-375, n = 17). Honey and pollen pots are elliptical or spherical, 7-7.2 mm high and have a remarkably transparent wall (so the colour of honey and pollen in side can be seen). Honey and pollen pots tended to be arranged in separate clusters. The number of honey and pollen pots ranges from 4 to 145 pots, and from two to 56 pots (n = 17) respectively. 3.1.2. Some observations on the behaviour of L. carpenteri For human observers, the guard bees are very timid, withdrawing even when a small stick is inserted in the entrance. Bees do not crawl in the hairs and 32
clothes, nor do they bite the skin when nests are opened. We observed a special behaviour with regard to waste disposal. Workers carried waste pellets from inside the nest to the outer rim of the entrance tube where they dropped the pellet immediately. As a result, we found a small waste dump was piled right under the entrance tube on the ground. 3.2. Trigona (Tetragonula) laeviceps Smith Trigona (Tetragonula) laeviceps is a jet-black species. The worker body length is 4.8-5 mm. Four nests were opened and hived for further observations. 3.2.1. Nest architecture and colony characteristics Unlike L. carpenteri, T. laeviceps nested mainly in living tree trunks of different tree species with trunks of about 30-50 cm in diameter. The hollow space they occupied apparently had been formed through a process of decay and rotting. Usually, a nest cavity was cylindrical with a of 8-11 cm, and a length of 40-90 cm. We also found nests of T. laeviceps in hollows between rocks. External and internal entrance tubes were not found in nests of T. laeviceps. The nest entrance consisted of a hole decorated with a thin rim of black sticky material. In trees, the nest-entrance was found at a level of 2-4 m above ground. The inner walls of the nest were lined with black material of 12 mm thickness. The oval brood cells (4.5-5 mm long) were arranged in amorphous clusters. The cells were connected to each other by pillars. New cells were brownish, but appeared yellow after the wax removal exposed the cocoons. Brood clusters were not covered with an involucrum. Several separate clusters of brood cells were found in the nests. There were also several clusters with food pots. Honey and pollen were stored in separate pots of the same size (5-7 mm , 12-15 mm high), but pollen pots were found closer to the brood masses. Several separate masses of plant resin and resinous material with a strong smell were found at the bottom of the nest. Males and workers were reared in identical cells. Queen cells, elliptical, were sporadically constructed at irregular positions in the brood nest. The number of queen cells was found from zero to seven. The volume of the nests was from 2.0 to 8.5 liters. The estimated number of brood cells was from 650 to 3000 cells and the number of adult bees was estimated from 487 to 1,150 bees. The total volume of stored (pollen and honey) food was estimated from 1.0 to 5 liters. 3.2.2. Some observations on the behaviour of T. laeviceps The temporal pattern of the mass provisioning and oviposition process was facultatively batched (c.f. Sakagami & Inoue, 1990). At some days young 33
workers (most of them bigger and paler than foragers) hovered above the entrance from 10:00 to 14:00 hrs. to make mass flights. After about half of an hour they returned inside the nest. In general, the defence behaviour of this species is mild, with some withdrawing, and often a mild thrusting with mandibles opened. When the nest was opened, several bees landed on hair and skin of the observer; they mildly bite the skin. 3.3. Trigona (Lepidotrigona) ventralis flavibasis Cockerell Trigona (Lepidotrigona) ventralis flavibasis is a bicolour type, with pale white in lateral sides of the abdomen and with pale black in dorsal sides. The worker body length of this species is 5.1-5.2 mm. Fourteen nests were opened and hived for further observations. 3.3.1. Nest architecture and colony characteristics This species constructed its nests in cavities of trunks of living trees and rarely in branches. Nests were found in different tree species as oak, longan, camellia, and lauraceous trees with a trunk diameter of about 30-60 cm. Usually, a hollow tree trunk provided a cylindrical cavity, of which the average diameter was 10.1 2.0 cm (range: 8.0-13.5, n = 14) and the length of 53.3 21.1 cm (range: 25-90, n = 14). The average volume was 4.8 3.3 liters (range: 1.3-10.7, n = 14). The wood surrounding the cavity had a thickness of 10-19 cm. Entrance tubes were found at a level of 1-6 m above the ground, and were slightly slanting downward. The tube was projecting 2-15 cm from the outside of the trunk, and it was formed by a very thin wax-like lamella. The average length of the tubes was 7.2 4.0 cm (n = 14). The tube aperture was funnel-shaped, soft, thin-walled (0.3-1 mm) with a smooth surface that unlike the tube of other species like Trigona terminata and T. itama was hardly sticky. The colour of the entrance tubes was brownish to yellow. The of the opening ranged from 1 to 4.2 cm with an average of 1.9 0.8 cm (n = 14). Fig. 1 presents illustrations of the nest entrance. The interior of the nests was decorated by a lining layer of resin and cerumen (1-2 mm). As all stingless bee species nests were divided into two major parts: a brood part and an area with storage pots. The brood part (12-40 cm long) was in the middle of the nest, and was nearly completely covered
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Figure 1. Architecture of external entrance tubes and inner nests in three sympatric species: Lisotrigona carpenteri (top), Trigona laeviceps (middle) and Trigona ventralis flavibasis (bottom)
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with some soft, thin, brownish-dark sheeted involucrum (2-4 layers). The inner layer (0.2-0.3mm) was thinner than the outer one (0.8-1.1 mm). The involucrum externally connected with the cavity wall by means of short pillars. There was always an internal tunnel (20-30 cm long) directly connecting the external entrance tube to the brood nest chamber. Brood cells (about 5-5.3 mm long, 2.4 mm ) were arranged in regular horizontal combs. Brood combs were round in shape or sometimes elliptical with an average of 7 1.9 cm (range: 3.2-9.2, n = 14). Some brownish pillars (1-1.5mm ) connected the different brood combs. The number of brood combs ranged from 4 to 32 combs with an average of 15.4 7.3 (n = 14). The total number of brood cells greatly varied in the different nests with an average of 15,118 12,320 cells (range: 659-43,586, n = 14). New cells were brownish, but turned yellow when becoming older. Workers and males were produced in similar cells and both were found in the same combs. Queen cells were elliptical (about 6 mm high, 4 mm ). They were mostly positioned at the margin of combs, but in rare cases some of them were found in the middle of the comb. Clusters with food storage pots were usually found both at the top and at the bottom of the nest. Storage food pots were spherical or oval; and the walls were soft, thin and dark-brown. Pollen and honey were stored in separate pots of similar shape. However, pollen pots were in general found closer to the brood. The volume of stored food greatly differed from colony to colony, with an average volume of 1.9 1.1 liters (range: 0.4-3.7, n = 14). Honey had a slightly acid taste, was hardly viscous when compared to honey of honeybees, and had a light yellow colour. Soft propolis-masses (10-12 mm high, 5-8 mm ), white or yellow in colour, were scatteredly deposited at various places in the nest. A remarkable observation was their striking variation in size of the colonies. The field estimate of adult bees ranged from 258 to 12,167 with an average of 4.221 3,348 bees. The number of brood cells was about 3-4 times higher and showed the same variation. Male adults were found in seven out of the 14 nests of T. ventralis. The analysis of mature brood samples showed that the percentage of male brood to female brood ranged from zero to 41.8 % (10.6 12.9 % on average). The number of queen cells present in a nest ranged from zero to 20 (6.4 6.6 on average), and up to six newly emerged gynes were found together with the mother queen. Detailed observations on production of sexuals in nests of T. ventralis have been described in another paper (Chinh & Sommeijer, chapter 5). Further quantitative characteristics of the colonies are listed in Tab. 2.
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3.3.2. Some observations on the behaviour of T. ventralis On summer afternoons, young workers sometimes performed mass flights in front of the entrance. These groups of bees consisted of about 50-120 young workers. Guard bees were normally sitting in the opening. When colonies were disturbed (e.g. by inserting small sticks inside their tubes) the worker bees quickly deposited lots of sticky resin droplets around the inner and outer sides of the opening. We also observed this behaviour when the colony was attacked by ants. When the nests were opened, numerous worker bees landed on the hairs and skin of the observer where they perform disturbing biting. At two times, once in May and once in August, we observed a group of about 14-25 males to hover over the entrance of a nest containing a mated laying queen and several gynes. This drone congregation was present from 12h to 17h, for a period of 5-6 days. Table 2. Characteristics of nest-architecture and colony measurements in 14 wild nests of T. ventralis Measurement criteria Range Means SD Occupied volume of nest site (liter) Length of external entrance tube (cm) of external entrance tube (cm) Volume of stored food (liter) Number of queen-cells Percentage of male brood (%) Number of brood combs of brood comb (cm) Total number of brood cells Field estimate of total adult bees 4.8 3.3 7.2 4.0 1.9 0.8 1.9 1.1 6.4 6.6 10.6 12.9 15.4 7.3 7.0 1.9 15,118 12,320 4,221 3,348 1.3 10.7 2.0 15.0 1.0 4.2 0.4 3.7 0.0 20.0 0.0 41.8 4.0 32.0 3.2 9.2 659 43,586 258 12,167
4. Discussion
L. carpenteri, T. laeviceps and T. ventralis from northern Vietnam, construct their nests in cavities. This is similar to most stingless bees; only very few species build exposed nests. Of the more than 60 species occurring in Costa Rica, only one (T. corvina) is building exposed nests. The three species studied in the Cuc Phuong forest were characterised by a different preference for nest substrates. T. ventralis has a strong preference for nesting in live tree trunks. T. laeviceps uses a somewhat wider variety of cavities, but mainly builds in trees. L. carpenteri constructs its nests in a wide variety of substrates and seems to adapt readily to available cavities in man-made structures. The flexibility for using other nest substrates appears in these species 37
related to colony size. T. ventralis has large colonies (up to around 10,000 adults), T. laeviceps is intermediate with colonies of up to 1200 adult bees, and L. carpenteri has by far the smallest colonies of only up to about 400 adult bees. The fact that colonies of L carpenteri are much smaller than those of the other two species, may allow for the use of all kinds of small cavities. The frequent occurrence of nests of L. carpenteri in diverse substrates may result from a focus on colonizing new holes, whereas the other two species described here focus more on colony defence and growth of the colony itself. This is in agreement with the common occurrence of L. carpenteri in this area. Of Tetragona angustula in Costa Rica (C.A.) and of Trigona nigra in Trinidad and Tobago (W.I.), two species that also use a wide variety of nest substrates, is known that both species swarm more frequently than other stingless bees, for example Melipona beecheii (Sommeijer, personal observation). It is obvious that the very common neotropical T. angustula, because of a great adaptableness to all sorts of cavities, of which many are man-made, is a frequently swarming species. This species is considered as a very weedy bee (D.W. Roubik pers. com.) We have as yet no information about the swarming frequency of L. carpenteri from Vietnam, but the occurrence of aggregations of this species may be a further indication for frequent colony division. The observation that a number of the colonies in the aggregation in the brick wall had very weak nests, may also be an indication for the colonising tendency of this species. Possibly, the aggregation of nests of L. carpenteri may not have a function in colony defence, as has been suggested for the aggregations of T. cupira nests (c.f. Seeley et al., 1981). The defensive behaviour of L. carpenteri was, at least during interference by human observers, very weak. Of T. nigra, which is very common in Trinidad and Tobago, also many nearby nests can be observed, when suitable nest cavities are available. For example, it is common to find tens of nests of this species in one abandoned building that provides ample nesting sites in hollow walls etc. Such occurrence of nest aggregations may result also from the typical colony multiplication of these bees: daughter colonies are established within the flight range of the mother colony from which food and nest materials are transferred to the new nest. The characteristic lacking of a distinct nest exit tunnel in L. carpenteri also resembles the situation in the neotropical T. nigra. The queenless colonies in the aggregation of L. carpenteri could have been the result of recent colony foundation. Virgin queens of recently established colonies could have died because of various reasons, for example they could have been lost in a nuptial flight by predation. The architecture of the comb builder T. ventralis demonstrates some 38
differences with that of the related T. terminata: T. ventralis covers the brood nest with a multi-layer involucrum, while T. terminata does not covers the brood nest with involucrum, but with a pillar systems (Sakagami et al., 1983). The involucrum is a general feature for all horizontal comb building species (Michener, 2000). In our study area in northern Vietnam, nests of T. ventralis flavibasis contain two to four layers of involucrum, whereas five to ten layers have been recorded in T. v. hoozana, from in Taiwan (Sakagami & Yamane, 1984). A higher number of involucrum layers may be related to harsh weather conditions and temperature may be the main factor for this. We observed in T. ventralis colonies that the number of involucrum layers was increased in winter and reduced in summer, which suggests that bees adjust the involucrum for temperature regulation. Unlike in Apis species, where the temperature of brood combs can be kept constant by clustering or fanning behaviour and by water vaporization (Seeley, 1985), thermo-regulation in stingless bees is limited (Michener, 1974; Wille, 1983; Sakagami et al., 1983). The lack of temperature homeostasis may be a factor that restricts the geographic distribution of stingless bees outside the tropics and that influences nest sites for some species (e.g. in large trunks providing thick wood protection). Lisotrigona carpenteri was only recently described by Engel (2000) from Nghe An and Ha Tinh province, northern Vietnam. In this study we report for the first time about the nest architecture of this species. Our study shows that many interesting biological data on social bees still have to be revealed, not only of rare species, but also of general ones that are expected to play important roles in the ecosystem.
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CHAPTER 4
Patterns of male production in the stingless bee Melipona favosa (Apidae, Meliponini)
Tong X. Chinh1, Gijs B.J Grob2, Franciscus J.A.J. Meeuwsen3 & Marinus J. Sommeijer3
Adapted from a publication in Apidologie 43 (2003): 161-170
Bee Research and Development Centre, 68 Nguyenhong, Langha, Dongda, Hanoi, Vietnam
Nationaal Herbarium Nederland, Leiden University, P.O.Box 9514, 2300 RA Leiden, The Netherlands Department of Social Insects, Utrecht University, P.O.Box 80.086, NL-3508 TB Utrecht, The Netherlands
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Summary. In many stingless bee species, laying workers oviposit trophic
eggs that serve as a component of the queens diet. Workers of some species also lay reproductive worker eggs that give rise to males. Male-producing workers can occur in queenright colonies. We studied male production by workers of Melipona favosa. In six colonies monitored under field conditions, we observed that males emerged during distinct Male Emerging Periods . Subsequently, we studied the laying of male eggs in laboratory observation hives with the use of video. We found that laying workers oviposited reproductive eggs in distinct Reproductive Laying Worker Periods and that this was followed by the clumped mergence of males afterwards. Behaviours of laying workers and of the ovipositing queen are preliminarily described.
1. Introduction
The haplo-diploid system in social Hymenoptera influences the relatedness of queens and workers to males and can contribute to conflict over male production. In the light of the kin selection theory (Hamilton, 1972), when a colony is headed by a single monandrous queen, workers are expected to favour their sons (relatedness, r = 0.5) or other workers sons (nephews) ( r = 0.375) above sons of the queen (brothers) (r = 0.25) (Hamilton, 1964; Ratnieks, 1988, Peters et al., 1999). In contrast, in colonies headed by polyandrous queens, worker-queen conflict over male production may be reduced because workers are then more related to the queens sons than to other workers sons (Ratnieks, 1988; Peters et al., 1999). In colonies of the honeybee, Apis mellifera, which are headed by a highly polyandrous queen, males are normally produced only by the queen (Seeley, 1985, Peters et al., 1999). In queenless colonies, honeybee workers can activate their ovaries and lay unfertilised eggs. Workers in queenright colonies generally do not activate their ovaries as a result of pheromonal control by the queen (Velthuis, 1970). However, it has recently been demonstrated that queen-right Apis mellifera workers sometimes do lay eggs but that their contribution to male production is low because of worker policing (Barron et al., 2001; Ratnieks, 1993; Visscher, 1989). In contrast to Apis, stingless bee (Meliponini) workers with active ovaries are common in queenright colonies (Sakagami, 1982). Moreover, worker oviposition in queenright colonies occurs widely among the various groups of stingless bees (Sakagami, 1982; Sommeijer & van Buren, 1992; ImperatrizFonseca & Kleinert, 1998; Koedam et al., 1999). However, in queenright colonies, Melipona workers may lay mostly trophic, not reproductive eggs (Sakagami, 1982; Sommeijer et al., 1984a). Trophic eggs are eaten by the queen prior to her oviposition, while reproductive eggs can give rise to 41
Chapter 4. Patterns of male production in Melipona favosa
offspring. In some species of non-Melipona stingless bees, e.g., in Scaptotrigona, reproductive workers commonly oviposit after queen oviposition (Beig, 1972; Bego, 1982; Sakagami, 1982), which leads to cells containing both a worker egg and a queen egg. In contrast, in Melipona, oviposition by reproductive laying workers generally precludes oviposition by the queen in the same cell (Sommeijer et al., 1984b; Sommeijer & van Buren, 1992). Reproductive workers of M. favosa may also oviposit after queen oviposition, however this phenomenon is very rare. Melipona trophic and reproductive worker eggs differ in their external morphology. Reproductive eggs in queenright colonies resemble reproductive worker eggs laid in queenless colonies (van Buren & Sommeijer, 1988), and have a well-developed chorion. In contrast, the chorion of trophic eggs is poorly developed. However, these differences are graded and not discrete (Sommeijer et al., 1984a). Besides a difference in trophic and reproductive egg morphology, there is a difference in the behaviour of workers that lay trophic and reproductive eggs, so the two categories of laying workers can be easily distinguished (Sommeijer & van Buren, 1992). We have studied the production of males in M. favosa. In a previous paper we showed that in M. favosa the workers are the main producers of males (Sommeijer et al., 1999). In this paper we describe the production of males in more detail and show that it occurs periodically both under field and laboratory conditions. In the discussion we link this clumped male production to oviposition behaviour of both the queen and the workers, and try to explain the observed patterns in light of existing theories.
2. Materials and methods
All colonies of M. favosa were kept in wooden box hives (WxLxH: 20.5x35.5x15 cm), which were covered by glass lids facilitating behavioural observation. The mature brood combs were partially separated from the major brood-nest by placing them in small annex boxes that were connected to the major nest box with plastic tubes. The annex served as a place where newly emerged bees could remain for about the first 24 hours after their emergence on the comb. Recently eclosed workers and males of M. favosa do not disperse from the comb of emergence. Upon the recording of daily-emerged adults, these were reintroduced into the main brood-nest compartment. Colony development parameters such as daily cell production, numbers of new cells and storage pots, etc., were measured weekly. The field studies were carried out from October 1994 to February 1995 on the island of Tobago (Trinidad & Tobago, West Indies). Six queenright colonies of Melipona favosa that earlier had been installed in a room in a 42
wooden building were used to study the temporal patterns of male production. These colonies had free flight to the outside in their natural habitat through plastic tubes leading through the wall. The colonies received no additional feeding. The colonies had been in this position for more than two years. The population of these colonies ranged from 129 to 204 bees, which is normal for this species. In 1997, four queenright colonies (referred to as 7, 8, 9 and 10) of M. favosa were housed in the room of our laboratory at the Faculty of Biology, Utrecht University, The Netherlands. These colonies originated from Trinidad and Tobago and their populations ranged from 109 to 270 bees1. Temperature was kept constantly at 24 degrees Celsius and RH was constantly around 75 %. In these lab colonies, all ovipositions were analysed to identify whether the queen or a worker oviposited in a cell. Red light and video were used to observe the ovipositions, comb development and adult emergence continuously and simultaneously from April to November. To identify the maternity of the emerged bees, the location of each oviposited cell was mapped and its development followed until emergence. 2.1. Classification of oviposition types The following classification of provisioning and oviposition processes (POPs) was made during this study: 1. Exclusive queen oviposition 2. Worker oviposition followed by queen oviposition. In this case the worker laid a trophic worker egg (TWE), which was followed by subsequent laying worker withdrawal, queen oophagy, and queen oviposition. 3. Reproductive worker oviposition. This is oviposition by a laying worker that does not withdraw after oviposition but operculates the cell immediately. This excludes queen oophagy and queen oviposition, and there is no obvious response of the queen to this. 4. Oviposition competition. In this case various laying workers are competing for oviposition and worker oophagy as they try to operculate after their own oviposition. 5. Worker oviposition after queen oviposition, followed by immediate operculation by this laying worker.
______________________________________________________________
1
The colonies studied in the Utrecht lab had an average population of 194 and were larger than the colonies studied in the field (average population 161), but this difference was not significant (P >0.05).
43
3. Results
3.1. Male Emerging Periods in the colonies under field condition The analysis of emerging bees of the different sexes and castes in the field revealed that males emerged regularly in repeated periods that we define as Male Emerging Periods (MEPs) (Fig. 2). In the six field -observed colonies,
Col. 6 Col. 5 Col. 4 Col. 3 Col. 2 Col. 1
26- 2- 9- 16- 23- 30- 7- 14- 21- 28- 4- 11- 18- 25- 1- 8- 15- 22Oct Nov Nov Nov Nov Nov Dec Dec Dec Dec Jan Jan Jan Jan Feb Feb Feb Feb Dates
Figure 2. Male Emerging Periods as they occurred in the different colonies observed in the field (black bars: MEPs). 11 MEPs were recorded, which occurred nonsynchronously in different colonies (Tab. 1). A typical MEP (Fig. 1) is characterised by a gradual increase of the daily number of emerging males. After reaching peak values (sometimes with 100% males emerging daily) the percentage of daily emerging males declines to zero. However, some MEPs were interrupted with short intervals from 1 to 9 days in which no males emerged. The occurrence of the MEPs differed in the colonies. The length of the MEPs varied from 10 to 74 days with an average of 32.8 days (SD = 18.3, n = 11) (Tab. 1, Fig. 2). Absolute and relative numbers of males emerging in a single MEP varied from day to day. The number of emerging males found on one day ranged from 1 to 12 with an average of 2.1 (SD = 2.6, n = 265) over all MEPs. The number of emerging males as a percentage of the total number of emerging 44
Table 1. Some parameters of all the fifteen observed Male Emerging Periods in the field and in the lab
MEP Colonies Start End 3 Jan 23 Feb 8 Dec 23 Feb 15 Feb 19 Dec 23 Feb 17 Nov 22 Jan 21 Feb 22 Jan 27 Jun 17 Oct 16 Jun 7 Dec Duration (day) 53 30 14 37 74 35 30 17 24 10 37 46 38 67 60 38.1 19.0 Males 153 92 8 62 88 19 79 43 12 14 69 422 163 940 192 157.1 % InterMales vals (day) 14.6 9 13.4 2 11.0 1 15.3 1 18.1 6 9.7 5 16.0 2 23.2 1 3.6 4 14.9 1 37.3 2 70.7 0 38.0 1 10.0 3 33.8 5 22.0 2.9 2.5 Abs. max 1 Dec 4 Feb 1 Feb 27 Jan 17 Jan 26 Dec 14 Feb 11 Dec 13 Jan 17 Feb 3 Jan 25 May 21 Sep 21 Apr 10 Oct Rel. max 10 Dec 8 Feb 1 Dec 26 Jan 17 Jan 18 Dec 1 Feb 14 Nov 4 Jan 11 Feb 2 Jan 17,19, 24 May 29, 23 Sep 23, 24, Apr; 3 Jun 10, 16, 17 Oct
1 1 11 Nov 2 1 24 Jan 3 2 24 Nov 4 2 17 Jan 5 3 19 Dec 6 4 15 Nov 7 4 24 Jan 8 5 31 Oct 9 5 29 Dec 10 5 11 Feb 11 6 16 Dec 12 7* 12 May 13 8* 8 Sep 14 9* 10 Apr 15 10* 9 Sep Average SD * Colonies in the lab
240.9 17.0
MEP: index number given to the Male Emerging Period. Start: the date on which the MEP starts, End: the date on which the MEP ends, Duration: The duration of the MEP in days, Males: the total number of males emerging during the MEP, %Males: the number of males emerging during the MEP as a percentage of the total number of emerging bees during the MEP. Intervals: the number of periods of no male production within MEPs of 1 day or more. Abs.max.: the date on which the absolute number of emerging males reaches its peak, Rel.max.: the day on which the % of emerging males reaches its peak.
bees per MEP ranged from 3.6 % to 37.3 %, with an average of 16.1 % (SD = 8.6, n = 11). The average percentage of emerging males over all MEPs was 15.6 % (SD = 19.2, n = 265). The total number of males emerging during one MEP ranged from 8 to 153, with an average of 58.1 (SD = 44.7, n = 11). The values for the individual colonies are presented in Tab. 1. The periods between the recorded MEPs in which exclusively females emerged ranged from 20 to 40 days with an average of 31.8 days (SD = 10.6, n = 5).
45
Percentage of emerging males
100 80 60 40 20 0
31-Jul 10-Aug 20-Aug 30-Aug 9-Sep 19-Sep 29-Sep 9-Oct 19-Oct 29-Oct 8-Nov
Dates
Figure 1. The typical pattern of an occurring MEP is presented for colony 10 as an example. The emerging males are given by percentages of daily emerging bees. 3.2. Distinct reproductive laying worker periods The comprehensive observation of ovipositions in the colonies at the Utrecht laboratory revealed that laying workers oviposited reproductive worker eggs (RWEs) in clearly distinct periods. We define such a period, which has a duration of about several weeks, as a Reproductive Laying Worker Period (RLWP). These RLWPs occurred in all observed lab colonies; during the observation time, a total number of four RLWPs occurred in the four colonies (Fig. 3). By definition, outside the RLWPs the workers laid no reproductive eggs at all; only trophic eggs were laid. In one complete period of 44 days outside the RLWP that we could observe in colony 8 the queen did not produce any males. In the other colonies we could not analyse the complete periods outside the RLWPs, but from the observed parts of these periods it appears that from the queen eggs operculated in these periods exclusively female bees emerged. Unfortunately, because of the limitation of our detailed observation time, we could not measure the whole length of all these nonRLWPs. In the RLWPs the daily number of RWEs varied and ranged from 0 to 35, with an average of 6.8 (SD = 6.7, n = 185 days). The daily number of oviposited cells by both the mated queen and the laying workers during RLWPs ranged from 2 to 40, with an average of 14.7 (SD = 6.9, n = 185 days). The RLWPs of Col.7, Col.8, Col.9 and Col.10 had a duration of 46, 47, 102 and 59 days respectively. 46
Col. 10 Col. 9 Col. 8
Col. 7
1- 15- 29- 12- 26- 10- 24- 7- 21- 5- 19- 2- 16- 30- 13- 27- 11- 25Mar Mar Mar Apr Apr May May Jun Jun Jul Jul Aug Aug Aug Sep Sep Oct Oct Dates
Figure 3. Reproductive Laying Worker Periods and Male Emerging Periods occurred in the different colonies observed in the lab (thin bars: RLWPs, thick bars: MEPs). 3.3. Oviposition rates fluctuate with RLWPs The average number of new cells per day was significantly higher in the RLWP (average = 14.7, SD = 6.9, range: 0-40, n = 185) than in the periods where only queen eggs were operculated (average = 10.1, SD = 6.0, range: 132, n = 67) (Anova, P < 0.0001). Furthermore, the average egg production by the queen in the RLWP (average = 7.9, SD = 5.5, range: 0-28, n = 185 days) was also significantly less (Anova, P < 0.01) than outside the RLWPs. In both these periods, trophic worker eggs (TWEs) were laid and the queen always ate such eggs. However, the daily percentage of TWEs per new cell in the RLWPs (average = 16.1, SD = 20.9%, range: 080 %, n = 185) was significantly lower than outside the RLWPs (average = 19.1, SD = 28.3 %, range: 0-200 %, n = 67) (ANOVA, P< 0.05). The daily number of TWEs (average = 1.6, SD = 2.5, range: 0-10, n = 185) positively correlated with the number of queen eggs (average = 7.9, SD = 5.5, range: 0-28), (Spearman-r = 0.68, P < 0.0001, n = 185 days,) whereas the daily number of RWEs was obviously negatively correlated with the daily TWE number (Spearman-r = 0.21, P < 0.01, n = 185 days) in the RLWPs. 3.4. Number of observations of the five classes of POPs Laying workers and the mated queen had a different oviposition time for releasing their various types of eggs. Laying workers took longer for the 47
release of RWEs, (average = 8.9s, SD = 2.6, range: 4-31 s, n = 571) than for ovipositing TWEs (average = 4.2s, SD = 1.4, range: 4-31 s, n = 295), (P < 0.001). Correlating the duration of the oviposition of the queen with the sex of her offspring it appeared that her oviposition act took a shorter amount of time for the release of a fertilised egg (average = 26.9s, SD = 6.9, range: 12-69 s, n = 1174) than for that of an unfertilised egg (average = 46.1s, SD = 35.8, range: 21-181, n = 33), (P < 0.01). In general, the number of observed instances of the five oviposition classes were: 1. Exclusive queen oviposition (n = 1698). 2. Worker oviposition followed by queen oviposition (n = 446). 3. Reproductive worker oviposition (n = 1248). 4. Oviposition competition (n = 26). 5. Worker oviposition after queen oviposition (n = 2). This was only observed twice and only in the RLWP of Col.9 in which the old queen died. In this colony, at the end of the recorded RLWP, a number of workers competed to various degrees for oviposition, for feeding on eggs of other workers, and for operculation in some cells Shortly after this, the old queen of this colony died and a new queen became accepted, developed physogastry and took over the ovipositions. This queen had exclusively male offspring, since she was unmated. In this colony, the old and the young queen were observed together in the brood nest for almost two weeks. 3.5. Reproductive laying worker periods (RLWPs) observed to be followed by male emergence periods (MEPs) In the colonies at our Utrecht laboratory we could confirm that RWEs were released in distinct RLWPs and that these were followed by the emergence of males during typical MEPs after the incubation time (see Fig. 3). In these MEPs, both the absolute and the relative number of emerging males varied from day to day. The absolute number of emerging males per day ranged from 0 to 45, with an average of 8.1 (SD = 9.3, n = 213) over all MEPs. In the MEPs of colonies Col.7 and Col.9, most of the emerging bees were males. On some days only males emerged. In contrast, in the MEPs of Col.8 and Col.10 most of the emerging bees were females. The total number of males produced during a single MEP ranged from 163 to 940 males, with an average of 429.3 (SD = 359.7, n = 4). The total number of males emerging in the four recorded MEPs was 1712. The relative number of emerging males as a percentage of the total number of emerging bees per MEP ranged from 33.8 to 70.7 %, with an average of 53.1 % (SD = 20.0, n = 4). The length of the MEP ranged from 38 to 67 days (average = 52.8, SD = 13.0, n = 4). In Col.8, two MEPs were 48
observed but the first MEP was in process upon the arrival of this colony at our lab, so its actual length was unknown (MEP recorded from May 12 to July 4). The values for the individual colonies are presented in Tab.1.
4. Discussion
Our results indicate that in M. favosa males are produced in distinct periods, the Reproductive Laying Worker Periods (or RLWPs), which lead to the clumped emergence of males in distinct Male Emerging Periods (or MEPs). This periodic pattern of male production occurs both in the field and in the laboratory, and thus occurs even in the absence of extranidal cues, like fluctuations in temperature, sunlight, food availability and the like. Lab conditions consisted of constant photoperiods for day and night and did not mimic field conditions. Another important observation is that under both laboratory and field conditions different colonies appear not to be synchronous in their male production. When certain colonies are producing males, others that are under the same condition are not, even when they are located in the vicinity of each other. Colonies appear to have their own rhythm for male production periods and a single colony may have a number of MEPs over the year. In addition, our results show that the brood cell production in the RLWP is higher than that outside these periods. This is because in the RLWP both the queen and laying workers lay reproductive eggs. Since bees were not individually marked, we could not determine the individual participation in cell construction, provisioning and worker oviposition. Nevertheless, besides this increase in brood production, we were not able to find any correlation between the occurrence of RLWPs and other intranidal factors such as the number of storage pots or production of queens. Finally, we observed that in M. favosa workers are the main producers of males (Sommeijer et al., 1999) and that, in agreement with Kerr, 1969, the queen-worker conflict is not expressed in overt aggression, with exception of queenless and nearly queenless colonies. What causes the internal rhythm of colonies in their male production? In the discussion below we will propose and evaluate a few hypotheses, in the light of circumstantial evidence and the worker-queen conflict over male parentage. 4.1. Male production in M. favosa is not seasonal In Tobago we recorded MEPs occurring from the beginning of the observation period in October to the end of the observation period in February. The recorded MEPs in colonies studied in the Netherlands were from March (immediately from the arrival of the colonies) through the end of October. From these results and from unpublished observations in long established 49
colonies in the natural habitat (Sommeijer & de Bruijn, personal observation), it appears that in M. favosa males are produced in batches throughout the year. M. favosa is not unique for not having a strict seasonal male production. M. beecheii (van Veen et al., 1999), Plebeia remota (van Benthem et al., 1995) and some Trigona species (Beig, 1972; Bego, 1982, 1990; Engels & Engels, 1984) are reported to produce males throughout the year as well, yet in different numbers, depending on the season. Van Veen et al. (1999) found a significant correlation between drone production and pollen storage in M. beecheii and male production decreased at the end of the rainy season due to floral scarcity in this period. We have no information about the age of the queens in the colonies studied in the natural condition and in the colonies transported to The Netherlands. The age of queens was also unknown in other, long established, colonies where similar periodicity of male production by workers was observed in 1998 (Sommeijer & de Bruijn, personal observation). This suggests that this periodicity may occur over the life of the queen. 4.2. The switch-point hypothesis In the bumble-bee, Bombus terrestris, queen-worker conflict over male parentage usually follows a switch point, which occurs when the queen starts laying haploid eggs (Duchateau & Velthuis, 1988; Bourke & Ratnieks, 2001). This could explain the observed patterns in M. favosa: the queen lays a haploid egg and becomes a competitor with the workers, who start laying reproductive eggs (the beginning of a RLWP). The RLWP may stop when the mass male production is at the expense of worker production. Worker production declines during a RLWP since the queen lays fewer diploid eggs. 4.3. Incomplete queen control hypothesis Foster et al. (2001) discuss how queen control in Dolichovespula colonies, headed by a monandrous queen, could result in fewer males being produced by workers than by the queen. However, the pheromonal queen control could be evolutionarily unstable, if workers were selected to ignore the pheromone (Keller & Nonacs, 1993; Foster et al., 2000). Sommeijer et al. (1984) hypothesized queen control in M. favosa. Diminishing queen control in colonies that are seasonally becoming larger would lead to the development of reproductive workers. However, in this study, reproductive workers were clearly observed in all colonies. Moreover, RWE laying did not occur in absence of the queen. Neither was there overt aggression between reproductive workers and the queen. The queen was present at most occurrences of RWE laying but she was far less involved in such POPs than during her own egg-laying or the laying of TWEs. From the observations it 50
appears that in the RLWPs the queen is much less motivated to play an active role in POPs than as she is outside these periods. The temporary occurrence of RLWs could result from incomplete queen control over reproductive development in workers. Perhaps the temporary worker -dominance in M. favosa is the arbitrary outcome of an arms race in favour of the workers (Foster et al., 2001). In this case it may very well be that closely related species do not show the same patterns (Foster et al., 2001). Indeed, M. beecheii seems to have a more seasonal male production (Moo-Valle et al., 2000; van Veen, 1999). In any case, it remains unclear why and how queen control would diminish frequently and during relatively short periods. 4.4. Queen-like worker hypothesis It has been established (Sommeijer & Velthuis, 1977) that in queenless colonies of M. favosa, some workers behave in a queen-like manner: they can start a POP and are responsible for many ovipositions. Through the genetictrophic caste determination system in Melipona (Velthuis & Sommeijer, 1991) it may be that, also in queenright colonies, some workers are genetically more fit to become reproductive than others. According to this hypothesis, only the limited number of workers with a genetic predisposition to develop into queens, could be responsible for all RWEs. Probably the individual oviposition performance of these reproductive workers can exceed the oviposition rate of workers that release TWEs (Sommeijer & van Buren, 1992). Most likely, the number of reproductive laying workers is limited per colony. It may be assumed that these queen-like workers are able to escape from queen pheromonal control. These workers cannot start a POP in queenright situations, but they can take one over. Pheromonal control may occur in these queen-like workers and prevent overt aggression by the queen and perhaps also by their fellow-workers. According to this hypothesis, the end of a RLWP is results from the cessation of activity by these specific laying workers and after some time, some new workers may be able to develop such queen-like reproductive behaviour. In contrast to many Hymenoptera, workers are the main producers of males in M. favosa. Apparently queen control of male production is less pronounced in this species, or worker reproduction is much less costly for colony reproduction in M. favosa than in other Hymenoptera. The general occurrence of reproductive workers is consistent with the hypothesis that M. favosa colonies are headed by monandrous queens. It remains to be tested how general this phenomenon is in Melipona and other stingless bees. It is important to study the proximate and ultimate function of this periodical male production by workers in these stingless bees.
51
CHAPTER 5
Production of sexuals in the stingless bee Trigona (Lepidotrigona) ventralis flavibasis Cockerell (Apidae, Meliponini) in northern Vietnam
Tong X. Chinh1 & Marinus J. Sommeijer2
Accepted for publication in Apidologie
Bee Research and Development Centre, Ngo 68 Nguyenhong, Langha, Dongda, Hanoi, Vietnam
1
Department of Social Insects, Utrecht University, P.O.Box 80.086, NL- 3508 TB Utrecht, The Netherlands
52
Summary. In a two-year study on the stingless bee Trigona ventralis under
field conditions in Vietnam, production of males and gynes was investigated in ten colonies. Male production in this species was found to be periodic. Typically, colonies produced a batch of males in a period of several weeks. At the population level, however, males were found throughout the year, since male production was not synchronized between colonies. Male production varied significantly among different months and between the rainy and the dry season. The number of males produced was positively correlated with numbers of brood cells, of food storage pots, and of emerging workers. Unlike the clumped male production in a certain colony, gynes were continuously produced in all colonies at low numbers, but seasonal effects on their numbers were found similar to effects on the numbers of males. Earlier the same periodical production of males in an individual colony without synchronization between colonies was found in a new world-species, Melipona favosa. This suggests that the mechanisms underlying allocation of resources over sexes may be general for a number of stingless bees.
1. Introduction
The highly eusocial group of stingless bees is widely distributed in tropical and southern subtropical areas throughout the world. The numerous species of these bees show considerable diversity in morphology, body size and nest architecture. However, all species have the characteristic of mass-provisioning brood cells prior to oviposition, as found in solitary bees. The typical provisioning and oviposition behaviour is described by Sakagami (1982). Of the presently described 374 species worldwide, belonging to 23 genera and 18 subgenera, about 43 species (two genera: Lisotrigona and Trigona comprising three subgenera: Heterotrigona, Homotrigona, and Lepidotrigona) occur in the Asian region (Michener, 2000). The subgenus Lepidotrigona prevails in this region with four species (Sakagami, 1984). Very little has been published on the biology of stingless bees of the Asian region. No information is available on the production of sexuals and on reproductive behaviour. From American and Asian species of Trigona we know that gynes are produced in typical "royal cells" that are distinctly larger than cells from which workers and males emerge (Sakagami, 1982; Imperatriz-Fonseca & Zucchi, 1995). Queen determination is nutritionally regulated through the quantity and the quality of larval food provisioned (Michener, 1974). Colonies of Trigona and related taxa bees rear continuously just a few gynes (Imperatriz-Fonseca & Zucchi, 1995; van Veen & Sommeijer, 2000). This is fundamentally different from Melipona colonies that permanently produce larger numbers of gynes (Kerr, 1950; Sakagami, 1982; Engels & ImperatrizFonseca, 1990; Imperatriz-Fonseca & Zucchi, 1995; Ratnieks, 2001; 53
Chapter 5. Production of sexuals in Trigona ventralis flavibasis in north Vietnam
Sommeijer et al., 2003a; Sommeijer et al. 2003b; Wenseleers et al., 2003). Workers with active ovaries are common in queenright stingless bee colonies. Moreover, workers feed the queen by laying trophic eggs, which occurs widely among the various groups of stingless bees (Beig, 1972; Bego, 1982; 1990; Sakagami, 1982). Besides producing unviable trophic eggs, workers also produce viable, reproductive eggs that develop into males (Sommeijer & van Buren, 1992; Imperatriz-Fonseca & Kleinert, 1998; Sommeijer et al., 1999, Chinh et al., 2003). Male production by workers in queenright colonies of stingless bees has been known for along time, since it was implied by early genetic studies which showed that a significant faction of males in several species were workers sons (Contel & Kerr, 1976; Machado et al., 1984), and this has been confirmed by more recent genetic studies (Tth et al., 2002; Paxton et al., 2003; Tth et al., 2003) and by behavioural studies (Koedam et al., 1999; Sommeijer et al., 1999; Chinh et al., 2003; Tth et al., 2004). Therefore, between the species studied so far male production by workers seems to be common. The influence of seasonal variations in food availability on male production in stingless bees has been a topic of study (Bego, 1990; van Benthem et al., 1995; van Veen et al., 1999; Grosso et al., 2000; Moo-Valle et al., 2001; Sommeijer et al., 2003a), but the precise relation is still unclear. Our study on male production in Melipona favosa established that males of this species are produced in distinct periods, asynchronously in different colonies. As a consequence, males are produced throughout the year at population level, but in clumped periods at colony level (Chinh et al., 2003). Most studies on this topic are either not detailed enough to reveal distinct periods of male production at colony level, or they do not include detailed observations throughout the year on a series of colonies to address the question whether or not male production is seasonal. To understand male and gyne production mechanisms in stingless bees it is significant to investigate the possible effects of environmental factors in areas where natural climatic conditions reveal more distinct seasonal variation than in areas where previous studies were carried out. The mountain-forest area of the northern part of Vietnam, where stingless bees are common and the climate varies considerably through the year, was considered suitable for such a study. In the present study, we investigated how resources are allocated over sexes and castes, and which colonial and climatic factors influence these processes under natural conditions in northern Vietnam.
Observations were done on ten nests of Trigona (Lepidotrigona) ventralis flavibasis Cockerell. In April 1999 the colonies were transferred from their 54
natural nest cavities in hollow trees to observation hives and installed at our field station. The research site is in the natural habitat of the bees. This study area is in the bufferzone of Cuc Phuong National Forest, which is a section of the primary tropical rain forest that remains on the limestone mountains near the Red River Delta. The Cuc Phuong forest comprises an area of 22,220 ha and is situated 100 km Southwest of Hanoi, Vietnam (20o14 -20o24 N and 105o29 -105o44 E, 350 m above sea level). The field data were collected from June 1999 to December 2000. To study the emergence of workers, gynes and males we used the technique developed at Utrecht University (Sommeijer and de Bruijn, 1990; Sommeijer et al., 2003a). The colonies were kept in observation hives (WxLxH: 22x40x17 cm), which were covered by glass lids to follow behaviour. The bees could freely fly out from the observation hive through a plastic tube of 7 mm diameter that passed though the wall. To each observation hive a small annex box was connected that served as a semiseparated place for the emergence of bees. Mature brood cells were regularly taken out of the brood nest and placed in the annex box. Adult workers of the major nest were able to move freely between major nest and annex box. Newly emerged bees that normally remain more than a day on the combs from which they emerge, were daily collected from the emergence box so that we could count their numbers and identify workers, gynes and males. After classifying the emerged adults, they were reintroduced into the main broodnest. Every fortnight, the total number of storage pots and number of brood combs were measured as an estimate of colony development. The number of brood cells was calculated by estimating the average radius of the circular combs that was measured by drawing circular comb outlines on a transparent plastic sheet. Then the number of cells for each comb was calculated using the formula: No = 20.5 x r2 (No: cell total number; 20.5: average cell number per square cm; r: average radius of a comb). The total number of cells per colony was corrected for the holes in mature combs with centrally emerging brood. Weather data were obtained from the National Hydrometeorology Station in Cuc Phuong forest, located about 100 m from our field laboratory. The climate in this area is characterized by a dry and a rainy season. The rainy season lasts from May to November with a daily average temperature of 25o C and a total precipitation of 1456 mm3 (86 % of the total annual rainfall). The dry season runs from December to April with a daily average temperature of 17o C and a total precipitation of 243 mm3 (see Fig. 1D for detailed climatic conditions). ANOVA tests were performed for the analysis of differences in male and gyne production across colonies and seasons. Linear regression tests were 55
used to investigate the relationship between the production of sexuals, colonial parameters and climatic factors.
3. Results
3.1. Colony development in T. ventralis In our colonies, most brood cells and stored food pots were found in the rainy season from May to September. In the dry season, both the brood cells and food stores diminished (Fig. 1A&1B), but the food reserves were more constant compared to the number of brood cells. The amount of stored food varied significantly between the rainy and the dry season (Anova, P = 0.05), over the months (Anova, P < 0.001), and between colonies (Anova, P < 0.001). The brood cell population varied significantly between colonies (Anova, P < 0.0001), between months (Anova, P < 0.0001), and between the rainy and the dry seasons (Anova, P = 0.0001). The fluctuating brood cell numbers were correlated with the numbers of stored food pots (Spearson (two-tailed)-r = 0.58, P = 0.05). 3.2. Male production at population level Of the total of 205,536 emerging bees examined, 6456 were males (3.1%). The production of males varied considerably between the colonies, but when the data of male production from the different colonies were combined it appeared that at population level males were produced during most of the year (Fig. 1B). The number of males produced in the rainy season was significantly higher than in the dry season (Anova, F = 16.876, df = 1, P < 0.01). The overall result of all the colonies combined indicated that a gradual increase from April to September was followed by a gradual decrease from November to March. The average monthly proportion of males to all emerging bees ranged from 0.0 % in December to 5.4 % September (1.9 1.8 %, n = 12 months), but the range was considerable explaining the high percentage of males (3.1 %) of the total emerged bees. The percentage of colonies that produced males ranged from 0.0 % in December to 100 % in May, with an average of 42.7 26.9 % (n = 12 months). Note that although all colonies produced males in May; this did not result in peak numbers of emerging males in that month (Fig. 1B). Per month, the production of maleswas correlated with the average temperature (Pearson-r = 0.78, P < 0.0001), the average precipitation (Spearson-r = 0.84, P < 0.0001) and the average hours of sunshine (Pearson-r = 0.73, P < 0.01), but not with the average relative humidity (Pearson-r = 0.097, P = 0.76).
56

14000 12000 10000 8000 6000 4000 2000 0 500 400 300 200 100 0 6 4 2 0 120
Average of temperature (oC) and humidity (%)
Number of cells and workers
cells workers
Number of males and pots
males pots
Number of gynes
gynes
600
Total of rainfall (mm) and sunshine (hrs.)
100 80 60 40 20 0 J F M A M J J A S O N D Month of the year temperature humidity rainfall
500 400 300 200 100 0
sunshine
Figure 1. Monthly average number (mean + SD) of brood cells, emerged workers and storage pots (A), of emerged males (B), and of emerged gynes (C); average (mean + SD) temperature (oC), relative humidity (%), rainfall (mm), and sunshine (hrs.) (D) 57
Regression analysis showed that the production of males was positively correlated with the production of gynes in a colony (F = 6.092, df = 1, P < 0.05). The monthly number of emerging males was positively correlated with that of closed honey and pollen pots (Pearson-r = 0.64, P < 0.05, n = 12), with the total number of brood cells (Pearson-r = 0.90, P < 0.0001, n = 12) and with the number of emerging workers (Pearson-r = 0. 79, P < 0.01, n = 12 months). Table 1. Detailed data on 26 MEPs that occurred in the observed colonies.
Colony and MEP name S1-MEP1 S1-MEP2 S2-MEP1 S2-MEP2 S2-MEP3 S3-MEP1 S3-MEP2 S3-MEP3 S4-MEP1 S4-MEP3 S5-MEP1 S5-MEP2 S5-MEP3 S5-MEP4 S6-MEP1 S6-MEP2 S7-MEP1 S7-MEP3 S7-MEP4 S9-MEP1 Start-date End-date Duration (days) 19 53 62 98 235 15 28 44 40 103 56 33 109 122 80 29 49 43 54 70 63 70 5 11 9 106 61.8 48.3 Total males 5 15 796 43 3597 7 4 11 35 5 269 31 13 90 27 3 4 124 754 3 14 584 2 5 4 7 248.2 720.6 Male Daily absolute No Daily relative No (%) (male) (%) Avera- SD Range Avera- SD Range ge ge 1.6 0.3 0.5 0-1 1.3 2.3 0 - 7.1 0.9 13.9 0.9 16.9 1.9 1.6 0.8 1.7 0.2 6.0 1.4 0.3 1.0 0.4 0.5 0.7 4.2 16.5 0.1 0.3 11.8 2.6 1.9 2.5 0.3 3.5 5.1 0.3 12.8 0.6 15.3 0.5 0.4 0.3 0.9 0.3 4.6 0.9 0.2 0.7 0.5 0.1 0.1 2.6 14.0 0.0 0.2 8.3 0.4 0.5 0.4 0.1 0.5 11.5 1.1 14.1 0.5 0.7 0.5 1.3 1.3 1.3 1.3 0.4 1.2 0.6 0.3 0.3 2.8 11.6 0.2 0.5 7.2 0.6 0.7 0.5 0.3 0-2 0 - 51 0-4 0 - 66 0-2 0-2 0-2 0-6 0-1 0 - 13 0-6 0-2 0-5 0-3 0-1 0-1 0 - 12 0 - 51 0-1 0-3 0 - 21 0-1 0-2 0-1 0-1 0.7 13.2 1.0 15.2 1.8 1.4 0.8 1.3 1.3 5.3 1.2 0.5 1.2 0.5 0.3 0.6 3.7 14.0 0.1 0.3 17.6 2.3 1.5 2.3 0.3 1.5 10.2 1.7 11.2 2.7 2.5 1.8 2.3 2.3 2.3 1.2 0.8 2.0 1.1 1.0 2.1 3.4 7.3 0.4 0.7 3.3 2.3 2.8 1.1 0 - 6.5 0 - 46.0 0 - 8.1 0 - 42.7 0 - 6.7 0 - 12.5 0 - 6.7 0 - 9.1 0 - 3.9 0 - 15.7 0 - 3.9 0 - 3.0 0 - 9.1 0 - 7.1 0 - 3.7 0 - 9.1 0 - 11.4 0 - 27.7 0 - 2.4 0 - 2.9 0 - 7.1 0 - 6.3 0 - 6.3 0 - 6.7
7-Aug-99 8-May-00 20-Jun-99 5-Nov-99 4-Apr-00 9-Aug-99 1-Nov-99 7-Apr-00 25-Jul-99 3-May-00 2-Aug-99 25-Oct-99 3-Mar-00 8-Aug-00 6-Aug-99 6-Nov-99 26-Jul-99 4-May-00 3-Aug-00 9-Aug-99
25-Aug-99 29-Jun-00 20-Aug-99 10-Feb-00 24-Nov-00 23-Aug-99 28-Nov-99 20-May-00 2-Sep-99 26-Feb-00 27-Jun-00 3-Sep-99 10-Feb-00 2-Jul-00 26-Oct-00 3-Sep-99 24-Dec-99 26-Jun-00 17-Sep-99 21-Feb-00 5-Jul-00 11-Oct-00 19-Aug-99 16-Aug-99 28-Jun-00
S4-MEP2 16-Nov-99
S6-MEP3 15-May-00 S7-MEP2 14-Dec-99
24.8 0 - 100.0
S8-MEP1 12-Aug-00 16-Aug-00 S10-MEP1 8-Aug-99 S10-MEP2 15-Mar-00 Mean SD
3.3. Male production at colony level and related factors At the colony level, male production occurred in distinct periods over the 58
year, in most of the colonies. These periods, referred to as Male Emergence Periods, MEPs (Chinh et al., 2003), alternated with short periods without male production. The MEPs were not synchronized between colonies (Fig. 2). From the start of a typical MEP, male production gradually reached peak values after several weeks (sometimes to 100 % of all daily emerging bees) and then the percentage of emerging males declined to zero again during several weeks (Fig. 2). During the observations from June 1999 to December 2000, a total number of 26 MEPs occurred in the ten colonies. During this period, the number of MEPs per colony ranged from 1 to 4, with an average of 2.6 (SD = 1.1, n = 10). All colonies produced males in summer, between May and July (Fig. 2). Detailed results of male production in the colonies are presented in Tab. 1 and Fig. 1B. 3.4. Queen cells and behaviour of gynes Similar to other Trigona species, T. ventralis constructed special queen cells for the production of young queens. These queen cells were egg-shaped, bigger and longer than the cells for males and workers. Queen cells were mainly constructed at the outmost edges of the brood combs. Normally, they were further supported by bars of cerumen, since they usually stayed on for several days after the original comb to which they had been attached had been dismantled. Sometimes, workers built queen cells at the centre of the comb, after which the tops and bottoms of queen cells clearly protruded from the upper and the lower comb surface. The newly constructed cells had a dark-brown and relatively thick wall. When reaching maturity they had a lighter colour and the wall was much thinner since workers removed cerumen from developing cells. However, we did not observe that workers helped queens to emerge. Instead, we observed that a gyne herself gnawed away the upper top of the cell from which she subsequently emerged. In contrast, we observed that workers and males were usually helped by workers to emerge from their cells. At the moment of emergence, virgin queens varied in their pigmentation, which ranged from pale-coloured to dark brown-coloured. The fuller pigmented, the more mobile a queen seemed to be, possibly because pigmentation depends on their age. After having emerged, we observed some fully pigmented gynes running to hide somewhere in between the combs or involucrum. In three cases, a mature queen cell contained a live gyne inside for a couple of days. We finally observed that the already damaged cell membrane was torn further and that the queen emerged by herself. The
59

20 10 0 50 25 0 20 10 Col.S3 Col.S2
Col.S1
50 100
20 10 0 20 10 0 20 10 0
j-99 j-99 j-00 a-99 o-99 n-99 d-99 s-99
j-00
f-00
a-00
j-00
a-00
o-00
n-00
m-00
Dates
Figure 2. Periods with and periods without male emergence that occurred in the different colonies observed in the field.
60
m-00
d-00
s-00
Pecentage of daily emerging males
0 20 10 0
20 10 0 20 10 0 Col.S7 Col.S6 Col.S5
Col.S4
Col.S8
Col.S9
Col.S10
already fully pigmented gyne was moving around quickly, vibrating her wings. In a few cases we observed clear worker aggression towards gynes. One young queen, just emerged from the cell, was bitten by a single worker in her wing. In a second case, two workers attacked a gyne. As a result of these attacks, some gynes had their wings damaged, other apparently escaped unharmed. Once we observed that a gyne was suddenly attacked by workers while she was walking around in the brood nest. During this observation the attacked gyne could finally escape. We could not observe whether newly emerged gynes were confined in special enclosures, e.g. constructed from involucrum material inside the nest (c.f. Imperatriz-Fonseca & Zucchi, 1995; Sakagami, 1982). 3.5. Queen cells containing dead queens Of the total of 138 queen cells observed in the annex, 41 (30%) contained dead queens. The phenomenon of queen cells containing dead adult gynes also occurred in natural nests. When opening a large natural nest from a tree in the forest, we encountered about 20 of these queen cells with live gynes next to five queen cells containing dead adult gynes. The natural brood nest of this species is difficult to open completely without seriously damaging the nest. Even in our observation boxes it was difficult to observe behaviour inside the brood nest. 3.6. Gyne production We observed that all virgin queens emerged from queen cells; no dwarf gynes were produced in worker cells, as occurs in some other Trigonine species (Imperatriz-Fonseca & Zucchi, 1995). At population level, gynes emerged from queen cells during most of the year (Fig. 1C). The number of queen cells varied significantly in different colonies (Anova, F = 5.5952, P < 0.0001). Unlike male production that occurred in distinct batches, gyne production at the colony level was more continuous. This made that gynes were produced together with males, but also in periods without male production in the same colony. In Fig. 3 the average production of gynes is presented. It is clear that the number of gynes is seasonal. Low number of gynes was found in January and February after which there is a gradual increase. After the high values in June and July (on average 2.5 gynes/colony per month in July), gyne numbers decreased gradually. No gynes were found in the winter months of November and December. Similar to the numbers of males, gyne numbers differed significantly over the months (Anova, F = 8.9034, P < 0.0001).
61
Per month, the average number of queen cells was positively correlated with the number of brood cells (r = 0.90, P < 0.0001), and the numbers of workers (Pearson-r = 0.79, P < 0.01). Unlike the numbers of males, the numbers of gynes did not correlate with the numbers of storage pots (Pearsonr = 0.38, P = 0.28). The numbers of queen cells were correlated with temperature (Regression, F = 13.744, df = 1, P < 0.01), but not with rainfall, sunshine and humidity (Regression, F = 1.209, df = 3, P = 0.367).
4. Discussion
4.1. At population level, males and gynes are synchronously produced over the season The production of sexuals in T. ventralis occurs over a long period of the year. Despite the considerable variation between the colonies we can conclude for both sexuals that their occurrence in the nests is common from March until September. In May we found already high values for gynes whereas male numbers are still low in that month. In September we observed the opposite tendency: male numbers were still high, but gyne numbers were already decreasing in that month. A seasonal production of sexuals is typical for Apis mellifera in temperate areas and some species of stingless bees have been reported to produce sexuals seasonally as well (Beig, 1972; Bego, 1990), M. subnitida (Sakagami, 1982), M. beecheii (van Veen et al., 1999; Moo-Valle et al., 2001) and T. angustula (Grosso et al., 2000; van Veen and Sommeijer, 2000). Our results show that the production of sexuals is highest in summer (correlations with temperature, rainfall and sunshine). The clear seasonality found is different from the production of sexuals in some tropical stingless bee species. In M. favosa, in Trinidad & Tobago, male and gyne production occurred in all months of the year (but no data for April and June) and variation in gyne production over the months was not significant (Sommeijer et al., 2003a). The significant correlation between male production and stored food in the nest is in agreement with other findings (van Veen et al., 1999; Moo-Valle et al., 2001; Grosso et al., 2000). Colonies apparently invest more in production of sexuals when there are more food reserves. There may even be a direct link between pollen reserves and production of males. In several species laying workers have been found to contribute to the production of drones. Egg-laying workers are much involved in pollen uptake from the storage pots, probably because pollen consumption is necessary to produce eggs (Sommeijer et al., 1984, 1985). When ample food is available workers may therefore be stimulated to lay viable eggs leading to production of males. However, the same correlation between pollen reserves and production of males has been found in Tetragonisca angustula, where the queen produces 62
all males (Grosso et al., 2000), which implies that another mechanism must be involved in production of males. In T. ventralis it is unclear if the workers contribute to the production of males, although they have been reported to lay trophic eggs that are eaten by the queen (Sakagami & Yamane, 1987). 4.2. Male production is in general clumped The periodic pattern of males emerging in clumps has earlier been found for M. favosa (Chinh et al., 2003), and also for T. postica (Bego, 1982, 1990), P. remota (Benthem et al., 1995) and M. subnitida (Koedam et al., 1999). For M. favosa we demonstrated that the clumped emergence of males results from the occurrence of reproductive (haploid) eggs laid by workers. These laying workers are active in repeating periods alternated with periods without male production (Chinh et al., 2003; Sommeijer et al., 2003a). The precise mechanism for the regulation of this rhythmic occurrence of reproductive laying workers in M. favosa is still unknown. Probably, the rhythmic occurrence of male production in T. ventralis is also the result of reproduction by laying workers. It is known that in various non-Melipona stingless bees, laying workers do contribute to the production of males (Beig, 1972; Bego, 1982, 1990; Koedam et al., 1999, Tth et al., 2004). In our previous study on male production in M. favosa, we have suggested several possible explanations for the regulation of the periodic male production phenomenon based on reproduction by laying workers (Chinh et al., 2003). This phenomenon may serve to prevent inbreeding. While certain colonies are producing males, others under the same conditions are not producing, even when they are situated very close to each other. At the population level, the asynchronous clumped male production phenomenon allows for the presence of males all over the season. However, if the clumped occurrence of males in the colonies is functional is still unclear. It may as well follow from dynamics inside the colony that have evolved for other reasons. Male production is correlated with food stores in the colony. Obviously, it is very costly for a colony to produce large numbers of males for a certain period. A colony may invest heavily in male production for some time, but it must allocate part of its resources to worker production in order to maintain a strong colony. It is evident from our results that only colonies with surplus food and with a large population of workers produce males for a long period in total over the year. 4.3. Gyne production and behaviour Gynes of T. ventralis are permanently present in the nest during most of the year, although no gynes were found in November and December. During the development of a gyne in her cell, workers treat the cell in the same way as the other brood cells: the cell wall material is continuously removed which 63
results in a gradually thinner cell wall. However, the workers apparently do not assist the gyne with emergence from her cell whereas they do assist other nest mates by opening the cells. Queen cells with completely developed gynes may therefore remain closed for some more days even when the gyne would be able to emerge. The common occurrence of dead gynes in the queen cells is a phenomenon not earlier reported for stingless bees. It probably reflects the fact that new queens are often not needed at moments they are available. Besides the absence of stimulating the emergence of gynes, workers may even prevent their actual emergence. If gynes do emerge, we have evidence that workers generally approach them in an agonistic manner. Some reports for other trigonine species indicate that gynes may be kept alive by workers in special enclosures to keep them available for moments when they might be needed (Engels & Imperatriz-Fonseca, 1990; Imperatriz-Fonseca & Zucchi, 1995). Gynes in such prisons are fed by the workers. This is clearly not the case in T. ventralis. The continuous presence of queen cells already guarantees that replacement gynes are present, which is necessary for colony survival if the old queen is lost. From the varying conditions at which queens emerge from their cells we conclude that queens often remain longer in their cells than would be necessary for their full development. Hence, their cells serve as prisons, but the gynes are not fed and therefore cannot survive for a long time. It is obvious that the colonies in this study showed considerable variation in the production of sexuals. Both internal processes in the colony and seasonal factors seem to play a role in the production of sexuals. Two main conclusions can be drawn: 1. in T. ventralis the production of sexuals occurs over most of the year, in varying numbers depending on the season; 2. population size and food reserves appear to be important factors for the production of males and gynes, whereas production of males is typically clumped per colony.
64
65
CHAPTER 6
Production of sexuals in the honeybee species Apis cerana Fabricius 1793 (Apidae, Apini) in northern Vietnam
Tong X. Chinh1, Willem J. Boot2 & Marinus J. Sommeijer3
Accepted for publication in Journal of Apicultural Research
Bee Research & Development Centre, 68 Nguyenhong, Langha, Dongda, Hanoi, Vietnam
Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EH Wageningen, The Netherlands
3
Department of Social Insects, Utrecht University, P.O.Box 80.086, NL-3508 TB Utrecht, The Netherlands
66
Summary. In 20 colonies of Apis cerana in northern Vietnam, observations
on colony growth, on production of drones and queens, and on swarming and supersedure were related to available flowers to forage on and climatic data. Despite the tropical setting of the study area with forage available year-round, production of sexuals (i.e. drones and queens) was restricted to two periods from March to July and from September to December. Most swarming occurred in May when forage was most abundant. Positive correlations between available forage, colony growth and production of sexuals suggest that the synchronized production of drones and queens is defined by the forage flow into the colony. If this flow is high, the colony starts growing; when the colony is large enough drones and queens are produced, and eventually the colony swarms. Production of sexuals is synchronized because foraging conditions are sufficient to allow growth for only part of the year. Patterns in drone and queen rearing by A. cerana are similar to patterns found in Apis mellifera. Variation probably reflects differences in environment rather than differences between species.
1. Introduction
In social insect societies only some of the individuals reproduce. Food resources are allocated to either adult workers or sexual offspring (Seeley, 1985; Hlldobler & Wilson, 1990). In colonies of the highly eusocial honeybees (Apini), drones and virgin queens are produced as sexuals. Colonies should allocate their resources in such a way that the chances for sexuals to contribute to the next generation are the highest. This contribution differs between drones and queens. Drones only need to search a virgin queen during her nuptial flight to mate with, whereas queens need a colony of workers to become successful (Velthuis, 1990). Therefore, the production of a successful young queen implies the production of a swarm or supersedure, where the old queen leaves with part of the bees and a young queen takes over the original colony. The size of the swarm and the moment it leaves the mother colony are important factors determining later success of the swarm and of the original colony. Hence, the number of successful young queens that a colony can produce is limited. In contrast, the number of drones produced can be very large because production costs are low and the more drones a colony can produce, the greater the possibility that some of them will mate with a virgin queen (Velthuis, 1990). To promote outbreeding, honey bees have developed a mating system in which insemination of the virgin queen occurs at drone congregation areas far from the colony, where drones from widely dispersed colonies congregate (Fletcher & Ross, 1985; Punchihewa et al., 1990; Koeniger & Koeniger, 2000).
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Chapter 6. Production of sexuals in Apis cerana in north Vietnam
In Western honeybees, Apis mellifera, the production of drones and queens is governed by a combination of external and internal factors, which has been comprehensively studied (Winston & Taylor, 1980; Ruttner, 1985; Winston, 1987). Much is known from A. mellifera colonies in temperate regions where flowering is present during a limited period of the year, which results in a defined seasonal appearance of sexuals and colony multiplication (Seeley, 1985; Winston, 1987). In contrast, tropical florescence is usually available over the entire year, potentially allowing the colonies to produce sexuals year-around (Allsopp & Hepburn, 1997; Hepburn & Radloff, 1998). The number of sexuals produced and the number of swarms issued mainly depends on food resource availability and the number of workers (Winston & Taylor, 1980; Winston, 1987, 1990; Allsopp & Hepburn, 1997). Moreover, other factors such as fecundity and age of the queen and genetic disposition are involved (Ruttner, 1983, 1985). Mechanisms and factors regulating the production of drones and queens may be similar (Allen, 1958; Winston, 1987). Currently, the honeybees (Apini) are considered to have nine species (Otis, 1997) that form a monophyletic group (Ruttner, 1988). Of these, A. mellifera, A. cerana (Ruttner, 1988), A. koschevnikovi (Tingek et al., 1988), A. nigrocincta (Hadisoesilo & Otis, 1996) and A. nuluenis (Tingek et al., 1996) form the cavity-nesting group of Apis. Bees in this group are probably rather similar in colony organization (Dyer & Seeley, 1991). Based on morphometric variation and geographic distribution, the Asiatic honeybee species, A. cerana, has also been classified into four subspecies: A. cerana cerana in northern Asia; A. cerana indica in southern Asia; A. cerana japonica in Japan; and A. cerana himalayana in the Himalayan region (Ruttner, 1988). Beekeeping with A. cerana is an ancient activity. In many natural and agricultural eco-systems A. cerana is the most important pollinator (Verma, 1990; Crane, 1991; Kevan, 1995). Since this species is distributed over a wide range of natural habitats and climates, in a vast geographic area of Asia ranging from Iran to China and from Japan to the south of Indonesia (Smith & Hagen, 1996), A. cerana would be expected to express basic differences in colony growth and reproduction in response to environmental differences. For example, the reproductive season is confined to three months in spring in the temperate region of Japan (Okada, 1970; Matsuura & Sakagami, 1973). In the tropical monsoon region of Northern Thailand, Southern India and Northern Pakistan colony reproduction usually occurs from March to October (Koeniger, 1976; Seeley et al., 1982; others reviewed in Seeley, 1985; Ruttner, 1988; Roubik, 1989) when no seasonality in colony reproduction has been found in the tropical rain forest of Sumatra (Inoue et al., 1990).
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Although, reproduction has been widely studied in A. mellifera, relatively little work has been done on the sister species A. cerana (Koeniger & Koeniger, 1991). For A. cerana, much of available data focuses on mating behaviour, physiology and reproductive isolation (Koeniger & Koeniger, 2000). Little information exists on seasonal cycles of colonial development, production of sexuals, swarming and supersedure. Mechanisms and factors regulating these processes are important not only for insight into the evolution of these social bees, but also for application in the breeding programs of the Asiatic honey bees in many Asian countries (Verma, 1990). In this paper we present data from a two-year study on the production of drones and queens of the native honeybee species Apis cerana in northern Vietnam. The influence of seasonal flowering and colony conditions on the production of drones, queens, swarming and supersedure has been comprehensively investigated. To understand production of sexuals in feral colonies, it is necessary to choose a place where A. cerana colonies and their environment are little or not influenced by beekeeping. The mountain-forest areas of the northern part of Vietnam have been chosen, because wild nests of A. cerana are common and primary tropical forest still exists.
This investigation was conducted from January 1999 to December 2000 in the buffer zones around Cuc Phuong National Forest, northern Vietnam (20.14o 20.24o N and 105.29o105.44o E, 300-400 m above sea level). Twenty queenright colonies of Apis cerana Fabricius 1793 were randomly selected in three apiaries in the bufferzone of Cuc Phuong forest and were kept in movable frame hives (WxLxH: 45x75x40 cm). To maintain the colonies as natural as possible, they were not managed. They were, however, fed with syrup water in winter to avoid risk of starvation. The colonies were provided with ample empty comb frames to construct either drone or worker cells (no limitation in hive space). A small band of wax foundation was fixed to the top bars of the frames to stimulate regular comb construction inside the frames. Every ten days, areas and brood (worker and drone), and areas of stored food (honey and pollen) were measured using a graduated grid. In addition, the average height of honey and pollen storage areas was estimated to calculate their volumes. At the same time, the colonies were inspected to see if swarming, supersedure or absconding (non-reproductive swarms) had occurred. The number of queen cups and occupied queen cells were counted. Newly mated queens were marked to know their age and to track supersedures. The abdominal margin of their hind wings was partly cut to prevent swarms from flying far away; all swarms were caught to examine the number of queens and workers. The original colonies (after having swarmed)
69
were observed to determine when their virgin queens emerged and started laying. Every twenty days at dusk, the size of the colonies was estimated by weighing the hives first with bees and thereafter without bees. To calculate the number of bees we assumed an average weight of 85 mg per bee. We defined flowering intensity as the number of bee plant species in bloom. It was calculated monthly from the records of Hanh (2000) in the study area (Fig. 2A). Weather data were obtained from the National Hydrometeorology Station in Cuc Phuong forest, located about 100 m from our field laboratory. Cuc Phuong has a dry and a rainy season. The rainy season lasts from May to November with a daily average temperature of 25o C and total precipitation of 1456 mm3. The dry season runs from December to April with a daily average temperature of 17o C and total precipitation of 243 mm3.
120
Average of temperature (oC) and humidity (%)
600
Total of rainfall (mm) and sunshine (hrs.)
100 80 60 40 20 0 J F M A M J J A S O N D Month of the year temperature humidity rainfall
500 400 300 200 100 0
sunshine
Figure 1. Average temperature (o C), relative humidity (%); rainfall (mm) and sunshine (hrs.) over the year. For further analysis all data were converted into monthly averages (Fig. 1). ANOVA tests were performed to analyse differences in the production of drones, queens, swarms and supersedures between colonies, between the dry and the rainy season and between two specific periods during the year during which sexuals were found to be produced. These periods lasted from March to June and from October to December. Linear regression tests were used to
70
investigate the relationship between the production of sexuals, swarms, supersedures, colony conditions and climatic factors.
3. Results
3.1. Seasonal production of sexuals, swarming and supersedure The production of drones and queens, and the occurrence of swarming and supersedure, were not evenly distributed throughout the year, but were synchronized into two discrete periods: the first one lasted from March to July and the second from September to December (Fig. 2). These two periods were closely associated with the two main flowering seasons (spring-summer and winter). Maximal production of drones and queens, and most swarming, occurred in May when flowering intensity was highest. No production of sexuals was found in the dearth periods in January-February and in August. During these periods three colonies absconded. The production of drones and queens as well as swarming varied significantly between the months of the year (Anova, p< 0.0001), between the colonies (Anova, p < 0.05), and between the two periods in which production of sexuals occurred (Anova, P < 0.0001). Average data of the two years are presented in Tab. 1. The production of drones was positively correlated with the production of queens (Pearson-r = 0.75, P = 0.005, n = 12) and with swarming (Pearson-r = 0.63, P < 0.05, n = 12). No correlation was found between swarming and supersedure, that is between the months of the year in which it occurred (Regression, F = 0.681, df = 1, P = 0.43). Production of drones always preceded production of queens, swarming and supersedure. In a considerable number of cases, drone rearing was not followed by swarming or supersedure afterwards. Of the colonies that produced drones, 95 % also produced queens in the spring-summer period, whereas only 24 % reared queens in the winter period. 3.2. Production of sexuals in relation to colony size and environmental factors We assume that the number of bee plant species flowering (flowering intensity) reflects the availability of nectar and pollen. In Cuc Phuong forest, flowering intensity was negatively correlated with sunlight (Pearson-r = -0.64, P < 0.05, n = 12), temperature (Pearson-r = - 0.71, P < 0.01, n = 12) and rainfall (Pearson-r = -0.64, P < 0.05, n = 12), but there was no correlation with humidity. Flowering is abundant in the period of March-June and OctoberNovember and has a peak in May, but it is reduced in December-February and in August. The production of drones (Pearson-r = 0.81, P = 0.02, n = 12), the production of queens (Pearson-r = 0.85, P < 0.0001, n = 12) were highly
71
Table 1. Demographic characteristics of 20 A. cerana colonies, flowering species and climatic factors at Cuc Phuong forest
Variable
Number of drone cells Number of drone brood Number of queen cups Number of queen cells Swarming frequency Superseding frequency Absconding frequency Number of worker brood Bee population Honey stored Pollen stored Flowering (No of species) Total of sunshine Average temperature Total of rainfall Average humidity
Months Jan. Feb. Mar. Apr. May. Jun. Jul. Aug. Sep.
0 0 0 0 0 0 0 0 0 0 0 0 0 0.06 524 322 510 349 718 465 408 123 38 20 0 0 0 0 0 0 0.03 4 4 0 0 0 0 0
Mean Oct.
159 110 0.04 0.04 0 0.01 0 5121
SD
263 169 0.67 0.42 0.05 0.01 0.02 1617 2124 129.5 48.9 9.7 45.1 4.4 112.7 3.0
n
20 colonies 20 colonies 20 colonies 20 colonies 20 colonies 20 colonies 20 colonies 20 colonies 20 colonies 20 colonies 20 colonies 1 year 2 years 2 years 2 years 2 years
Nov. Dec.
419 291 0.19 0.15 0.01 0.01 0 6464 28 10 0 0 0 0.02 0 3756 234 141 0.41 0.26 0.03 0.00 0.01 4606 794.8 98.7 17.5 124.7 22.6 141.6 86.0
0.88 0.42 2.07 1.28 0.03 0.51 0.28 1.33 0.79 0.03 0.02 0.05 0.16 0.12 0.02 0 0 0 0 0.01 0.01 0 0 0 0
3633 1550 5233 5428 7039 5728 4523 2202 4600
10325 8986 15346 14675 16135 14762 13811 11814 11815 12497 14102 13694 13164 871.5 542.5 864.6 884.0 953.3 861.8 894.4 715.0 910.7 636.0 133.0 13.6 100.1 106.4 145.6 89.6 49.4 45.7 53.6 12 17 14 18 33 36 28 10 5 10 71.7 52.6 59.2 99.3 125.3 168.1 186.8 141.9 178 16.8 19.7 24.2 25.5 27.5 28.1 26.9 25.7 18.1 24.2 37.2 131.3 15 139.3 23.7 87.5 715.1 688.6 166.1 149.7 16 20.1 38.5 82.7 13 16.5 38.8 79.0 141.6 132.6
125 195.2 163.4 228.5 271 358.6 200.6
84.8 87.4 89.9 87.9 85.5 85.1 85.4 89.7 87.1
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1200
Number of drone brood
40
1000 800 600 400 200 0 3.0
flowering
30 20 10 0
Number of queen cells
queen
8000 6000 4000 2000 0

Number of worker brood Number of worker adults
2.5 2.0 1.5 1.0 0.5 0.0
B
C
worker
S warming and supersedure frequency
0. 30 0. 20 0. 10 0. 00
30 20 10 0
1200
Volume of honey & pollen (cubic cm)
honey
pollen
worker adults
20000 15000 10000 5000 0
1000 800 600 400 200 0 J
Mont hs of the year
Figure 2. Monthly average production of drones (A), queens (B), swarming and supersedure (C) in relation to colonial conditions (D)
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Number of flowering species
0. 40
swarming supersedure flowering
40
Number of flowering species
drone
correlated with flowering intensity, but interestingly they were not correlated with the amount of honey and pollen stored. Similarly, the amount of worker brood (Pearson-r = 0.68, P = 0.01, n = 12) and the number of workers (Pearson-r = 0.65, P = 0.02, n = 12) were correlated with flowering intensity, and not with the amount of honey and pollen stored, except for the correlation between the amount of worker brood and pollen stores (Pearson-r = 0.65, P = 0.02, n = 12). Worker brood production was positively correlated to drone rearing (Pearson-r = 0.78, P = 0.003, n = 12), queen rearing (Pearson-r = 0.67, P = 0.01, n = 12), and swarming (Pearson-r = 0.63, P = 0.03, n = 12), but not with supersedure. Similar correlations were found between the number of worker bees and drone rearing, queen rearing and swarming (Pearson-r = 0.76, P = 0.04; r = 0.70, P = 0.01; r = 0.65, P = 0.02, respectively). The production of drones, new queens, swarming and supersedure were not correlated with sunlight, temperature, rainfall and humidity. Thus in general, the flow of nectar and pollen collected by the bees seemed to be the main driving force behind colony growth and production of sexuals. 3.3. Drone rearing Most of the bee colonies annually constructed new drone cells in the abovementioned active periods (Fig. 2A). Generally, all new drone cells were immediately occupied with eggs following their construction, and no new drone cells were built when drone brood rearing had stopped. In fact, the majority of old drones cells were gnawed away by workers. Drone cells were mainly built at corners and the lower edges of the comb. Very few combs were found to consist mainly of drone cells. The percentage of drone cells to all cells in a colony ran from 0 % (in January, February and August) to 7.1 % (in May), with an average of 2.0 2.3 % (n = 12 months). The actual amount of drone brood reared made up 5.7% of the total brood present in a colony (in May), with an average of 2.0 2.3 % (n = 12 months).To investigate the impact of the queens age to drone rearing, we divided the dataset into a group with a young queen (1-6 months old) and a group with an old queen (more than one year old). The former produced about three times more drones than the latter (Anova, P < 0.05). 3.4. Queen rearing The total number of queen cups produced by a colony (in one active period) ranged from 1 to 21 cups, with an average of 6.80 4.95 (n = 66). The number of queen cups constructed by a colony before swarming was about seven times higher than that before supersedure (F = 9.809, df. = 1, P < 0.01). A colony produced between 2 and 13 sealed queen cells for swarming, with an 74
average of 6.03 2.80, (n = 35 swarms). For supersedure just 1 to 2 sealed cells were produced, with an average of 1.33 0.52 (n = 5 supersedures). Of 21 colonies, the age of the queen was known when the colony swarmed or superseded the queen (Tab. 2). The age of the queen in the swarming colonies was much younger (on average 8.0 5.8 months, range: 1.4 15.6 months, n = 15 events) than that of superseded queens (13.9 3.8, range: 7.3 19.1 months, n = 6 events) (F = 4.669, df. = 1, P < 0.05) (Tab. 3). Table 2. Naturally occurring queen production in 20 unmanaged colonies of A. cerana over two years at Cuc Phuong forest
Reproduction mode 1. Reproduction swarming Variations Incidence
1.1. Old queen swarms 1.2. Young queen swarms
32 3 5 23 3
2. Supersedure 3. Failed reproductive events1 4. Absconding2

1 2
Queen cells that were aborted and did not result in either swarms or supersedures Absconding was not necessarily associated with production of queens.
Table 3. Queen age in relation to swarming, supersedure and absconding in colonies of A. cerana over two years at Cuc Phuong forest Queens Number swarmed Number superseded Number absconded 1-6 8 0 3 Months 6 12 3 1 0 13+ 5 4 0
3.5. Swarming Summed for the 20 colonies of A. cerana over two years, a total of 63 attempts to rear queens were made, of which 40 were successful and 23 failed 75
(Tab. 2). Only three colonies issued an afterswarm, seven, eleven and 19 days after the prime swarm. Swarming colonies (just before swarming) contained from 13,680 to 27,370 bees, with an average of 21,000 3,800 (n = 35 colonies). The size of prime swarms ranged from 6,840 to 14,740 bees, with an average of 11,020 bees 1,960 (n = 32 prime swarms), whereas that of after swarms was on average of 7,220 bees 1,400 (range: 5,630 8,950, n = 3 afterswarms). After swarming, about half of the bee population in the original colony was left behind, with an average of 9,840 2,110 bees (range: 6,420 -12,630, n = 35 colonies). The amount of worker brood left behind in the original colonies at the moment of swarming was on average of 7,780 1,930 (1,370-12,360, n = 35 colonies). The time at which the colonies issued swarms (n = 32) was recorded: 72 % of swarming occurred between 8:00 h and noon, and 28 % between noon and 16:00. The stage of queen rearing was quite variable at the moment of swarming. Six percent of the queen cells contained eggs, 37 % were open and contained a larva, 38 % were sealed, and 19 % of the young queens had already emerged. The latter were found in six out of 32 colonies. The average interval between the issuing of the prime swarm and the emergence of the queen was 5 days (range: 0-14 days). Young queens started egg-laying after 8.0 days on average (n = 35, range: 5-15days). Twenty-five of 35 young queens were mated successfully.
4. Discussion
Despite the tropical setting of our study area with forage availability yeararound, the colonies of A. cerana swarmed only once or twice a year. Moreover, production of sexuals and swarming was strongly synchronized in two periods of the year, of which the period from March to June was the most important by far. The synchronized production of sexual offspring has also been found for A. cerana in other regions, both temperate (Okada, 1970; Masuura & Sakagami, 1973) and tropical (Sharma, 1969; Seeley et al., 1982; others reviewed in Seeley, 1985; Ruttner, 1988; Roubik, 1989). In addition, the pattern is rather similar to what has been described for A. mellifera both in temperate (Seeley, 1985; Winston, 1987) and tropical regions (McNally & Schneider, 1994; Hepburn & Radloff, 1995; Otis, 1990; Allsopp & Hepburn, 1997; Hepburn & Radloff, 1998). The correlations between flowering intensity, colony growth and production of drones and queens suggest that the synchronized production of drones and queens is defined by the forage flow into the colony. If the flow is high, the colony starts growing, then drones and queens are produced and eventually the colony swarms. Apparently, foraging conditions are not good enough during large parts of the year in our study area, which leads to synchronization. If foraging conditions were good during 76
much longer periods, honey bees would swarm more often (up to twelve swarms and after swarms per year) and the synchronized production of sexuals would be lost (Winston, 1990; Otis, 1990). We found no correlation between the production of sexuals and the amount of nectar and pollen stored inside the colony, showing that the bees react directly on forage availability. Under temperate conditions this link between available forage and production of sexuals may be weaker than under tropical conditions, because at the end of the active season colonies have to store large amounts of food to survive the long winter. A. mellifera and A. cerana are sister species with similar traits of social structure and behaviour (Seeley, 1985; Dyer & Seeley, 1991). Both species have evolved to exploit flower patches for maintaining their colonies and subsequently to invest as much as possible in production of sexuals (Seeley, 1985; Velthuis, 1990). Therefore, it is not surprising that the general pattern of seasonality in colony growth and production of sexuals is similar between A. cerana and A. mellifera. In our A. cerana colonies we found that in favorable conditions, on average of 8.2 % of the cells constructed were drone cells. This is similar to the 7.2 % found in wild nests of the eastern bee A. c. indica (Inoue et al., 1990). A. mellifera colonies devote a similar percentage to drone cells. A percentage of 5.9 have been reported for Africanised bees (Ratnieks et al., 1991), 8.1 % has been reported for European bees (Lee & Winston, 1985), and 8.3 % for native African bees (McNally & Schneider, 1994). Variation may result from the different environment the bees live in, rather than from fundamental differences between the species. For example, in A. mellifera, temperate-evolved bees invest a lower proportion of drone comb at relatively small colony sizes than tropical evolved bees, but more drone comb in larger colonies (Lee & Winston, 1985; Winston, 1987; Winston, 1990). In agreement with previous studies in A. cerana (Inoue et al., 1990) and in A. mellifera (Lee and Winston, 1985; Winston, 1990; Allsopp & Hepburn, 1997), we established that colony size affects production of sexuals. Colonies initiated drone and queen rearing when their population was larger than ten thousand bees, which is similar to earlier studies of A. c. indica (Sharma, 1969; Inoue et al., 1990) and A. mellifera (Free & Williams, 1975; Lee & Winston, 1985). In our study, the sex investment ratio was 554 males to one queen, the same as found before in A. c. indica (556:1, Inoue et al., 1990). In A. mellifera similar ratios have been reported (750-833:1, reviewed by Seeley, 1985; Winston, 1987; Roubik, 1989). Colony multiplication in honey bees takes place through swarming. Queens are produced in a small number and appropriate to the needs of the colony, while 100-1000 times more drones are produced. This ratio probably reflects the relative costs a colony has to invest to get equal fitness returns. 77
The efficiency of drone production defined as the chance for a drone to mate seems to vary much throughout the year; however, most of the drones produced in the winter period were chased away from the hives by workers and died soon after their production. In addition the number of young queens produced was very low (Fig. 2). Why did our colonies produce so many drones when they were killed shortly afterwards and when very few virgin queens were available to mate with in winter time? Unfavourable conditions after the start of drone rearing apparently make production inefficient in winter. It indicates that rearing of drones is the expression of a certain surplus capacity in brood rearing (Velthuis, 1990), and immediately responds to resource availability. An interesting observation on the construction of drone cells was made. Colonies of A. mellifera re-use their constructed drone cells many times whereas colonies of A. cerana, at least in this study, only seem to re-use newly constructed cells during the same period of drone production. Old cells are destroyed during the dearth periods. The question is why these bees destroy empty drone cells when they can re-use them only about one month later. During the dearth periods, colonies minimize brood rearing in response to unfavourable conditions. Workers cluster at the centre of the colony. We observed that the comb quickly get old, black and brittle when it is empty. Workers probably gnaw out unused cells to prevent infestation of wax moth, a common pest in the tropics. Because the drone cells are usually on the outer rims of the comb, they are destroyed earlier than the worker cells. In this study basic data related to production of sexuals in A. cerana colonies have been presented. The data are similar to data from the much better studied Western honey bee, A. mellifera, suggesting that the same mechanisms govern rearing of sexuals. In both species bees seem to respond quickly to changes in floral resources by adjusting the extent of brood rearing. This affects colony growth which in turn affects production of sexuals and eventually swarming. Because during large parts of the year floral resources are often not sufficient to warrant colony growth, seasonal patterns in the production of sexuals appear.
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79
CHAPTER 7
Reproductive strategies in sympatric species of eusocial bees
Tong X. Chinh1, Willem J. Boot2 & Marinus J. Sommeijer3
Bee Research and Development Centre 68 Nguyenhong, Langha, Dongda, Hanoi, Vietnam
Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EH Wageningen, The Netherlands
3
Department of Social Insects, Utrecht University, P.O.Box 80.086, NL-3508 TB Utrecht, The Netherlands
80
Summary. In tropical areas many species of eusocial bees are found. We
review differences in provisioning of the brood, in the general swarming process, in which caste (worker or queen) produces the males, in how caste is determined and in seasonal production of sexuals. These differences are related to general strategies for reproduction and swarming. We focus on three eusocial bee species occurring in the same forest in Vietnam. The honey bee Apis cerana is well adapted to exploit seasonal or temporal resources, because it responds quickly to changing environmental conditions and swarms can travel far from the original nest. Stingless bees respond much slower and new nests are started as satellites in the vicinity of the old nest. Hence, it is expected that in general stingless bees will flourish most in stable environments. However, some differences can be observed among sympatric (stingless bee) species. Species with large colonies like Trigona ventralis focus more on colony defence and growth of the colony itself. Species with small colonies like Lisotrigona carpenteri probably focus more on colonizing many new nest sites. Together with resource partitioning and specialization on various nest sites, the different reproductive strategies help explaining why so many eusocial bee species are living sympatrically.
1. Introduction
Social bees play a mayor role in ecosystems as pollinators since they specialize on only two food sources: pollen and honey. In tropical areas many species of eusocial bees are found. Both honey bees (Apini) and stingless bees (Meliponini) live in perennial colonies and have distinct female castes: queen (or gyne if not yet mated) and worker. All honeybee species live in tropical areas and the distribution of Apis mellifera and Apis cerana extends into temperate regions. The distribution of the Meliponini is confined to the tropical and southern subtropical areas throughout the world. Stingless bees are particularly common in large areas in tropical America and in Southeast Asia. Colony size in eusocial bees is diverse and ranges from a few dozen to 100,000 or more individuals (Michener, 2000). Although the general way of life in eusocial bees is similar and many species use the same floral resources, numerous species may occur sympatrically. Since in eusocial insect societies only part of the individuals reproduce, food resources are allocated to either workers or sexual offspring (Seeley, 1985; Holldobler & Visscher, 1990). Colonies should allocate their resources in such a way that the chances for sexuals to contribute to the next generation are the highest. This contribution differs between males and gynes. Males only need to search a gyne to mate with, whereas gynes also need a colony of workers to become a successful queen (Velthuis, 1990). Therefore, the production of a successful young queen implies the production of a swarm. In 81
Chapter 7. Reproductive strategies in sympatric species of eusocial bees
honey bees, the old queen leaves with part of the bees and a young queen takes over the original colony. The swarm with the old queen has to find a new nesting site. In stingless bees, a suitable new nest site is found first, after which a young queen moves to the new nest with part of the workers. The size of the swarm and the moment it leaves the mother colony are important factors determining later success of the swarm and of the original colony. Hence, the number of successful young queens that a colony can produce is limited. In contrast, the number of males produced can be large because production costs are low and the more males a colony can produce, the greater the possibility that some of them will mate with a gyne (Velthuis, 1990). Since many eusocial bees occur sympatrically, nature has apparently found many solutions for eusocial bees to exploit floral resources in a certain habitat. Here we review the reproductive strategies applied by different species in relation to the specific colony organization found. Finally, we focus on three species occurring in the same forest in Vietnam: the Asiatic honey bee Apis cerana and the stingless bee species Lisotrigona carpenteri and Trigona ventralis.
2. Differences in provisioning of the brood
Honey bees and stingless bees have a different way of rearing their brood. In honeybee colonies, nursing workers feed larvae progressively in open cells arranged in combs. The cells are capped when the larvae stop feeding in order to pupate. In this system, workers have the possibility to exert control over caste determination through the quantity and quality of the larval food they provide. In addition, the colony can quickly react to unfavourable environmental conditions by eating the young brood (Woyke, 1977). Different from the progressive provisioning system of honey bees, stingless bees mass provision cells first after which egg laying and capping of the cell follows immediately (Sakagami, 1982). Hence, larvae are reared in closed cells with no direct contact between the larvae and workers. Closing the cells immediately after oviposition may help colonies to prevent parasites and diseases (Sommeijer & de Bruijn, 1994), but it also implies that no caste control can be exerted after egg-laying and the brood nest cannot be adjusted directly to environmental conditions.
3. Differences in the swarming process
Honey bees and stingless bees differ in the age of the queen that leaves the original colony. In honeybees, the old queen leaves the nest with about half of the bees. From that moment contact between the two halves is lost. The swarm has to find another nesting place that can be far away from the original colony. In stingless bees, a daughter queen departs from the mother colony to 82
a new nest site. This nest can be regarded as a satellite and contact may remain for some time between the original nest and the new nest (Inoue et al., 1984; Engels & Imperatriz-Fonseca, 1990; van Veen & Sommeijer, 2000). The proportion of workers that leaves the original colony for a new home seems to be smaller than in honeybees. In Trigona angustula 10 % of the bees left to the new nest (van Veen & Sommeijer, 2000) and 30 % left in Trigona laeviceps (Inoue et al., 1984). Swarming to relatively small satellite nests probably reduces the risk of mortality of both the original colony and the new colony, since the original colony remains strong and the new colony can rely on the original one for resources. It also limits dispersal, however. In general, stingless bees seem to swarm much less frequent than honeybees in the same habitats. Normally, a stingless bee colony swarms only once a year or even less frequent, whereas colonies of Apis mellifera issue 4-5 swarms yearly (Bego, 1982; Roubik, 1989). Under favourable conditions up to twelve swarms per year have been reported (Winston, 1990). Little information on swarming frequency in stingless bees is available, however, and there may be large differences between the many species. In Costa Rica, Slaa (2003a) found on average 0.05 swarms per year in colonies of 14 different species taken together. For Scaptotrigona petoralis and Tetragonisca angustula she found frequencies of 0.06 and 0.52, respectively (Slaa, 2003). One swarm yearly was reported for Trigona minangkabau in Sumatra, Indonesia (Inoue et al., 1993). So swarming in stingless bees seems to be rare when compared with honey bees, but this does not threaten their existence since colonies are very resilient. Yearly colony mortality rates in a natural population have been reported from 6.7 % (Slaa, 2003) to 13.5-15 % (Eltz et al., 2002).
4. Production of males by workers or by the queen?
When a colony is headed by a single monandrous queen, workers are on average more related to their own sons (r = 0.5) and to other worker sons (nephews, r = 0.375) than to the queens sons (brothers, r = 0.25) (Hamilton, 1964; Ratnieks, 1988, Peters et al., 1999). Therefore, a conflict between the queen and the workers may occur over the production of males: the workers will favour to produce the males themselves in stead of leaving it to the queen. In colonies headed by a polyandrous queen, this worker-queen conflict over male production may be reduced because workers are in that case more related to the queens sons (r = 0.25) than to sons of other workers with a different father (half-sisters, r = 0.125). Note that sons of workers with the same father (full-sisters) are still more related (r = 0,375), but since most of the workers are half-sisters even when only a few patrilines occur, the average relatedness with sons of other workers will be below 0.25 (Ratnieks, 1988; Peters et al., 83
1999). Queens of stingless bees typically mate once, although exceptions occur (Imperatriz-Fonseca et al., 1998; Paxton et al., 1999), whereas honey bees mate multiple times. In queenright colonies of the polyandrous honey bees the queen normally produces all drones (Seeley, 1985; Winston, 1987; Peters at al., 1999). Usually, Apis workers do not activate their ovaries at all as a result of pheromonal control by the queen (Velthuis, 1970; Free, 1987). If they do activate their ovaries and lay eggs, however, their contribution to the drone population is low because other workers remove the eggs laid by their sisters. This worker policing has been demonstrated in queenright colonies of A. mellifera (Visscher, 1989; Ratnieks, 1993; Barron et al., 2001) and Apis cerana (Oldroyd et al., 2001). In contrast to honey bees, workers in queenright colonies with active ovaries are common in stingless bees (Sakagami, 1982; Crespi, 1992), and they often lay eggs (Sakagami, 1982; Sommeijer & van Buren, 1992; Koedam et al., 1999; Chinh et al., 2003). Not all of the eggs laid are viable, however. In many cases, workers lay trophic eggs that are eaten by the queen before she lays her own egg (Beig, 1972; Bego, 1982; 1990; Sakagami, 1982; Sommeijer et al., 1984; Chinh et al., 2003). In some species of Trigona, e.g. Scaptotrigona postica, reproductive worker eggs are normally laid after the queens oviposition (Beig, 1972; Bego, 1982; Sakagami, 1982), which leads to cells containing both a queen and a worker egg. Worker produced males emerge as a consequence of the queens larvae being killed (Beig, 1972). In contrast, reproduct ive workers in Melipona generally preclude oviposition by the queen in the same cell after they have laid an egg (Sommeijer et al., 1984a; Sommeijer & van Buren, 1992; Chinh et al., 2003). Reproductive workers of M. favosa may also lay after oviposition by the queen, but this phenomenon is rare (Chinh et al., 2003). Reproductive workers of M. bicolor sometimes reopen and oviposit in earlier capped cells in which the queen had laid an egg (Velthuis et al., 2002, 2003). In the stingless bees, the contribution of the queens and the workers to the production of males has often been studied, but no general pattern readily appears (Contel & Kerr, 1976; Machado et al., 1984; Green & Oldroyd, 2002; Kellie et al., 2002; Palmer et al., 2002; Tth et al., 2002a, 2002b, 2003; Paxton et al., 2002). By continuous observation of groups of cells from oviposition until its emergence in queenright colonies of M. favosa, it was shown that reproductive egg-laying by workers is clustered in certain periods. When these cells emerge, almost all males found are worker produced, whereas in other periods in the same colony all emerging males are produced by the queen. Depending on the time when measured, estimates for the contribution of workers to the production of males in queenright colonies 84
range from virtually none to 95% (Sommeijer et al., 1999; Chinh et al., 2003; Sommeijer et al., 2003a). This pattern may occur more often since the same was recently found in a complete different species: Trigona ventralis (Chinh & Sommeijer, chapter 5).
5. Caste determination in Apis, Trigona and Melipona bees
In the social Hymenoptera, distinct difference in size and morphology between female castes (gynes and workers) generally results from differences in quantity and quality of larval food that female larvae receive from nursing workers (Michener, 1974). In honey bees, gynes are reared in larger cells and fed progressively with much more food than workers (Free, 1987; Winston, 1987; Engels & Imperatriz-Fonseca, 1990). Similar to honey bees, gynes of Trigona bees are also reared in typical royal cells that are distinctly larger than cells from which workers and males emerge. They are provisioned with more food than worker cells, but unlike honey bees they are capped directly after oviposition by the queen. Different from honey bees and Trigona bees, gynes of Melipona bees are reared in the same cells as workers, because caste determination in Melipona is regulated by a combination of genetic and trophic factors (Kerr, 1997; Velthuis & Sommeijer, 1991). This means that 25% of the female larvae have the genetic disposition to become a queen, but the real number is lower due to incomplete food supply in the cells. The different caste determination systems probably affect when gynes are produced and how many gynes are produced. In honey bees just enough gynes are produced to provide for new queens after swarming and it occurs predominantly in periods when swarming occurs (Seeley, 1985, Winston, 1987; Hepburn & Radloff, 1998). If the old queen dies in between, honey bees can rear a new queen from existing female larvae that were originally destined to become workers. Because cells are immediately capped after oviposition, Trigona colonies have to rear gynes continuously, but a few gynes are enough to warrant colony survival after death of the queen. This is fundamentally different from Melipona colonies that produce permanently larger numbers of gynes (Sakagami, 1982; Ratnieks, 2001; Sommeijer et al., 2003a, 2003b; Sommeijer & de Bruijn, 2003). Their caste determination system may be a constraint for a more efficient production of gynes.
6. Differences in seasonal production of sexuals in sympatric species
European honey bees (Apis mellifera) live in temperate regions where floral resources are available in a limited period of the year. This results in seasonal production of sexuals closely related to colony multiplication through swarming (Seeley, 1985; Winston, 1987). In tropical regions, floral resources are available throughout the year, but are not always sufficient to warrant 85
colony growth and subsequent production of sexuals and swarming. Since honey bees seem to respond quickly to availability of floral resources, seasonal patterns may still appear (Chinh et al., chapter 6; Hepburn & Radloff, 1998). In contrast, the production of sexuals in stingless bees occurs throughout the year (Moo-Valle et al., 2001; Chinh & Sommeijer, chapter 5; Sommeijer et al., 2003a; van Veen et al., 1999). Partly, the more continuous production of sexuals is needed for rearing gynes as a safeguard in case the queen dies. In addition, stingless bees have a much longer development time than honey bees. Workers of honeybees take about 20 days to develop from egg to adult (Winston, 1987), whereas Melipona stingless bees take about 40 days (Chinh, 1998). Therefore, colonies of stingless bees cannot respond as quickly as honey bees can to changing environmental conditions, and numbers of sexuals produced are only gradually adjusted. So from their life histories it is expected that seasonal production of sexuals due to variable environmental conditions will be less pronounced in stingless bees when compared to honey bees. Information on how stingless bees and honey bees actually behave in the same habitat is rare, however. A two-year study on seasonal production of sexuals in the Asiatic honeybee species Apis cerana and in the stingless bee species Trigona ventralis in the tropical monsoon environment of northern Vietnam revealed that both sympatric species show seasonal variation (Chinh & Sommeijer, chapter 5; Chinh et al., chapter 6). However, A. cerana appears to respond faster to varying availability of floral resources than T. ventralis by adjusting the size of the brood nest in general and therefore also the numbers of sexuals produced (Fig. 1). In this habitat, a relative shortage of flowering occurs in August (Hanh, 2000), to which A. cerana responds by reducing the brood nest and stopping sexual production (Chinh et al., chapter 6), while in T. ventralis the size of the brood nest is maintained and sexuals are produced in large number (Chinh & Sommeijer, chapter 5). Two-three months later in November, A. cerana uses the abundant flowering (Hanh, 2000) to produce a marked number of sexuals, while the production in T. ventralis is reduced. A similar pattern emerges from data on swarming in a study site in Costa Rica, where Africanised honey bees (hybrids of African and European subspecies of Apis mellifera) live in the same area as a guild of about 15 species of stingless bees (Slaa, 2003a). Swarming of the honeybees was concentrated in a few months, whereas the swarming in the stingless bees (data of 13 species taken together) occurred throughout the year (Fig. 2).
86

3 2 1 0
8 7 6 5 4 3 2 1 0 J-F M-A M-J J-A S-O N-D Period of ye ar (m onths ) Stingless bees
500 400 Honey bees
1.40 1.20 1.00 0.80 0.60
Honey bees
Figure 1. Seasonal variation in production of sexual offspring in the sympatric species Apis cerana and Trigona ventral in North Vietnam (after chapter 5&6)
The resilience of stingless bees to unfavourable environmental conditions is reflected by their enormous food stores. A relatively short period without floral resources is therefore not necessarily a constraint for their development (see also Inoue et al., 1993). We observed that wild nests of T. ventralis had large food stores, the volume of which was about half the volume of their nest cavity (Chinh et al., chapter 3). The far-most part of pollen reserves was already in a hard and brittle state, suggesting that it had been stored for years. Hence, stingless bees can survive extended periods of unfavourable conditions before the colony dies (Inoue et al., 1993).
7. General reproductive strategies of sympatric eusocial bees in northern Vietnam
Although their general way of life is similar and many species use the same floral resources, numerous species of eusocial bees often occur sympatrically. These species can live together because resources are partitioned based on competition, and because morphology of the distinct flowers often limits availability to part of the bees. For instance in Costa Rica, considerable differences in size occur among the more than 60 sympatric stingless bee 87
Number of immature males and queens
300 200 100 0

males 140 120 100 80 60 40 20 0 J F M A M J J A S O N D Months of the year Stingless bees gynes
Number of swarms
0.40 0.20 0.00

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
Figure 2. Seasonal variation in swarming in stingless bees and honey bees in Costa Rica (after Slaa, 2003).
species, which are related to the flower species the bees prefer (Slaa et al., 2003). In addition, nesting sites used by eusocial bees differ much. All kind of cavities in trees or rocks is used by almost all stingless bees and the cavitydwelling honey bees. Dwarf honey bees build nests in the open between shrubs and around branches, and giant honey bees build exposed nests under high cliffs or branches (Seeley, 1985; Trung et al., 1996). Besides differences in the floral resources and the nesting sites the bees use, eusocial bees also differ in their general strategy for colonial reproduction, which can be illustrated by the species we studied in Cuc Phuong forest in the north of Vietnam: Apis cerana, Lisotrigona carpenteri and Trigona ventralis (Chinh et al., chapter 6, chapter 3; Chinh & Sommeijer, chapter 5; respectively). Above we reviewed numerous differences in colony organization between stingless bees and honey bees, especially differences in how they organize the production of males and gynes. From these differences a general picture emerges describing the nature of different eusocial bee species. The honey bee species A. cerana is a relatively opportunistic species. Honey bees respond quickly to changing environmental conditions. Their short development time allows a quick population growth under favourable conditions. If conditions deteriorate, their progressive feeding of the larvae allows reuse of resources already invested in brood by eating the larvae (Woyke, 1977). Colonies swarm without concern for suitable nest sites in the neighbourhood. New nest sites will be explored after swarming, and swarms may travel far from the original nest if no suitable new site is found (Seeley, 1985). In fact, if conditions are unfavourable, the whole colony leaves the nest and absconds to find a better place (Seeley, 1985; Winston, 1987; Hepburn & Radloff, 1998). Seasonal migrations occur in various honeybee species, e.g. giant honey bees have been reported to migrate over hundreds of kilometres (Seeley, 1985). Hence, honey bees are well adapted to exploit seasonal or temporal resources. In contrast, stingless bees like T. ventralis, of which many colonies were found in Cuc Phuong forest (Chinh et al., chapter 3; Chinh & Sommeijer, chapter 5), behave conservative to changes in environmental conditions. Their development time is much longer than that of honeybees. Since brood cells are immediately capped after egg-laying, resources invested in the brood nest will not be reallocated. A good nesting site seems to be very important, in which they store large amounts of food to survive unfavourable conditions. Because new nests start as satellites from the original nest, stingless bees only colonize new sites in the neighbourhood of the old nest (see above). Hence, it is expected that stingless bees will flourish most in stable environments. T. ventralis has large colonies with up to 10,000 adults, which restricts the availability of large enough nest sites. In Cuc Phuong forest, perhaps the most commonly found stingless bee is L. carpenteri, which has colonies of 88
only about 400 adult bees at most. Therefore, this species can use all kinds of small cavities as nest sites. Stingless bees with relatively small nests may focus on colonizing new sites, whereas species with large nests focus on colony defence and growth of the colony itself. Of Tetragona angustula in Costa Rica (C.A.) and of Trigona nigra in Trinidad and Tobago (W.I.), two species that also use a wide variety of small cavities, it is known that both species swarm more frequently than other stingless bees, for example Melipona beecheii (Sommeijer, personal observation). As yet, the swarming frequency of L. carpenteri has not been studied. However, finding seven broodless colonies out of 17 inspected ones, six of which were queenless and one contained a gyne or young queen (Chinh et al., chapter 3), is a clue for a high swarming frequency, because colonies are most likely to become broodless and queenless when a young queen is lost during her nuptial flight. Because of the focus on colony propagation, stingless bee species like L. carpenteri may be better adapted to exploit dynamic environments than T. ventralis. Nevertheless, unlike honey bees, they will still be restricted in their dispersal because nest sites have to be close to the original nest.
89
Samenvatting: Voortplanting bij eusociale bijen (Apidae: Apini, Meliponini)

1. Voortplanting op individueel niveau
In dit proefschrift worden enkele sleutelaspecten van de regulerende mechanismen van kolonievermeerdering in honingbijen en angelloze bijen behandeld. Er wordt speciale aandacht gegeven aan de vraag hoe geslachtsdieren (koninginnen en darren) worden geproduceerd en hoe deze processen worden gestuurd. De honingbijen (Apini) en de angelloze bijen (Meliponini) zijn hoog-sociale ("eusociale") bijen. De soorten van beide groepen leven in meerjarige kolonies. De kolonies van deze bijen zijn gekenmerkt door morfologisch verschillende vrouwelijke kasten (koninginnen en werksters), het voorkomen van een taakverdeling op het gebied van de voortplanting en een overlap van generaties bij de leden van de kolonie (Eigener, 1974). Op individueel niveau is de voortplanting gericht op de productie van bijen, nieuwe leden ter versterking van de kolonie. Door groei van de kolonie kunnen er nieuwe kolonies worden afgesplitst ("zwermen"). De leggende koninginnen van bestaande kolonies worden indien nodig vervangen en jonge koninginnen worden tijdens een bruidsvlucht gensemineerd door mannetjes. De overdracht van genen bij de paring van de geslachtsdieren kan dus gekoppeld zijn aan vervanging van een koningin en aan het zwermproces.
2. Kolonievermeerdering
Afwijkend van de situatie bij de hommels (Bombini) worden nieuwe kolonies bij de angelloze bijen en de honingbijen gevestigd door afsplitsing van een deel van de moederkolonie. Bij geen van deze groepen zijn individuele bijen in staat om afzonderlijk te overleven en de koninginnen kunnen niet zelfstandig foerageren. Bij het zwermen vertrekt een cohort van werksters uit de moederkolonie en gaat op zoek naar een nieuwe nestplaats. Bij de honingbijen vertrekt de eerste zwerm met de oude koningin, waarna andere zwermen kunnen volgen met jonge koninginnen. Het aantal zwermen dat zich kan afscheiden houdt geen verband met het aantal geproduceerde koninginnen, maar met het aantal bijen. De overlevingskansen van een volk worden namelijk in hoge mate bepaald door zijn grootte. Bij de angelloze bijen komt een afwijkend zwermgedrag voor. Hier wordt gedurende een langere tijd door werksters gewerkt aan de inrichting van een dochternest, waarbij geleidelijk nestmateriaal en voedsel van het moedernest wordt
90
overgebracht naar het nieuwe nest. Pas na enkele dagen vliegt een jonge koningin over naar het nieuwe nest. Het zwermgedrag is hier sterk gekoppeld aan de capaciteit om werksters te produceren en aan de hoeveelheid opgeslagen voedsel in het nest.
3. Het onderzoek van dit proefschrift
In ons onderzoek hebben wij ons gericht op de vraag hoe geslachtsdieren (koninginnen en darren) worden geproduceerd en hoe deze processen worden gestuurd. Daartoe is allereerst in Trinidad & Tobago de productie van mannetjes bij Melipona favosa onderzocht. De productie van mannetjes bleek opvallend golfsgewijs voor te komen. De verschillende kolonies bleken hierbij niet in fase te zijn. Aansluitend is in het laboratorium in Utrecht onderzocht hoe de koningin en de leggende werksters deelnemen aan de productie van mannetjes door het leggen van haplode eieren. Vervolgens is het proces van de productie van mannetjes, koninginnen en het optreden van zwermgedrag onderzocht in sympatrisch voorkomende Vietnamese eusociale bijensoorten: Trigona (Lepidotrigona) ventralis en Apis cerana. De bevindingen bij verschillende soorten zijn vergeleken en fundamentele verschillen en overeenkomsten in de voortplantingsstrategie van de Apini en Meliponini zijn besproken en de adaptieve betekenis van deze kenmerken is behandeld. 3.1. Drie sympatrisch voorkomende angelloze bijensoorten en hun nestbouw De nestarchitectuur en de kolonieopbouw van drie sympatrische angelloze bijen is onderzocht in het grote natuurgebied Cuc Phuong Forest ten zuiden van Hanoi. Trigona (Lepidotrigona) ventralis and Trigona (Heterotrigona) laeviceps bouwen een nest in bomen met een stamdiameter van minstens 35 cm. De soort Trigona (Lisotrigona) carpenteri gebruikt vooral kleine holtes in een grote diversiteit aan substraten. Deze soort maakt bovendien veelvuldig gebruik van holtes in muren en andere niet-natuurlijke substraten en materialen. De drie soorten hebben kenmerkende nestuitgangen. T. ventralis bouwt een uitstekende nestuitgang in de vorm van een trechtervormig pijpje van zacht materiaal. Bij T. carpenteri is de nestuitgang een klein bros cilindervormig pijpje en bij T. laeviceps bestaat de nestuitgang slechts uit een verdikte ring rond het gat van de nestholte. Bij T. ventralis blijkt het formaat van de uitwendige uitgangsbuis positief gecorreleerd met de omvang van de kolonie. Bij T. ventralis zijn de broedcellen in horizontale raten gerangschikt en bij de twee andere soorten zijn de cellen gerangschikt in onregelmatige clusters. 3.2. Periodieke productie van mannetjes in Melipona favosa 91
Samenvatting
De productie van mannetjes bij angelloze bijen vindt plaats gedurende het hele jaar, hoewel de aantallen kunnen variren afhankelijk van het seizoen (van Veen et al., 1999; Moo-Valle et al., 2001; Sommeijer et al., 2003). Om dit proces te onderzoeken werden observaties uitgevoerd aan zes kolonies van M. favosa in de natuurlijke habitat. Het voorkomen van mannetjes bleek in deze kolonies zeer periodiek zijn; mannetjes verschijnen golfsgewijs en de verschillende kolonies, hoewel naast elkaar levend, zijn hierbij niet in fase (hoofdstuk 4). Er is nog geen duidelijke correlatie gevonden met eventuele koloniecondities, hoewel in de literatuur gesuggereerd is dat voedselopslag een belangrijke factor is (Bego, 1990; Gross et al, 2000; Moo-Valle et al., 2001). 3.3. Het periodiek voorkomen van mannetjes is het resultaat van het voorkomen van leggende werksters De hypothese dat het ritmisch voorkomen van mannetjes het gevolg is van eileg door reproductieve werksters werd onderzocht in Utrecht. Door middel van gedetailleerde observaties in kolonies waar alle cellen bij de eileg in kaart werden gebracht en waar met permanente video-opname het leggedrag van koningin en werksters werd vastgelegd, was het mogelijk te achterhalen of mannetjes door werksters of door de koningin geproduceerd werden. De resultaten (hoofdstuk 4) ondersteunen de bovengenoemde hypothese. Het was duidelijk dat het overgrote deel van de mannetjes geproduceerd wordt door de werksters. De werksters leggen ook voedingseieren die steeds een belangrijk bestanddeel van het dieet van de koningin vormen. De productie van reproductieve werkstereieren is negatief gecorreleerd met de productie voedingseieren. Het belang van de voedingseieren voor de voortplanting van de koningin werd aangetoond. 3.4. Hypotheses voor de periodieke productie van mannetjes bij Melipona favosa De achtergrond voor de periodieke productie van mannetjes bij M. favosa is nog niet geheel duidelijk. Verschillende hypotheses worden gepresenteerd (hoofdstuk 4). De asynchrone productie van mannetjes in de kolonies is mogelijk een mechanisme ter voorkoming van inteelt. Dit kan van betekenis zijn bij soorten met slechts een beperkte dispersie ten gevolge van het typische zwermgedrag en dientengevolge met een hoge verwantschap tussen kolonies in hetzelfde gebied. 3.5. Seizoensgebonden productie van geslachtsdieren in Trigona ventralis Voor de neotropische soorten is informatie beschikbaar over de productie van geslachtsdieren en de kolonievermeerdering. Zoals hierboven is aangegeven, 92
kan in deze tropische gebieden de productie van mannetjes en koninginnen gedurende het gehele jaar voorkomen (van Veen et al., 1999; Moo-Valle et al., 2001; Sommeijer et al., 2003). Echter, de beschikbare informatie is nog erg fragmentarisch. Over het voortplantingsgedrag van de angelloze bijen in Afrika en Azi was nog geheel niets bekend. Om de voortplanting van angelloze bijen uit het Aziatisch continent te bestuderen hebben we veldonderzoek uitgevoerd in Vietnam. Tien kolonies van Trigona (Lepidotrigona) ventralis werden geobserveerd om de productie van geslachtsdieren over het jaar te onderzoeken in relatie met verschillende factoren. Het blijkt dat ook in dit gebied de productie van mannetjes en koninginnen in het grootste deel van het jaar plaats vindt. De fluctuatie van de aantallen blijkt afhankelijk van het jaarlijks verloop van de bloei van voedselplanten. Op het niveau van de individuele kolonies blijkt, evenals we vonden voor de neotropische soort M. favosa, dat de productie van mannetjes ritmisch verloopt en dat in n kolonie ook meerdere opeenvolgende periodes van mannenproductie voorkomen. De productie van koninginnen heeft een hiervan afwijkend patroon. Zij worden in lage aantallen, verspreid over het hele seizoen, geproduceerd. In de winterperiode, waarin weinig planten bloeien, worden geen geslachtsdieren geproduceerd. De productie van geslachtsdieren was positief gecorreleerd met voedselopslag en kolonieomvang. 3.6. Het gedrag van pas geboren koninginnen bij Trigona ventralis Het gedragsrepertoire van pasgeboren koninginnen ("gynes") wordt beschreven in hoofdstuk 5 waarbij speciale aandacht wordt gegeven aan interacties tussen gynes en werksters. Werksters vertonen agressief gedrag naar de gynes. Gynes proberen hieraan te ontsnappen door zich te verbergen onder neststructuren. Een zeer opmerkelijke waarneming betrof het voorkomen van dode gynes in nog gesloten koninginnencellen. Zowel in kolonies in observatiekasten als in de kolonies in de natuurlijke behuizing in het bos werden dode gynes in koninginnencellen aangetroffen naast koninginnencellen met levende koninginnen. Dit verschijnsel is nog niet eerder waargenomen in angelloze bijen met typische koninginnencellen. Een verklaring kan gevonden worden in het zeer typische broedsysteem van angelloze bijen. Door de volledige bevoorrading van broedcellen met larvaal voedsel voorafgaand aan de eileg kunnen deze bijen geen nieuwe koningin ( "emergency queen") produceren op het moment van verlies van de leggende koningin. Dit is in scherp contrast met het koninginnenproductiesysteem van de honingbij. Hier kunnen werksters door de typische progressieve voeding van de larven de ontwikkeling van werksterlarven ombuigen voor de productie van "emergency queens". (Dergelijke koninginnencellen worden 93
Samenvatting
door de imker "redcellen" genoemd.) Het productiesysteem voor koninginnen bij angelloze bijen komt er in het algemeen op neer dat er, als reserve, permanent volwassen gynes beschikbaar moeten zijn (zie Sommeijer 7; de Bruijn 2003, Sommeijer et al. 2003a and 2003b). Het merendeel van deze reserve gynes is echter overbodig en moet door de werksters doorlopend opgeruimd worden. De dode gynes in koninginnencellen van T. ventralis houden zeer waarschijnlijk verband met dit typische systeem van koninginnenproductie. 3.7. De regulatie van de productie van darren bij Apis cerana In hoofdstuk 6 wordt onderzoek beschreven naar de regulatie van de productie van mannetjes bij de Aziatische honingbij A. cerana. Hiervoor zijn in Vietnam 20 kolonies onderzocht in 1999 en 2000. Mannetjes worden jaarlijks in twee periodes geproduceerd en dit is gekoppeld aan de twee periodes van bloei van voedselplanten. De betekenis van deze bloeiperiodes voor de jaarlijkse broedproductie werd vastgesteld. Vers verzameld stuifmeel en nectar doet de kolonies snel groeien en als respons worden in alle kolonies synchroon mannetjes geproduceerd. 3.8. De regulatie van de productie van koninginnen bij Apis cerana Bij de honingbijen (Apini) worden nieuwe koninginnen geproduceerd voor kolonievermeerdering of voor de vervanging van de oude koningin. Als de leggende koningin plotseling verloren gaat kan er een "emergency queen" geproduceerd worden (zie punt 3.6). In hoofdstuk 6 wordt onderzoek beschreven waarbij is gevonden dat de koninginnen, evenals de mannetjes, bij A. cerana jaarlijks in twee perioden werden geproduceerd. De meeste koninginnen werden in de eerste productieperiode geboren. Er werd gevonden dat het aantal geproduceerde koninginnen op populatieniveau gecorreleerd is met het aantal geproduceerde werksters evenals met het aantal bloeiende plantensoorten in de omgeving. Koninginnenproductie leidt niet altijd tot zwermgedrag of koninginnenvervanging. Dit geeft aanwijzingen voor regulatie door niet-stabiele stimuli voor de aanzet van koninginnenproductie. Het aantal koninginnen dat geproduceerd wordt voor kolonievermeerdering is ongeveer zes keer het aantal dat geproduceerd wordt voor een vervanging van de oude koningin. 3.9. Kolonievermeerdering bij A. cerana In totaal werden 35 zwermen en acht koninginnenvervangingsprocessen in A. cerana waargenomen (hoofdstuk 6). De A. cerana kolonie komt in de zwermfase als de populatie een omvang van ongeveer 21000 bijen heeft bereikt en als ongeveer 90 % van de cellen op de raten is bezet. De 94
verhouding van werksterbroed en volwassen werksters is dan ongeveer 56 %. Ongeveer 97% van de zwermen en 87 % van de koninginnenvervangingen vond plaats in de eerste koninginnenproductieperiode. Per kolonie werd er gemiddeld n keer per jaar gezwermd. Zoals de productie van darren het eerste signaal is van het begin van het complexe zwermproces, zo is het vertrek van de zwerm de afsluiting hiervan. In hoofdstuk 6 wordt besproken hoe op populatieniveau de synchrone productie van darren en koninginnen van betekenis is voor het reproductief succes en de overleving van kolonies. 3.10. Vergelijking van voortplantingsstrategien bij honingbijen en angelloze bijen Gebaseerd op de reeds beschikbare informatie en de eigen resultaten worden de belangrijkste overeenkomsten en verschillen in de voortplantingsstrategien van beide groepen sociale bijen vergeleken in hoofdstuk 7. De achtergronden van de belangrijkste verschillen met betrekking tot de productie van geslachtsdieren in de sympatrische soorten Apis cerana, Trigona ventralis en Lisotrigona carpenteri worden besproken.
4. Conclusies en opmerkingen over de voortplantingsstrategien van sympatrische eusociale bijen in noordelijk Vietnam
In dit proefschrift worden enkele belangrijke verschillen in kolonieorganisatie van de angelloze bijen en de honingbijen besproken, met nadruk op de verschillen in organisatie van de productie van mannetjes en koninginnen. Uit deze verschillen komt een algemeen beeld naar voren. De honingbij A. cerana is een opportunistische soort. Deze soort kan snel op veranderende milieuomstandigheden reageren. De korte ontwikkelingstijd resulteert in een snelle koloniegroei bij gunstige situaties. Als de omstandigheden verslechteren kan door het systeem van progressieve voeding van de larven de energie die al in broed genvesteerd was hergebruikt worden door de larven op te eten. Kolonies zwermen zonder de noodzaak voor nieuwe nestruimte direct in de omgeving. Nieuwe nesten worden gezocht na het vertrek van de zwerm uit het moedernest. Zwermen kunnen zich, indien nodig, ver verspreiden. Bovendien kunnen kolonies bij zeer slechte omstandigheden het oude nest verlaten en als complete kolonie naar een andere plaats vertrekken. Bepaalde soorten honigbijen (b.v. de "giant honeybee") zijn ook in staat om jaarlijks over grote afstanden te migreren. Het is dus duidelijk dat honingbijen in staat zijn om zich aan te passen aan seizoensafhankelijke of tijdelijke verandering van de bronnen in hun omgeving. Angelloze bijen, bijvoorbeeld T. ventralis die in grote aantallen in Cuc Phuong Forest voorkomt, blijken zich veel conservatiever te gedragen ten opzichte van veranderingen in hun milieu. Hun ontwikkelingstijd is veel 95
Samenvatting
langer dan bij de honingbij. Vanwege het typische broedsysteem waarbij de broedcellen direct afgesloten worden na de eileg, wordt energie die in broed genvesteerd is niet makkelijk herverdeeld. Een goede nestplaats blijkt essentieel voor deze bijen. Hierin worden grote hoeveelheden voedsel opgeslagen om ongunstige omstandigheden te overleven. Omdat nieuwe nesten als satellieten van het oude nest worden gesticht kunnen angelloze bijen slechts de nabije omgeving van het oude nest koloniseren. Het is dus te verwachten dat angelloze bijen het best kunnen leven in stabiele milieus. T. ventralis komt voor in grote kolonies tot 10.000 volwassen dieren. Dit beperkt de beschikbaarheid van nestholtes. De meest algemene angellozebijensoort in Cuc Phuong Forest is L. carpenteri. Deze soort heeft slechts kolonies van maximaal 400 bijen en kan dus gebruik maken van allerlei soorten kleine holtes als nestplaats. Angelloze bijen met relatief kleine kolonies kunnen hun energie vooral steken in het koloniseren van nieuwe nestholtes, terwijl de soorten met grote kolonies kunnen specialiseren op nestverdediging en koloniegroei. In de neotropen komen soorten voor die een voorbeeld zijn van bovengenoemde kolonisatiestrategie (b.v. Tetragonisca angustula in Costa Rica, en Trigona nigra in Trinidad & Tobago). Deze soorten die aangepast zijn aan kleine en onregelmatige nestholtes blijken tevens een relatief frequent zwermgedrag te vertonen. De zwermfrequentie van de Vietnamese L. carpenteri is nog niet onderzocht, maar de waarneming van 7 broedloze kolonies onder de 17 genspecteerde nesten, waarvan er zes koninginnenloos waren en er n slechts een gyne bevatte, kan een aanwijzing zijn voor frequent zwermgedrag (kolonies lopen tijdens het zwermen en de daaropvolgende bruidsvlucht het risico om de koningin te verliezen). Vanwege deze veronderstelde frequentere kolonievermeerdering in L. carpenteri kan deze soort beter aangepast zijn aan dynamische omstandigheden dan de soort T. ventralis. Maar, in tegenstelling tot de honingbij, zijn deze angelloze bijen beperkt in hun dispersie, vanwege het feit dat nieuwe nestplaatsen dicht bij het originele nest gelegen moeten zijn.
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174-189. Sakagami S F; Inoue T (1990) Oviposition Behaviour of Two Sumatran Stingless Bees, Trigona (Tetragonula) laeviceps and T. (T) fuscobalteata. In: Sakagami S F; Ohgushi R; Roubik D W (eds) Natural History of Social Wasps and Bees in Equatorial Sumatra. Hokkaido University Press; Sapporo, Japan; pp 20-216. Sakagami S F; Yamane S (1984) Notes on taxonomy and nest architecture of the Taiwanese Stingless Bees Trigona (Lepidotrigona) ventralis hoozana. Bulletin of the Faculty Education, Ibaraki University, Natural Science 33: 37- 48. Sakagami S F; Yamane S; Hambali G G (1983) Nests of some Southeast Asian Stingless Bees. Bulletin of the Faculty of Education, Ibaraki University, Natural Sciences: 32: 1-2. Schwarz H F (1937) Results of the Oxford Sarawak (Borneo) expedition: Bornean stingless bees of the genus Trigona. Bulletin of the American Museum of Natural History 73: 281-328. Schwarz H F (1939) The Indo-Malayan species of Trigona. Bulletin of the American Museum of Natural History 76: 83-141. Schwarz H F (1948) Stingless bees (Meliponinae) of the Western Hemisphere. Bulletin of the American Museum of Natural History 90: 1-546. Seeley T D (1985) Honeybee ecology. Princeton University Press; Princeton, New Jersey. USA; 201 pp. Seeley T D; Seeley R H; Akratanakul P (1982) Colony defense strategies of the honey bees in Thailand. Ecological Monograph 52: 43-63. Sharma P L (1969) Observation on the swarming and mating habits of the India honeybee. Bee World 41: 121-125. Slaa E J (2003a) Community ecology of stingless bees in forested and deforested habitats in Guanacaste, Costa Rica. In: Slaa E J (ed) Foraging ecology of stingless bees: from individual behaviour to community ecology. PhD thesis, Department of Behavioural Biology, Faculty of Biology, Utrecht University, The Netherlands. Budde Elinkwijk (ISBN: 90-393-3254-1); pp: 29-57. Slaa E J; Michiel B; Biesmeijer J C; Sommeijer M J (2003b) Social foraging in stingless bees: recruitment patterns and competitive dominance hierarchies. In: Slaa E J (ed) Foraging ecology of stingless bees: from individual behaviour to community ecology. PhD thesis, Department of Behavioural Biology, Faculty of Biology, Utrecht University, The Netherlands. Budde Elinkwijk (ISBN: 90-393-3254-1); pp 133-151. Smith D R; Hagen R H (1996) The biogeography of Apis cerana as revealed by mitochondrial DNA sequence data. Journal of the Kansas Entomological Society 69(4): 294-310. 111
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Senckengergiana Biologica 76(1/2): 115-119. Tingek S; Mardan M; Rinderer T; Koeniger N; Koeniger G (1988) Rediscovery of Apis vechti (Maa, 1953): The Saban honey bee. Apidologie 19(1): 97-102. Tth E; Queller D C; Dollin A; Strassmann J E (2004) Conflict over male parentage in stingless bees. Insectes Sociaux 51: 1-11. Tth E; Queller D C; Imperatriz-Fonseca V L; Strassmann J E (2002a) Genetic and behavioural conflict over male production between workers and queens in the stingless bee Paratrigona subnuda. Behavioural Ecology and Sociobiology 53: 1-8. Tth E; Strassman J E; Imperatriz-Fonseca V L; Queller D C (2003) Queens, not workers, produce males in the stingless bee Schwarziana quadripuncta quadripuncta. Animal Behaviour 66(2): 359-368. Tth E; Strassmann J E; Nogueira-Neto P; Imperatriz-Fonseca V L; Queller D C (2002b) Male production in stingless bees: variable outcomes of queenworker conflict. Molecular Ecology 11(12): 2661-2667. Trung L Q; Dung P X; Ngan T X (1996) A scientific note on first report of Apis laboriosa F Smith, 1871 in Vietnam. Apidologie 27(6): 487-488. van Benthem F D J; Imperatriz-Fonseca V L; Velthuis H H W (1995) Biology of the stingless bee Plebeia remota (Holmberg): Observations and evolutionary implications. Insectes Sociaux 42: 71-87. van Veen J W; Sommeijer M J (2000) Colony reproduction in Tetragonisca angustula (Apidae, Meliponini). Insectes Sociaux 47: 70-75. van Veen J W; Sommeijer M J (2000) Observations on gynes and drones around nuptial flights in the stingless bees Tetragogisca angustula and Melipona beecheii (Hymenoptera, Apidae, Meliponinae). Apidologie 31: 47-54. van Veen J W; Sommeijer M J; Arce A H G (1999) The role of colony development and resource availability in the regulation of queen production in Melipona beecheii (Apidae, Meliponini). In: van Veen J W (ed) Colony reproduction in stingless bees. PhD- thesis, Utrecht University; Litografia Imprenta Lil. S.A. San Jos; pp 80-87. Velthuis H H W (1970) Ovarian development in Apis mellifera worker bees. Entomological Experiments and Applications 13: 377-394. Velthuis H H W (1990) Regulation of honey bee reproduction at colony level. In: Hoshi M; Yamashita O (eds) Advances in invertebrate reproduction 5. Elsevier Science Publishers B V; The Netherlands; pp 489-493. Velthuis H H W (1997) The biology of stingless bees. Utrecht University, Utrecht, The Netherlands, 33 pp. Velthuis H H W; Alves D de A; Imperatriz-Fonseca V; Duchateau M J (2002) Worker bees and the fate of their eggs. Proceedings of the Section 113
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Advancement of Apiscultural Science and Technology in China. In: Verma L R (ed) Honeybees in Mountain Agriculture. Oxford & IBH Publishing Co. Ltd; New Delhi, India; pp 1-148.
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Acknowledgements
This thesis is the result of fruitful collaboration between various research institutes and scientists from The Netherlands and Vietnam. In the first place I would greatly express my gratitude to Dr. Marinus J. Sommeijer, my supervisor, who often points at the keys to the obscure gates of science and whose work has added significantly to my scientific career thanks to his never-ending enthusiastic supervision, valuable suggestions and critical comments on all chapters in this thesis. I also wish to thank him for introducing me to the stingless bees, and giving me the opportunity to work with these bees at the laboratory of the Tropical Bees Research Unit, of the Behavioural Biology Group, Faculty of Biology, Utrecht University. He also helped with obtaining funds for my following studies in Cuc Phuong, Ninhbinh, Vietnam. Special thanks are due to Dr. Willem J. Boot, of Wageningen University, my second co-supervisor, for his sharp and critical scientific discussion and comments on all parts of my thesis, and for encouraging the development of my ideas about the ecological ethology of social bees. His collaboration has been essential for the completion of this thesis. I am grateful to Prof. Dr. Jan A.R.A.M. van Hooff for being my promotor and providing critical comments on my thesis. During my work in the Netherlands, I had wonderful colleagues and I want to thank these for their help, encouragement and friendship: Frans Meeuwsen, Frouke Hofstede, Bert Agterhuis, Koos Biesmeijer, Judith Slaa Han de Vries, Marie-Jos Duchateau, Rene van Gelderen, Paul Wester, Edwin Martens and Zjeffrie Pereboom. I also owe thanks to Tung, Edwin, Tra My, Kim Anh, Dai, Hung, Nga, Tu Minh, Linh, Tu, Minh, Rafael, Pedro, Victor, Desalegn, Robert, Ingrid and all the other M.Sc. bee students that I met at the Lab for their encourage and friendship. Thanks are given to Mr. Marcel Kramer and Mr. Rob van Weerden for their help with computer and network problems and to Bert Agterhuis for important administrative help in the final phase of this work while I was in Vietnam. I cordially thank the Directorate Board of the Bee Research and Development Centre: Mr. Dinh Quyet Tam, Mr. Nguyen Thong Dap and Dr. Phung Huu Chinh for their support and assistance. Thanks go to all my colleagues at the Bee Research and Development Centre for their help and encouragement. I personally thank Dr. Pham Van Lap, Faculty of Biology, 116
Hanoi National University, Vietnam for his help with important administrative formalities. Pham Hong Thai is thanked for using his documents. I highly appreciate the assistance and cooperation of the staff of Cuc Phuong National Forest, of Cuc Phuong Commune, Nho Quan district, Ninh Binh province, Vietnam, during my fieldwork. I personally would like to give special thanks to the family of Mr. Do Cao Chi for their enthusiastic help when I stayed for two years (1999-2000) in their house during my field work in Cuc Phuong. Finally, I personally give many thanks to my wife Trinh Thi Thuy and my son Tong Van Minh, for their silent dedication and sacrifices during a long period that I have been so far away from them to carry out this research project. Their great support and encouragement are the dynamics for my work. I am greatly indebted to my uncle Tong Xuan Trinh and his wife for their help and stimulation. I acknowledge the support from various institutions that made it possible for me to do these studies. The research projects were financially supported by the Royal Netherlands Embassy, Hanoi, Vietnam. Mr. Wijnand J.van Ijssel, first secretary, is thanked for his interest in this project. The Netherlands Expertise Centre for Tropical Apicultural Resources (NECTAR) is thanked for moral support. I also received scholarships from the NUFFIC (Netherlands Universities Foundation for International Cooperation) in The Hague, from the Utrecht University Scholarship Programme, and from the United Nations University. Babette Berrocal and Berthe Schoonman, of the Utrecht University International Relations Office, have continuously shown a lively interest in my work and I am very grateful to them.
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Curriculum Vitae
Tong Xuan Chinh was born on May 16, 1965 in Tong Xuyen village, Thai Hung commune, Hung Ha district, Thai Binh province. In 1985 he started his studies at the Faculty of Biology, the Hanoi University (now the University of Science, Hanoi National University) in Hanoi city, Vietnam, and obtained a B. Sc degree of Genetics in 1990. From 1990 to 1994, he took up a post as bee-researcher and beekeeping lecturer at the Bee Research and Development Centre, Hanoi, Vietnam. In 1994, he attended a special one-month course on Tropical Bees and Queen Rearing at Pertania University, Malaysia. From 1995 to 1996, he worked on a project on Instrumental Insemination for Apis cerana Queens at the Division of Genetics, the Faculty of Biology, Hanoi National University, Hanoi, Vietnam. In 1996, he was granted a fellowship by NUFFIC to attend the Utrecht University International MSc Programme Tropical Bees and Beekeeping in Tropical Climates. He obtained the diploma in 1998. In 1998, he received training in Field Research Methodology of Stingless Bees on the island of Tobago, Trinidad & Tobago W.I. working there together with the Utrecht bee researchers. From 1999 to 2000, he could carry out field studies in Vietnam with a grant from the Royal Netherlands Embassy in Hanoi. This work was part of his PhD research project on Reproduction in eusocial bees. The present thesis is essentially based on this research project in Cuc Phuong National Forest, Ninh Binh, Vietnam. From 2001 to 2003, he prepared his manuscripts for publication in international journals and wrote his PhD thesis at the Utrecht University. This work was possible thanks to fellowships from the Utrecht University Scholarship Programme and from the United Nations University. His research interests are the biology, ethology and ecology of tropical colony-living bees, with special interest in their reproductive biology. His further interests are honeybee breeding and honeybee diseases, topics on which he has worked for several years.
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Publications
Chinh P H; Thang D V; Hanh P D; Ngoc P V; Chinh T X; Toan T V; Lap P V (2001) Evaluation of a closed population breeding programme with Apis cerana cerana after six years of selection. Proceedings of the third AAA Conference on Bee Research and Beekeeping development (October 6-10, 1996) pp. 47. Chinh T X; Grob G B; Meeuwsen F J A J; Sommeijer M J (2003) Patterns of male production in the stingless bee Melipona favosa (Apidae, Meliponini). Apidologie 42: 161-170. Chinh T X; Sommeijer M J (accepted) Production of sexuals in the stingless bee Trigona (Lepidotrigona) ventralis flavibasis Cockerell (Apidae, Meliponini) in northern Vietnam. Apidologie. Chinh T X; Sommeijer M J; Michener (submitted) Nest architecture and colony characteristics of three stingless bees in north Vietnam with the first description of the nest of Lisotrigona carpenteri Engel (Hymenoptera: Apidae, Meliponini). Journal of the Kansas Entomological Society. Chinh T X; Willem J B; Sommeijer M J (accepted) Production of sexuals in the honeybee species Apis cerana Fabricius 1793 (Apidae, Apini) in northern Vietnam. Journal of Apicultural Research. Chinh T X; Willem J B; Sommeijer M J (being submitted) Reproductive strategies in sympatric species of eusocial bees. Chinh T i T Khoa h K nh Ong. Nh xu ,H n tr. 13; 44; 49-50; 264. Lap, P V; Chinh P H; Ha T D; Chinh T X; Hanh P D; Ngoc P V (1992) Some biological characteristics of Apis cerana queen in Vietnam. In: Asian Apiculture. Proceeding of 1st International Conference on the Asian Honey Bee and Bee mites. Changmai Thailand, pp: 117-123. Pham L V; Chinh T X (2001) Instrumental insemination for Apis cerana (Farb) queens. Proceedings of the third AAA Conference on Bee Research and Beekeeping development (October 6-10, 1996; Hanoi, Vietnam) pp. 34-39. Sommeijer M J; Chinh T X; Meeuwsen F (1999) Behavioural data on the production of males by workers in the stingless bee Melipona favosa (Apidae, Meliponinae). 46: 92-93. Insectes Sociaux
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120

150 Reproduccion Abejas Eusociales

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Reproduction in eusocial bees

(Apidae: Apini, Meliponini)

Reproduction in eusocial bees

Voortplanting bij eusociale bijen

(met een samenvatting in het Nederlands)

Tong Xuan Chinh geboren op 16 mei 1965, te Thai Binh, Vietnam

Dr. Willem J. Boot Laboratory of Entomology, Wageningen University

References Acknowledgements Curriculum vitae Publications

104 116 118 120

Introduction and summary: reproduction in eusocial bees (Apidae: Apini, Meliponini)

Reproduction in eusocial bees

1. Reproduction at the individual level

Chapter 1. Introduction and summary

3. The research of this thesis

Chapter 1. Introduction and summary

4. Reproductive strategies of honeybees and stingless bees

General methods: the bees and the study sites

Reproduction in eusocial bees

Chapter 2. General methods

Reproduction in eusocial bees

Chapter 2. General methods

Reproduction in eusocial bees

Chapter 2. General methods

2. The study sites

Reproduction in eusocial bees

Chapter 2. General methods

Reproduction in eusocial bees

Tong X. Chinh1, Marinus J. Sommeijer2, Willem J. Boot3 & Charles D. Michener4

Submitted to Journal of the Kansas Entomological Society

Reproduction in eusocial bees

Summary. In the tropical primary forest of Cuc Phuong National

Reproduction in eusocial bees

Reproduction in eusocial bees

Reproduction in eusocial bees

Reproduction in eusocial bees

Reproduction in eusocial bees

Reproduction in eusocial bees

Adapted from a publication in Apidologie 43 (2003): 161-170

Reproduction in eusocial bees

Summary. In many stingless bee species, laying workers oviposit trophic

Chapter 4. Patterns of male production in Melipona favosa

2. Materials and methods

Reproduction in eusocial bees

Chapter 4. Patterns of male production in Melipona favosa

Reproduction in eusocial bees

Chapter 4. Patterns of male production in Melipona favosa

Percentage of emerging males

Reproduction in eusocial bees

Col. 10 Col. 9 Col. 8

Chapter 4. Patterns of male production in Melipona favosa

Reproduction in eusocial bees

Chapter 4. Patterns of male production in Melipona favosa

Reproduction in eusocial bees

Tong X. Chinh1 & Marinus J. Sommeijer2

Accepted for publication in Apidologie

Reproduction in eusocial bees

Summary. In a two-year study on the stingless bee Trigona ventralis under

Chapter 5. Production of sexuals in Trigona ventralis flavibasis in north Vietnam

2. Materials and methods

Reproduction in eusocial bees

Chapter 5. Production of sexuals in Trigona ventralis flavibasis in north Vietnam

Reproduction in eusocial bees

Number of cells and workers