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Investigations into the biology, behaviour and phylogeny of a potential crop pollinator: the Australian stingless bee, Austroplebeia australis

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Abstract

This research was conducted with a view to increasing knowledge about a little known Australian stingless bee, Austroplebeia australis Friese (Hymenoptera: Apidae, Meliponinae). Bees are known to be superior pollinators due to the fact that they actively collect pollen and nectar in large amounts for the purpose of raising their young. There has been limited research into the utilisation of alternative pollinators to the European honey bee (Apis mellifera) in Australian horticultural crops. Au. australis is a eusocial bee that can be managed in transportable artificial hives and has, therefore, the potential to be managed as a crop pollinator. During this project I investigated aspects of the biology, behaviour and phylogeny of Au. australis, with a view to better understanding this species, as well as to determine whether it could be utilised as a greenhouse and / or field crop pollinator. To develop a context for my studies, an online survey was conducted to assess the current status of the Australian stingless bee industry and its recent development. This was a follow up survey conducted one decade after the first study, by Heard and Dollin in 1998. It showed that the Australian industry had grown over the past ten years but is still underdeveloped. The majority of stingless bee keepers are hobbyists, who own only one colony. There is a high demand for Australian stingless bee colonies and their honey, but with less than 250 bee keepers currently propagating colonies, and many of them on a small scale, it is difficult to meet this demand. Pollination services are provided by a small number of stingless bee keepers; however, there is an urgent need for research into this as well as many other aspects of stingless beekeeping in Australia. Public education is also needed to increase community awareness and, ultimately, to improve efforts in conserving these native species. Pollination studies were initially conducted using Au. australis in vegetable seed crops within an experimental insectary and greenhouse, at the University of Western Sydney, NSW. Au. australis colonies acclimated to the greenhouse enclosures and visited crops, with seed set in celery being significantly higher (76.25%) than the control (no pollinator) (0.02%). As a result, further investigations were undertaken with Au. australis in commercial field and greenhouse vegetable seed crops, located xxv at Musk, Vic. These studies, were less successful, due to the cool climatic conditions experienced in this area (Central Victoria), even during summer. These latter results suggest that Au. australis colonies do not adapt well to cool climate areas and low ambient and in-hive temperatures negatively influence their flight activity. The above hypothesis was tested in a subsequent investigation, which assessed the influence of abiotic climatic factors on flight activity of Au. australis. The entrance activity of five colonies was monitored and compared to the prevailing weather factors: ambient temperature, in-hive temperature, relative humidity, light intensity and cloud cover. Entrance activity commenced when in-hive temperatures were ≥ 18.6°C and the corresponding ambient temperature was ≥ 20°C. Optimal flight activity was observed when ambient temperatures were ≥ 26°C. Light intensity did not influence flight activity as much as temperature, and relative humidity had a negative impact. Heavy cloud cover and rain periods resulted in Au. australis foragers returning to their colonies and ceasing flight activity. It was unclear whether Au. australis was able to thermoregulate its nest, as reported in some other bee species. Au. australis colonies naturally occur in northern regions of Australia but some populations experience extreme temperature ranges, including sub-zero temperatures. Thermoregulation enables social insects to produce offspring throughout the year, thus giving them an advantage over solitary insects. To investigate this research question, the in-hive temperatures and brood temperatures of six Au. australis colonies were monitored over 12 months. Brood production and development was assessed, along with visible signs of pre-winter preparation. This study showed that colonies of Au. australis demonstrated ectothermic characteristics and were unable to warm the brood chamber when temperatures were < 15°C. Brood production, however, continued throughout the cold seasons and developing offspring survived and emerged even after exposure to prolonged periods of sub-zero temperatures, as well as to temperatures > 37°C. Au. australis, thus, shows a remarkable ability to survive temperature extremes. Adaptation to unique environmental characteristics may enable one species to survive over another. To investigate this, comparative studies on the foraging behaviour of three species of Australian stingless bee were conducted. The aim was xxvi to better understand the mechanisms by which Au. australis survives during prolonged periods of drought, when floral resources become scarce. The foraging behaviours of Au. symei, Au. australis and Trigona carbonaria were observed within the confines of an experimental greenhouse, and then compared. Fresh cut floral resources were used instead of artificial feeders, to allow observation of the foragers‘ behaviour within natural floral structures. Both Austroplebeia species were significantly more efficient than T. carbonaria. Hovering time was significantly lower in Austroplebeia (~ 8%) compared to Trigona carbonaria (> 30%). The number of flowers visited by Austroplebeia foragers, during a set time period, was greater than for T. carbonaria. Austroplebeia foragers demonstrated solitary foraging strategies, while T. carbonaria foraged in groups. There were no significant differences between the two Austroplebeia species. The different foraging strategies demonstrated by the two different stingless bee genera illustrate adaptive behaviours that may have evolved under different environmental conditions. Until the current study, little was known about the biology and lifecycle of Au. australis. The ontogenic times for developing offspring, as well as the longevity of adults, drive the overall life cycle of a eusocial colony. The developmental times for brood within cluster-building species, such as Au. australis, were yet to be reported. In order to observe developing Au. australis brood cells for investigations, it was necessary to separate sections of brood from the main brood cluster. A technique was developed where ‗donor‘ brood cells were ‗grafted‘ into hive annexes. This allowed colony members to access the brood ‗grafts‘ for maintenance, while enabling observation of their development. The mean ontogenic time for Au. australis workers, maintained at temperatures ~ 26°C, was 55 days, similar to that reported for other stingless bee species. This extended time, compared to A. mellifera, may be linked to lower brood incubation temperatures. In order to determine the longevity of Au. australis workers, bees within a single ‗closed‘ colony, as well as six ‗foraging‘ colonies, were marked and observed. ‗Foraging‘ colonies were able to freely access external resources, thus being exposed to a high ‗rate of living‘ and the risk of predation. Workers within both the ‗closed‘ and ‗foraging‘ colonies demonstrated extended longevity, compared to A. mellifera. The mean maximum longevity of workers was used to determine the longevity of xxvii cohorts within the foraging colonies. Au. australis workers are small (~ 4mm) and cannot be marked with an individual-identification colour scheme. As a result, cohorts of workers within each colony were colour marked and the longest lived bee within each cohort was recorded as the last individual remaining. The maximum longevity of each cohort was used to estimate the mean maximum longevity of all of the cohorts, within the six foraging colonies. The mean maximum longevity of workers from the ‗foraging‘ colonies was 161 days, with the longest lived bee being 240 days old. Within the ‗closed‘ colony, over 20% of the cohorts lived for at least 200 days and the longest lived bee reached 289 days old. The combination of long ontogenic period and extended longevity indicates that individual bees within colonies of Au. australis may live as long as seven months. Natural nest density of social bees is dictated by the availability of both nest substrate and floral resources. A focus survey was conducted to ascertain the nest density of Au. australis within an area of south-east Queensland that was known to support substantial bee populations. It was found that nest densities, which ranged from 0.1 to 3.0 nests / ha (mean 0.6 nests / ha) were low, compared to some others reported social bee species. The nest characteristics of Au. australis showed that this species prefers narrow cavities within dead trees but does not discriminate between tree taxa. Brood populations range from 2,000 to 13,000 (mean 5,000) in natural nests, within its native range. Colonies under threat of destruction can be easily extracted from their natural cavity and adapt well to artificial hive boxes. Au. australis are well adapted to the arid environment of south-east Qld. During my studies, it became apparent that the current descriptions for the species within the genus Austroplebeia are inadequate as a tool for the identification of specimens in either the field or the laboratory. As a result, a triangulated approach was undertaken in an attempt to better delimit morphologically identified groups within Austroplebeia. First, morphological data, based on worker bee colour and size were analysed. Drones collected from nests representing morphologically similar groups were dissected and their genitalia were imaged using scanning electron microscopy. Next, data for the geometric morphometric analysis of worker wing venations were obtained. Finally, molecular analysis, using mitochondrial rDNA segment 16S, was conducted on workers from representative nests for each group xxviii which displayed morphological similarities. Data from the four datasets were compared, resulting in the separation of two distinct species, with a large unresolved species complex. My research results contribute to the previously limited pool of knowledge on Austroplebeia spp. in general and Au. australis in particular. Its conservative recruitment of foragers for low level floral resources, combined with the efficiency with which foragers harvest these resources, reduces worker mortality within a colony. In the presence of abundant floral resources, forager recruitment is high and resource harvesting and storage is likely to be greater than the energy expenditure of the colony as a whole. Low energy expenditure within the colony, through thermoconformity, reduced nest architecture and efficient foraging strategies enable colonies to conserve precious resources. This combination of behaviours has enabled this species to survive, in harsh environmental conditions.
Investigations into the biology, behaviour
and phylogeny of a potential crop
pollinator: the Australian stingless bee,
Austroplebeia australis
by
Megan Therese Halcroft
A thesis submitted in fulfilment of requirements for the degree
of Doctor of Philosophy
University of Western Sydney
March 2012
Statement of Authentication
The work presented in this thesis is, to the best of my knowledge and
belief, original except as acknowledged in the text. I hereby declare that
I have not submitted this material, whether in full or in part, for a degree
at this or any other institution.

Megan Therese Halcroft
March 2012
Dedication
I dedicate this thesis to my family. To Steven and Elen Ruttley, for their patience and
genuine interest in my project; most of the time!
My sincere thanks to my daughter Elen, for her support over the many, many years in

writing up. Also for her occasionally feigned interest in my projects.
To my husband Steve, for his IT technical support and his support in the field. Also
for the photographic work undertaken during the trip to Tara (Section 6.4). But most
of all, for his unwavering support throughout the entire time of my PhD candidature
and for his understanding of the enormity of this task, including the never ending
write-up period. And for the moral, domestic and culinary support, without which we
may have starved.
I also dedicate this thesis to Allan Beil, Tara Qld, and his wonderful Austroplebeia
bees. For his generosity of time and knowledge. I wish to sincerely thank Allan for
his contagious enthusiasm and energy, and for the time and effort he committed to
locating and marking wild colonies. For his patience with us in the field and when
rescuing colonies. I wish to also acknowledge Allan for his dedication to his bees,
which he loves so much.
Acknowledgements
I would like to sincerely thank my supervisors, Assoc. Professor Robert Spooner-
Hart and Dr Tony Haigh, the School of Science and Health, University of Western
Sydney, for their support and guidance throughout my project. I would especially
like to thank Tony for his patience and guiding support in the still-bewildering world
of statistics.
The survey on the Australian stingless bee industry, one decade on: I would like to
thank Dr Anne Dollin, ANBRC and Dr Tim Heard, CSIRO, Qld, for their support in
designing the follow-up survey for the stingless bee industry survey. Special thanks
: the UWS
media unit, especially Danielle Roddick and Kristy Gleeson for their efforts to
broaden the media releases and to the many bee keepers who gave generously of
their time and knowledge in regard to stingless bee management and for trying to
answer the many constant questions I threw at them. These bee keepers include;
Allan Beil, John Klumpp, Thomas Carter, Frank Adcock, Mark Grosskopf, Russell
Zabel, Bob Luttrell and Peter Clarke.
For the pollination trials: Thank you to Rijk Zwaan Australia, Musk, Vic. for their
financial support throughout the two year trial. Also for their hospitality and support
during the Musk trial period. I would especially like to thank Lea Hannah for her
technical support during the UWS trial and for her kindness and hospitality at Musk.
Thanks to: Andy Ryland, Integrated Pest Management Consulting, NSW, for his
technical support in regard to the beneficial insect regimes; Linda Westmoreland,
UWS, for her technical support with the ever troublesome greenhouse enclosures;
Michael Duncan for his technical support in the meliponary and for helping me set
up the bee shed. Also for his input during our many casual brain-storming sessions.
For the phylogenetic placement studies: Thanks go to Rute Brito, University of
Sydney, NSW
region in Qld and to Lewis Robert for collecting the bees and sharing his knowledge.
I wish to, again, thank Anne Dollin for her unerring support during this time. Her
vast knowledge in regard to the morphology and distribution of our Australian
stingless bees was invaluable. The supply of her precious bees for the molecular
experiments was proffered with some tentativeness but we managed to shed some
light on this long term puzzle. Anne also provided technical support in the fine
tuning of phylogenetic tree for the final results. I wish to also thank Markus Riegler,
Hawkesbury Institute for the Environment, UWS, for his technical support, advice
and guidance. Thank you to: Jen Morrow, Aiman Jajo and Jocelyn King for their
continued lab support; Tiago Francoy, University of São Paulo, São Paulo, Brazil,
for his guidance and technical support in the wing geometric morphometry and his
expertise in their analysis; to Paul Smith and Adriyan Milev, UWS, for their
technical support in preparation and imaging of the bees using the SEM; to Michael
Batley for his guidance on specimen dissection and storage; to Liz Kabanoff for her
technical support and guidance with the microscopy aspect of this and other studies
and to John Rhodes for his photograph of honey bee sperm and for his advice in this
area.
For the nest density, distribution and characteristics study: Thank you to John
Klumpp, Brisbane, Qld, for helping and supporting us during our expedition around
 followed
Allan around the scrub and to Laurie and Helen Carmody, Queensland Primary
Industries and Fisheries, Dalby, Qld, for their hospitality and guiding support during
our expedition around the local Tara forestry areas. I wish to also acknowledge Allan

expedition and Allan 
life. His dream of being able to map the nest locations has been made possible with
the help of our IT support person, Kelvin Nicholson, Sydney, NSW.
Thank you to Michael Hornitzky, Department of Primary Industries, NSW, for his
technical support in helping to diagnose the mystery of the brood damage within
some of the colonies.
i
Table of Contents
Statement of Authentication ..................................................................................... 2
Dedication ................................................................................................................... 3
Acknowledgements ..................................................................................................... 4
Table of Contents ........................................................................................................ i
List of Figures .......................................................................................................... viii
List of Tables ........................................................................................................... xvi
List of Appendices ................................................................................................. xviii
List of Figures and Tables in Appendices ............................................................. xix
Abbreviations .......................................................................................................... xxi
Glossary .................................................................................................................... xxi
Thesis summary ..................................................................................................... xxiv
CHAPTER 1 - Literature review, scope and aims of thesis ................................... 1
1.1 General introduction ...................................................................................... 1
1.2 Alternative bee pollinators to A. mellifera .................................................... 3
1.3 Stingless Bees ................................................................................................ 7
1.4 Meliponiculture ............................................................................................. 9
1.4.1 Native plant and agricultural crop pollination by stingless bees .......... 10
1.4.2 Meliponiculture in Australia ................................................................ 13
1.5 Australian stingless bee classification ......................................................... 14
1.5.1 Trigona ................................................................................................. 15
1.5.1.1 Species description ....................................................................... 15
1.5.1.2 Nest architecture ........................................................................... 16
1.5.1.3 Colony population and brood structure ........................................ 17
1.5.2 Austroplebeia ....................................................................................... 18
1.5.2.1 Species description ....................................................................... 18
1.5.2.2 Nest architecture ........................................................................... 19
1.5.2.3 Colony brood structure ................................................................. 19
1.6 Colony life cycle ......................................................................................... 20
1.7 Geographic origin, distribution and density of stingless bees ..................... 21
1.7.1.1 Natural distribution of Australian stingless bees .......................... 23
ii
Trigona ................................................................................................... 23
Austroplebeia ................................................................................................. 23
1.8 Stingless bee biology ................................................................................... 24
1.8.1 Ontogeny .............................................................................................. 25
1.8.2 Longevity ............................................................................................. 26
1.8.3 Colony reproduction in stingless bees.................................................. 27
1.9 Stingless bee behaviour ............................................................................... 28
1.9.1 Nest dynamics ...................................................................................... 28
1.10 Temperature regulation ............................................................................... 29
1.11 Foraging and communication ...................................................................... 29
1.12 The general scope and aims of this thesis ................................................... 30
CHAPTER 2 - The Australian stingless bee industry .......................................... 33
2.1 Introduction ................................................................................................. 33
2.2 Materials and methods ................................................................................. 34
2.3 Results ......................................................................................................... 36
2.3.1 Overall comparison between surveys................................................... 36
2.3.2 Detailed analysis and comparisons ...................................................... 38
2.3.2.1 Summary of bee keepers and their stingless bee colonies ............ 38
2.3.2.2 Honey production ......................................................................... 41
2.3.3 Colony propagation .............................................................................. 41
2.3.4 Pollination ............................................................................................ 43
2.4 Discussion ................................................................................................... 44
2.4.1 Hive ownership for enjoyment ............................................................. 44
2.4.2 Colony propagation .............................................................................. 45
2.4.3 Honey production ................................................................................. 47
2.4.4 Pollination services .............................................................................. 48
2.4.5 Bee keepers and the future of the industry ........................................... 49
2.5 Key findings ................................................................................................ 50
iii
CHAPTER 3 - Studies on the pollination efficacy of Au. australis in greenhouse
research chambers and the field ............................................................................. 52
3.1 Introduction ................................................................................................. 52
3.1.1 Trial crops ............................................................................................ 53
3.1.1.1 Carrot ............................................................................................ 53
3.1.1.2 Celery ............................................................................................ 54
3.1.1.3 Leek .............................................................................................. 54
3.1.1.4 Lettuce .......................................................................................... 55
3.1.2 Trial aims ............................................................................................. 55
3.2 General materials and methods ................................................................... 56
3.2.1 General observations pertaining to Au. australis colonies ................... 61
3.3 Greenhouse chamber pollination study ....................................................... 61
3.3.1 Modifications to the stingless bee comparative study ......................... 61
3.3.2 Materials and methods ......................................................................... 63
3.3.3 Orientation to the greenhouse chamber ............................................... 65
3.3.3.1 Materials and methods .................................................................. 65
3.3.3.2 Results ........................................................................................... 66
3.3.4 Forager activity and flower visiting behaviour .................................... 66
3.3.4.1 Materials and methods .................................................................. 67
3.3.4.2 Results ........................................................................................... 68
3.3.5 Stingless bee behavioural observations ................................................ 69
3.3.5.1 Materials and methods .................................................................. 69
3.3.5.2 Results ........................................................................................... 69
3.3.6 Crop yield ............................................................................................. 73
3.3.6.1 Materials and methods .................................................................. 73
3.3.6.1.1 Leek ............................................................................................ 73
3.3.6.1.2 Celery .......................................................................................... 73
3.3.6.1.3 Carrot .......................................................................................... 75
3.3.6.1.4 Lettuce ........................................................................................ 75
3.3.6.1.5 Data analysis ............................................................................... 76
3.3.6.2 Results ........................................................................................... 76
3.3.6.2.1 Overview of pest and crop management problems at UWS site 76
3.3.6.2.2 Leek ............................................................................................ 77
3.3.6.2.3 Celery .......................................................................................... 77
3.3.6.2.4 Carrot .......................................................................................... 77
iv
3.3.6.2.5 Lettuce ........................................................................................ 78
3.4 Key findings in UWS pollination study ...................................................... 78
3.5 Greenhouse and field crop pollination studies ............................................ 79
3.5.1 Crop layout and pollinator setup .......................................................... 79
3.5.1.1 Materials and methods .................................................................. 79
3.5.2 Pollinator behaviour ............................................................................. 82
3.5.2.1 Materials and methods .................................................................. 83
3.5.2.2 Results ........................................................................................... 84
3.6 Key findings in greenhouse and field studies .............................................. 92
3.7 Discussion ................................................................................................... 93
3.8 Key findings ................................................................................................ 95
CHAPTER 4 - Phylogenetic placement of Au. australis within the genus
Austroplebeia ............................................................................................................. 96
4.1 Introduction ................................................................................................. 96
4.1.1 Collection of specimens ....................................................................... 99
4.1.1.1 Materials and methods .................................................................. 99
4.1.2 Head width and colouration analysis ................................................. 102
4.1.2.1 Materials and methods ................................................................ 103
4.1.2.2 Results ......................................................................................... 104
4.1.3 Au. cincta morphology ....................................................................... 110
4.1.3.1 Materials and methods ................................................................ 110
4.1.3.2 Results ......................................................................................... 110
4.1.4 Drone morphology ............................................................................. 111
4.1.4.1 Materials and methods ................................................................ 112
4.1.4.2 Results ......................................................................................... 118
4.1.5 Geometric morphometric wing analysis ............................................ 122
4.1.5.1 Materials and methods ................................................................ 123
4.1.5.2 Results ......................................................................................... 124
4.1.6 Molecular analysis ............................................................................. 129
4.1.6.1 Materials and methods ................................................................ 129
4.1.6.1.1 DNA extraction, amplification and sequencing ........................ 129
4.1.6.1.2 Sequence alignment .................................................................. 133
4.1.6.1.3 Phylogenetic methods and parsimony analysis ........................ 134
v
4.1.6.2 Results ......................................................................................... 135
4.2 Discussion and key findings ...................................................................... 142
4.3 Key findings .............................................................................................. 145
CHAPTER 5 - Ontogeny and longevity ............................................................... 146
5.1 Introduction ............................................................................................... 146
5.1.1 Ontogeny ............................................................................................ 148
5.1.1.1 Materials and methods ................................................................ 148
5.1.1.2 Results ......................................................................................... 151
5.1.2 Longevity and life table ..................................................................... 151
5.1.2.1 Materials and methods ................................................................ 151
5.1.2.1.1  ................................... 152
5.1.2.1.2  ................................................... 153
5.1.2.2 Results ......................................................................................... 154
5.1.2.2.1  ................................... 154
5.1.2.2.2  ................................................... 157
5.2 Discussion ................................................................................................. 157
5.3 Key findings .............................................................................................. 162
CHAPTER 6 - Colony dynamics and forager behaviour ................................... 163
6.1 Climatic factors influencing flight activity ............................................... 163
6.1.1 Introduction ........................................................................................ 163
6.1.2 Methods .............................................................................................. 164
6.1.3 Results ................................................................................................ 165
6.1.4 Discussion .......................................................................................... 167
6.2 Nest temperature and colony dynamics ..................................................... 169
6.2.1 Introduction ........................................................................................ 169
6.2.2 Materials and methods ....................................................................... 175
6.2.3 Results ................................................................................................ 177
6.2.4 Discussion .......................................................................................... 187
6.3 Paralleling study on foraging behaviour ................................................... 191
6.3.1 Introduction ........................................................................................ 191
6.3.1.1 Study background ....................................................................... 192
vi
6.3.2 Materials and methods ....................................................................... 195
6.3.2.1 Enclosure set up .......................................................................... 195
6.3.2.2 Colony orientation ...................................................................... 196
6.3.2.3 Preliminary observations ............................................................ 197
6.3.2.4 Forager observations ................................................................... 200
6.3.2.5 Digital imaging ........................................................................... 200
6.3.3 Results ................................................................................................ 201
6.3.3.1 Worker bee losses ....................................................................... 201
6.3.3.2 Hive entrance activity ................................................................. 201
6.3.3.3 Forager behaviour ....................................................................... 203
6.3.3.4 Resource collection ..................................................................... 205
6.3.4 Discussion .......................................................................................... 207
6.3.4.1 Hive entrance activity ................................................................. 207
6.3.4.2 Forager behaviour ....................................................................... 209
6.4 Nest density, distribution and characteristics ............................................ 211
6.4.1 Introduction ........................................................................................ 211
6.4.1.1 Australian stingless bee habitat................................................... 212
6.4.2 General materials and methods .......................................................... 215
6.4.3 Nest density and nest sampling within the focus study site ............... 219
6.4.3.1 Materials and methods ................................................................ 219
6.4.3.2 Results ......................................................................................... 223
6.4.4 Nest distribution and nest characteristics ........................................... 225
6.4.4.1 Materials and methods ................................................................ 225
6.4.4.2 Results ......................................................................................... 227
6.4.5 Nest characteristics............................................................................. 228
6.4.5.1 Materials and methods ................................................................ 228
6.4.5.2 Results ......................................................................................... 233
6.4.6 Discussion .......................................................................................... 236
6.5 Estimating brood population size .............................................................. 244
6.5.1 Introduction ........................................................................................ 244
6.5.2 Method ............................................................................................... 245
6.5.3 Results and discussion ....................................................................... 246
6.6 Overall discussion ..................................................................................... 246
6.6.1 Current distribution of Au. australis .................................................. 249
6.7 Key findings .............................................................................................. 253
vii
CHAPTER 7 - General discussion ........................................................................ 254
7.1 Introduction ............................................................................................... 254
7.2 Au. australis and its environment .............................................................. 255
7.3 Behavioural adaptations in Au. australis ................................................... 256
7.4 Phylogeny of species within the genus Austroplebeia .............................. 258
7.5 Possible effects of climate change on Au. australis distribution ............... 258
7.6 Implications of my findings for industry ................................................... 262
7.6.1 Potential pollinator management........................................................ 262
7.6.2 Au. australis colony management and industry development ........... 263
7.7 Implementation of my research findings for stingless bee conservation .. 265
7.8 A practical research output from my studies ............................................. 267
7.9 Recommendations for further research ..................................................... 268
7.10 Final conclusion ........................................................................................ 270
References ............................................................................................................... 272
Appendices .............................................................................................................. 307
viii
List of Figures
1.1 Typical Trigona horizontal brood structure (T. carbonaria). .............................. 18
1.2 Typical, healthy Austroplebeia brood cluster (Au. australis). ............................. 20
1.3 Au. australis queen, easily identified on the brood cluster. ................................. 25
2.1 Increase in bee keepers and their nests between 1998 / 99 and 2010. ................. 37
2.2 Species owned by bee keepers. ............................................................................ 37
2.3 Number of colonies owned by bee keepers.......................................................... 38
2.4 Bee keepers residing in NSW and Qld in 1998 / 99 and 2010. ............................ 39
2.5 Areas where hives were located. .......................................................................... 40
2.6 Number of years of stingless beekeeping experience of respondents. ................. 40
2.7 Number of bee keepers producing honey and total annual honey production. .... 41
2.8 Number of bee keepers participating in hive propagation. .................................. 42
2.9 Number of hives propagated. ............................................................................... 42
3.1 External entrance tube to a hive housed in the bee shed. ..................................... 57
3.2 Hive entrances protruding from the bee shed. Entrances were marked with
different colours to aid in colony orientation. ............................................................ 58
3.3 Feeder-float inside hive and float within an external feeder. ............................... 59
3.4 OP connected between Au. australis hive and external entrance. ........................ 60
3.5 Layout of vegetable seed plants in each greenhouse chamber at UWS. .............. 64
3.6 Au. australis forager walking over the celery flowers. ........................................ 68
ix
3.7 Pollen grains attached to the body hair of Au. australis nectar forager and on a
celery flower stigma. .................................................................................................. 69
3.8 Number of Au. australis workers entering the hive in and those working in the
crop over two consecutive 2 min periods, during the UWS trial period. ................... 71
3.9 Number of active Au. australis and its relationship to light intensity in the UWS
greenhouse.................................................................................................................. 72
3.10 Celery flowers showing primary and secondary umbels, as well as pedicels. ... 74
3.11 Developing fruit in the male sterile flowers of the cross-pollinated female line
of carrot. ..................................................................................................................... 78
3.12 Hives set up on steel platforms attached to uprights in greenhouse. ................. 80
3.13 OATH boxes attached to star pickets and positioned on the west side of the field
crops. .......................................................................................................................... 82
3.14 Entrance activity of worker and drone bees during the 4 h video sessions which
monitored a single Au australis hive entrance, RZA greenhouse site. ...................... 86
3.15 Ambient temperature compared to in-hive temperatures of heated and unheated
hives in the RZA commercial greenhouse, over 24 h. ............................................... 88
3.16 Hive entrance activity compared to in-hive and ambient temperatures, within the
heated hives and the unheated hive in the RZA greenhouse. ..................................... 89
3.17 Entrance activity correlated with in-hive and ambient temperatures. On a cold,
sunny day with delayed in-hive temperature increases and on a warm, sunny day,
with steadily increasing in-hive temperatures. Mean light intensity for the two days.
.................................................................................................................................... 90
3.18 Honey bee hive entrance activity. ...................................................................... 91
4.1 Locations of holotype specimen collection, between 1898 and 1935. ............... 100
4.2 Distribution of collected Austroplebeia specimens............................................ 101
x
4.3 Maximum head width measurements, used in scoring calculations. ................. 103
4.4 Scatter graph showing the clustering of the Austroplebeia groups in relation to
head width vs. thorax colour. ................................................................................... 107
4.5  ................... 108
4.6 Scatter graph showing the grouping of 14 Austroplebeia nests from Duaringa,
Qld, in relation to head width vs. thorax colour. ...................................................... 109
4.7 Austroplebeia groups.
.................................................................................................................................. 111
4.8  ................................................. 111
4.9 Ventral view of dehydrated whole bee. Specimens were sputter coated with gold.
.................................................................................................................................. 113
4.10 KOH-treated drone abdomen showing ventral (tegumen) surface; dissected
metasomal segments and apical segments and genitalia of an Austroplebeia drone.
.................................................................................................................................. 114
4.11 Sternal segments and tegumen segments mounted in anatomical order. ......... 115
4.12 Drone genitalia and sternal segment, S6 showing some structural measurements.
.................................................................................................................................. 116
4.13 SEM images of intact metasomal segments S7 and T7 were uninformative due
to mounting problems. ............................................................................................. 116
4.14 
edges of S7, and laser-dissected S7 segment, outlined to show tissue edges. ......... 117
4.15 Drone metasomal apex with genitalia in situ. .................................................. 119
4.16 Drone genitalia showing distortion associated with eversion. ......................... 119
4.17 Metasomal segment morphology and hair patterns on S4 and S5 were found to
be the same for all groups. ....................................................................................... 120
xi
4.18 
produce conflicting data. .......................................................................................... 120
4.19 Curved gonostylus of drones collected from WA and Cobourg Peninsula NT
 .................................................. 121
4.20 
large degree of gonostylus tip curvature. Compared to the straight gonostylus of the
 ............................................ 121
4.21 Pinned drone specimens showing intact gonostyli. Note the curved angle of the
'curved' group and the straight tip of the 'symei' group. ........................................... 122
4.22 Landmarks on wings used in geometric morphometric analysis. .................... 124
4.23 Principal component analysis of the six Austroplebeia groups with Cartesian
coordinates of each landmark after alignment. ........................................................ 125
4.24 Dendogram of morphological proximity of the Austroplebeia groups,
constructed using neighbour-joining methodology, based on the Mahalanobis square
distances between the centroids of the groups. ........................................................ 126
4.25 Discriminant analysis of Austroplebeia groups using discriminant functions 1
and 3. ........................................................................................................................ 127
4.26 Discriminant analysis of Austroplebeia groups using discriminant functions 1
and 2. ........................................................................................................................ 128
4.27 
forward primer, resulted in a very poor quality chromatogram with multiple peaks.
.................................................................................................................................. 131
4.28 Electrophoresis time was extended from 1 hto 2.5 h 
product...................................................................................................................... 132
4.29 Gel bands prior to cutting.. ............................................................................... 133
4.30 Phylogenetic reconstruction of dataset by Neighbor-Joining. ......................... 136
xii
4.31 Neighbor-Joining phylogenetic tree constructed from typical sequences for the
'symei', 'intermediate', 'australis' and 'striped' groups within the genus Austroplebeia.
.................................................................................................................................. 141
5.1 Mother colony with OPs attached to house brood grafts. .................................. 149
5.2 Tooth pick positioned on the leading edge of the brood cluster. ....................... 150
5.3 Grafted brood cells, attached to tooth pick, in OP. ............................................ 151
5.4 Survivorship and mortality rate curves for the Au. australis 
colony, plotted against hypothetical Type II survivorship curve ............................. 156
6.1 Entrance activity compared in-hive temperatures, light intensity and relative
humidity. .................................................................................................................. 166
6.2 Schematic diagram of 'typical' stingless bee nest structures within a tree cavity,
and a T. carbonaria nest showing similar nest structures. ....................................... 172
6.3 Pollen pots stacked near hive entrance and multiple stores of honey within a hive.
.................................................................................................................................. 172
6.4 Au. australis hives, protected from severe weather elements but exposed to
ambient conditions. .................................................................................................. 175
6.5 Involucrum brood-coverage of 30% (late February) and > 90% (late April). ... 176
6.6 Ambient and hive cavity temperatures measured during early stages of study
period, from 27 December 2009 12 January 2010. ............................................... 178
6.7 Cavity and brood temperatures experienced by colonies of Au. australis over a 12
month period.. .......................................................................................................... 180
6.8 Minimum and maximum temperatures of the Au. australis brood and hive cavity
during winter, 2010. ................................................................................................. 181
6.9 Minimum and maximum brood and cavity temperatures during summer, 2010.
.................................................................................................................................. 182
xiii
6.10 Differences between daily minimum brood temperature and minimum cavity
temperature over 12 month period from December 2009 to December 2010. ........ 183
6.11 Au. australis brood and cavity temperatures, showing diurnal fluctuations
during three consecutive summer and winter days. ................................................. 184
6.12 Trends for brood growth & involucrum coverage over brood clusters, compared
to mean temperatures and trends for stored pollen and honey from December 2009 to
December 2010. ....................................................................................................... 186
6.13 T. carbonaria flight activity during UWS pollination trial. Hive entrance
activity compared to activity within the crop. .......................................................... 194
6.14 Au. australis flight activity during UWS pollination trial. Hive entrance activity
compared to activity within the crop. ....................................................................... 194
6.15 Au. symei 
orange on her wings, from the dusting of fluorescent powder. Au. australis with
obvious cream coloured markings on scutellum. T. carbonaria showing dense
thoracic hair patterns and finer, more prominent thoracic rim................................. 199
6.16 Entrance activity of T. carbonaria was higher than Au. symei or Au. australis.
.................................................................................................................................. 202
6.17 Entrance activity of T. carbonaria was significantly greater than either Au.
symei or Au. australis, even when floral resources were old and depleted. ............. 203
6.18 Percentage of time the three bee species, Au symei, Au australis and T
carbonaria, spent hovering vs. time on flowers in a 60-second observation period.
.................................................................................................................................. 204
6.19 Five T. carbonaria foragers attending a single flower. .................................... 204
6.20 T. carbonaria foragers hovering near an already heavily populated flower. ... 205
6.21 Percentage of foragers of Au. symei, Au. australis and T. carbonaria collecting
nectar and pollen resources. ..................................................................................... 206
xiv
6.22 Pollen forager collecting nectar. ...................................................................... 206
6.23 Known areas of distribution where Austroplebeia and Trigona colonies have
been reported. Tara is in the focus study site. .......................................................... 212
6.24 A single Austroplebeia guard at the nest entrance and a returning forager. .... 216
6.25 This tree is located on the roadside and is marked with the base of an aluminium
drink can, to notify council personnel of the existing nest inside. ........................... 217
6.26 Austroplebeia nest with a cerumen extension built at the entrance. ................ 217
6.27 A 5 mm entrance hole, 5 m up a tree is very difficult to spot as well as access.
.................................................................................................................................. 218
6.28 Allan Beil patiently surveying the length of a potential nesting tree. .............. 220
6.29 Sampling an easily accessed Austroplebeia nest entrance with a pooter. ........ 220
6.30 Focus study site was determined by the nest site coordinates within the areas of
density and Google 
guides to map out sites of interest within 14 ha areas. ............................................. 221
6.31 Nest Density site D15, showing the Extent of Occurrence (EO) within the Area
Sampled (AS). Pink balloons represent the nest locations. ...................................... 222
6.32 The Areas of Exclusion (AE) were removed from the AS. ............................. 223
6.33 Nest sites located within the Areas of Occupancy (AO).................................. 223
6.34 Trends in mean and standard error of nest density of entire sample data. This
lls or quadrats would need to be sampled in order to obtain
consistent data. ......................................................................................................... 225
6.35 Extent of occurrence (EO) for the surveyed site. The area of distribution
encompassed the nests within the EO. ..................................................................... 226
6.36 Rescued nests in logs. Note nest entrances. ..................................................... 229
6.37 The log was split in two and the pieces pried apart, exposing the nest inside. 230
xv
6.38 Brood was extracted with a long, thin knife blade. .......................................... 230
6.39 Brood and pollen stores relocated into the artificial hive box. ........................ 231
6.40 Honey stores rated 1 on the right side but 2 on the left.................................... 232
6.41 Relocating newly boxed colony close to original position. ............................. 232
6.42 Workers entering new hive. Note resin around the entrance to improve
orientation. ............................................................................................................... 233
6.43 Percentage of nest cavities filled with nest structures, including honey, pollen
and brood. The remaining sections were empty or occupied by empty storage pots.
.................................................................................................................................. 234
6.44 Austroplebeia nest with severely depleted honey stores and air spaces between
pots of stores. ........................................................................................................... 235
6.45 Small sections of involucrum over brood surface. ........................................... 236
6.46 Density site D15 located adjacent to an unimproved section of forest. ........... 239
6.47 Holes which have been 'marked' by Austroplebeia workers. ........................... 240
6.48 Random sampling technique. ........................................................................... 243
6.49 Adaptive sampling technique. .......................................................................... 243
6.50 Au. australis colony.
.................................................................................................................................. 245
6.51 Days / year that meet the climatic requirements for Au. australis flight activity.
.................................................................................................................................. 251
7.1 Predicted changes in foraging opportunities, between 1990, 2030 and 2070, as a
reasult of climate change. ......................................................................................... 261
7.2 Beetracker website with icons representing registered wild nests. .................... 267
xvi
List of Tables
1.1 Managed bee species for crop pollination. ............................................................. 5
1.2 Comparative description of Trigona spp. and Austroplebeia spp. ....................... 17
1.3 Developmental stages and their length of time in some Melipona species and A.
mellifera. .................................................................................................................... 26
2.1 Reasons for keeping stingless bees. ..................................................................... 39
2.2 Reported crops which benefit from stingless bee pollination services. ............... 43
3.1 Biological control agents used in the crops.......................................................... 65
3.2 Rating of Au. australis hive entrance activity within the greenhouse and field .. 84
4.1 Abbreviated comparative descriptions of currently named Austroplebeia species.
.................................................................................................................................... 97
4.2 Allocated colour grades and their colour proportions (%) based on grades
illustrated in Table 4.III............................................................................................ 105
4.3 Thorax colour grading system for identification of Austroplebeia groups within
the sampled nest, using the proportion of markings on the thorax. ......................... 106
4.4 The p values for centroids differences within the Mahalanobis analysis........... 125
4.5 Pairwise distances between the six groups, based on differences in 16S rDNA
nucleotide sequences. ............................................................................................... 135
4.6 Nests and GenBank sequences demonstrating identical 16S sequences within
each group and their geographical separation. ......................................................... 138
4.7 Autapomorphic sites within the consensus sequences for Austroplebeia. ......... 139
5.1 Summary of Au. australis cohort life tabclosed colony. .................. 155
5.2 Longevity of previously studied social bee species. .......................................... 159
xvii
6.1 Threshold and optimal ambient temperatures for flight activity of previously
studied stingless bee species. ................................................................................... 168
6.2 Species of social bee studied to assess nest regulation capabilities. .................. 173
6.3 Climate details of areas where Au. australis colonies naturally occur* and areas
to which experimental colonies had been relocated. ................................................ 174
6.4 Approximations of honey and pollen pot volumes, based on area. ................... 177
6.5 Nest density for each of the 17 Density sites, within the focus study site, as well
as site description. .................................................................................................... 224
6.6 Tree species used as nesting sites by the stingless bee species. ......................... 227
6.7 Austroplebeia nesting site within dead or living Poplar box cavities. ............... 228
6.8 Nest tree characteristics for cluster-building and comb-building stingless bee
species. ..................................................................................................................... 242
6.9 Table summarising effects of some environmental factors on Au. australis
entrance activity. ...................................................................................................... 248
xviii
List of Appendices
Appendix 1 Summary and of the responses to the 1998 and 2010 surveys. 
Appendix 2 The use of hive weights as a management tool to assess stingless bee
colony health. ..311
Appendix 3 Drone populations and possible maturity.
Appendix 4 Austroplebeia nest codes, their locations and the associated holotype for
that location. ...........319
Appendix 5 Full list of the sampled nests used for colour, HW analysis. .321
Appendix 6 Improved marking techniques for Au. australis. .
Appendix 7 Age related worker behaviour. ...
Appendix 8 Failed queen or brood disease? .326
Appendix 9 Gyne production, introduction and imprisonment. ..
Appendix 10 Brood production and overwintering. ...
Appendix 11 A follow-up survey on the Australian stingless bee industry, one
decade on. ..342
Appendix 12 Sequences produced from the 36 successfully amplified specimens.

xix
List of Figures and Tables in Appendices
Figure A-3a Proportion of drones to workers emerging in Hive 1 during the division
of labour study. 
Figure A-3b Drones congregating within the OP. 
Figure A-3c Drone at hive entrance, just prior to leaving the nest. 
Figure A-3d Single drone, easily distinguished by his cream-coloured markings,
resting on a structure outside newly relocated Au. australis colonies. 
Figure A-3e Mean sperm counts for each drone as well as the overall mean of the
samples. 
Figure A-3f Au. australis sperm compared to A. mellifera 
Figure A-3g Drone feeding himself while in captivity. 
Figure A-8a Damaged brood cells due to chill injury caused by the failure of the
heating unit. 
Figure A-8b Damaged brood from possible queen failure. 
Figure A-8c Desiccated brood cells from Au. australis colony. 
Figure A-8d Physogastric lony. 
Figure A-8e 
Au. symei colony. 
Figure A-9a Pupating queen and worker cells. 
Figure A-9b Plebeia remota royal chamber still closed and chamber opened to show
virgin queen inside. 
Figure A-9c Partially emerged gyne. Note the orange colouring of the front legs and
antennae. 
Figure A-9d Gyne inside queen cage, introduced into queenless colony. 
xx
Figure A-9e Au. australis workers gather over the brood cells and blanket it so as to
obscure for view their possible manipulation of the cells. 
Figure A-10a Empty honey pots from starved Au. australis colony. 
Figure A-10b Mean brood diameter for each treatment of the feeding regime. 
Table A-1 Summary and of the responses to the 1998 and 2010 surveys. 
Table A-4 Austroplebeia nest codes, their locations and the associated holotype for
that location. 
Table A-5 Full list of the sampled nests used for colour, HW analysis. 
Table A-7 In-hive tasks performed by the different age groups of workers within the
two Au. australis   
Unless otherwise stated, the photographs and images within this dissertation
were produced by Megan Halcroft.
xxi
Abbreviations
CT controlled temperature.
ID internal diameter of tube.
OATH Original Australian Trigona Hive.
OD outer diameter of tube.
OP observation platform.
POP provisioning and oviposition process. Provisioning of a
prepared brood cell, with brood food, and the subsequent
laying of a single egg by the queen onto the food.
RZA Rijk Zwaan Australia.
S pertaining to the sternal (ventral) segments of the metasoma,
e.g., S4, S5.
T pertaining to the tergum (dorsal) segments of the metasoma,
e.g., T6, T7.
UWS University of Western Sydney.
Glossary
Batumen made of cerumen, resin, and sometimes vegetable material.
Plates that form the walls and lining of nests. Mostly seen in
Trigona spp. and less common in Austroplebeia spp.
Brood nursery for rearing young.
Callow newly emerged adult, up to one week old and distinguishable
by its lighter body colour.
Cerumen wax and resin mix.
Cloudy day a cloudy day is recorded when the mean of the 09:00 and
15:00 cloud observations is greater than or equal to 6 oktas
(see below).
Endotherm an animal that is dependent on, or capable of, the internal
generation of heat. Often contrasted with ectotherm.
Ectotherm an animal that is dependent on external sources for body heat.
Often contrasted with endotherm. Compare with poikilotherm.
xxii
Feeder-float vessel used to supplement carbohydrate source for colonies. A
full description is contained in Chapter 3, Section 3.2 .
Gyne reproductive, unmated queen. Virgin queen.
Indo-Pacific comprising the tropical waters of the Indian Ocean, the
western and central Pacific Ocean, and the seas connecting the
two in the general area of Indonesia, including Australia.
Involucrum multiple layers of cerumen around brood chamber.
Imago final developmental stage prior to emergence as an adult.
Imagoes, imagines plural.
Monandrous only mating with one male. Stingless bee species.
Neotropic the biogeographic region of the New World that stretches
southward from the Tropic of Cancer and includes southern
Mexico, Central and South America, and the West Indies.
Regions in which stingless bees are naturally found.
Oktas cloud cover observations, are measured in oktas (eighths). The
sky is visually inspected to produce an estimate of the number
of eighths of the dome of the sky covered by cloud. A
completely clear sky is recorded as zero okta, while a totally
overcast sky is 8 oktas.
Ontogeny the development of an individual from oviposition to emerging
adult.
Paleotropical comprising Africa, tropical Asia, New Guinea, and many
Pacific islands (excluding Australia and New Zealand).
Physogastric queen a mated queen.
Poikilotherm an organism that cannot regulate its body temperature except
by behavioural means such as basking or burrowing.
Dependent upon the temperature of its environment.
Pollen trays a vessel for providing protein supplement to colonies. A full
description is contained in Chapter 3, Section 3.2 .
Polyandrous referring to a mating system in which a female mates with
several males during one breeding season.
Potted pollen Au. australis collected pollen, stored by bees in cerumen pot.
A full description is contained in Chapter 3, Section 3.2 .
Queenless a queenless colony that does not have a functional queen. This
includes a colony with no physogastric queen, one with only a
virgin queen or one with a queen which is not laying viable
eggs.
xxiii
Queen-right a colony is queen-right if it contains a physogastric queen
which has been accepted by the colony and is laying viable
eggs.
Requeen the introduction of a queen cell or live queen in an effort to
complete the colony.
Trophallaxis the mutual exchange of regurgitated liquids between adult
social insects or between them and their larvae.
xxiv
Thesis summary
This research was conducted with a view to increasing knowledge about a little
known Australian stingless bee, Austroplebeia australis Friese (Hymenoptera:
Apidae, Meliponinae). Bees are known to be superior pollinators due to the fact that
they actively collect pollen and nectar in large amounts for the purpose of raising
their young. There has been limited research into the utilisation of alternative
pollinators to the European honey bee (Apis mellifera) in Australian horticultural
crops. Au. australis is a eusocial bee that can be managed in transportable artificial
hives and has, therefore, the potential to be managed as a crop pollinator. During this
project I investigated aspects of the biology, behaviour and phylogeny of
Au. australis, with a view to better understanding this species, as well as to determine
whether it could be utilised as a greenhouse and / or field crop pollinator.
To develop a context for my studies, an online survey was conducted to assess the
current status of the Australian stingless bee industry and its recent development.
This was a follow up survey conducted one decade after the first study, by Heard and
Dollin in 1998. It showed that the Australian industry had grown over the past ten
years but is still underdeveloped. The majority of stingless bee keepers are hobbyists,
who own only one colony. There is a high demand for Australian stingless bee
colonies and their honey, but with less than 250 bee keepers currently propagating
colonies, and many of them on a small scale, it is difficult to meet this demand.
Pollination services are provided by a small number of stingless bee keepers;
however, there is an urgent need for research into this as well as many other aspects
of stingless beekeeping in Australia. Public education is also needed to increase
community awareness and, ultimately, to improve efforts in conserving these native
species.
Pollination studies were initially conducted using Au. australis in vegetable seed
crops within an experimental insectary and greenhouse, at the University of Western
Sydney, NSW. Au. australis colonies acclimated to the greenhouse enclosures and
visited crops, with seed set in celery being significantly higher (76.25%) than the
control (no pollinator) (0.02%). As a result, further investigations were undertaken
with Au. australis in commercial field and greenhouse vegetable seed crops, located
xxv
at Musk, Vic. These studies, were less successful, due to the cool climatic conditions
experienced in this area (Central Victoria), even during summer. These latter results
suggest that Au. australis colonies do not adapt well to cool climate areas and low
ambient and in-hive temperatures negatively influence their flight activity.
The above hypothesis was tested in a subsequent investigation, which assessed the
influence of abiotic climatic factors on flight activity of Au. australis. The entrance
activity of five colonies was monitored and compared to the prevailing weather
factors: ambient temperature, in-hive temperature, relative humidity, light intensity
and cloud cover. Entrance activity commenced when in-hive temperatures were


not influence flight activity as much as temperature, and relative humidity had a
negative impact. Heavy cloud cover and rain periods resulted in Au. australis
foragers returning to their colonies and ceasing flight activity.
It was unclear whether Au. australis was able to thermoregulate its nest, as reported
in some other bee species. Au. australis colonies naturally occur in northern regions
of Australia but some populations experience extreme temperature ranges, including
sub-zero temperatures. Thermoregulation enables social insects to produce offspring
throughout the year, thus giving them an advantage over solitary insects. To
investigate this research question, the in-hive temperatures and brood temperatures of
six Au. australis colonies were monitored over 12 months. Brood production and
development was assessed, along with visible signs of pre-winter preparation. This
study showed that colonies of Au. australis demonstrated ectothermic characteristics
and were unable to warm the brood chamber when temperatures were < 15°C. Brood
production, however, continued throughout the cold seasons and developing
offspring survived and emerged even after exposure to prolonged periods of sub-zero
temperatures, as well as to temperatures > 37°C. Au. australis, thus, shows a
remarkable ability to survive temperature extremes.
Adaptation to unique environmental characteristics may enable one species to
survive over another. To investigate this, comparative studies on the foraging
behaviour of three species of Australian stingless bee were conducted. The aim was
xxvi
to better understand the mechanisms by which Au. australis survives during
prolonged periods of drought, when floral resources become scarce. The foraging
behaviours of Au. symei, Au. australis and Trigona carbonaria were observed within
the confines of an experimental greenhouse, and then compared. Fresh cut floral

behaviour within natural floral structures. Both Austroplebeia species were
significantly more efficient than T. carbonaria. Hovering time was significantly
lower in Austroplebeia (~ 8%) compared to Trigona carbonaria (> 30%). The
number of flowers visited by Austroplebeia foragers, during a set time period, was
greater than for T. carbonaria. Austroplebeia foragers demonstrated solitary foraging
strategies, while T. carbonaria foraged in groups. There were no significant
differences between the two Austroplebeia species. The different foraging strategies
demonstrated by the two different stingless bee genera illustrate adaptive behaviours
that may have evolved under different environmental conditions.
Until the current study, little was known about the biology and lifecycle of
Au. australis. The ontogenic times for developing offspring, as well as the longevity
of adults, drive the overall life cycle of a eusocial colony. The developmental times
for brood within cluster-building species, such as Au. australis, were yet to be
reported. In order to observe developing Au. australis brood cells for investigations,
it was necessary to separate sections of brood from the main brood cluster. A


enabling observation of their development. The mean ontogenic time for
Au. australis workers, maintained at temperatures ~ 26°C, was 55 days, similar to
that reported for other stingless bee species. This extended time, compared to A.
mellifera, may be linked to lower brood incubation temperatures.
In order to determine the longevity of Au. australis workers, bees within a single

being exposed
to a high  
A. mellifera.
The mean maximum longevity of workers was used to determine the longevity of
xxvii
cohorts within the foraging colonies. Au. australis workers are small (~ 4mm) and
cannot be marked with an individual-identification colour scheme. As a result,
cohorts of workers within each colony were colour marked and the longest lived bee
within each cohort was recorded as the last individual remaining. The maximum
longevity of each cohort was used to estimate the mean maximum longevity of all of
the cohorts, within the six foraging colonies. The mean maximum longevity of
g

200 days and the longest lived bee reached 289 days old. The combination of long
ontogenic period and extended longevity indicates that individual bees within
colonies of Au. australis may live as long as seven months.
Natural nest density of social bees is dictated by the availability of both nest
substrate and floral resources. A focus survey was conducted to ascertain the nest
density of Au. australis within an area of south-east Queensland that was known to
support substantial bee populations. It was found that nest densities, which ranged
from 0.1 to 3.0 nests / ha (mean 0.6 nests / ha) were low, compared to some others
reported social bee species. The nest characteristics of Au. australis showed that this
species prefers narrow cavities within dead trees but does not discriminate between
tree taxa. Brood populations range from 2,000 to 13,000 (mean 5,000) in natural
nests, within its native range. Colonies under threat of destruction can be easily
extracted from their natural cavity and adapt well to artificial hive boxes. Au.
australis are well adapted to the arid environment of south-east Qld.
During my studies, it became apparent that the current descriptions for the species
within the genus Austroplebeia are inadequate as a tool for the identification of
specimens in either the field or the laboratory. As a result, a triangulated approach
was undertaken in an attempt to better delimit morphologically identified groups
within Austroplebeia. First, morphological data, based on worker bee colour and size
were analysed. Drones collected from nests representing morphologically similar
groups were dissected and their genitalia were imaged using scanning electron
microscopy. Next, data for the geometric morphometric analysis of worker wing
venations were obtained. Finally, molecular analysis, using mitochondrial rDNA
segment 16S, was conducted on workers from representative nests for each group
xxviii
which displayed morphological similarities. Data from the four datasets were
compared, resulting in the separation of two distinct species, with a large unresolved
species complex.
My research results contribute to the previously limited pool of knowledge on
Austroplebeia spp. in general and Au. australis in particular. Its conservative
recruitment of foragers for low level floral resources, combined with the efficiency
with which foragers harvest these resources, reduces worker mortality within a
colony. In the presence of abundant floral resources, forager recruitment is high and
resource harvesting and storage is likely to be greater than the energy expenditure of
the colony as a whole. Low energy expenditure within the colony, through
thermoconformity, reduced nest architecture and efficient foraging strategies enable
colonies to conserve precious resources. This combination of behaviours has enabled
this species to survive, in harsh environmental conditions.
1
CHAPTER 1
Literature review, scope and aims of thesis
1.1 General introduction
Worldwide, over 200,000 plant species depend on more than 100,000 species of
pollinators. The importance of these pollinators, with regard to agricultural outputs,
is finally gaining recognition (Loughlin 2008). In 2000, pollination services provided
by the European honey bee were estimated to be worth $A1.7 billion / yr, for the 35
most responsive crops in Australia (Gordon & Davis 2003). However, if the flow-on
effects to all Australian horticultural and agricultural industries are taken into
account, this figure expands to $4 6 billion / yr (Thomson 2007). Unfortunately,
globally, the honey bee industry is in crisis, due to pest and disease-induced colony
collapse. reliance on this industry demonstrates the importance of
exploration into and utilization of alternative insect pollinators, such as native bees.
Pollination is essential for fruit and seed production, with most horticultural and
many agricultural crops being dependent upon insect vectors. Plants producing
scented and coloured floral structures attract insect pollinators, thus enabling or
enhancing pollination (Free 1970). Incidental pollination by nectar feeding insects
can add to overall pollination; however, pollen collecting insects, such as bees,
contribute the highest quality pollination service. They are also capable of carrying
pollen from one flower to another, thus facilitating cross-pollination. While insect
visitation may not be essential for seed set in some crops, the presence of large
numbers of pollinators can improve yield and quality as well as produce earlier and
more uniform crops (Free 1970; McGregor 1976; Delaplane & Mayer 2000). Many
vegetable seed crops are hybrid cultivars and require insect pollinators for effective
cross-pollination and seed set (Banga & Banga 1998).
Bees belong to in the order Hymenoptera and make up a large component of the
superfamily Apoidea (Michener 2000). They depend on pollen as a protein source to
raise their young (brood) and the evolutionary development of dense, branched,
electrostatic hairs increases pollen attraction and facilitate its collection while bees
2
forage on flowers (Thorp 1979; Michener 2000). These features make bees, by far,
the best pollinators of cultivated crops (Free 1970; Delaplane & Mayer 2000;
Michener 2000). As foragers move among the flowers, collecting pollen and nectar,
pollen grains are attracted to the hairs and transferred to the stigmatic surface. This
facilitates self-pollination or cross-pollination within the crop. There are over 18,000
described species of bee worldwide (Michener 2000) but the European honey bee,
Apis mellifera Linnaeus (Hymenoptera: Apidae), is the most well understood and
most important bee for pollination of cultivated crops (Delaplane & Mayer 2000).
The native range of A. mellifera extends from northern Europe, down through the
Middle East and throughout the arable regions of Africa (Ruttner 1988). As
European colonists spread throughout the world, beginning in the early 17
th
century,
so too did the honey bee (Delaplane & Mayer 2000). Since the successful
introduction of honey bees into Australia in 1822 (Hopkins 1886) feral populations
have spread. Oldroyd et al. (1994; 1997) reports up to 150 colonies per km
2
in some
nectar-rich native forests. All of these feral colonies contain foragers that collect
nectar and pollen from a variety of floral species, including native and agricultural
plants, pollinating as they visit. The pollination services these bees provide are free,
abundant and available to any farmer in the area.
Bradbear (1988) published a comprehensive document listing the ten major honey
bee pests and diseases and their distribution throughout the world. This list was
updated by Ellis and Munn (2005) and of the named threats, only the bee louse,
Braula coeca Nitzsch (Diptera: Braulidae), and the three parasitic mites: tracheal
mite, Acarapis woodi Rennie (Acari:Tarsonemidae), varroa mite, Varroa destructor
Anderson and Trueman (Acari: Paraditidae), and tropilaelaps mite, Tropilaelaps
clareae Delfinado and Baker (Acari: Laelapidae), had not reached Australian shores.
Ellis and Munn (2005) broadened their studies to include viruses that affect honey
bees and of the 12 viral diseases suffered by A. mellifera only three were absent from
Australia. The loss of valuable pollinators, including feral populations, through
increasing pressure from introduced pests and diseases is predicted to have
devastating effects on world food supplies and a major impact on Australian
agricultural and horticultural industries (Allen-Wardell et al. 1998; Anderson &
Lawrence 2007; RIRDC 2007). For example, African small hive beetle (Aethina
3
tumida Murray (Coleoptera: Nitidulidae) infestations caused the loss of > 4,500
managed colonies in New South Wales (NSW) between 2002 and 2006 (Rhodes &
McCorkell 2007) and > 10,000 colonies in Queensland (Qld) in 2009 and 2010
(Williams 2011).
The most immediate threat comes from the varroa mite, as it occurs in all of
nation
services (van Engelsdorp et al. 2008). Anderson and Lawrence (2007) consider that it
is only a matter of time before this destructive pest breaches Australian quarantine
borders. This is especially concerning with the recent incursions of Asian honey bees
(Apis cerana Fabricius (Hymenoptera: Apidae)), a natural host of varroa mite, into
Australia (PIF 2012). Continued reliance on honey bees for crop pollination is risky
when the threat of population decline is imminent (Somerville 2005; Holden 2006).
Currently, Australian honey bee keepers have the capacity to manage approximately
500,000 colonies. Only 100,000 of these are used for paid pollination services, as
feral honey bee populations freely supply services to many growers (RIRDC 2009).
Demand for pollination services in the booming almond industry will see
approximately 100,000 managed honey bees being migrated south to the almond
orchards (RIRDC 2012). It has been estimated that if varroa mite kills all the feral
honey bee colonies, which would take less than four years from its initial incursion
(Anderson & Lawrence 2007), the demand for managed honey bee colonies would
increase to 480,000. This number would be required at the beginning of the season,
i.e., September, and would peak at 750,000. This is beyond the current capacity of
the Australian honey bee keepers (RIRDC 2010a). To provide essential pollination
services for the horticultural and agricultural industries, one option is that other
species of insect pollinators be recruited.
1.2 Alternative bee pollinators to A. mellifera
Animal pollinators contribute to the production of one-third of the world dietary
input (McGregor 1976; Richards 1993; Klein et al. 2007), including nuts, fruit, seed
oil, vegetables, crop seed, meats and dairy (Losey & Vaughan 2006), with non-Apis
bee species being included in this group. The management and utilization of
4
alternative pollinators for crop pollination is practised worldwide (reviewed by
Bohart 1972; Heard 1999; Ruz 2002). This is, however, not the case in Australia.
Some bee species are intentionally introduced into regions for their pollination
services, while others are introduced accidentally, through the transport of building
materials, utilised as nesting media by solitary bee species (Bohart 1972). Many non-
Apis species are effective pollinators but providing these bees in large quantities is
extremely difficult (Bohart 1957). Except for bumblebees (described later in this
chapter), the most commercially reared alternative pollinators are solitary bees that
can nest in man-made materials, thus enabling mass rearing (Bosch & Kemp 2004).
A list of some managed bee species is contained in Table 1.1.
The alkali bee, Nomia melanderia Cockerell (Halictidae), is a solitary bee which
nests gregariously in the ground and is utilised in alfalfa (lucerne), Medicago sativa
Linnaeus (Fabales: Fabaceae), pollination (Bohart 1972; Richards 1993). Although
native to North America its assisted spread by man has had varying degrees of
success. It is difficult to transfer nesting material (blocks of soil) any great distance
and success is limited by the new conditions presented to the over-wintering larvae.
The alkali bee has several predators and parasites and its management can be time
consuming and expensive. It is, however, a very effective pollinator of alfalfa crops
that occur nearby nesting sites (Bohart 1972).
The alfalfa leaf-cutter bee, Megachile rotundata Fabricius (Megachilidae), is a more
manageable solitary bee and nests gregariously in naturally occurring and man-made
cavities. It can be encouraged to nest in large drilled boards, which provide 2,000
nests / board (Bohart 1972). Management varies from provision of drilled boards
only to cocoon harvesting, incubation and storage. This also includes sanitization of
nesting material. Management methods have been devised to reduce pests and
predators as well as to manipulate bee emergence times through incubation (Bohart
1972). Attempts to introduce this bee into Australia have been minimally successful,
although further research is planned by the Rural Industries Research and
Development Corporation (RIRDC) and Lucerne Australia (Clarke 2008; D.
Anderson, CSIRO, pers. comm., 2008).
5
Table 1.1 Managed bee species used in crop pollination.
A. mellifera
N. melanderi
M. rotundata
Osmia spp.
Bombus spp.
Meliponini spp.
Common name
Honey bee
Alkali bee
Alfalfa leafcutter
bee
Mason, Orchard bee
Bumble bee
Stingless bee
Social / solitary
Social
Solitary
Solitary
Solitary
Social
Social
Nest formation
Perennial colony
Ground /
aggregations
Borer holes or
manmade nests
Borer holes or
manmade nests
Annual colony
Perennial colony
Diapause period
None
Winter mid-
Summer
Autumn - Spring
Autumn - Spring
Winter Spring
None
No workers /
aggregation or
colony
Up to 50,000
Up to 2,000 / m
2
Up to 2,000 /
nesting board
750-1000 / nesting
board
100 to 400
500-10,000
Pest/parasite
SHB*, mites, wax
moth, bee louse,
Nosema
Parasitic wasps
Parasitic wasps
Mites
Mites, beetles, flies,
nematodes
Syrphid &
phorid flies
Diseases
Chalk and sac
brood, AFB*,
EFB*, numerous
viruses
Brood virus
Chalk brood
Unknown
Chalk brood
Unknown
Native country
Europe
USA
Worldwide
Japan, USA
Europe, USA
Neotropics
Commercial
introductions
World wide
New Zealand
Worldwide
Northern
hemisphere & New
Zealand
Worldwide except
Africa & Australian
mainland
Neotropics
Data compiled from Holm 1966; Wille 1983; Greer 1999; Richards & Kevan 2002; Spiewok & Neumann 2006; Huntzinger et al. 2008; NBII 2008
*SHB: Small hive beetle, AFB: American foul brood, EFB: European foul brood
6
Osmia spp. Panzer (Megachilidae) pollinate apple, Malus spp. Miller (Rosales:
Rosaceae); pear, Pyrus spp. Linnaeus (Rosales: Rosaceae); almond, Prunus dulcis
Miller (Rosales: Rosaceae); plum and cherry, Prunus spp. Linnaeus; as well as
blueberry and cranberry, Vaccinium spp. Linnaeus (Ericales: Ericaceae) blossoms in
North America, Japan and Spain (Bohart 1972; Richards 1993; Stubbs & Drummond
1997; Greer 1999; Vicens & Bosch 2000). The bees are managed and maintained in
open-ended boxes filled with hollow reeds or straws. These are distributed
throughout the orchard to maximize pollination and fruit set (Richards 1993).
Bombus spp. Latreille (Apidae) have been domesticated and utilised in numerous
crops for many years (Velthuis and van Doorn 2007). In 1985 bumblebees were
found to be effective buzz pollinators of greenhouse tomato, Solanum lycopersicon
Miller (Solanales: Solanaceae) crops. Since then, Bombus spp. have been introduced
into other solanaceous crops with good success (Velthuis & van Doorn 2007).
Bombus spp. are also utilized as pollinators of stock fodder crops in regions where
large populations naturally occur (Holm 1966). Bombus have a large number of pests
including parasitic nematodes, fly maggots, hive beetles and mammals (Holm 1966;
Macfarlane et al. 1995; Antonelli & Glass 2004; Spiewok & Neumann 2006).
Stingless bees (Apidae) are important pollinators of many tropical crops including:
choko, Sechium edule (Jacquin) Swartz (Violales: Cucurbitaceae); coconut, Cocos
nucifera Linnaeus (Arecales: Arecaceae); mango, Mangifera indica Linnaeus
(Sapindales: Anacardiaceae); carambola, Averrhoa carambola Linnaeus (Geraniales:
Oxalidaceae) (Heard 1999); coffee, Coffea spp. Linnaeus (Rubiales: Rubiaceae)
(Klein et al. 2003) and macadamia nut, Macadamia integrifolia Maiden & Betche
(Proteales: Proteaceae) (Heard 1993). Coffee plantations located near tropical forests
benefit from visiting stingless bees, resulting in higher crop yields (Klein 2009).
Stingless bees have also been successfully introduced into greenhouse enclosures for
pollination of crops such as strawberries, Fragaria spp. Linnaeus (Rosales:
Rosaceae) (Kakutani et al. 1993; Slaa et al. 2000; Malagodi-Braga & Kleinert 2004;
Roselino et al. 2009), tomatoes (Cauich et al. 2004; Del Sarto et al. 2005) and
capsicum, Capsicum spp. Linnaeus (Solanales: Solanaceae) (Cruz et al. 2005; Cauich
et al. 2006; Greco et al. 2011). They have a variety of natural enemies, depending
7
upon their native ranges. More detailed information on all aspects of stingless bees
will be discussed later in this chapter.
In Australia, investigation of alternative pollinators to A. mellifera has been limited.
Blue banded bees, Amegilla spp. Friese (Hymenoptera: Apidae), and carpenter bees,
Xylocopa spp. Latreille (Hymenoptera: Apidae), have been investigated for their

to introducing the invasive bumblebee, Bombus terrestris Linnaeus (Hymenoptera:
Apidae), (Hingston & McQuillan 1999). Buzz pollinators are able to sonicate the
poricidal anthers of tomatoes, promoting the release of pollen, and resulting in
increased fruit quality and yield (Hogendoorn 2000; Bell et al. 2006; Hogendoorn et
al. 2007). Problems are still being encountered with regard to perfecting nesting
substrates (K. Hogendoorn, pers. comm., 2010). Stingless bees have been used in
macadamia nut crops with good results (Heard 1994).
1.3 Stingless Bees
There is much needed change regarding classification of the genus / subgenus group
name of Trigona (Heterotrigona), which includes a portion of the native Australian
Meliponini. At present, according to Michener (1990) species of Trigona that occur
in the Indoaustralian regions are of the subgenus Heterotrigona. Recent molecular
studies, and also morphology, suggest this taxonomic classification is incorrect and
that Australian species previously named Trigona (subgenus Heterotrigona) should
be changed to the genus Tetragonula Moure, 1961 (Rasmussen and Cameron, 2007;
Rasmussen and Cameron, 2010). There are many species and subgenera to consider
in Asia and Australia, with 15 species in Australian comprising two genera. I do not,
therefore, believe it is my place to make such changes when referring to this genus.
As such, I have chosen to abide by the rules of nomenclature set down by the
International Commission on Zoological Nomenclature (ICZN 1999) and refer to the
group name as Trigona (Heterotrigona) in this dissertation.
The greatest diversity of stingless bees is found in the Neotropical regions of South
America, with 412 described species (Camargo & Pedro 2012). Subtropical and
tropical regions in Africa, Madagascar, Asia, New Guinea and Australia are also
home to many species of stingless bee (Michener 1979). This compares with six to
8
eight species of Apis Linnaeus (Hymenoptera: Apidae) worldwide (Michener 2000).
All stingless bees are in the order Hymenoptera and family Apidae
1
. They are then
classified into the subfamily Meliponinae which is comprised of two tribes:
Meliponini and Trigonini (Wille 1979). These tribes are characterised by
morphological differences, and reinforced by some biological and nesting
characteristics. Meliponini are more robust in body shape and size than Trigonini and
have shorter wings and more dense pubescence (Wille 1983; Michener 1990). Most
stingless bees are smaller than honey bees and their wings are reduced in size and
venation (Winston & Michener 1977). Stingless bees have a vestigial, functionless
sting (Rayment 1935; Wille 1983; Michener 2000); however, they have substantial
mandibles, connected to comparatively larger muscles than in Apis (Sakagami 1982),
that are effectively used for defence. There is a penicillum (long, stiff bristle) on the
outer apical margin of their hind tibiae (Wille 1983) and wax glands are located on
the dorsal side of the abdomen in stingless bees (Sakagami 1982), whereas they are
ventrally located in Apis (Snodgrass 1956). The most extensive description of South

Bees (Camargo & Pedro 2012).
Stingless bees live in social, perennial colonies comprised of a single queen (except
in Melipona bicolor Lepeletier (Velthuis et al. 2006) and Melipona quadrifasciata
Lepeletier (Alves et al. 2011)), a variable number of males (drones) and hundreds to
thousands of female workers. Colony populations vary greatly in stingless bees;
Lindauer and Kerr (1960, cited in Michener 1974) estimate that M. quadrifasciata
populations are 300 to 400; Trigona capitata Smith 1,000 to 1,500 and Trigona
spinipes Fabricius from 5,000 to 180,000 workers.
Stingless bee nests contain elaborate structures made from cerumen, a mixture of
collected plant resin and wax produced by the bees (Wille & Michener 1973). The
most common brood structures are horizontal combs (Wille 1983) and these are
covered with thin sheets of involucrum, as an insulative layer against temperature
extremes (Michener 2000). Cerumen pillars support resin pots in which honey and
pollen are stored. The strength of the supports and the colour of these structures are
1
Within this dissertation, reference will be made to a large number of stingless bee species. In order to
assist the flow of text, the order and family (Hymenoptera: Apidae) will not be included with each
new species reference.
9
governed by the proportion of resin to wax. The more resin in the mixture, the
harder, darker and stronger is the structure (Michener 2000). Some Meliponini
species incorporate mud and plant debris into the nest structure (Wille 1983). Resins
have varying levels of flavonoid compounds, depending on their botanical origin,
and have been shown to possess antimicrobial properties (Burdock 1998; Kujumgiev
et al. 1999)perties remain active, as part of the
cerumen, within the nest (Velikova et al. 2000; Patricio et al. 2002). Some stingless
bee honeys possess similar flavonoid profiles to extracts of plants found in the same
geographical region as their nests (Vit & Tomás-Barberán 1998). These compounds
may aid in the preservation of honey and pollen within pots constructed of cerumen.
1.4 Meliponiculture
Meliponiculture is the practice by which bee keepers reproduce stingless bee
colonies, of various species, for profit. This profit may be in the form of honey,
cerumen, resin and nucleus colonies (Heard & Dollin 2000). Meliponiculture is
practiced by communities throughout South America, Central America and Mexico,
as well as in small areas of northern Australia (Cortopassi-Laurino et al. 2006). The
ancient Mayans considered the stingless bees worthy of a place in their religious
worship, including a god of honey in their ensemble of deities (Cortopassi-Laurino et
al. 2006). Stingless bees have been highly regarded by indigenous northern
Australians for many centuries and their hive products played an important part in
their culture (Akerman 1979; Souza et al. 2006). Today, the aboriginal people
manage bees in artificial hives and sustainably harvest products such as honey,

health food stores and restaurants. There has been an increasing awareness and

(Cortopassi-Laurino et al. 2006; Souza et al. 2006). Currently, only a few of these
enterprises are run by indigenous communities.
Stingless bee honey is used medicinally in the treatment of gastro-intestinal upsets,
ocular complaints, ulcers and wounds and coughs (Vit & Tomás-Barberán 1998; Vit
et al. 2004; Cortopassi-Laurino et al. 2006). The honey has a higher moisture content
(20 42%) (Souza et al. 2006; Persano Oddo et al. 2008) than A. mellifera honey (18
10
20%) (Bijlsma et al. 2006) and usually requires pasteurisation or refrigeration to
avoid fermentation (Cortopassi-Laurino et al. 2006; R. Zabel, pers. comm., 2006).
Moisture content and viscosity of Australian stingless bee honey is reported to be
variable (R. Zabel; T. Heard; R. Raymond, pers. comm., 2008) and is dependent on
(Vit et al. 1997).
Limited research has been conducted on Australian honeys; however, Trigona
carbonaria Smith honey has been shown to have similar composition to those of
other Meliponine honeys (Persano Oddo et al. 2008). Preliminary studies on its
antioxidant and antimicrobial activities show some promise for nutritional and
pharmaceutical uses (Irish et al. 2008; Persano Oddo et al. 2008; Boorn et al. 2010).
Worldwide, limited research has been carried out on the chemical composition of the
honey, pollen and yeasts contained within stingless bee colonies (Sommeijer et al.
1983; Fernandes-da-Silva & Serrao 2000; Cortopassi-Laurino et al. 2006).
1.4.1 Native plant and agricultural crop pollination by stingless bees
As with all members of the corbiculate Apidae family, stingless bees have
morphological adaptations which enable them to gather and safely store pollen whilst
foraging on flowers (Rayment 1935; Snodgrass 1956; Michener 2000). The
corbiculae enable bees to forage and collect large loads of pollen at a time. A small
amount of nectar is added to the pollen mix to aid in its packing (Rayment 1935).
Stingless bees also use the pollen basket to transport resin, for use in nest structures
(Rayment 1935; Patricio et al. 2002). In Austroplebeia Moure, the inner surface of
the hind tibial keirotrichiate area is broad and almost reaches the upper margin of the
tibia; this is not so for Trigona Jurine (Michener 1990; 2000).
Stingless bees perform pollination services, transferring pollen grains to the stigmas,
while foraging amongst a wide variety of native and exotic flowers (Sommeijer et al.
1983; Adams & Lawson 1993; Heard & Exley 1994; Heard 1999; Cruz et al. 2005;
Cortopassi-Laurino et al. 2006). They can be seen foraging high atop rainforest trees
and their contribution to forest biodiversity is considerable within their native ranges
(Wille 1983).
Of the 1,000 or more plant species cultivated in the tropics, approximately 250 are
compatible with stingless bee pollination (Heard 1999). Pollination services provided
11
by stingless bees help increase the yield of a number of cultivated crops (Section 1.2
). Many economically significant, cultivated crops originate from regions where
A. mellifera do not naturally occur (Heard 1999).
Stingless bees have the ability to visit and pollinate a large variety of plants, with
high floral constancy (Heard & Hendrikz 1993; Ramalho et al. 1994; Hilário et al.
2000; White et al. 2001). This, together with their low susceptibility to European
honey bee pests and diseases (Delfinado-Baker et al. 1989), makes the exploitation
of these bees an attractive agricultural and horticultural activity. Artificial hives
containing T. carbonaria or Trigona hockingsi Cockerell have been introduced into
Australian macadamia orchards, with good results (Heard 1999). The fact that
eusocial stingless bees are perennial (Wille 1983) ensures the presence of pollinators
throughout the year, while environmental conditions are favourable (Heard &
Hendrikz 1993). This is particularly important in year-round greenhouse crop
production (Amano 2004; Malagodi-Braga & Kleinert 2004). They are successful
foragers within the confines of a greenhouse (Kakutani et al. 1993; Slaa et al. 2000;
Malagodi-Braga & Kleinert 2004; Roselino et al. 2009) and, being stingless, they are
also less harmful to the humans tending the crops (Heard 1999).
Although some stingless bee species are capable of 1.5 km flight distances (Roubik
& Aluja 1983), Trigona and Austroplebeia are thought to forage only one to two
hundred metres from their nests (Dollin 2010a), to a maximum of 500 m (Bartareau
1996). This is advantageous for crop pollination, as stingless bees are more likely to
forage within the crop than venture further afield in search of more attractive floral
resources, as is the case with honey bees (Graham 1992). When located in
macadamia crops, T. carbonaria usually forage a mere 25 to 30 m from their hive
(F. Adcock, pers. comm., 2008). As a result, hive placement is important and 15 to
20 hives / ha (comthe crop,
especially when cross-pollination is required (Heard & Dollin 1998; F. Adcock, pers.
comm., 2008; T. Carter, pers. comm., 2010). The distance a stingless bee is prepared
to fly depends on how attractive the resource is (Heard 1999) and the relative size of
the bee (Michener 1974).
The Australian stingless bee pollination industry had its beginnings in the late 1980s
when it was found that yields of macadamia nut, M. integrifolia grown near remnant
12
native vegetation were noticeably higher than for those crops situated near cleared
land (Heard 1988a; Heard & Exley 1994). The main pollinators of macadamia are
honey bees and stingless bees (Vithanage & Ironside 1986) and the presence of these
insects is extremely important for maximum nut set (Wallace et al. 1996). Although
the temperature threshold for T. carbonaria (Heard &
Hendrikz 1993), resulting in shorter foraging days compared to honey bees (7 h vs.
10 h / day) (Heard & Exley 1994), Trigona are efficacious pollinators of macadamia
flowers (Heard 1994; T. Carter, pers. comm., 2009). Their small bodies are able to
make more intimate contact with the stigmas while they collect pollen (Heard 1994),
optimising pollen transfer. Heard (1987) also demonstrated that Trigona foragers
returned to hives with 100% macadamia pollen, compared to honey bees with only
24%. Interestingly, Trigona prefer warm flowers (Norgate et al. 2010) and this is
demonstrated by their attraction to flowers on outer, sunny racemes (Heard & Exley
1994). Macadamia also benefits from varietal interplanting for cross-pollination
(Rhodes 1986) as their flowers are mostly self-incompatible and protandrous
(Sedgley et al. 1985).
Crops other than macadamia can also benefit from stingless bee pollination.
Anderson et al. (1982) showed stingless bees to be effective pollinators of mango
(M. indica) and anecdotal accounts of increased crop quality and yield have been
reported for other crops such as lychee, Litchi chinensis Sonnerat (Sapindales:
Sapindaceae); avocado, Persea americana Miller (Laurales: Lauraceae) and
watermelon, Citrullus lanatus (Thunberg) Matsumura & Nakai (Violales:
Cucurbitaceae) (T. Carter, pers. comm., 2009). Although no scientific studies have
been conducted on the effectiveness of stingless bees as pollinators in field crops in
Australia other than macadamia and mango, improved crop yields have been
reported by one bee keeper and his associated growers (T. Carter, pers. comm.,
2009). Stingless bees have also been introduced into blueberry, Vaccinium
corymbosum Linnaeus (Ericales: Ericaceae), crops and are able to collect pollen and
nectar more efficiently than honey bees (F. Adcock, S. Maginnity, M. Grosskopf,
pers. comm., 2010) because blueberry flowers are small, with a deep corolla and
narrow terminal orifice (Rhodes 2006). Unfortunately, there are no experimental
designs or statistical analyses associated with these trials. Although the role of
stingless bees in pollination of native flora is well documented, their efficacy in
13
horticultural and agricultural crops of Australia needs further study (Heard 1999;
Slaa et al. 2006).
1.4.2 Meliponiculture in Australia
The practice of meliponiculture in Australia was almost non-existent in 1984.
However, a survey conducted in 1998 / 99 showed considerable growth in its
popularity to that date. It was predicted that meliponiculture would steadily increase
over the next 20 to 30 years (Heard & Dollin 2000). Since then, interest in stingless
bee keeping has increased with conservation groups being established, especially
along the eastern regions of the country.
More recently, it was estimated that the number of professional service providers was
probably six. Even so, pollination service fees were only a secondary income, and
some of these providers were orchardists, keeping colonies for pollination of their
own crops (Cortopassi-Laurino et al. 2006). A small number of colony producers
transferred and split colonies for sale to enthusiasts, honey producers and pollination
service providers (Cortopassi-Laurino et al. 2006).
The production of honey and cerumen for niche markets has been only very small in

was < 100 kg / yr. Prices for the honey increased from $A40 / kg in 1998 to
$A50 / kg in 2005 (Heard & Dollin 2000; Cortopassi-Laurino et al. 2006). However,
these prices were less than with inflation, representing an actual depreciation in the
value of the product. Production was low and costs were high and, as a result, it was
thought that in order to commercialise the industry commodity honey prices needed
to increase (Cortopassi-Laurino et al. 2006).
Stingless bees are harmless and are an attractive tool which can be used to
demonstrate the wonders of nature. These include sociality, pollination and, of
course, entomology (Cortopassi-Laurino et al. 2006). Schools, gardens, universities
and museums have started to utilise these fascinating creatures in this way.
Workshops on stingless bee keeping are often run by enthusiasts in many areas and
websites have been set up to help the community increase their knowledge in this
area. The Australian Native Bee Research Centre has a website
14
(www.aussiebee.com) and the Australian native bee interest group
(www.australiannativebees.com) is well populated.
To date, while there has been little organised research conducted on Australian
stingless bees, the wealth of knowledge held by stingless bee enthusiasts is
invaluable. Further scientific studies are needed to support these bee keepers and to
help improve techniques in colony propagation, queen rearing, drone rearing and,
possibly, artificial insemination. The reported successes in pollination services
provided by stingless bees overseas (see Section 1.2) have yet to be realised in
Australia.
Colony propagation is the driving factor for the stingless bee industry. Large
numbers of colonies are required for honey production and pollination services. A
strong colony can provide only 1 to 1.5 kg of honey / yr and three times as many
colonies as honey bees are required to pollinate the same area. Other aspects
requiring research include honey preservation post-harvest, education of farmers to
reduce bee losses through pesticide use and collation of research material to enable
efficient whole-community education and industry training (Cortopassi-Laurino et al.
2006).
1.5 Australian stingless bee classification
The stingless bees of Australia belong to two genera, Trigona (Jurine 1807) and
Austroplebeia (Moure 1961) and are in the sub-tribe Trigonini (Wille 1979). They
were first described by Hockings (1883) 
respectively, The genus Trigona contains over 100 species worldwide
and is divided into ten subgenera. All of the Australian Trigona spp. are currently
classified in the subgenus Heterotrigona. The Austroplebeia spp. were also
previously placed in the genus Trigona until Michener revised the family Apidae in
1990. The bees within both genera are small (< 4.5 mm) and black; however,
Austroplebeia can be distinguished from Trigona by its coloured body markings,
thoracic shape and nest architecture. Austroplebeia has small creamy, yellow
markings on the scutellum and axillae of the thorax and the face, whereas Trigona is
completely black (Michener 1990; Klumpp 2007). The dorsal rim of the thorax in
Trigona is more angular than in Austroplebeia (Dollin 2010b). Below is the key to
15
(2000)
:
1. Scutellum and usually face and scutum with well-developed yellow markings;
inner surface of hind tibia with keirotrichiate area broad, nearly reaching upper
margin of tibia ……Austroplebeia
_. Head and thorax without distinct yellow markings; inner surface of hind tibia with
strong longitudinal keirotrichiate ridge above which is a broad depressed, shining
marginal area ……Trigona
1.5.1 Trigona
Australia is home to six species of Trigona; however, the most commonly
domesticated and studied species are T. carbonaria and T. hockingsi. The drones of
Trigona are difficult to identify within the nest, without the aid of a magnifying
glass, as they have no defining markings (Dollin 2010b). They frequently form drone
swarms outside colonies and aggregate on foliage or other structures at night. These
aggregations and swarms can be seen for a number of days when seasonal conditions
are favourable (Klumpp 2007).
1.5.1.1 Species description
The currently described Australian Trigona are classified into three species-groups,
namely:
iridipennis group (Sakagami 1978)
Trigona (Heterotrigona) clypearis Friese 1908
laeviceps group (Sakagami 1978)
Trigona (Heterotrigona) sapiens Cockerell 1911
carbonaria group (Dollin et al. 1997)
Trigona (Heterotrigona) carbonaria Smith 1854
Trigona (Heterotrigona) hockingsi Cockerell 1929
Trigona (Heterotrigona) mellipes Friese 1898
Trigona (Heterotrigona) davenporti Franck 2004
(Dollin et al. 1997; J. Klumpp, A. Dollin, pers. comm., 2010).
16
Identification of Australian Trigona spp. is very difficult in the field and some
species, especially T. carbonaria, can vary considerably in size according to their
geographic location (Dollin et al. 1997). The largest Australian Trigona is
T. hockingsi, measuring ~ 4.5 mm in length, while the smallest is T. clypearis, at
3.5 mm (Klumpp 2007). Species within the carbonaria species-group are difficult to
separate on their body size or morphology. Thus, nest architecture is an invaluable
tool in the accurate identification of species within the Trigona genus (Dollin et al.
1997).
1.5.1.2 Nest architecture
Tree cavities are the most commonly chosen nest substrate for Trigona spp. They can
also be found inside water meter boxes, stone walls, beneath concrete foot paths and
within wall cavities (Dollin et al. 1997). T. mellipes has also been reported to nest
within termite mounds (R. Zabel, pers. comm., 2007). Nest entrance modifications
vary, depending on species although environmental factors, such as weather and
predators, can also influence these structures (Dollin et al. 1997). T. carbonaria often
daub the area around the entrance with substantial amounts of resin, whereas
T. hockingsi and T. davenporti generally leave their entrances unadorned (Dollin
2010b). T. mellipes, T. sapiens and T. clypearis are capable of building entrance
tubes of varying sizes (Table 1.2), although they do not always do so (Dollin et al.
1997).
17
Table 1.2 Comparative description of Trigona spp. (Dollin et al. 1997; Klumpp 2007) and
Austroplebeia spp. (Michener 1961).
Species
Entrance characteristics
& tube length
Mean nest
cavity dia.
Brood structure
T. hockingsi
None. Seldom smears
entrance with resin
145 mm
Horizontal steps/terraces.
Hexagonal comb.
T. carbonaria
None. Usually smears
entrance with resin
198 mm
Flat spiral, single layer.
Hexagonal comb.
T. mellipes
16 mm (mean)
82 mm
Similar to T. hockingsi but
smaller.
T. sapiens
6 mm (mean)
58 mm
Irregular, horizontal or
diagonal layers. No hexagonal
comb.
T. clypearis
28 mm (mean)
78 mm
Roughly arranged in diagonal
rows. No hexagonal comb.
Austroplebeia except
Au. cincta
3 10 mm
65 mm
Clustered.
Au. cincta (PNG)
20 80 mm
45 mm
Irregular concentric layers of
one cell thickness.
1.5.1.3 Colony population and brood structure
It has been estimated that a strong colony of T. carbonaria has a population of
approximately 11,000 workers (Hoffmann, unpublished data). Brood volume can
vary from 940 to 3,535 mL in T. carbonaria and from 1,100 to 2,550 mL in
T. hockingsi (Dollin et al. 1997); however, T. hockingsi is able to build much larger
nests if provided with the appropriate nest cavity (A. Dollin, pers. comm., 2010).
Both T. davenporti and T. hockingsi build brood areas with similar structure;
however, T. davenporti has a smaller adult population. T. mellipes, T. sapiens and T.
clypearis have much smaller nests and average brood volumes measure 595 mL, 224
mL and 464 mL, respectively (Dollin et al. 1997).
Most stingless bee species build regular, horizontal brood comb, which is located
near the centre of the nest (Wille 1983). All Australian Trigona build elongated,
vertically-oriented brood cells in regular, or nearly-regular, structures (Figure 1.1)
(Dollin et al. 1997). There are distinguishing features within these structures that can
aid in species identification (Table 1.2). T. carbonaria builds single layers of
hexagonal comb, arranged in a horizontal spiral. Brood cells are constructed on the
18
outer rim of up to three spirals at a time. T. hockingsi builds a regular, horizontal
brood structure with hexagonal comb, which is best described as terraced or stepped
and is not in a single layer. Both T. davenporti and T. mellipes build similar brood
comb to that of T. hockingsi but the brood comb area of T. mellipes is considerably
smaller (J. Klumpp, pers. com., 2010). Neither T. sapiens nor T. clypearis have a
hexagonal comb structure and individual cells are arranged in irregular horizontal or
diagonal layers (Dollin et al. 1997).
Figure 1.1 Typical Trigona horizontal brood structure (T. carbonaria).
1.5.2