ORIGINAL RESEARCH ARTICLE Australian stingless bees improve greenhouse

Capsicum production

Mark K Greco1,2,3*, Robert N Spooner-Hart1, Andrew G A C Beattie1, Idris Barchia4 and Paul Holford1 1Centre for Plants and the Environment, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 2751, Australia. 2Universität Bern, Departement für klinische Veterinärmedizin, Vetsuisse Fakultät, Länggassstrasse 124, Postfach 8466, Germany. 3INVERT Group, Department of Electrical and Electronic Engineering, University of Bath, BA2 7AY, UK. 4Industry and Investment NSW, Elizabeth Macarthur Agricultural Institute, Private Bag 4008, Narellan, NSW 2567, Australia. Received 3 December 2009, accepted subject revision to 25 October 2010, accepted for publication 2 March 2011. *Corresponding author: Email: M.K.Greco@bath.ac.uk

Summary Australian stingless bees contribute to the pollination of some commercially important field crops, but it is unclear whether they can increase

crop production reliably in the greenhouse environment. Three 20 week trials were therefore conducted, each using a different hive of

Austroplebeia australis Friese and Trigona carbonaria Smith placed in separate glasshouses containing Capsicum annuum L. A third glasshouse

contained C. annuum but no bees. In the third week of each trial, the numbers of pollen grains present on stigmatic surfaces and pollen tubes

growing along styles were determined. Changes in brood volume were assessed by x-ray computerised tomography at weeks 1, 10 and 20.

Additionally, the hives were weighed at these times. At the end of each trial, fruit diameter and length and their fresh and dry weights were

measured as were seed fresh and dry weights. Bee behaviour was recorded in the third trial. T. carbonaria foraged less sporadically on C.

annuum flowers than did A. australis, and pollination by both bee species showed their potential to increase fruit yield and quality. The effects

of pollination by either species were, however, not consistent among the three trials. Hive weights and brood volumes for all colonies

increased, so it is considered that both species thrived whilst being able to pollinate the plants. Both species therefore have the potential to

improve fruit yield and quality within the greenhouse environment. It was noted that A. australis caused damage to the styles in each trial.

This may be attributed to the foraging strategies employed by this species and further work is needed to determine optimum bee to flower

stocking rates.

Las abejas sin aguijón australianas mejoran la producción de

pimiento en invernaderos Resumen

Las abejas sin aguijón australianas contribuyen a la polinización de campos de cultivo comerciales muy importantes; sin embargo, no está

claro si pueden aumentar de manera fiable la producción de cultivos dentro del invernadero. Por lo tanto, se llevaron a cabo tres pruebas de

20 semanas cada una usando diferentes colmenas de Austroplebeia australis Friese y de Trigona carbonaria Smith en invernaderos separados

en los cuales había Capsicum annuum L.; en un tercer invernadero había también C. annuu, pero no abejas. En la tercera semana del ensayo,

se determinó el número de granos de polen presentes tanto en la superficie del estigma como en los tubos polínicos en el interior del estigma.

Los cambios en el volumen de cría fueron evaluados mediante tomografía computerizada de rayos x en las semanas 1, 10 y 20. Además, en

las mismas semanas se pesaron las colmenas. Al término de cada prueba, se midió el diámetro y la longitud del fruto y su peso fresco y seco,

también se midió el peso fresco y seco de las semillas. El comportamiento de las abejas se registró en el tercer ensayo. T. carbonaria pecoreó

menos esporádicamente en las flores de C. annuum que A. australis, y la polinización de ambas especies mostró su potencial para aumentar

el rendimiento y la calidad de este cultivo. Sin embargo, los efectos de la polinización por ambas especies no fueron consistentes en ninguno

de los tres ensayos. El tamaño de las colmenas y el volumen de cría aumentaron en todas las colonias; por lo tanto, se considera que ambas

especies prosperaron siendo capaces de polinizar las plantas. De este modo, ambas especies tienen potencial para incrementar la calidad y el

Journal of Apicultural Research 50(2): 102-115 (2011) © IBRA 2011 DOI 10.3896/IBRA.1.50.2.02

Introduction

Plants, which provide around 35% of our diet, are directly or indirectly

dependent on insect pollinators (Richards, 1993; Klein et al., 2007).

Estimates by Martin (1975), Levin (1983), Robinson et al. (1989) and

Southwick and Southwick (1992) on the value of pollination in the

USA range up to US$ 40 billion per annum and, in Canada, up to CN$

1.2 billion per annum. Winston and Scott (1984) and Gill (1991)

estimated that Australia benefits by approximately AU$ 156 million per

annum from pollination and, more recently, Gordon and Davis (2003)

suggested that the value of pollination in Australia was worth AU$ 1.8

billion per annum when feral bees are included in the estimations.

Many of these insect pollinators are bees, predominately the European

honey bee (Apis mellifera L.). The global decline of the European

honey bee industry due to pests such as the mites Varroa destructor

and Acarapis woodi (Buchmann and Nabhan, 1996), Africanisation of

North American Apis species (Delaplane and Mayer, 2000) and the

decline of natural populations due to insecticides (Kevan and

Plowright, 1995) is, however, creating the need for alternative bee

pollinators. In the past decade, beekeepers in Europe, the USA and

China have been confronted with severe annual colony losses (>

€150,000,000 p.a. collectively), which have in recent years occurred

more frequently, at a greater magnitude and with different symptoms

such as those of Colony Collapse Disorder (Foster et al., 2007).

Globally, the benefits from non-Apis, native bee pollinators are

increasing, as the honey bee industry continues to decline (Richards,

1993, Gallai et al., 2008).

Meliponiculture, the management of stingless bees (Hymenoptera:

Apidae: Meliponinae), is a widely practiced activity in large parts of

the tropics (Batra, 1995), particularly in Central America (Munn,

2000). The neotropical stingless bees, Nannotrigona testaceicornis

Lepeletier, N. perilampoides Cresson and Melipona quadrifasciata

Lepeletier, and the South East Asian stingless bee, T. minangkabau

Sakagami, have been used for greenhouse crop pollination (Kakutani

et al., 1993; Cruz-Lopez et al., 2001; Cauich et al., 2004; Del Sarto et

al., 2005). Additionally, the African stingless bee, Hypotrigona

gribodoi Magretti, has also been shown to forage on crops

(Byarugaba, 2004).

In Australia, meliponiculture is still in its infancy (Heard and Dollin,

2000). Stingless bees have been used effectively to pollinate a range

of crops such as macadamias (Macadamia integrifolia F. Muell.;

Proteales: Proteaceae), carambola (Averrhoa carambola L.;

Oxalidales: Oxalidaceae), litchi (Litchi sinensis J.F. Gmel.; Sapindales:

Sapindaceae ) and avocados (Persea americana Fernández de Enciso;

Laurales: Lauraceae) resulting in improvements in yield and / or

quality (Heard, 1999). Two of Australia’s stingless bees, Austroplebeia

australis Friese and Trigona carbonaria Smith, are potentially the most

promising pollinator species because: they are able to survive

temperatures between 18–36°C (Dollin, 2000a, 2000b); are harmless

to greenhouse workers; are active throughout the year; can be

transported easily; and do not pose an environmental risk by invading

natural habitats if they escape (Amano et al., 2000). No studies have,

however, been concluded on the suitability of Austroplebeia or

Trigona spp. as pollinators in the greenhouse environment. There is

continued interest in the year-long, greenhouse cultivation of crops

such as (Capsicum annuum L.) in countries that experience cooler

winter climates (Jarlan et al., 1997) and these crops have the need

for adequate greenhouse pollination services (Jarlan et al., 1997). We

hypothesized that Australian stingless bees of the species A. australis

and T. carbonaria could increase yield and quality of fruit for

greenhouse-grown plants. We tested this by introducing colonies of

these bees into chambers within a glasshouse at the University of

Western Sydney, Hawkesbury Campus, Richmond, Australia

containing plants of the Grossum Group of C. annuum.

Materials and methods Experimental design

Three 20 week trials (S1, winter/spring; S2, spring/summer; S3,

summer/winter) were conducted from 7 May 2004 to 1 July 2005 at

Richmond, Australia (33°35’ S, 150°45’ E). For each trial, three 3 m

wide, 5 m long and 4 m high temperature-controlled glasshouses

were used. A hive of A. australis was placed in one glasshouse, a hive

of T. carbonaria in the second and the third contained no bees and

acted as a control. These treatments were assigned to different

glasshouses in each trial. Different hives of the two species of bees

were used in each of the three trials.

Plants of C. annuum ‘Aries’ were potted in 300 mm pots and

arranged in five rows on a 500 mm high bench. C. annuum was

selected because it is a commercially important self-pollinating crop

(McGregor, 1976; Aleemullah et al., 2000; Delaplane and Mayer,

2000; Dollin, 2000b) that benefits from the activity of external

pollinating agents (de Oliveira Cruz et al., 2005), and because wind

and rain do not pollinate it (Crane and Walker, 1984). ‘Aries’ was

selected because it is commonly sold in supermarkets and grocery

Australian stingless bees improve capsicum 103

rendimiento de este cultivo dentro de invernaderos. A. australis causó daños en los estilos en cada ensayo. Esto puede ser atribuido a la

estrategia de pecoreo utilizada por esta especie y es necesario seguir trabajando para determinar la abeja óptima para las tasas medias de las

flores.

Keywords: Austroplebeia, Capsicum, Trigona, greenhouse, pollination, stingless bee

stores (Leonardo Pintos, pers. comm.), and the bushes are high

yielding with uniform fruit set carried on compact bushes to 0.5 m in

height (Bill McCarthy, pers comm.). The rows were spaced 300 mm

apart with a spacing of 300 mm between plants within rows and with

20 plants per glasshouse. The plants were approximately six weeks

old when the trials started with flower buds about one week from

anthesis. All plants were fertilised with 100 g of liquid complete

fertiliser (Aquasol, 23:4:18, Yates Ltd.) once per week and checked

and treated (if required) for diseases weekly using bee-compatible

methods. The day temperature was set at 27°C and the night

temperature at 20°C giving minimum temperatures during the night

of 14°C and maximum daytime temperatures of 35°C. Humidity

fluctuated at external, ambient levels for each glasshouse.

Temperature and humidity were monitored using data loggers

(Tinyview Plus, Hastings Data Loggers; Port Macquarie, Australia). An

automated watering system was set to water the plants three times

per day for five minutes each time.

To train the bees to forage in the glasshouse environment, prior

to initial anthesis, five feeders each containing 200 g of pollen and a

40–50% raw sugar (sucrose) solution were placed in glasshouses

containing bees (Jones, 1981). Three drops of tea tree oil were added

to the sugar solutions to encourage foraging (Slaa et al., 1997). A 5 l

plastic container containing approximately 500 g of resinous material

(Thorpe, 1981; Dollin, 1996) was also placed in the glasshouses

containing bees. The resinous material was made from a combination

of sap from local turpentine trees, Syncarpia glomulifera (Sm.), and

cerumen from salvaged stingless bee hives. The feeders were

removed after anthesis (7–10 days after potting) to encourage the

bees to forage on the flowers. Fresh drinking water was provided by

means of a shallow tray (20 mm in depth) that was fitted with ramps

for the bees to gain access to the water without drowning.

Assessment of pollination, pollen tube growth

and stigmatic necrosis

In the third week of each trial, approximately 1 h after anthesis, the

stigmas of ten randomly-selected flowers from each glasshouse were

microscopically examined using epifluorescence resulting from

excitation by near UV wavelengths (Martin, 1959, cited in Clark, 1981)

to determine the numbers of pollen grains present on stigmatic

surfaces. Using another 10 randomly selected flowers from each

glasshouse selected 6 h after anthesis, the numbers of pollen tubes

growing along styles was assessed. To do this, the styles were

macerated in 1 M NaOH at 60°C for 2 h, then stained with 0.5%

decolorized aniline blue in 0.1 M K3PO4 for 3–12 h before being

observed using a fluorescence microscope. Stigmatic damage was

assessed on the latter group visually using a dissecting light

microscope to check for brown, desiccated tissue indicating cell death

(necrosis).

104 Greco, Spooner-Hart, Beattie, Barchia, Holford

Assessment of fruit yield and quality

Fruits were harvested from each glasshouse at weekly intervals for 14

weeks commencing six weeks after the first flower completed

anthesis. Fruits were deemed to be ripe when the first red blush

appeared (Leonardo Pintus, pers comm.). Fruits were weighed to

determine fresh weights, and their basal circumference and length

recorded. Fruit that weighed greater than 80 g, had a basal

circumference greater than 150 mm and a length greater than 110

mm were designated as Grade 1, fruit that weighed greater than 80 g,

had a basal circumference between 150 to 100 mm and a length

between 110 to 50 mm were designated as Grade 2 and all other

fruit, including large, deformed (non-bell shaped) fruit, were

designated as Grade 3: Grade 3 fruit were not considered marketable

(Leonardo Pintus, pers comm.). The number and weight of seeds

produced per fruit and per plant was recorded for each glasshouse.

The seeds and the fruit were placed in paper bags and dried at 80°C

until fully desiccated and then weighed.

Colony health

To assess changes in brood volume (used as an indicator of colony

health) during the trial, all hives were scanned by x-ray computerised

tomography (CT) at weeks 1, 10 and 20 of each trial using a single

slice human body scanner (General Electric HiSpeed, General Electric

Company; Fairfield, CT, USA) (Greco et al., 2005). This procedure was

non-invasive and enabled accurate measurements of all hive

structures and components without damaging the involucrum which

surrounds and supports the brood structure in Trigona species (Dollin,

1996). Three-dimensional images of hives from both species were

reconstructed to visually assess all hive structures. To calculate brood

volume, the brood was measured in vertical, horizontal and

orthogonal planes using on-screen linear callipers. Images were

stored on DVD and hard copies were printed on 350 mm by 430 mm,

20 frames formatted, laser film. As another indicator of colony health,

all hives were weighed at weeks 1, 10 and 20 of each trial to assess

whether there was any variation in total hive weight during the trial.

At week one, all hives were healthy, approximately one third full of

nest structure and increasing in strength.

Bee behaviour

Bee flight and foraging behaviours were recorded under different

ambient conditions for 10 min intervals at five times per day (sunrise,

mid-morning, mid-day, mid-afternoon and sunset) for three days per

week during S3 from 1 June 2005 to 1 July 2005. Light intensity was

measured using a Sekonic illuminometer (Handi Lumi 246, Sekonic

Corporation, Japan). Bee behaviour categories were assigned as

follows: 1. Short flights (SF), flights of approximately 1m from the

hive or less which included orientation flights and that did not involve

any foraging activity; 2. Long flights (LF), flights of over 1m from the

hive that did not include any foraging activity; 3. Collecting pollen

(CP), bees working anthers and ignoring nectaries; 4, Collecting

nectar (CN), bees with tongues extended and working the bases of

corollas; 5. Collecting resin and/or mud (CR), bees foraging in pots

containing resin, or potting mix or on leaf edges; 6. Imbibing water

(IW), bees imbibing water from the irrigation system; and 7.

Removing rubbish (RR), bees removing rubbish from the hive.

Statistical analyses

Data were modelled using a linear random model assuming all factors

affecting the response variables had random effects. Each hive of

bees was considered as a random effect due to likely variation from

hive to hive. Prior to analysis, data were appropriately transformed as

necessary to reduce the variance heterogeneity. The model used can

be written as follows:-

Data = Random (Trial + Treatment + Trial*Treatment + Plant) + error

The variance components of treatments and the two contrasts

were tested using likelihood ratio tests (Kendall and Stuart, 1973).

Since treatment (bees) and experimental effects were assumed to be

random, treatment means were predicted using the best linear

unbiased predictor (BLUP) method (Henderson, 1975). Bivariate

correlations between seed number and grade, basal circumference,

fruit length, fruit weight and between seed weight and grade, basal

circumference, fruit length, fruit weight were explored using Pearson

correlation.

Grade data (values 1, 2 or 3) were analysed using an ordinal data

analysis. A generalized linear model was fitted to the data and the

errors assumed to follow a multinomial distribution. Likelihood ratio

values were calculated for each pair of the treatments (McCullagh and

Nelder, 1989). The structure of this multinomial data does not permit

the testing of the interaction between the treatments and trial.

The bee behaviour data (short flight, long flight, collect pollen,

collect nectar, collect mud, imbibing, remove rubbish and guarding)

were analysed using the principal component (PC) analysis on the

Australian stingless bees improve capsicum 105

variance-covariance matrix (Anderson, 1984). The PC equations

derived from the first two largest components were used to generate

the scores which were then associated with the possible causal factors

(light intensity, temperature and relative humidity). Clusters of

observations were identified visually and the 95% confidence areas

from each cluster centre were drawn to determine differences

between clusters.

Results Pollination and pollen tube growth, stigmatic

necrosis and seed production

Significant differences (P < 0.05 in all cases) were found in mean

numbers of pollen grains, pollen tubes and seeds among the

treatments (Table 1 and Fig. 1). With respect to pollen numbers on

stigmatic surfaces, the relative numbers of grains varied within the

three experiments. In S1, more grains were found on the stigmas of

plants in the glasshouse containing T. carbonaria than on the plants in

the other two treatments. In this trial, there was no difference in

pollen numbers between the other two treatments. In experiments S2

and S3, the numbers of pollen grains differed significantly among all

three treatments with the plants in the glasshouses containing T.

carbonaria having the greatest numbers and the control the least.

This difference between experiments led to a small but statistically

significant trial by treatment interaction (χ2 = 4.31, P < 0.038). For

the three experiments as a whole (Table 1), the presence of bees

significantly increased the number of pollen grains on stigmatic

surfaces, with more grains on stigmatic surfaces of flowers pollinated

by T. carbonaria.

With respect to pollen tubes within styles, in trial S1, there were

more tubes in the styles of plants in the glasshouse containing

T. carbonaria; the numbers of tubes did not differ significantly

between the other two treatments. In trials S2 and S3, the styles of

plants pollinated by A. australis contained more tubes than the control

Pollen grains Pollen tubes Square root number of seeds per fruit

Terms Variance component Χ2 P Variance

component χ2 P Variance component χ2 P

Seasons 30.1 0.02 0.8875 18.7 0.05 0.8231 5.8 7.76 0.0053

Among all treatments 1864.1 7.66 0.0055 891.2 6.52 0.0107 7.8 8.81 0.0030

Trial * treatment 334.6 4.31 0.0379 113.2 2.70 0.1003 0.0 0.0 1.0000

Residual 1367.8 644.4 11.4

Table 1. Variance components, chi-square values obtained from likelihood ratio tests and significance of the random terms of the model fitted

to the number of pollen grains present on stigmatic surfaces, pollen tubes present in styles and mean number of seeds in fruit of plants of C.

annuum grown in three separate trials.

whilst, in turn, plants pollinated by T. carbonaria contained more

pollen tubes than those pollinated by the other species. There was no

significant interaction between trials and treatment when all

treatments were considered. Regarding necrosis of stigmatic surfaces

(Fig. 2), there was no difference in the amount of damage among

trials. In each trial, A. australis caused damage to the stigmatic

surface resulting in an average damage (necrosis) of 0.8 on all three

occasions; in the other two treatments, the damage scores were 0.1

in all trials. The numbers of seeds per fruit varied with trial and with

treatment; however, there was no significant trial by treatment

interaction (Table 1). In each trial, there were approximately equal

numbers of seeds in the fruits of bee-pollinated plants with more

seeds in these treatments than in the control.

Greco, Spooner-Hart, Beattie, Barchia, Holford

Fruit yield and quality

When the data from all three trials were considered together (Table

2), there was no significant affect of bee pollination on fruit size and

weight parameters. There were, however, highly significant

interactions between trial and treatments mainly due to the

interaction between A. australis and T. carbonaria by trial. In S1,

pollination by T. carbonaria increased fruit circumference, fresh and

dry fruit weight and fresh and seed weight over control levels whereas

pollination by A. australis reduced these parameters below those of

the control (Table 3). In S2 and S3, there was a trend for bee-

pollinated plants to have higher fresh fruit weights as well as higher

fresh and dry seed weights. In addition, in S2, bee pollination

increased dry fruit weight. In S3, pollination by both species increased

basal fruit circumference whilst in S2 circumference was only

increased by pollination by A. australis. Fruit length was little affected

by bee pollination in any of the three experiments.

For the combined data from the three experiments, the mean

quality of the fruit (as assessed by grade) was also increased by

pollination with T. carbonaria (Table 4) but not A. australis. In S1,

however, pollination by this latter species increased the weight of

total, marketable and Grade 1 yields (Fig. 3) whilst in the other

experiments these yields were either similar to or below those of the

control. Pollination by T. carbonaria increased marketable and Grade

1 yields in S2 and total, marketable and Grade 1 yields in S3, with the

increases in this latter trial being most marked. Plants pollinated with

T. carbonaria produced a higher number of marketable and Grade 1

fruit in the final two experiments but there was no difference from the

control in trial S1. In contrast, plants pollinated by A. australis

produced higher marketable and Grade 1 fruit in trial S1 but similar or

lower yields than the control in the other two trials (Fig. 3).

Pearson’s correlations for combined treatments showed positive

correlations between the dimensions of the fruits and fruit and seed

weights. Grade, however, was negatively correlated with these

parameters (Table 5). It should be noted that a low grade score is

associated with high quality. All correlations were significant at

P = 0.05.

106

S1

Num

bers

50

100

150

200

250

S2

Num

bers

50

100

150

200

250

S3

Pollen grains Pollen tubes Seeds

Num

bers

50

100

150

200

250 Control A. australisT. carbonaria

b b

a

b b

a

b

aa

c

b

a

cb

a

b

aa

c

b

a

c

b a

b

aa

Fig. 1. Mean number (estimated by best linear unbiased prediction)

of pollen grains present on stigmatic surfaces, pollen tubes present

in styles and mean number of seeds in fruit of plants of C. annuum

grown three in separate trials (S1, S2 and S3). For each trial, the

plants were either provided with a hive of A. australis or T. carbonaria;

no bees were provided to the plants in the third treatment which

acted as a control. Bars within each cluster and labelled with the

same letter are not significantly different according to LSD test at

P = 0.05.

Fig. 2. Light micrograph showing (a) the surface of an undamaged

stigma and (b) necrosis on the stigma of a flower visited by A. australis.

Australian stingless bees improve capsicum 107

Basal circumference

Log length of fresh fruit

Sq. root weight of fresh fruit

Sq. root weight of dry fruit

Sq. root weight of fresh seeds

Log weight of dry seeds

Terms VC χ2 P VC χ2 P VC χ2 P VC χ2 P VC χ2 P VC χ2 P

Seasons 922.5 471.0 <0.0001 0.0 0.0 1.000 2.27 201.2 <0.0001 0.137 166.4 <0.0001 0.0055 56.9 <0.0001 0.0145 92.8 <0.0001

Among treat-ments

0.0 0.0 1.000 0.0 0.0 1.000 0.0 0.0 1.000 0.0024 0.22 ns 0.6390 0.003 0.32 0.5716 0.0 0.0 1.000

Trial treatment 469.1 139.6 <0.0001 0.00094 42.0 <0.0001 1.62 86.0 <0.0001 0.0679 14.7*** 0.0001 0.1976 174.7 <0.0001 0.1156 125.4 <0.0001

Residual 942.3 0.00617 5.000 0.3342 0.2554 0.2341

Table 2. Variance components, chi-square values obtained from likelihood ratio tests and significance of the random terms of the model

fitted to the grade, dimensions and weights of fruits and seeds of C. annuum plants grown in three separate experiments., VC = variance

component.

Trial Treatment Basal circumference (mm)

Log fruit length (mm)

Sq. root fresh fruit weight (g)

Sq. root dry fruit weight (g)

Sq. root fresh seed weight (g)

Log dry seed weight (g)

Mean Std error Mean Std

error Mean Std error Mean Std

error Mean Std error Mean Std

error

1 Control 183.8 b 3.49 2.080 (120.3) b 0.009 10.37

(107.6) b 0.25 2.52 (6.37) b 0.06 1.01

(1.01) b 0.06 -0.588 (0.26) b 0.055

1 A. australis 169.9 c 2.88 2.010

(102.4) ab 0.007 9.01 (81.2) c 0.21 2.26

(5.10) c 0.05 0.51 (0.26) c 0.05 -1.124

(0.075) c 0.045

1 T. carbonaria 223.8 a 4.09 2.113 (129.7) a 0.010 12.97

(168.3) a 0.30 3.08 (9.51) a 0.08 1.75

(3.05) a 0.07 -0.084 (0.824) a 0.064

LSD 9.90 0.025 0.72 0.18 0.16 0.0169

2 Control 231.3 b 3.30 2.095 (124.5) a 0.008 12.41

(153.9) b 0.24 2.71 (7.33) b 0.06 1.26

(1.58) c 0.05 -0.612 (0.24) c 0.052

2 A. australis 251.2 a 3.66 2.102 (126.6) a 0.009 13.33

(177.6) a 0.27 2.90 (8.40) ab 0.07 1.82

(3.32) a 0.06 -0.286 (0.52) b 0.058

2 T. carbonaria 225.8 b 3.71 2.081

(120.6) a 0.009 12.68 (160.7) ab 0.27 2.96

(8.74) a 0.07 1.56 (2.45) b 0.06 -0.251

(0.56) a 0.058

LSD 10.04 0.025 0.73 0.19 0.166 0.0164

3 Control 240.1 b 3.83 2.083 (121.1) a 0.010 13.57

(184.2) b 0.28 3.38 (11.40) a 0.07 1.27

(1.62) b 0.06 -0.289 (0.51) c 0.060

3 A. australis 279.0 a 3.92 2.062 (115.3) a 0.010 14.74

(217.3) a 0.28 3.45 (11.91) a 0.07 1.94

(3.75) a 0.06 0.034 (1.08) a 0.062

3 T. carbonaria 251.0 b 4.51 2.079

(119.8) a 0.011 13.97 (195.2) ab 0.33 3.36

(11.31) a 0.08 1.42 (2.02) b 0.07 -0.175

(0.67) b 0.071

LSD 11.52 0.028 0.83 0.21 0.19 0.019

Table 3. Mean (estimated by the best linear unbiased predictor (BLUP) method) dimensions and weights of fruits and seeds of C. annuum

plants pollinated by either A. australis or T. carbonaria compared to self-pollinated plants (control) during 3 x 20 week trials held from 7 May

2004 to 1 July 2005. Within an trial, means followed by the same letter are not significantly different from each other according to LSD tests

at P = 0.05. Data in parenthesis are the back transformed means.

(a) Pair-wise comparison Chi square Probability (b) Treatment Average grade

Control vs A. austroplebia 2.24 0.134 Control 1.76b

Control vs T. carbonaria 14.77 <0.001 A. austroplebia 1.64b

A. austroplebia vs T. carbonaria 7.08 0.008 T. carbonaria 1.47a

Table 4. (a) Pairwise comparisons of grade data from ordinal data analysis and (b) average grades for the capsicum fruit over the three trials.

Means followed by the same letter are not significantly different.

Greco, Spooner-Hart, Beattie, Barchia, Holford 108

Colony health

Brood volumes (BV) for both species increased in all trials (Table 6).

The percentage increases in BV for A. australis (20–40%) were

greater than for T. carbonaria (12–20%). However, the sizes of BV for

A. australis were smaller than for T. carbonaria being approximately

110 ml at the end of the trials compared to 1130 ml for T. carbonaria.

The honey, pollen and cerumen volumes of all hives also increased.

Surprisingly, for each of these parameters, there was little difference

in the percentage increases between species or trial with each hive

increasing by approximately 9.3, 41 and 9.3% in these volumes for

S1, S2 and S3, respectively. These increases in volumes were

associated with increased hive weight with most hives increasing by

1.5–3%. The exception to this was the hive of A. australis in trial S3,

which increased by 5.5%.

For both species, there was a strong, positive correlation between

BV and pollen volume (PV) (r = 0.72, P < 0.029, n = 9 for A. australis

and r = 0.90, P < 0.001, n = 9 for T. carbonaria). For A. australis, PV

was also correlated with honey volume (HV) (r = 0.70, P = 0.035).

For T. carbonaria, further positive correlations existed between HV

and BV (r = 0.67, P = 0.048), cerumen volume (CV) and HV (r = 0.68,

P = 0.043) and hive weight and CV (r = 0.99, P < 0.001).

Bee behaviour

There were highly-significant, linear correlations among light intensity,

RH and temperature (light vs. temp., r = 0.96; light vs. RH, r = 0.88;

and temp vs. RH, r = 0.91, P < 0.05 in all cases) within the

glasshouse during the collection of the behavioural data. To simplify

the presentation of these data, multi-axis graphs were therefore used

S1

Yiel

d (k

g)

2

4

6

8

10

12

14 Control A. australisT. carbonaria

S2

Yiel

d (k

g)

2

4

6

8

10

12

14

S3

Total Marketable Grade 1

Yiel

d (k

g)

2

4

6

8

10

12

14

Fig. 3. Total, marketable and Grade 1 yield of C. annuum grown in

separate three trials (S1, S2 and S3). For each trial, the plants were

either provided with a hive of A. australis or T. carbonaria; no bees

were provided to the plants in the third treatment as a control.

Grade of mature fruit

Basal circumference

Length of mature fruit

Weight of mature

fresh fruit

Weight of mature dry fruit

Weight of fresh seeds

Weight of dry seeds

Grade of mature fruit

Basal circumference -0.55

Length of mature fruit -0.47 0.30

Weight of mature fresh fruit -0.65 0.87 0.54

Weight of mature dry fruit -0.58 0.74 0.53 0.88

Weight of fresh seeds -0.46 0.73 0.29 0.68 0.52

Weight of dry seeds -0.50 0.70 0.40 0.77 0.73 0.79

Number of seeds -0.44 0.62 0.37 0.64 0.56 0.84 0.86

Table 5. Pearson correlations analysis of the relationships between fruit quality parameters. Values are correlation coefficients (r) and all

relationships are significant at P = 0.05.

(Figs 4 and 5). The activities that occurred over the greatest

temperature range were short and long flights and guarding. For both

species, short and long flights mainly occurred when the temperature

was > 20ºC, the LI was > 13000Lx and the RH > 47% with the

greatest activity between 21–26ºC, 13000–23000Lx and 46–52% RH.

Outside of this range, flights of A. australis were more limited than

those of T. carbonaria, especially at the cooler temperatures

experienced during the trial. Guarding was least influenced by

environment, with significant numbers of bees of both species

guarding their hives at all assessment times. The collection of pollen,

nectar and mud/resin occurred over a more limited range of

conditions. Although these activities commenced under similar

environmental conditions as flights the number bees collecting these

Australian stingless bees improve capsicum

material tended to be greatest at temperatures > 25ºC, LI >

23,000Lx and at RH > 56%. For nectar and pollen collection, the

highest number of bees of A. australis performing these activities was

at ~27–28ºC, whilst for T. carbonaria, maximum activity was at 29–

31ºC (Figs 4 and 5). Imbibing water was the activity most constrained

by environmental conditions, with this activity only occurring when the

temperature exceeded 25ºC, the LI was >23000 Lx and the RH > 56%.

A. australis foragers normally emerged in large numbers (> 200

individuals) and foraged for approximately 30–40 min and then

returned to the hive for several hours before re-emerging. In contrast,

T. carbonaria foragers emerged gradually but regularly and foraging

continued all day but with less intensity than A. australis. The

maximum number of long flights for an A. australis individual in one

109

Fig. 4. Behaviour of A. australis during pollination trials from 1 June

2004 to 1 July 2005.

Fig. 5. Behaviour of T. carbonaria during pollination trials from 1 June

2004 to 1 July 2005.

110

Greco, Spooner-Hart, Beattie, Barchia, Holford

Austroplebia australis Trigona carbonaria

Brood volume

(ml)

Honey volume

(ml)

Pollen volume

(ml)

Cerumen volume

(ml)

Hive weight (g)

Brood volume

(ml)

Honey volume

(ml)

Pollen volume

(ml)

Cerumen volume

(ml)

Hive weight

(g)

S1 Initial wt 63 435 82 475 2954 986 514 72 155 3402

% increase in wk 10 54.0 5.1 30.5 5.1 2.4 12.5 5.1 30.6 5.2 1.0

% increase in wk 20 68.3 10.3 69.5 10.3 2.8 14.3 10.3 69.4 10.3 2.2

S2 Initial wt 83 321 65 552 3102 985 482 61 103 2896

% increase in wk 10 9.6 5.0 30.8 5.1 1.0 2.1 5.0 29.5 4.9 0.4

% increase in wk 20 37.3 10.3 69.2 10.3 1.6 14.2 10.2 68.9 10.7 3.1

S3 Initial wt 86 454 87 673 2915 943 531 59 333 5404

% increase in wk 10 5.8 5.1 29.9 5.1 2.7 6.8 5.1 30.5 5.1 0.6

% increase in wk 20 24.4 10.4 69.0 10.3 5.9 24.3 10.2 69.5 10.2 1.5

Table 6. Volume of hive components and hive weights for A. australis and T. carbonaria during three 20 week trials (S1, S2 & S3) measured

at weeks 1, 10 and 20 of each trial.

Fig. 6. Bi-plot of the first and second principle component scores and the axes of all behavioural variables used in the analysis. Ellipses

represent the areas covered by the 95% confidence intervals drawn from each cluster centre. A = A. australis, T = T. carbonaria.

day was 267 whereas it was only 50 for T. carbonaria. Most long

flights were by bees that settled on the far walls or roof of the

glasshouse. Most short flights were orientation flights undertaken by

both species each day at sunrise. Both species behaved similarly

whilst collecting resin or removing rubbish from the hive. These

activities commenced approximately 1 h after sunrise and continued

until sunset. In this third trial, approximately equal numbers of bees

of A. australis were observed to be collecting nectar and pollen (43

and 36 bees, respectively, per observation period (~10 min)). For T.

carbonaria, however, nearly three times as many bees were collecting

Australian stingless bees improve capsicum

nectar (102 per observation period) as were collecting pollen (35

bees).

To determine which environmental factors were influencing

behaviour, the behavioural data were subjected to principal

component analysis. The first two components accounted for more

than 95% of all variation and presented clear separations of

observations into 6 clusters (Fig. 6). Collecting nectar and pollen

accounted for most of the variation in PC1 whilst long flights

accounted for most of the variation in PC2 (Table 7). Using the

confidence areas of the clusters, it can be seen that the two species

of bees behaved differently at corresponding light intensities, except

at the lower range (below 700Lx). Visual inspection of the range of

the environmental values of each of the six clusters (Table 8)

suggested that the clusters are more closely defined and

differentiated by light intensity than by temperature or RH.

Discussion The results showed that although T. carbonaria foraged less sporadically

on C. annuum flowers than did A. australis over the sixty weeks of the

three trials, both species of Australian stingless bee have the potential

to improve fruit yield and quality within the greenhouse environment.

The effects of the bees, however, differed markedly between the

trials. A. australis was able to increase total marketable and Grade 1

yields in the first trial but these yields were either similar to or lower

than those of the controls in the other two trials. T. carbonaria caused

a significant increase in quality (as assessed by grade) throughout the

trials, with increases in total, marketable and Grade 1 yield in the

second and third trials. Kristjansson and Rasmussen (1991) tested

111

Latent vector

Variables PC1 PC2

Short flights 0.0876 0.1663

Long flights 0.1028 0.9657

Collecting pollen 0.3192 0.1172

Collecting nectar 0.9352 -0.1609

Collecting mud 0.0274 0.0036

Imbibing water 0.0640 -0.0112

Removing rubbish 0.0189 -0.0084

Latent roots 10968 8094

% variation 54.91 40.53

PC Group Species

Light intensity

(Lux)

Temperature (°C)

Relative humidity

Short flights

Long flights

Collecting pollen

Collecting nectar

Collecting mud

Imbibing water

Removing rubbish Guarding

1 Aa 277 (210-650)

17.6 (16-20)

40.7 (38-43) 2.0 1.7 0 0 0.5 0 1.6 4.5

1 Tc 277 (210-650)

17.5 (16-19)

40.7 (39-43) 19.3 10.6 0 0 0 0 1.1 3.5

2 Tc 15922

(12000-25000)

23.36 (21-25)

47.0 (42-55) 49.6 36.6 19.7 54.8 5.4 2.8 7.2 6.7

3 Aa 39500

(38500-40000)

28.8 (27-31)

54.7 (47-60) 15.6 106.8 85.0 72.8 6.9 11.0 2.0 7.3

4 Aa 15867

(12000-25500)

23.46 (21-26)

47.0 (42-55) 66.4 255.1 47.5 37.8 3.5 1.9 2.4 7.1

5 Tc 39409

(38000-40000)

27.6 (36-29)

52.1 (47-56) 28.2 31.5 79.6 184.8 6.2 19.6 11.1 7.5

6 Tc 39409

(38000-40000)

30.0 (29-31)

58.9 (56-60) 60.6 46.4 107.6 385.6 12.0 20.7 7.4 7.6

Table 7. Latent vectors and roots of the first two largest principle

components derived from the behavioural data from A. australis and

T. carbonaria.

Table 8. Average behaviour variable values calculated from the principal component analysis and the mean number of bees within each cluster.

Osmia cornifrons on greenhouse Capsicum and also found variances

in yields within seasons. They found that yields increased early in the

season rather than later and attributed these variances to shorter

daylight hours and lower temperatures later in the season. Shipp et

al. (1994) also found similar seasonal variances for greenhouse

Capsicum pollinated by Bombus impatiens Cresson, and attributed

these variations to plant stress during periods of shorter day length

rather than bee influences. As Capsicum growers in Australia are,

however, paid for their produce according to grade and fresh fruit

weight (Leonardo Pintus, pers. comm.), any increases in yield and

quality (grade) due to pollination by both Australian species are likely

to increase income from the crop. The use of stingless bees, however,

requires the purchase or renting of hives, so this cost needs to be

considered in financial calculations before adoption of this strategy.

The increases in yield recorded in these trials associated with

pollination by T. carbonaria are comparable to increases reported for

neotropical (Amano, 2004, Cauich et al., 2004, Malagodi-Braga and de

Matos Peixoto Kleinert, 2004, de Oliveira Cruz et al., 2005) and Asian

(Kakutani et al., 1993) stingless bees.

A. australis caused substantial damage to the styles in all three

trials. This may be attributable to the foraging strategies employed by

this species at different times of year. A. australis inhabits the more

arid areas of regions occupied by stingless bees (Dollin, 1996). They

do not forage until suitable light (13000Lx), temperature (21°C) and

relative humidity (42%) conditions are reached but, when these

occur, the bees forage en masse (personal observations). This

behaviour and the resultant flower damage that it caused may explain

the reductions in yield recorded in S2 and S3. Despite the damage,

however, the number of pollen tubes in the styles was either higher

(S2 and S3) or the same (S1) as in control flowers. In addition, seed

numbers in flowers pollinated by A. australis were higher in each trial

as was mean fresh fruit weight. Thus, it seems that either pollen

germination or tube growth are not affected by necrotic tissue

damage observed or that pollen germination occurred prior to tissue

necrosis. In contrast to A. australis, T. carbonaria’s natural habitat is

more diverse, and typically has floral resources available for longer

periods throughout the day. T. carbonaria are generalist foragers

(Heard, 2001) and foraged for approximately 2 h longer each day

than A. australis. As a result, T. carbonaria caused little or no physical

damage to stigmas during this trial and the level of tissue necrosis

(10% of stigmas showing damage) was the same as in the control

treatment. It appears that over-pollination occurred with A. australis

so further work is needed to determine optimum bee to flower

stocking rates.

In C. annuum, seed set is limited by pollen load (Marcelis and

Baan Hofman-Eijer, 1997). In the current study, bee pollination

increased the pollen load on the stigmas. In addition, positive

correlations were found between fresh and dry seed weights with fruit

weight and size. Improvement in fruit size in Capsicum due to

112

increases in pollen load have been reported by a number of authors

(Rylski, 1973, de Ruijter et al., 1991, Shipp et al., 1994, Marcelis and

Baan Hofman-Eijer, 1997). The increase in size is likely due to auxin

production by the seeds (Leopold and Kriederman, 1975) leading to

increased metabolic activity (Varga and Bruinsma, 1976, Goodwin

1978) which, in turn, increases sink strength. Additionally, increased

fruit weight from this trial may be from xenic effects (Denney, 1992)

due to pollen brought to the stigmas by the bees. Various

phytohormones, such as gibberellins, cytokinins and auxins within the

pollen grains (Singh and Sawhney, 1992), may be influencing

maternal tissue and thus improving characteristics such as grade and

fresh fruit weight.

Although Capsicum flowers produce both nectar and pollen

(Rabinowitch et al., 1993), the amount of each may not be sufficient

for the long-term maintenance of honey bee colonies (Free, 1993),

and Kalev et al. (2002) found that pollen supplementation was

needed. To establish whether Australian stingless bees could survive

during lengthy pollination programmes within greenhouses, it was

necessary to assess colony health and vigour. When total hive weights

and brood volumes remain constant or increase, it is considered that

the colonies are in good health and vigour (Heard, 1988). Because

hive weights and brood volumes for all colonies increased during this

trial, it is considered that they all survived well and thrived whilst also

being able to pollinate the crops.

CT scanning enabled the determination of a large, positive

correlation between brood volume and hive weight. Therefore, by

simply weighing the hives, we are now able to assess brood volume

and, thus, colony health. In future greenhouse pollination

programmes, CT scanning of the hives (Greco et al., 2005) to monitor

brood, cerumen, honey or pollen volumes could be considered as a

diagnostic technique if hive weights are reducing due to some

unknown cause.

The light intensity threshold for pollen and nectar collecting was

approximately 13000Lx for A. australis and 12000Lx for T. carbonaria

and, in general, light seemed to be the major factor controlling

behaviour. A. australis would emerge from the hive approximately 1 h

later and cease flying 1 h earlier than T. carbonaria each day,

irrespective of temperature. These findings are similar to those of

open field experiments with T. carbonaria (Heard and Hendrikz, 1993)

except for the light intensity threshold in their trial (approximately

10000 Lx) which was much lower. Our experiments were conducted

under greenhouse conditions and this closed environment could have

an influence on light intensity thresholds for flight activity compared

to open field experiments due to changes in light spectrum and

polarity. Our experiments suggest that T. carbonaria requires a

brighter, hotter and more humid environment than A. australis to

forage abundantly. Since A. australis were most abundantly foraging

at 18000 Lx, 27.5°C and a RH of 49–56% and T. carbonaria were

most abundantly foraging at 30500–42500 Lx, 31°C and an RH of

Greco, Spooner-Hart, Beattie, Barchia, Holford

56–60%, greenhouse crop producers could modify the environment to

suit either species for optimal pollination activity or select the most

appropriate species for their greenhouse conditions.

Pollination to increase fruit and seed yields of greenhouse grown

C. annuum by A. mellifera, is standard practice in some countries (de

Ruijter et al., 1991) and certain flies (Delaplane and Mayer, 2000) and

managed colonies of B. terrestris (Shipp et al., 1994) are also used.

This study indicates that whilst both A. australis and T. carbonaria

have potential for improving yield of C. annuum in greenhouses, the

less sporadic foraging strategy of T. carbonaria is likely to be more

suitable for improving fresh weight and grade of C. annuum fruit. It is

recommended that further studies be conducted with both species to

develop reliable pollination programmes. This should include

determination of optimum bee stocking rates and continued

assessment of colony health to determine long-term effects of

glasshouse confinement on these two Australian stingless bees.

Acknowledgements The authors wish to thank Macarthur Diagnostic Imaging for donating

time on the CT scanner.

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