Morphometric characterization of stingless bees (Trigona iridipennis Smith) in Kerala

Table of contents

Table of figures

Table of tables

List of abbreviations

Morphometric characterization of stingless bees (Trigona iridipennis Smith) in Kerala: an overview

1. Introduction

2. Materials and Methods
2.1 Study area
2.2 Study design and data collection
2.2 Morphometric measurements
2.3 Statistical analysis

3. Results
3.1 Descriptive analysis
3.2 Corelation analysis
3.3 Principal component analysis

4. Discussion

5. Conclusions

Acknowledgements

References

Table of figures

Figure 1. Morphometric characters considered for the analysis of worker and drone bees of stingless bees in Kerala: 1 = head length, 2 = head width, 3 = proboscis length, 4 = distance between two dorsoal ocelli, 5 = dorsal ocello-ocular distance, 6 = antennal length, 7 = fermur length, 8 = tibia length, 9 = metatarsus length, 10 = metatarsus width, 11 = forewing length, 12 = forewing width, 13 = pterostigma length, 14 = pterostigma width, 15 = hind wing length, 16 = hind wing width, 17= number of hamuli, 18 = tergite length, 19 = sternite length, 20 = width of sternum, 21 = mandible length, 22 = mandible width.

Figure 2. Map of Kerala showing various sample collection points marked as a star symbol during the study.

Figure 3. a) stingless bee hive showing pollen, honey and eggs; b) bee hive made of wooden box; c) bee hive kept on earthen pots; d) entrance tube with fresh resin on a stone wall; e) stingless bee drone (top) and worker (bottom).

Figure 4. a) stingless bee worker foraging on Euphorbia flower; b) antenna drone (left) and worker (right); c) pterostigma; d) mandible drone (left) and worker (right).

Figure 5. a) stingless bee brood; b) worker tongue; c) head of drone (top) and worker (bottom); d) worker fore-wing and hind wing; e) worker bee with pollen basket.

Figure 6. Schematic representation of head length width ratio (HLW), thorax metatarsus length with ratio (TMLW) and thorax forewing length width ratio (TFWLW) among stingless bee workers across different districts in Kerala, India.

Figure 7. Schematic representation of head length width ratio (HLW), thorax metatarsus length with ratio (TMLW) and thorax forewing length width ratio (TFWLW) among stingless bee drones across different districts in Kerala, India.

Figure 8. Scree plot for finding the various components to be extracted during Principal Component Analysis among stingless bee workers in Kerala.

Figure 9. Component plot in rotated space during principal component analysis among stingless bee workers in Kerala.

Figure 10. Distribution of factor scores among stingless bee workers across various locations in Kerala.

Figure 11. Scree plot for finding the various components to be extracted during Principal Component Analysis among stingless bee workers.

Figure 12. Component plot in rotated space during principal component analysis among stingless bee drones.

Figure 13. Distribution of factor scores among stingless bee drones across various locations in Kerala.

Table of tables

Table 1. Description of the morphometric variables used to classify Trigona iridipennis Smith in Kerala, India.

Table 2. Site description of the selected farms (W1-W21) across Kerala, India for collecting worker bees during 2011-2013.

Table 3. Site description of the selected farms (D1-D9) across Kerala, India for collecting drone bees during 2011-2013.

Table 4. Description of Trigona iridipennis Smith worker and their morphometric characteristics.

Table 5. Description of some morphometric of Trigona iridipennis Smith worker and their characteristics.

Table 6. Description of the various habitat of Trigona iridipennis Smith drone and their characteristics.

Table 7. Description of the various habitat of Trigona iridipennis Smith drone and their characteristics.

Table 8. Pearson correlation coefficients between morphometric characters of Trigona iridipennis Smith workers (n=84) in the various districts across Kerala, India during 2011-2013.

Table 9. Pearson correlation coefficients between remaining morphometric characters of Trigona iridipennis Smith workers (n=84) in the various districts across Kerala, India during 2011-2013.

Table 10. Pearson correlation coefficients between morphometric characters of Trigona iridipennis Smith drones (n=71) in the various districts across Kerala, India during 2011-2013.

Table 11. Pearson correlation coefficients between remaining morphometric characters of Trigona iridipennis Smith drones (n=71) in the various districts across Kerala, India during 2011-2013.

Table 12. Eigenvalues and percentage of variance explained by the first four components in a PCA of Trigona iridipennis Smith workers in Kerala.

Table 13. Component matrix in a PCA of Trigona iridipennis Smith workers in Kerala.

Table 14. Rotated component matrix in a PCA of Trigona iridipennis Smith workers in Kerala.

Table 15. Component score coefficient matrix in a PCA of Trigona iridipennis Smith workers in Kerala.

Table 16. Eigenvalues and percentage of variance explained by the first four components in a PCA of Trigona iridipennis Smith drones in Kerala.

Table 17. Component matrix in a PCA of Trigona iridipennis Smith drones in Kerala.

Table 18. Rotated component matrix in a PCA of Trigona iridipennis Smith drones in Kerala.

Table 19. Component score coefficient matrix in a PCA of Trigona iridipennis Smith drone in Kerala.

List of abbreviations

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ACKNOWLEDGEMENT

Firstly we thank God Almighty whose blessing were always with us and helped us to complete this research work successfully.

The first author is extremely grateful to Dr. Sajeshkumar N.K (Head of the Department, Biotechnology) for the valuable suggestions, support and encouragements.

The first author would wish to thank beloved Manager Rev. Fr. Dr. George Njarakunnel, respected Principal Dr. V.J. Joseph, Bursar Shaji Augustine, Vice Principal Fr. Joseph Allencheril, and the Management for providing all the necessary facilities in carrying out the study.

We lovingly and gratefully indebted to our teachers, parents, siblings and friends who were there always for helping us in this project.

Prem Jose Vazhacharickal and Sajan Jose K

Morphometric characterization of stingless bees (Trigona iridipennis Smith) in Kerala: an overview

Prem Jose Vazhacharickal1* and Sajan Jose K2

* premjosev@gmail.com

1Department of Biotechnology, Mar Augusthinose College, Ramapuram, Kerala, India-686576

2Department of Zoology, St. Joseph’s College, Moolamattom, Kerala, India-685591

Abstract

The major species of stingless bees in Kerala is Trigona iridipennis Smith which are easy to domesticate due to low temperament and easy adaption to various habitats. Little is known about the variations exist among them. We therefore conducted morphometric studies among workers and drones across 14 districts in Kerala with a view to characterize them. A total of 188 and 71 samples of drones and workers collected and analyzed various morphometric characters including head length (HL), head width (HW), proboscis length (PL), distance between two dorsal ocelli/lower intercocular distance (DBO), dorsal ocello-ocular distance (DOOD), antennal length (AL), thorax femur length (TFL), thorax tibia length (TTL), thorax metatarsus length (TML), thorax metatarsus width (TMW), thorax forewing length (TFWL), thorax forewing width (TFWW), pterostigma width (PtStW), pterostigma length (PtStW), hind wing length (HWL), hind wing width (HWW), number of hamuli (HAM), abdominal tergite length (AbTL), abdominal sternum width (AbSW), mandible length (MdL) and mandible width (MdW), head length width ratio (HLW), thorax metatarsus length width ratio (TMLW) and thorax forewing length width ratio (TFWLW). Our studies revealed that most of the worker morhopometric characters were positively correlated across all the districts in Kerala. However, there is not much significant differences among drones across various districts. Based on our studies, even though we detected significant differences among worker morhphotmetry we could not to find distinguishing and remarkable differences among collected samples. They may be interpreted as nutritional, seasonal and climatic factors.

Keywords: Stingless bees; Meliponiculture; Principal component analysis; Rotated component matrix.

1. Introduction

Despite of role in pollination and ecosystem services, honey bee population is drastically decreasing due to destruction of natural habitat, intensive agriculture, diseases and climate change ( et al., 2008; Stingless bees originated in the earth 65 million years ago and are limited to tropics and subtropics with diversity in species and share morphological and behaviour pattern especially in nest building and resources exploitation patterns (. They belong to five different genera; Melipona, Trigona, Meliponula, Dectylurina and Lestrimelitta which plays a vital role in plant pollination ( with an estimation that 30% of the human food is derived from bee pollinated crops ( et al., 2006). Stingless beekeeping is known as meliponiculture undertaken by traditional communities ( Unlike most bee species that live solitary lives, the stingless bees (Apidae, Meliponini) and the true honey bees (Apidae, Apini) are highly social (‘eusocial’) and have complex and long lasting colonies (Michener, 1974). The stingless bee found in Kerala is Trigona iridipennis Smith also called ‘dammer bees’ locally known as ‘Cherutheneecha’ in Malayalam (Singh, 2013).

Trigona iridipennis Smith are kept in India for centuries for the high medicinal value of honey as well as propolis and bee wax (Rasmussen, 2013; Cortopassi-Laurino et al., 2006; Virkar et al., 2014; Kumar et al., 2012; Andualem, 2013; Choudhari et al., 2013; Choudhari et al., 2012; Rasmussen, 2013) and serves a high demand from pharmaceutical sector (Kumar et al., 2012). Impact of anthropogenic influences on honey bees were already reported by Basavarajappa (2010). Recent studies also showed the various nesting behaviour of Trigona iridipennis Smith in natural habitat as well as its adaptability various antroropoegnic habitats (Virkar et al., 2014; Singh, 2013; Nair and Nair, 2001; Kumar et al., 2012; Jose and Thomas, 2012; Jose and Thomas, 2013). A wide range of variations especially worker and drone body size and measurement were reported (Pignata et al., 1996; Vijayakumar and Jayraj, 2013; Danaraddi, C. S., & Viraktamath, 2010; Danaraddi et al., 2012). Morphometric and geometric analysis provide a valuable tool for the discriminating variations among various honey bees and stingless bees (Vijayakumar and Jayraj, 2013; Wappler et al., 2012; Monteiro et al., 2002; Hernández et al., 2007; Francoy et al., 2011; Quezada-Euán et al., 2011; Hartfelder and Engels, 1992). These tools were sucessfuly utilized for the genetic lineage, geographic origin, heritability of shape, gender identification, differtiation and conservation (May-Itzá et al., 2012; de Jesús May-Itzá et al., 2010; Quezada-Euán et al., 2007; Francoy et al., 2011; Francoy et al., 2009; Hernández et al., 2007; Monteiro et al., 2002; Rattanawannee et al., 2010; Combey et al., 2013). Further possibilities of phylogentic relationships were explored using advanced molecular biology tools especially DNA microsatellites, AFLP markers, mitochondrial DNA and nuclear DNA (Arias and Sheppard, 2005; Francisco et al., 2008; Arias et al., 2006; Theeraapisakkun et al., 2010a; Theeraapisakkun et al., 2010b; Theeraapisakkun et al., 2011; May-Itzá et al., 2012). A little is reported so far about the morphometric studies of the Trigona iridipennis Smith as across various districts of Kerala. Based on these back ground, our objectives of this study were to 1) to characterize the morhometric aspects of drones and worker in Trigona iridipennis Smith 2) to identify the various similarities and differences existed among based on factor and principal component analysis.

2. Materials and Methods

2.1 Study area

Kerala state covers an area of 38,863 km2 with a population density of 859 per km2 and spread across 14 districts (Figure 2). The climate is characterized by tropical wet and dry with average annual rainfall amounts to 2,817 ± 406 mm and mean annual temperature is 26.8°C (averages from 1871-2005; Krishnakumar et al., 2009). Maximum rainfall occurs from June to September mainly due to South West Monsoon and temperatures are highest in May and November (Figure 3).

2.2 Study design and data collection

Twenty one different locations (panchayaths) were selected across the 14 districts in Kerala using information’s collected from various agricultural departments, farmers and initial baseline survey. A total of 188 and 71 samples of drones and workers collected respectively. The bees were caught using a self-designed net trap of size (200 µm) and the bees were immediately paralysed using chloroform solution (70%v/v) and later stored in eppendof vials with 70% alcohol. Non-destructive methods were followed using sample collection process. The drones were collected during aggressive swarming/mating swarming/from flower. The samples were labelled according to the location and stored in airtight boxes until morphometric analysis were conducted. The positions of the locations were recorded with the help of a Trimble Geoexplorer II GPS (Trimble Navigation Ltd, Sunnyvale, CA, USA).

2.2 Morphometric measurements

The bees are dried at 60°C for 24 h in a hot air oven (Labtech 112, lantech, Mumbai) to obtain their dry weight. After the weighing, the bees were rehydrated in glycerol solution (70%v/v) to soften their body. The head and thorax were dissected, and size of the following variables were recorded: head length (HL), head width (HW), proboscis length (PL), distance between two dorsal ocelli/lower intercocular distance (DBO), dorsal ocello-ocular distance (DOOD), antennal length (AL), thorax femur length (TFL), thorax tibia length (TTL), thorax metatarsus length (TML), thorax metatarsus width (TMW), thorax forewing length (TFWL), thorax forewing width (TFWW), pterostigma width (PtStW), pterostigma length (PtStW), hind wing length (HWL), hind wing width (HWW), number of hamuli (HAM), abdominal tergite length (AbTL), abdominal sternum width (AbSW), mandible length (MdL) and mandible width (MdW). Head length width ratio (HLW), thorax metatarsus length width ratio (TMLW) and thorax forewing length width ratio (TFWLW) were also determined. The morphometric measurements were made using steromicroscope (Leica E24D, Leica Microsystems, Switzerland) and analysed using LAS EZ version 1.4.0 (IBM Inc., CA, USA).

2.3 Statistical analysis

Descriptive statistics using SPSS 12.0 (SPSS Inc., Chicago, IL, USA) were conducted to summarize the data and graphs were generated using Sigma Plot 7 (Systat Software Inc., Chicago, IL, USA). The data were checked for normal distribution using Kologovic-Smiroff (KS) test. A comparison size metrics among the samples collected from different locations was conducted by means of Principal Component Analysis (PCA). Value scores for each worker and drone bees for each principal component were obtained from the first three components of PCA. Data were analysed by comparing the means at univariate level using ANOVA and multivariate approach (PCA). Colony scores from PCA were compared by means of ANOVA and plotted against pairs of factors.

A principal component analysis (PCA) was performed on the morphometric variables for the data reduction and obtain single measure of size (Wiley, 1981; Pignata and Diniz-Filho, 1996; Quezada-Euán et al., 2007). The PCA was carried out based on a correlation matrix (verified by means of Keiser-Meyer-Olkin measure of sampling adequacy and Bartlett’s test of sphericity) and later analysed using VARIMAX axis rotation. The correlation matrix was selected as the derived principal component 1. A final measure of body size was given by the PC scores that were calculated for each individual as the product of the resulting coefficients in each PC (Quezada-Euán et al., 2007).

3. Results

3.1 Descriptive analysis

The cropping pattern of the collected sites vary with a wide variety plantation crops were natural rubber dominates all the other crops (Figure 2). Majority of the samples were collected from wooden boxes followed by bamboo and earthen pots. The morhometric characters of workers and drones varied widely across all the districts (for workers) and selected districts for drones. Significant variations in the morphometric characters were observed for workers expect for HL; PL; ML (Figure 2 and 3). Highly significant difference (P< 0.001) were observed for HW; DBO; AL; TFL; TTL; TMW;ML; TFWL; TFWW; PtStW; HWL; HWW; AbTL and AbSW. In the case of the drone bees, highly significant differences were seen only for AL and ML (Figure 2 and 3).

The lowest HW among workers was 1.42 ± 0.05 (Kozikode) while highest up to 1.65 ± 0.12 (Kottayam). The lowest AL was seen in Kasargod (1.91 ± 0.05) and highest in Kottayam and Thiruvanathapuram (2.01 ± 0.06 and 0.07 respectively). The lowest MdL and MdW were 0.54 ± 0.05 and 0.18 ± 0.02 respectively. ANNOVA of morphometric characters among workers and drones across various districts in Kerala showed difference in statistical significance among the two castes. Even though numerical significance were observed in HL, PL and MdW but not able to prove their significance statistically.

In the case of drones, the lowest AL is in Kannur (2.17 ± 0.09) and highest in Kasargod (0.44 ± 0.00). Even though numeric significance were shown, most of the morpometric characteristics of drones except AL and MdL were statistically non significant.

3.2 Correlation analysis

Person correlation analysis showed considerable correlation among various morphometric characters in worker and drone bees across Kerala. Highly significant positive correlations were observed among HL and HW, MdL and HW, TFL and HW, TTL and HW, MdL and AL, TFL and AL, TTL and TFL, HWL and TML, PtStL and TMW, AbTL and TMW, AbSL and TMW, TFWW and TFWL, PtStW and TFWW, HWL and TFWW, HWW and TFWW, AbTL and TFWW, HWL and PtStW, AbSL and PtStL, HWW and HWL, and AbTL and HWL. In the case of drones, strong positive correlations were observed among following morphometric characters; AL, DBO, MdL, MdW, TTL, TFL, PtStW, TMW, TFWW, TFWL, HWW, AbTL, AbSL, PtStW and HWL. Despite of strong positive correlations, strong negative correlation was observed among TFL and MdL in the case of worker bees.

3.3 Principal component analysis

The validity PCA of 21 morphometric characters was justified as indicated by the Kaiser-Meyer-Olkin measure of sampling adequacy of 0.65. Bartlett’s test of sphericity was significant (<0.001), indicating that the matrix was equally distributed and thus, suitable for PCA. A PCA was performed using a correlation matrix was performed on selected 16 characters which seems to be significant in the study. The components were decided using scree plot and fixed to 5 and 4 components in workers and drones respectively. Eigenevalues and percentage of variance were considered only for the above mentioned components as they include the largest variation in the sample. The first three PCs explained 56.1, 12.2 and 12.5% of the total variation in the morphometric data respectively.

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Figure 1. Morphometric characters considered for the analysis of worker and drone bees of stingless bees in Kerala: 1 = head length, 2 = head width, 3 = proboscis length, 4 = distance between two dorsoal ocelli, 5 = dorsal ocello-ocular distance, 6 = antennal length, 7 = fermur length, 8 = tibia length, 9 = metatarsus length, 10 = metatarsus width, 11 = forewing length, 12 = forewing width, 13 = pterostigma length, 14 = pterostigma width, 15 = hind wing length, 16 = hind wing width, 17= number of hamuli, 18 = tergite length, 19 = sternite length, 20 = width of sternum, 21 = mandible length, 22 = mandible width.

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Figure 2. Map of Kerala showing various sample collection points marked as a star symbol during the study.

Table 1. Description of the morphometric variables used to classify Trigona iridipennis Smith in Kerala, India.

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Table 2. Site description of the selected farms (W1-W21) across Kerala, India for collecting worker bees during 2011-2013.

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Table 3. Site description of the selected farms (D1-D9) across Kerala, India for collecting drone bees during 2011-2013.

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Table 4. Description of Trigona iridipennis Smith worker and their morphometric characteristics.

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Numbers represent means ± one standard deviation (SD) of the mean.

Table 5. Description of some morphometric of Trigona iridipennis Smith worker and their characteristics.

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Numbers represent means ± one standard deviation (SD) of the mean.

Table 6. Description of the various habitat of Trigona iridipennis Smith drone and their characteristics.

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Numbers represent means ± one standard deviation (SD) of the mean. * Indicate no statistics conducted

Table 7. Description of the various habitat of Trigona iridipennis Smith drone and their characteristics.

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Numbers represent means ± one standard deviation (SD) of the mean. * Indicate no statistics conducted

Table 8. Pearson correlation coefficients between morphometric characters of Trigona iridipennis Smith workers (n=84) in the various districts across Kerala, India during 2011-2013.

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*Significant at the level of P< 0.05.

**Significant at the level of P< 0.01.

Table 9. Pearson correlation coefficients between remaining morphometric characters of Trigona iridipennis Smith workers (n=84) in the various districts across Kerala, India during 2011-2013.

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*Significant at the level of P< 0.05.

**Significant at the level of P< 0.01.

Table 10. Pearson correlation coefficients between morphometric characters of Trigona iridipennis Smith drones (n=71) in the various districts across Kerala, India during 2011-2013.

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*Significant at the level of P< 0.05.

**Significant at the level of P< 0.01.

Table 11. Pearson correlation coefficients between remaining morphometric characters of Trigona iridipennis Smith drones (n=71) in the various districts across Kerala, India during 2011-2013.

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*Significant at the level of P< 0.05.

**Significant at the level of P< 0.01.

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Figure 3. a) stingless bee hive showing pollen, honey and eggs; b) bee hive made of wooden box; c) bee hive kept on earthen pots; d) entrance tube with fresh resin on a stone wall; e) stingless bee drone (top) and worker (bottom).

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Figure 4. a) stingless bee worker foraging on Euphorbia flower; b) antenna drone (left) and worker (right); c) pterostigma; d) mandible drone (left) and worker (right).

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Figure 5. a) stingless bee brood; b) worker tongue; c) head of drone (top) and worker (bottom); d) worker fore-wing and hind wing; e) worker bee with pollen basket.

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Figure 6. Schematic representation of head length width ratio (HLW), thorax metatarsus length with ratio (TMLW) and thorax forewing length width ratio (TFWLW) among stingless bee workers across different districts in Kerala, India.

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Figure 7. Schematic representation of head length width ratio (HLW), thorax metatarsus length with ratio (TMLW) and thorax forewing length width ratio (TFWLW) among stingless bee drones across different districts in Kerala, India.

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