Research Article |
Corresponding author: Leopoldo Cruz-López ( lcruz@ecosur.mx ) Academic editor: Christopher K. Starr
© 2023 Karen Espadas-Pinacho, Julieta Grajales-Conesa, Julio C. Rojas, Leopoldo Cruz-López.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Espadas-Pinacho K, Grajales-Conesa J, Rojas JC, Cruz-López L (2023) Melipona beecheii (Hymenoptera, Apidae) foragers deposit a chemical mark on food to attract conspecifics. Journal of Hymenoptera Research 96: 155-166. https://doi.org/10.3897/jhr.96.98127
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Stingless bees have a sophisticated system of chemical communication that helps conspecifics find food sources. In this study, we investigated whether Melipona beecheii foragers deposit a chemical mark on food to recruit conspecifics. Our results showed that foragers preferred to visit the feeders visited previously by conspecifics over clean feeders. We also found that foragers preferred visiting feeders baited with labial gland extracts over those baited with mandibular extracts or hexane. Labial gland extracts elicited higher forager antennal responses compared with those evoked by the mandibular gland extracts or hexane. Labial gland extracts and extracts from feeders visited by foragers contain a mixture of unsaturated hydrocarbons, followed by straight chain hydrocarbons and small quantities of esters. The main component is a mixture of alkene isomers C27:1.
CG-MS, cuticular hydrocarbons, labial gland, stingless bees
Eusocial bees, such as Apis mellifera and stingless bees, use communication mechanisms such as vision, smell for food search and mechanical signals (Dyer 2002;
A typical method of communication happens when stingless bee foraging workers arrive at their nest with food; then they release chemical signals to stimulate other workers to go out to the field in search of food sources (
We used five M. beecheii (Hymenoptera, Apidae, Meliponini) colonies obtained from a meliponary in Tuxtla Chico (14°56'25"N, 92°10'08"W), Chiapas, Mexico. The experiments were conducted from April to October 2021 at El Colegio de la Frontera Sur gardens, Tapachula (14°54'39.86"N, 92°15'51.55"W), Chiapas, Mexico. Annual rainfall in this region is approximately 3843 mm, with September as the wettest month and February as the driest month. The temperature normally ranges between 29 °C and 35 °C. The colonies were free of fungi and parasites.
The forager bees were trained to collect a solution of 3 M sucrose ad libitum from an artificial feeder, consisting of a Petri dish (100 × 10 mm) containing a small cotton ball drenched in the sucrose solution in the center. The feeder was placed 5 m from the beehives. The training was conducted between 08:00 and 13:00 h.
We extracted the compounds deposited by M. beecheii foragers on a glass feeder (100 × 10 mm) (
To prepare the gland extracts, we captured foragers bees that arrived at the feeder during training. Bees were frozen at -20 °C before dissection and analysis. The glands of the foragers were carefully dissected in distilled water with two pairs of fine tweezers under a stereoscopic microscope. The gland extracts were prepared by carefully dissecting the labial and mandibular glands of 10 foragers in 1000 μL of solution. So, 100 μL of the prepared solution is the amount corresponding to 1 labial gland equivalent (LGE), 50 μL of solution corresponds to 0.5 LGE and 10 μL to 0.1 LGE; and 100 μL of the prepared solution is the amount corresponding to 1 mandibular gland equivalent (MGE), 50 μL of solution corresponds to 0.5 MGE and 10 μL to 0.1 MGE. Five gland extracts were prepared.
In a first experiment, we evaluated whether the forager bees leave odor marks on the feeders in a two-choice tests. We offered to foragers two feeders: one with chemical marks (previously visited by foragers) and the other clean (not visited by foragers). The feeders were placed at the site where the bees were trained. Feeders were placed 30 cm from each other. We placed a few drops of the 3M sucrose solution in the entrance to stimulate the visits. We recorded the number of bees that visited the marked feeder and the clean feeder. A visit was counted when the bee landed and extended its proboscis to feed. All bees were marked with a fine brush with acrylic paint not toxic and captured on their first visit to avoid counting the same bee more than once (
In a second experiment, we assessed the effect of the labial gland extracts on food searching in two choice tests. Here, using pieces of 1cm2 filter paper placed on feeders, one feeder was sprinkled with 10 μL of labial gland extract at the beginning of the experiment and the other feeder was sprinkled with 10 μL of hexane as a control. The gland extracts were evaluated at 0.1, 0.5, and 1 LGE. In total, 10 replications were performed for each gland equivalent extract.
In a third experiment, we evaluated foragers preference for labial gland extract, mandibular gland extract, or solvent in three-choice tests. Three feeders were placed in the training site: control (hexane), labial gland extract, and mandibular gland extract. The feeders were placed 20 cm from each other. Care was taken not to count bees that visited the feeder while other bees were there to avoid the phenomenon of social facilitation. The position of the feeders was interchanged every 5 min to avoid position bias. The gland extracts were evaluated at 0.1, 0.5, and 1 LGE or MGE. In total, 10 replications were performed for each gland equivalent extract.
We collected forager bees from three established colonies, bees were frozen for one minute to numb them before dissection, subsequently their antennae were carefully removed. The base of the antenna was inserted into the tip of the glass capillary logging electrode. The signals generated by the antenna passed through a high impedance amplifier (NL 1200; Syntech, GmbH) and visualized with the software Syntech to process the EAG signals. We used a stimulus Flow controller (CS-05, Syntech) to generate stimuli at intervals of one minute. A constant current of pure humidified air (0.7 L min-1) was directed toward the antenna (
The experimental procedure consisted of depositing the treatment (1 LGE extract, 1 MGE extracts or solvent, in this order) onto 1.5 × 1.5 mm pieces of filter paper (Whatman no. 1, Whatman, Maidstone, England) exposed to air for 20 s to allow the solvent to evaporate, inserted into a glass Pasteur pipette, and left for 40 s before applying. The application of the stimulus consisted of inserting the tip of the pipette that contained the piece of filter paper in a hole at the end of the glass tube through which the current of air blew continuously on the antenna. The waiting time between one stimulus and the next was one minute. The treatment was carried from the filter paper to the antenna on the controlled air current (0.5 L/min). The duration of the stimulus was 1 s. The continuous flow of pure air was maintained through the tube to assure that the odors were removed immediately. We used one antenna of the bee per treatment, and at least thirty five bees were used.
Extracts were analyzed in a gas chromatograph (Shimadzu GC-2010 Plus) coupled with a quadrupole mass spectrometer (Shimadzu, TQ8040), using a capillary column of non-polar silica SPB-1 (30 m long × 0.25 mm interior diameter) (Supelco, Toluca, Mexico). The initial temperature was 50 °C (held for 2 min), increased 15 °C/min up to 280 °C (held 10 min). Helium was the carrier gas and the injector temperature was 250 °C. Ionization was achieved by electron impact at 70 eV. The compounds were identified by comparison with those registered in the NIST 2014 library (software GCMS-solution), the retention index, the mass spectra, and retention times of available synthetic standards. The relative percentage of the components was calculated from the sum of the recorded peaks.
We evaluated the mixture of synthetic compounds with some compounds identified in the labial gland secretion. The synthetic blend evaluated was prepared in accord with the natural proportions of the M. beecheii labial gland using hexane as solvent. The compounds evaluated were heneicosane (100 ng), tricosane (100 ng), and pentacosane (25 ng). These compounds were chosen as they were available in supplies. Unfortunately, C27:1 isomers the main components in the labial gland secretion were not commercially available. We recorded the number of bees that visited a feeder baited with the synthetic blend, or a feeder with solvent. The feeders were placed where the bees were trained to visit. The distance between the feeders was 30 cm. The position of the feeders was changed every 5 min to avoid position bias. In total, 10 replications were performed in this experiment.
All data were analyzed using R software (
M. beecheii foragers preferred the feeders visited previously by their conspecifics over the clean feeder (χ2 = 56.783, df = 1, p < 0.001) (Fig.
In the two-choice bioassays, foragers preferred to visit the feeders marked with extracts of 0.5 LGE (χ2 = 134.38, df = 1, p < 0.001) and 1 LGE (χ2 = 71.676, df = 1, p < 0.001) over those treated with hexane. However, they did not show a preference for the 0.1 LGE or the control (χ2 = 0.53236, df = 1, p = 0.4656) (Fig.
In the three-choice bioassays, more foragers preferred visiting feeders with labial gland extracts (0.1 LGE: χ2= 13.335, df = 2, p<0.01; 0.5 LGE: χ2= 81.747, df = 2, p < 0.001; 1 LGE: χ2 = 23.929, df = 2, p < 0.001) over other feeders with mandibular extracts or the control (Fig.
Mean (± SEM) responses of M. beecheii foragers to labial gland and mandibular gland extracts at different concentrations. Different small letters (P < 0.05) indicate significant differences between treatments. Ten replications were performed for each gland equivalent extract. LGE=labial gland equivalent, MGE=mandibular gland equivalent, C=control.
Analysis of EAG data revealed significant differences in the antennal response of forager bees to the different extracts evaluated and to the solvent (control) (F = 13.24, df = 2, P > 0.001). The antennal responses of the foragers was greater with 1 eq/µL of labial gland extract than with the mandibular gland extract or with the control (Fig.
Electroantennographic (EAG) response in mV of M. beecheii forager bee antennae to labial gland extract, mandibular gland extract and the control (C). Different small letters (P < 0.05) indicate significant differences between treatments. N=35. LGE=labial gland equivalent, MGE=mandibular gland equivalent, C=control.
Chemical analysis showed that the labial gland extracts and extracts from feeders visited by foragers contain a mixture of unsaturated hydrocarbons, followed by straight chain hydrocarbons and small quantities of esters. The main components are a mixture of alkene isomers C27:1 (Table
Average value (%) ± standard error of the proportion of the compounds found in the labial glands of M. beecheii forager bees. N=Five gland extracts.
Peak | RT | RI | Compound | Proportion in labial gland | Proportion on feeder |
---|---|---|---|---|---|
1 | 13.53 | 1778.50 | Alcohol | 2.07±0.40 | ND |
2 | 15.57 | 2100.00 | Heneicosane (C21)* | 0.26±0.15 | ND |
3 | 15.85 | 2124.75 | Methyl stearate * | 0.4±0.07 | ND |
4 | 15.97 | 2143.48 | Geranyl palmitate ** | 2.79±0.48 | ND |
5 | 16.11 | 2166.93 | 2,3-Dihydro farnesyl hexanoate** | 1.89±0.31 | ND |
6 | 16.25 | 2189.90 | Unknown 1 | 1.28±0.22 | ND |
7 | 16.33 | 2202.45 | Farnesyl butanoate** | 1.88±0.30 | ND |
8 | 16.74 | 2274.56 | Alkene C23:1 (1) | 0.63±0.26 | 0.81±0.47 |
9 | 16.78 | 2281.22 | Alkene C23:1 (2) | 0.61±0.18 | 0.12±0.07 |
10 | 16.88 | 2300.00 | Tricosane (C23)* | 0.26±0.07 | 0.30±0.18 |
11 | 17.90 | 2474.00 | Alkene C25:1 (1) | 7.09±0.60 | 0.74±0.42 |
12 | 17.94 | 2481.16 | Alkene C25:1 (2) | 2.17±0.20 | 0.19±0.11 |
13 | 18.04 | 2500.00 | Pentacosane (C25)* | 0.6±0.05 | 0.24±0.14 |
14 | 18.20 | 2570.25 | Unknown 2 | 3.37±0.75 | ND |
15 | 19.29 | 2672.08 | Alkene C27:1 (1) | 8.33±0.87 | 1.21±0.70 |
16 | 19.33 | 2684.35 | Alkene C27:1 (2) | 8.05±0.81 | 1.38±0.80 |
17 | 19.39 | 2698.00 | Alkene C27:1 (3) | 11.2±1.10 | 1.37±0.79 |
18 | 19.51 | 2700.00 | Heptacosane (C27)* | 2.89±0.27 | 5.52±3.19 |
19 | 21.07 | 2853.12 | Alkene C29:1 (1) | 3.95±0.34 | ND |
20 | 21.14 | 2875.78 | Alkene C29:1 (2) | 1.92±0.17 | 0.61±0.35 |
21 | 21.34 | 2883.39 | Alkene C29:1 (3) | 5.52±0.58 | 3.02±1.74 |
22 | 21.43 | 2897.22 | Alkene C29:1 (4) | 7.93±0.96 | 1.06±0.61 |
23 | 21.58 | 2900.00 | Nonacosane (C29)* | 1.23±0.14 | 1.21±0.70 |
24 | 23.86 | 3065.26 | Unknown 3 | 2.81±2.13 | ND |
25 | 24.05 | 3076.78 | Alkene C31:1 (1) | 0.95±1.76 | 0.08±0.5 |
26 | 24.19 | 3089.48 | Alkene C31:1 (2) | 1.01±0.12 | 0.60±0.35 |
27 | 24.35 | 3096.23 | Alkene C31:1 (3) | 1.92±0.21 | 1.14±0.66 |
The chromatographic profile of the hexane feeder wash was similar to the labial gland extract profile, but different from that of the mandibular gland extract (Fig.
Foragers did not show a preference for the feeders treated with a three-component blend and those treated with hexane.
In this study, we demonstrated that M. beecheii foragers preferred to visit feeders that had been previously visited by their conspecifics over a clean feeder. This behavior has been observed in several species of the genus Melipona, such as M. favosa (
Interestingly, during the analysis of the labial gland extracts (data no shown) we found a group of samples with the same composition but in different proportions. According to the literature, M. beecheii bees first fly at the age of 33 days, probably to orient themselves in the environment; the first foraging flight is at 40-days-old, although at 20 days of age, a constant proportion of bees leave the hive to feed (
When the mandibular gland extract was evaluated, foragers behavior was more aggressive. Bee antennae responded to the labial and mandibular gland extracts similarly. The responses to the labial gland were stronger, while responses to the mandibular gland were weak, but stronger than the control. The M. beecheii mandibular gland possesses rose oxide isomers, which cause high levels of defense behavior, as do geraniol and farnesyl acetate that, when used at levels similar to those of the mandibular extract, cause more pronounced defense reactions than the rose oxide isomer (
In sum, our results indicate that M. beecheii foragers prefer to visit feeders have been previously visited by their conspecifics. Labial gland secretion may contribute to chemicals left behind by foraging bees at food sources, and more sophisticated analyses are needed to come to a definite conclusion. The secretion found at feeding sites is composed of a mixture of unsaturated hydrocarbons, straight chain hydrocarbons, and small quantities of esters. The main components are a mixture of alkene C27:1 isomers. Further studies are needed to identify and synthetize if required the compounds used by M. beecheii foragers to recruit conspecifics toward the food resources.
KEP thanks the Consejo Nacional de Ciencia y Tecnología (CONACYT, Mexico) for postgraduate scholarship.