Abstracts

INTRODUCTION

The Stingless Bee (Hymenoptera: Meliponini) Tetragonisca angustula (Latreille, 1811) defends its nests with resins collected from different plants (Armbruster 1984Armbruster WS. 1984. The role of resin in angiosperm pollination: Ecological and chemical considerations. Am J Bot 71: 1149-1160., Marsaioli et al. 1999Marsaioli AJ, Porto ALM, Gonçalves RAC, Oliveira CMA, Manfio GP and Bittrich V. 1999. The Ecosystem of Microorganisms, Bees, and Clusia Floral Resin and Oils, from the Chemistry Point of View. 2nd IUPAC Conference on Biodiversity.) which are stored in deposits within their nests (Roubik 1989Roubik DW. 1989. Ecology and natural history of tropical bees. New York: Cambridge University Press, 514 p., Nogueira-Neto 1997Nogueira-Neto P. 1997. Vida e criação de abelhas indígenas sem ferrão. São Paulo: Editora Tecnapis, 445 p.). When intruders like the ant Camponotus sericeiventris (Guérin, 1834, Hymenoptera: Formicinae) enter the nest, worker bees form small pebbles of resin from these deposits and stick them onto the intruder's extremities until legs and antennas are tied up causing immobilization (Radtke 1994Radtke R. 1994. Die kleptoparasitische neotropische Biene Lestrimelitta limao: Verhalten und chemische Kommu-nikation bei Raubzügen auf Nester anderer Stachellosen Bienen. Ph.D. thesis, University of Tübingen, 172 p.). These pebbles are formed and transported by the bee's mandibles (Gastauer et al. 2011Gastauer MM, Campos LAO and Wittmann D. 2011. Handling sticky resin by Stingless Bees. Rev Bras Ent 55: 234-240.).

It has been observed that the resin, once attached on the intruder's extremities, clings very tight to its cuticle, while it is removed easily and without residues from the bee's mandible (Gastauer et al. 2011Gastauer MM, Campos LAO and Wittmann D. 2011. Handling sticky resin by Stingless Bees. Rev Bras Ent 55: 234-240.). Resin seems to stick lesser to the bee's mandible than to other insect's surfaces.

The aim of this paper is to test the hypothesis that resin sticks lesser to the bee's mandible than to the extremities of typical intruders because of specialized surface structure or composition. The mandible of T. angustula and the trochanter of the hind leg of C. sericeiventris have been selected as representative surfaces, on which structure and adhesive properties were analyzed.

MATERIALS AND METHODS

Scanning Electron Microscopy (SEM)

Mandibles of T. angustula as well as the trochanter of the hind leg of C. sericeiventris have been examined with the scanning electron microscope Hitachi S-2460, Japan, at voltages varying between 20 and 25 kV.

Individuals of both species had been killed by means of acetic ether. The body parts used for analysis had been dissected and immediately cleaned in a solution of dish washing liquid, kept in alcohol 70% for five days, then dehydrated in an ascending alcohol series. Afterwards the samples were transferred into acetone and subsequently cleaned for 10 minutes in a bath of ultrasound (Bandolin Sonorex). The samples were mounted on pin stubs and sputtered with a 40 nm layer of gold.

Vouchers of both species have been incorporated in the collections of the Entomological Museum of the Universidade Federal de Viçosa (UFV-B).

Adhesion Force

To measure the adhesive properties of the cuticles, these were mounted to fine, pulled-out glass pins (P₁) being withdrawn from a similar glass pin P₂ covered with resin (Figure 1). The adhesive properties were measured as forces necessary to provoke adhesive failure. These forces are called adhesive forces in the following.

Production of Glass Pins

The pins P₁ and P₂ were produced from glass bar fragments (diameter 1 mm, length 20 cm). With the Puller P-97 from Sutter Instruments Co the midpoint of the bars was heated up to 560°C. With an acceleration of 110 m/s², the pins were stretched; the resulting diameters of the pins' tips ranged from 1 and 10 µm. For pin types P₁, diameters of 3 to 4 µm were selected, types P₂ of 10 µm.

Fixation of the Cuticle Sample on P₁

For analyzing the adhesive properties of the cuticles of the trochanter of C. sericeiventris and the mandible of T. angustula, the specimen were captured, knocked out with CO₂ and dissected immediately.

All samples were fixed to the tip of P₁ with warmed dental wax (65°C). P₁ was mounted on the micromanipulator (MM in Figure 1) and resin was installed on P₂ immediately.

Installation of Resin on P₂

The dichloromethane cleaned P₂ was dipped 2 - 3 mm into a mass of 2 g resin collected from a deposit of a colony of T. angustula. When the tip of P₂ was covered completely by a drop of resin (length: 20-50 µm, width: 10-20 µm, see Figure 2), it was clipped perpendicular to P₁ on the object table of the microscope MC (Figure 1). The behavior of the attached resin is described in Figure 2.

Measurement

All treatments and measurements with resin were conducted at 19°C.

The adhesion forces were measured at the moment, when P₂ was covered with only a thin, but still complete layer of resin (Figure 2, middle). P₁ was slowly moved upwards to P₂ until the cuticle sample touched the resin on P₂. The two glass pins were kept in this position for 5 seconds so that resin and cuticle contacted each other. Then P₁ was slowly withdrawn from P₂ with a constant speed of 62.5 µm/s (corresponding to one turn of the micromanipulator's fine adjustment), until resin and cuticle disconnected. The distance s (Figure 1) between position of contact and position of separation describes the deformation of P₁ and depends on the stiffness of P₁ as well as the adhesion forces between resin and cuticle.

Calibration of P₁

The adhesion forces were calculated from the stiffness of P₁. Each P₁ that was used for measurement was carefully pressed with the micromanipulator onto a digital balance in steps of 62.5 µm. Step by step the weight indicated by the balance was recorded.

Calculation of the Adhesive Properties

The adhesion forces F_A is given by the product of mean distance s between position of contact and position of separation and the stiffness c of P₁.

Statistical Evaluation

To proof the results statistically, the non-parametric Mann-Whitney Test has been used. Probability values lesser than 1% were considered as highly significant.

RESULTS

The cutting edge of the mandible of workers of T. angustula is composed of a large blade and a pointed tooth. The surface of these structures are smooth (Figure 3) without microstructures. On several mandibles we found distinct traces of use like scratches or broken edges. On the other hand, the trochanter of the ant C. sericeiventris is scaled (Figure 4).

Calibration trials of all P₁ show linear relation (R² ≥ 0.998) between deformation in µm of P₁ and weight in µg indicated by the balance (Figure 5), so that adhesion forces had been interpolated by linear regression.

During measurement of the adhesion forces, hydrophobic interaction has been observed between resin and the tested surfaces. The resin attached on P₂ attracts the tested cuticles and the cover slip on P₁ with a jerk as soon as it reaches a critical distance somewhere between 1.5 µm and 1 µm.

Figure 6 shows that adhesion of resin on the structured cuticle of the trochanter of C. sericeiventris is six times smaller than on the smooth cuticle of the tip of the mandible of T. angustula. These differences are highly significant using the Mann-Whitney-Test for independent samples.

DISCUSSION

The results of the adhesion force measurement refute our hypothesis that resin sticks lesser to the bees' mandible than to other insects' surfaces – even if the contact area between resinous and insect's surface in experiment is smaller than in reality.

Adhesion on rough surfaces like the scaled trochanter of C. sericeiventris is lower than on smooth ones like the mandible of T. angstula (Kendall 2001Kendall K. 2001. Molecular Adhesion and Its Applications: The Sticky Universe. New York, Boston, Dordrecht, London, Moscow: Kluwer Academic, 440 p.). Major contact areas covering hairs and various scales on the ant's trochanter surface, will reduce adhesion further. As such adhesion reducing structures are lacking on the bee's mandible, resin should stick more on the bee's mandible than on the trochanter of the ant when contact area is increased.

However, these findings do not explain why resin, once attached to the mandible of T. angustula, is removed easily and without leaving residues behind. To manage that, the bee should possess mechanisms to reduce adhesion of resin on the mandible. T. angustula might change the adhesive properties temporarily by lubricating their mandibles with adhesion reducing substances. Lubricating substances seem to be common within insects. The body surface of the mirid bug Pameridea roridulae (Reuter 1907) is covered by a lipid layer avoiding the bug getting stuck in the resinous surface of its host plant (Voigt and Gorb 2008Voigt D and Gorb S. 2008. An insect trap as habitat: cohesion-failure mechanism prevents adhesion of Pameridea roridulae bugs to the sticky surface of the plant Roridula gorgonias. J Exp Biol 211: 2647-2657.). In stingless bees such lubricating substances might be secretions of the mandibular, salivary or other cephalic glands as proposed by Santos et al. (2009)Santos CGA, Megiolaro FL, Serrão JE and Blochtein B. 2009. Morphology of the Head Salivary and Intramandibular Glands of the Stingless Bee Plebeia emerina (Hymenoptera: Meliponini) Workers Associated with Propolis. Ann Ent Soc Am 102: 137-143.. As production of secretions is a complex and energy consuming processes, worker bees might even regurgitate nectar from the nectar crop. The liquid could run down the ventral side of the mandible via a small groove as illustrated in Figure 7. Once distributed, it reduces the adhesion of resin on the complete mandible.

This groove on the base of the ventral side of the mandible, also observed in other stingless bee species (Figure 8, Stort et al. 1986Stort AC, Bueno de Moraes MM and Barelli N. 1986. Scanning electron microscopy observations of the mandibles of Scaptotrigona postica workers (Hymenoptera, Apoidea). J Apic Res 25: 65-70.) and the honey bee Apis mellifera, is discussed by Goodman (2003)Goodman J. 2003. Form and function in the Honey Bee. Cardiff: International Bee Research Association, 220 p. as a feature to channel liquids to the tips of the mandibles. The groove might be an adaptation of fsecbees to alter adhesive properties of the mandibles in order to form, cut and knead resins without sticking to them.

Further investigations have to test, if adhesion of resin is reduced on mandibles greased with nectar or honey. To prove our hypothesis, chemical research activities have to attest the existence of residues of the adhesion reducing substances like honey, nectar or sugar in resins treated by Stingless Bees.

Thanks to Bernd Hoffmann and Wolfgang Rubner from the Institute of Bio- and Nanosystems, Institute 4 (Biological Layers), Research Center Jülich, Germany, for their help in developing a transportable apparatus to measure adhesion forces. We are grateful to Karin Ulmen from the Museum König in Bonn, Germany, to grant access to the SEM. Some parts of this research were supported by the DFG and the DAAD.

REFERENCES

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