HIGH-PERFORMANCE GEL FILTRATION PATTERNS OF VENOMS FROM THREE SPECIES OF WASPS OF THE GENUS Polybia (HYMENOPTERA, VESPIDAE)

Original paper

HIGH-PERFORMANCE GEL FILTRATION PATTERNS OF VENOMS FROM THREE SPECIES OF WASPS OF THE GENUS Polybia (HYMENOPTERA, VESPIDAE).

M. S. PALMA , M. F. MANZOLI-PALMA , N. GOBBI ²

1 Department of Biology, Institute of Biosciences of Rio Claro, State of São Paulo, Brazil, 2 Department of Ecology, Institute of Biosciences of Rio Claro, State of São Paulo, Brazil.

ABSTRACT. The profiles of high-performance gel filtration of venoms from Polybia paulista, Polybia ignobilis and Polybia occidentalis occidentalis showed 13 peaks distributed among the three species. These profiles presented similarities that permitted the chromatographic characterization of the genus Polybia and differences that permitted the identification of each species studied. Thus, the comparative analysis of chromatographic profiles of high-performance gel filtration of venoms may be used as an auxiliary tool in taxonomic studies of Polybia wasps.

KEY WORDS: vespidae, chromatographic, sac of venom, Polybia.

INTRODUCTION

Wasp venoms have been characterized by repeated cycles of conventional chromatographic and electrophoretic procedures(1,2,6). These studies concentrated on wasps of the genera Dolichovespula, Vespula and Polistes. Due to the difficulty in obtaining venoms from vespid wasps in sufficient quantity to carry out the conventional techniques, it is not easy to purify and characterize the venom components(3,4,6). Thus, there is little information available about most wasp venoms(5). In addition, it is a hard task to detect and quantify the small peptidic components by the use of classical experimental procedures. In this work, we describe the fractionation of venoms from three species of Polybia wasps by high-performance gel filtration. The results shown in this paper suggest that Polybia venoms are a complex mixture of high-molecular polipetide and small peptidic components.

MATERIAL AND METHODS

P. paulista, P. ignobilis and P. o. occidentalis were collected in Rio Claro, SP, southeast of Brazil.

Five workers of each species were captured from three different nests, dissected and the sting apparatus removed. The venom sacs were cut off, suspended in 0.1M sodium phosphate, pH 6.6, containing 0.1% SDS and washed vigorously by stirring at 40ºC for 1 min. The extract was centrifuged at 10,000g and the debris discarded. The supernatant was submitted to high-pressure gel filtration. The analysis for each nest was performed separately.

The system used for chromatographic analysis included a CG model 480 liquid chromatograph equipped with a Reodhyne model 7125 sample injector, a Brownlee Aquapore Sec OH-300, 10µ, gel filtration column (25cm x 4.6mm I.D.) preceded by a precolumn (4cm x 2mm I.D.) of the same packing material. A CG model 350 variable wavelength detector and a CG model 300 reenster integrator were also used. The elution was performed at 30ºC using 0.1M sodium phosphate, pH 6.6, containing 0.1% SDS (w/v) as mobile phase at a flow rate of 500µl/min. The detection was performed by monitoring the column eluate at 210nm.

A calibration curve was constructed using the following molecular weight markers: alcohol dehydrogenase (MW 140,000) bovine serum albumin (MW 67,000), albumin chicken egg (MW 43,000), myoglobine (MW 16,900), cytochrome C (MW 12,400), aprotinin (MW 6,500) and bacitracin (MW 1,800).

RESULTS AND DISCUSSION

The high-performance gel filtration patterns of venoms from the three species of Polybia wasps (Figure 1) showed 13 different peaks with MW from 800 to 140,000 daltons. The profiles showed five peaks common to the three species suggesting that these may be characteristic of the genus Polybia: peaks 1, 5, 8, 12 and 13 (MW 140,000, 40,000, 19,000, 950 and 800 respectively). Peaks 4 and 7 (MW 48,000 and 23,000 respectively) characterized the venom from P. o. occidentalis; peak 2 (MW 55,000) characterized P. ignobilis, while P. paulista presented one characteristic peak (No 11; MW 4,500). In addition to this, both P. paulista and P. ignobilis presented peaks 3 and 6 (MW 51,000 and 38,000 respectively) in common; P. paulista and P. o. occidentalis presented peak 9 (MW 12,000) in common; P. o. occidentalis and P. ignobilis presented peak 10 (MW 8,500) in common.

FIGURE 1.
Profiles of high-performance gel permeation of venoms of different species of Polybia wasps on Aquapore SEC OH-300 column (25cm x 4.6mm I.D.), 10 preceded by a pre column (4cm x 2mm I.D.) of the same packing material. The elution was performed using 0.1M sodium phosphate buffer, pH 6.6, containing 0.1% SDS (m/v) as mobile phase at a flow rate of 500µl/min at 30ºC.

The reduced quantity of proteins make the isolation of venom components relatively difficult. In addition, the collection of pure venom is laborious and generally requires the manipulation of a large number of insects. The electrical milking is frequentely accompained by digestive and/or fecal material(3) and commercial venom sacs present great contamination from the sac tissue(4 ) In order to avoid contamination, we used dissection followed by washing of sacs. The presence of SDS in the extraction buffer improved the solubilization of high-molecular-weight material.

The use of HPLC in analytical scale permitted the manipulation of a reduced number of individuals. The high-performance gel filtration with a column packed with silica spheres modified with glyceryl propyl silanes permitted both the use of high salt concentrations and ionic detergent (SDS), improving the resolution of fractionation.

The general pattern of high-performance gel permeation profiles of the venoms show the possibility of the chromatographic characterization of the genus Polybia and the identification of the 3 species investigated. Although this procedure should be extended to other species of this genus, the comparative analyses of profiles could be used as an auxiliary tool in future taxonomic studies of wasps.

06 WOOD CL., HOFFMAN DR. Two-dimensional polyacrylamide gel electrophoresis of Hymenoptera venom and venom sac extracts. Toxicon., 1983, 21, 291-9.

• 01 ABE T., KAWAI N., NIWA A. Purification and properties of presynaptically acting neurotoxin, mandarotoxin from hornet (Vespa mandarina). Biochemistry, 1982, 24, 1693-7.
• 02 EINARSSON R., RENCK B. Ion-exchange chromatographic characterization of stinging insect Vespid venoms. Toxicon, 1984, 22, 154-60.
• 03 HOFFMAN DR. Allergens in Hymenoptera venom XII: Isolation and purification of protein components from three species of vespid venoms. J. Allergy Clin. Immunol., 1985, 5, 599-605
• 04 HOFFMAN DR., WOOD CL. Allergens in Hymenoptera venom XI: Isolation of protein allergens from Vespula maculifrons (yellow jacket) venom. J. Allergy Clin. Immunol., 1984, 74, 93-103.
• 05 NAKAJIMA T. Pharmacological biochemistry of vespid venoms. In: PIEK T. Ed. Venoms of the Hymenoptera - Biochemical, pharmacological and behavioral aspects London: Academic Press, 1986, 309-27.
• 06 WOOD CL., HOFFMAN DR. Two-dimensional polyacrylamide gel electrophoresis of Hymenoptera venom and venom sac extracts. Toxicon., 1983, 21, 291-9.  

CORRESPONDENCE TO:

N. GOBBI - Departamento de Ecologia do Instituto de Biociências, Caixa Postal 199, CEP 13506-900, Rio Claro, São Paulo, Brasil.

Publication Dates

• Publication in this collection
  08 Jan 1999
• Date of issue
  1996