POLLEN GRAINS OF SOME POISONOUS BEE PLANTS OF ETHIOPIA

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1 POLLEN GRAINS OF SOME POISONOUS BEE PLANTS OF ETHIOPIA A. Nuru 1, H.R. Hepburn 2 1 Holetta Bee Research Centre P. O. Box 17, Tel , Ethiopia, current address: Oakdene house, private Bag 1025, Rhodes University, Grahamstown 6140 South Africa gooa3639@cumpus.ru.za Tel Department of Zoology & Entomology, Rhodes University, Grahamstown 6140, South Africa. R.Hepburn@ru.ac.za Tel Abstract Several cases of poisoning of people and bees due to honey from poisonous plants have been reported. In this work an attempt was made to analyse the pollen grains of plants, which are reported as poisonous to bees and honeys from such plants poisonous to humans. Samples of ripe pollen grains were collected from the field and herbarium specimens and prepared using Louveaux et al. (1978) protocols and sputter coating techniques for light and scanning electron microscopy observations respectively. Important morphological features like symmetry, form, size, shape, aperture (type, number and diameter), surface pattern, sexine and exine thickness were examined and described using both light and scanning electron microscopy. The average sizes of the pollen grains were determined by measuring 20 pollen grains each for polar axis and equatorial diameters. The photographs of the pollen grains were taken both in light and scanning microscopes. Accordingly, pollen grains of 9 species of poisonous bee plants from the families of Ranunculaceae, Solanaceae, Acanthaceae, Euphorbiaceae and Plantaginaceae were analysed and documented. Introduction A number of cases of poisoning humans after consuming toxic honeys and deaths of bees due to such poisonous bee plants have been reported (Maurizio, 1975; Majak et al., 1980; Jonathan et al., 1981; Sharma, et al., 1986). Some plant species from the families of Ericaeae, Euphorbiaceae, Ranculaceae, Solanaceae, Plantaginaceae and Acanthaceae are well known for their toxicity. In Ethiopia the diversified ecological situations create conditions conducive for the growth of 7000 species of flowering plants. A high proportion of these flowering plants are endemic to the country (Edwards, 1976) and high proportions of these plants are also foraged by honeybees. In the country, a number of plant species are reported to be poisonous to honeybees and livestock and some honeys from such plants are also reported to be toxic to humans. As a result of this, cases of poisoning and hospitalisation of individuals, family members and neighbours, after consuming such honey and partial or total death of colonies due to poisonous plants have been repeatedly reported. Burning sensations, acute vomiting and diarrhea are some of the major reported symptoms of poisoning in humans. Since, the pollen grains of each plant species has a special structural pattern that enables us to differentiate the pollen grains of one plant species from the other, knowledge of the pollen morphology becomes very important for many practical applications. Based on the analysis of pollen grains from the guts of forager bees (Sharma et al., 1986), the stomach contents of a dead person (Erdtman, 1969), and a toxic honey (Kerkvliet, 1981) they have been able to detect and confirm the botanical origin of the poisonous honeys. To identify and confirm any cases of poisoning of humans and honeybees and to analyse the pollen spectrum of a honey, it is of primary importance to have references and information on the morphology of pollen grains of bee plants of an area. In this preliminary work an attempt was made to collect, analyse and document the pollen grains of some bee plants, which are reported to be poisonous to bees and honeys from such plants poisonous to humans. Proceedings of the 37 th International Apicultural Congress, 28 October 1 November 2001, Durban, South Africa APIMONDIA 2001 To be referenced as: Proc. 37 th Int. Apic. Congr., 28 Oct 1 Nov 2001, Durban, South Africa ISBN: Produced by: Document Transformation Technologies Organised by: Conference Planners

2 Materials and methods Samples of ripe pollen grains were collected from mature flower buds, directly from the field after the plants have been confirmed as bee plants by close observations that bees are foraging on the plant either for nectar or pollen or both. Along with pollen sample collection, the necessary plant parts of specimens were collected and their scientific names were determined in the National Herbarium of Addis Ababa University. For some plants, pollen grain samples were also taken from already identified herbarium specimens at the Holetta Bee Research Center. For light microscope analyses the pollen grains were prepared based on Louveaux et al. (1978) protocol. In the case of scanning electron microscopy, dried pollen grains were directly dusted on to brass stubs provided with double side tape and coated with gold for 2 minutes using Balzers sputtering device. The important morphological features of the pollen grains were described mostly based on the light microscope preparations, under 1000 magnification and supported with the SEM observations. In the description of pollen grains characters like symmetry, form, size, shape, aperture (type, number and diameter), surface pattern, sexine and nexine thickness, were considered. The shapes of the pollen grains were expressed in terms of the ratio of the length of polar axis to the equatorial diameter of the pollen grains using the standard of Erdtman (1969). The average sizes of the pollen grains were determined by measuring 20 pollen grains each for polar axis and equatorial diameter using a calibrated ocular micrometer. In the case of prolate pollen grains, it was not always possible to get as many pollen grains in polar views as required. In this case the average of fewer measurements was taken. In the case of aperture diameter, sexine and nexine thickness, an average of 10 measurements was taken. The pictures of the pollen grains were taken under both light and scanning electron microscopy. Results The morphological description of each species with their photographs is presented as follow. Clematis hirsuta Perr. & Guill. (Ranunculaceae), (Plate 1, A F) Symmetry and form: isopolar simple. Shape: spheroidal, circular outline in polar view. Dimension: P = 24(22-28) µm, E = 23(21-25) µm. Aperture: tricolporate; colpi long with 2-3 µm. in diameter at equator and fairly uniform through out the length. Each pore with 3-4 µm in diameter, circular. Sxine: is with fine verrucae and baculated. Sexine µm thick, nexine 1 µm thick. Clematis simensis Fresen (Ranunculaceae), (Pate 1, M P) Symmetry and form: isopolar, simple. Shape: spheroidal, circular outline in polar view. Dimension: P = 20(18-21) µm, E = 22(20-28) µm. Aperture: tricolporate, colpi long and narrow with 2 µm. in diameter at equator, each pore with 2 µm. in diameter, circular. Sxine is with relatively scattered and fine warts (verrucae), baculated. Sexine, µm. thick, nexine 1um thick Croton macrostachyus Del. (Euphorbiaceae), (Plate 2, A D) Form, simple. Shape: spheroidal. Dimension: D = 73 (64-80) µm. Aperture: not observed. Sxine, is provided with warts very dense warts (gemmae), baculated. Sexine, 4 µm thick, nexine 1 µm thick. Datura stramonium L. (Solanaceae), (Plate 2, E I) Symmetry and form: isoplar, simple. Shape spheroidal, circular to subangular outline in polar view. Dimension: P = 46(43-50) µm, E = 47(43-51) µm. Aperture: tricolporate, colpi, fairly uniform with 2 µm in diameter at equator, each pore with µm. in diameter, circular. Sxine, irregularly straited and the straie are branched, it is also baculated. Sexine 2 µm. thick, nexine 1-5 µm. thick. Discopodium penninervium Hochst. (Solanaceae), (Plate 1, K L) Symmetry and form: isopolar, simple. Shape: spheroidal, circular to subangular outline in polar view. Dimension: P = 27(27-28) µm, E = 29(27-32) µm. Aperture: tricolporate; colpi with 2 µm in diameter, relatively narrow, fairly uniform through out their lengths; each pore with 5 µm in diameter, circular. Sxine baculated; Sexine, µm. thick, nexine 0.5 µm thick.

3 Euphorbia abyssinica Gmel. (Euphorbiaceae) (Plate 3, G I) Symmetry and form: isopolar, simple. Shape: spheroidal, circular to semiangular outline in polar view. Dimension: P = 35 (31-36) µm, E = 39 (35-42) µm. Aperture: tricolporate; colpi µm wide at equator, fairly uniform through out their remaining length; each pore with 5 µm in diameter, circular. Intine protrudes at aperatures. Sxine baculated. Sexine 2 µm thick, nexine 1 µm thick. Justicia schimperiana T.Anders. (Acanthaceae), (Plate 3, A C) Symmetry and form: isopolar, simple. Shape: prolate, circular outline in polar view and some of the pollen grains are constricted at the equator. Dimension: P = 66 (60-76) µm, E= 47 (45-48) µm. Aperture: dicolporate; colpi long with µm and fairly uniform throughout their length; each pore with 6-10 µm in diameter and the entine is protruded at pore. Sxine, finely reticulated and baculated. Sexinec 1.5 µm thick, nexine 1 µm thick. Phytolacca dodecandra L Herit (Phytolaccaceae), (Plate 1, G - J Symmetry and form: isopolar, simple. Shape: spheroidal, circular outline in polar view. Dimension: P = 27 (26-28) µm, E = 30 (27-31) µm. Aperture: tricolporate; colpi 4-5 µm wide at equator, long and narrow; each pore with 4-5 µm in diameter, circular. Sxine, is with relatively dense verrucae and baculated. Sexine 2 µm thick, nexine 1 µm thick. Ranunculus multifidus Forssk. (Ranunculaceae), (Plate 3, D F) Symmetry and form: isopolar, simple. Shape subobate, circular outline in polar view. Dimension: P = 30(26-32) µm, E= 35(32-39) µm. Aperture: triporate, sometimes tetra porate; each pore with 5 µm in diameter. Sexine is provided with verrucae and baculated. Sexine 1 µm thick, nexine µm thick. Discussion The pollen grains of J. schimperiana are easly recognizable by their prolate shape and dicolporate appartures and also differ from other pollen grains of the same genus of Justicia like, J. striata, J. hetrocarpa, and J. cufodontii by their larger size. The pollen grains of Ranunculaceae species: Clematis simensis, and C. hirsuta are morphologically very similar, both in light and scanning electron microscopic observations; however, they vary in polar axis, equatorial and colpi diameters. The surface pattern of Ranunculus multifidus shares similarities with the Clematis species, however differ much in aperture type and dimensions. The surface pattern and aperture types of Phytolacca dedocandra pollen grains are similar to Clematis species but larger in size. The pollen grains of C. simensis, C. hirsuta, and Phytolacca dedocandra are prolate in SEM preparations and change to spheroidal when they are treated with ether and glycerine jelly for light microscope observations while the pollen grains of J. schimperiana remain more or less similar in both preparations. The pollen grains of Croton macrostachys are peculiar in that, they do not have apertures and the surface is covered with dense warts (Gemae). Honey from Justicia schimperiana is well known for its toxicity to man; even, the nectar of this plant causes vomiting and diarrhea when sipped by children (Nuru, 1996), but a poisonous effect on bees has not been reported. The other plant species presented here are reported to be poisonous to honeybees. Ranunculus multifidus is also reported to be poisonous to livestock. On the other hand all of these plants, which are reported as poisonous, are also widely used in traditional healing for different medications (Gelahun et al., 1976), which, indicates that the plants produce some active ingredients, which could be toxic when they are consumed in large amounts. Moreover, Justicia shimperiana honey is used as a purgative substance. From the pollen morphological analyses of these plants it would be possible to indicate the botanical origin of poisoning cases of bees and humans. However, besides the pollen grain analyses of these plants, identifying the chemical substances, which are responsible for the toxicity, would help to support the confirmation of poisoning cases.

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6 References Edards Sue (1976) Some wild flowering plants of Ethiopia. Addis Ababa University press, Addis Ababa, Ethiopia. Erditman G (1969) Hand book of palynology. Hafnear publishing Company. New York. Gelahun A, Tewolde B.G. E, Mesfin T (1976) A study of the medicinal plants of Ethiopia Part 1 A- The identity of some of the plants. The faculty of Science, Department of Biology, Addis Ababa University, Addis Ababa, Ethiopia. Jonathan W, White Jr (1981) Natural honey toxicants. Bee World 62(1): Kerkvliet J D (1981) Analysis of toxic Rhododendron honey. Journal of Apicultural Research 20 (4): Louveaux J, Maurizio A, Vorwohl G (1978) Methods of melissopalynology. Bee World 59 (4): Majak W, Neufeld R, Corner J (1980) Toxicity of Astragalus miser V. serotinus to the honeybee. Journal of Apicultural Research 19 (3): Maurizio A (1975) How bees make honey. pp from Honey: a comprehensive survey. ed. E. Crane. London: Heinemann in cooperation with International Bee Research Association. Nuru A. (1996) Poisonous honey source plant (Justicia shimperiana) in Agew Mider area in northwest part of Ethiopia (case study report). Holetta Bee Research Centre, Holetta, Ethiopia. Sharma O P, Raj D, Garg R (1986) Toxicity of nectar of tea (Comellia Thea L). to honeybees. Journal of Apicultural Research 25 (2):

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