Comp.Biochem.Physiol.Vol.70B,pp. 607 to 609, 1981
0305-0491/81/110607-03502.00/0 Copyright © 1981PergamonPressLtd
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MALATE DEHYDROGENASE AND NON-SPECIFIC ESTERASE ISOENZYMES OF EGGS OF THE HONEY BEE (APIS MELLIFERA L.) R. A. NUNAMAKERand W. T. WILSON* Honey Bee Pesticides/Diseases Research, Agricultural Research, Science and Education Administration, U.S. Dept. of Agriculture, Laramie, WY 82071, U.S.A.
(Received 16 March 1981) Abstract--1. lsoelectric focusing on polyacrylamide gels was used to detect malate dehydrogenase and non-specific esterase isoenzymes in diploid honey bee (Apis mellifera L.) eggs of several known ages. 2. It was determined that the number of MDH and EST isoenzymes increased as development proceeded with an apparent increase in the quantity of some of the isoenzymes.
MATERIALS A N D METHODS
Tripathi & Dixon (1969) found two and three malate dehydrogenase (MDH) isoenzymes in the hemolymph of queen and worker honey bees (Apis mellifera L.), respectively, throughout larval development; within each caste, the bands differed only in intensity, which decreased with age. In a similar study, Tripathi & Dixon (1968) observed a marked difference in the hemolymph pattern of non-specific esterases (EST) of queen and worker larvae: in 48-hr-old queen larvae there were two major and two minor bands that gradually decreased in intensity until the larvae were 108 hr old, at which time one major and one minor band disappeared. In 48-hr-old worker larvae there were two major and four minor bands that also decreased in intensity until only one major and one minor band were present at 108 hr of age. Subsequently, Nunamaker & Wilson (submitted for publication) used isoelectric focusing on polyacrylamide gels and found three major and two minor M D H isoenzymes in the hemolymph of the worker honey bee larva and seven EST isoenzymes in the 48-, 60and 72-hr-old larva, Other scientists have reported differences in the electrophoretic distribution of esterases during insect development (Clements, 1967; Cook & Forgash, 1964; Salkeld, 1965; Laufer, 1960 and 1961). In addition, Gilliam & Jackson (1972) found two M D H and two EST isoenzymes in the hemolymph of adult worker honey bees. However, Nunamaker & Wilson (1980) had also observed as many as seven M D H and seven EST isoenzymes in the hemolymph of newly-emerged ( < 6 hr old) adult workers. Little is known about the multiple forms of M D H and EST in eggs of insects, but isoenzyme patterns are now being used in insect taxonomy. We, therefore investigated the M D H and EST isoenzyme patterns of honey bee eggs of various known ages.
A single colony consisting of a mostly heterozygous line of honey bees was reared out-of-doors during the summer months near Laramie, Wyoming (elevation approx. 2181 m). Before the test, an artificially inseminated marked queen was confined to a specific area of brood comb in the colony for 3 hr by using the specialized cage described by Rothenbuhler & Thompson (1956). Thus, eggs were of known age and ancestry. The investigation of the isoenzymes of MDH and EST was thus made with eggs that were 3, 12, 21, 30, 39, 48, 57 and 66 hr old.
* Also research associate and adjunct professor, respectively, University of Wyoming, Laramie, Wyoming, U.S.A. * Mention of a commercial or proprietary product does not constitute an endorsement by the USDA. 607
Samples One hundred eggs of the same age were placed on a small watch glass and macerated. From this, 5 #1 samples (3 replications) were drawn into a graduated Accupette®. *
Isoelectric focusing in thin-layer polyacrylamide gel The electrofocusing analyses were performed with an LKB Multiphor System, 2117 (LKB Producktor, Bromma, Sweden). Polyacrylamide gels (PAG-plates) in the 3.5-9.5 pH range were purchased from LKB. The gel composition was 10.0ml 29.1% acrylamide (w/v), 10.0ml 0.9% NN'methylene bisacrylamide (w/v), 36.0 ml distilled water in which 7.5 g sucrose had been dissolved, 2.8 mi Amphohne pH 3.5-10, 0.4 ml Ampholine pH 9.0-11.0, 0.2 ml Ampholine pH 4.0-6.0 and 0.2 ml Ampholine 5.0-7.0. The fluid from the macerated eggs was applied directly to the gel surface and electrofocused for 105 min at 9°C by using the LKB 2190 Power Supply. Voltage and amperage were 300V/90mA at the onset of the experiment and 1300 V/20 mA at the completion.
Location of isoenzymes The MDH isoenzymes were located by using the enzymespecific staining technique of Hunter & Markert (1957), i.e. nitro-blue tetrazolium was used as an artificial hydrogen aceeptor. The sites of enzyme activity were detectable by the deposits of insoluble blue formazan in the gels. The components for the MDH stain were 100ml of 0.1 M Tris-HCI buffer, pH 8!3, containing 24 mg of malic acid, 12 mg of nitro blue tetrazolium, 16 mg of phenazine methosulfate, 24 mg of nicotinamide adenine dinucleotide and 8 mg of potassium cyanide. Esterase bands were located by using ~- and fl-naphthyl acid phosphate as a substrate and Fast Red TR salt as a dye coupler (Tripathi & Dixon, 1968). The components of
R.A. NUNAMAKERand W. T. WILSON
Fig. 1. Malate dehydrogenase lsoenzymes in honey bee eggs. (Age in hr.) the stain were 1So ~t-fl-naphthyl acetate (1 g ~t-naphthyl acetate. 1 g fl-naphthyl acetate, and 87 ml H20). All samples were incubated in the dark with continuous shaking for one hour at 37°C. The controls contained all the chemical components listed---except the substrate. Immediately afterwards, the gels were rinsed with distilled water and photographed. RESULTS
The isoenzyme patterns of MDH and EST found by studying eggs of eight different ages are depicted in Figs 1 and 2. The relative activity at each site is indicated by shading in the diagrams.
Malate dehydrogenase The distribution patterns of MDH showed an increase in both the number and intensity of isoenzymes in eggs between three and 66 hr old. From 3 to 12 hr there was one band present. From 21 to 66hr the band became even more intense, and two additional bands were apparent (Fig. 1).
Non-specific esterases The distribution patterns of EST showed an overall increase in the number of isoenzymes in eggs between three and 66 hr old. Some also increased in intensity as the egg aged. Between three and 30 hr, one of the three major bands became more intense, but two of the minor bands remained unchanged throughout the three to 66-hr period. At 30 hr, one additional major band and one minor band appeared; this minor band became more intense as the egg aged (Fig. 2). DISCUSSION
Only one to three MDH isoenzymes were found in any stage of Drosophila (Hubby & Lcwontin, 1966)
and Culex pipiens L. (Trebatoski & Haynes, 1969). Changes in MDH electrophoretic patterns were reported to occur in all developmental stages of Cecropia and Cynthia (Laufer, 1961). Salkeld (1965) noted 14 bands of esterase activity in the 2-day old egg of the large milkweed bug (Oncopeltusfasciatus (Dallas)). Other attempts to separate the esterases of insect eggs by electrophoretic methods include studies by Laufer (1960) on eggs of unknown age of the moth Hyalophora cecropia (L). He found only four esterases that hydrolyzed ~-naphthyl butyrate, three that migrated to the anode and one to the cathode. Hudson (unpublished data), using :t-napthyl acetate as substrate, was able to identify five anodic esterases and two cathodic ones in a sample of eggs of mixed ages from another moth, the tomato hornworm, Manduca quinquenaculata (Haworth). In contrast, only one esterase was found to hydrolyze fl-naphthyl acetate in eggs of unknown age from the house fly, Musca domestica L. (Menzel et al., 1963). Our studies indicate that development of the female (diploid) eggs of Apis mellifera is accompanied by changes in malate dehydrogenase and esterase activity. The number of MDH and EST isoenzymes increased as development proceeded, and there was an apparent increase in the quantity of some of the isoenzymes. It is quite possible that some isoenzymes that appeared suddenly during development are, in fact, present in concentrations too low to be detected at an earlier stage of development. Currently, we are investigating the MDH isoenzymes patterns in worker bees from around the world. We agree with Sylvester's (1976) conclusion that the MDH locus is controlled by three alleles (a, b, c) with heterozygous bees possessing three bands (i.e.: MDH is a dimeric molecule an(t homozygous bees have one band). Thus, the heterozygous bees used in this study were of the MDH 5°/L°° genotype.
MDH and EST isozymes of Apis
:':':~:r:~ ......,... :.:;:.:.:;:;
Fig. 2. Non-specific esterase isoenzymes in honey bee eggs. (Age in hr.)
Earlier investigations showed that as the worker honey bee larva aged, the number of M D H and EST isoenzymes decreased and the minor bands became less intense and eventually disappeared (Nunamaker & Wilson, 1981). Our data from both studies would seem to indicate that more isoenzymes are present (and at high levels) during morphogenesis from the embryonic to the larval stage because of active tissue formation and rearrangement. Then, during the early larval stadia, all the major integral systems undergo changes, with some becoming functional for the first time. Also during these stages (egg and early larval) the bulk of biochemical activity is high because of the formation of new tissue. Thus, a large number of isoenzymes are present during this interval. Then, the major changes are in metabolism and growth; consequently, the isoenzymes that were important during morphogenesis are no longer present. Acknowledgements--We would like to thank Genetic Systems Inc., LaBelle, Florida, for providing the heterozygous line of Apis mellifera L. used in this study. We would also like to thank Viola Gibson and Janett Hutsell for their help in the preparation of the manuscript.
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