Insect Biochem. Vol. 17, No. 2, pp. 303-308, 1987 0020-1790/87 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1987Pergamon Journals Ltd DEVELOPMENTAL CHANGES OF ISOENZYMES CATALYSING GLYCOLYTIC AND ASSOCIATED REACTIONS IN LOCUSTA MIGRATORIA IN RELATION TO THE REARING DENSITY OF HATCHLINGS D. J. COLGAN Department of Population Biology, Research School of Biological Sciences, Australian National University, G.P.O. Box 475, Canberra City, A.C.T. 2601, Australia (Received 24 February 1986; revised and accepted 11 June 1986) Abstract--More than half of the enzymes associated with glycolysisin the acridine grasshopper Caledia captiva differ in electrophoretic phenotype between embryonic and adult stages. This paper reports the results of a survey of 23 enzymes of the locust, Locusta migratoria which was intended to ascertain whether a similar pattern of changes occurs in this species. Ten of the enzymes (including seven associated with glycolysis) were shown to be developmentally variable. Six of the enzymes were studied in first-instar nymphs reared at high or low densities to test the hypothesis that this variation is in some way related to the potential of some acridid grasshoppers to undergo phase transformation. There were two major findings. Firstly, the aldolase phenotype found in the initial survey and high rearing-density hoppers was replaced by a novel isoenzyme in the low density group. Secondly, the levels of the two glycerol-3- phosphate dehydrogenase isoenzymeswere higher in the low density group than in the high density group. These results suggest an opportunity for further study which promises the first entr6e to the elucidation of the molecular effects of phase polymorphism in locusts. Key Word Index: Isoenzymes, glycolysis, Locusta, rearing density INTRODUCTION It has recently been discovered in this laboratory that there is extensive developmental variation in the soluble proteins of the Australian acridine grasshop- per, Caledia captiva. One such protein has been revealed by general stains (Colgan, 1985). However, most of the variation occurs in the enzymes of glycolysis and its associated pathways. More than half of these enzymes show major changes between the isoenzymic phenotypes of the embryos and the adults (Colgan, 1986). The changes occur near the time of hatching for all enzymes. This was the first report of widespread, temporally-coincident, devel- opmental variation in the isoenzymes of a biochem- ical pathway. The organism in which this situation was found is a member of the acridid family of which 20 or more species show the remarkable plasticity of phase polymorphism (Uvarov, 1966). It is notable that the period of isoenzymic changes is also one of those critical for the development of phase-specific characteristics (Uvarov, 1966). The migratory locust, Locusta migratoria was chosen to test whether there is a relation between these two examples of onto- genetic variability. To date it has not proven possible to find an entr6e to the genetic basis of phase polymorphism. It was hoped that the glycolytic sys- tem might provide such an entr6e. This is not to say that the genes in the pathway "cause" the poly- morphism. Rather, their changes in expression may be subject to the factors controlling polymorphism and they may be more accessible to study at the molecular level. Initially, an electrophoretic survey was carried out to confirm the existence of differences between various life-stages in the phenotypes of the isoenzymes of glycolysis (and associated biochemical reactions) in L. migratoria. This paper describes the results of the survey and a subsequent densitometric analysis of the effects of rearing density of first and second instar nymphs on the isoenzymic phenotypes of six of the systems which differ most strikingly between stages. Rearing density was selected as a variable in this experiment because it is the best known environmental factor which influences phase determination (Uvarov, 1966) and because its effects begin to be seen relatively shortly after a change in conditions (Uvarov, 1966). MATERIALS AND METHODS Locusts were maintained in the manner described for Caledia captiva by Shaw and Wilkinson (1980) except that the egg pods were not removed from the pots of sand in which they were laid. The locusts were provided from a culture maintained at this institution which originates from the Australian subspecies. Samples for electrophoresis were prepared by hand- grinding in fresh buffer (pH 7.0) comprising I00 mM "Iris, I mM Na2EDTA, 0.5 mM NADP and fl-mercaptoethanol (50/~I/I00 nil). Eggs were ground in 30/~ I of this buffer and hatchlings in 50/zl. Adult tissues were ground I:I w/vin the buffer and spun for 3 rain in an Eppendorff centrifuge to remove cellular debris. Embryos were analyzed a few days prior to hatching, when eye pigmentation was becoming visible and nymphs a few days after hatching. Two adult females and one male were divided into four tissues (head, thoracic muscle, hind leg muscle and abdomen) which were separately scored. Only leg muscles were run for other adults. To assess the effect of rearing density on phenotypes, nymphs were allocated to groups less than an hour after hatching. The low density group was reared individually in 303