Corap. Biochem. Physiol., 1976, Vol. 53B, pp. 393 to 397. Pergaraon Press, Printed in Great Britain ENZYME CHANGES DURING DEVELOPMENT OF HOLO- AND HEMI-METABOLIC INSECTS JOHN C. AVISE AND JOHN F. McDONALD Department of Genetics, University of California, Davis, CA 95616, U.S.A. (Received 12 November 1974) Abstract--l. Several carbohydrate metabolizing enzymes were examined spectrophotometrically and electrophoretically in four life stages of the holometabolic fruit fly, Drosophila pseudoobscura, and in three life stages of the hemimetabolic pea aphid, Acyrthosiphon pisum. 2. Drosophila pupae exhibit significantly lower enzyme activities than third instar larvae. Most enzymes recover to larval levels or higher in adults, but some continue to decrease following eclosion. 3. Enzyme activities remain relatively unchanged during development of Acyrthosiphon. 4. The same isozymic forms of each enzyme are usually represented in all life stages of Drosophila, and in all life stages of Acyrthosiphon. 5. Enzyme levels parallel the distinct morphological and physiological changes characterizing devel- opment of representative holo- and hemi-metabolic insects. INTRODUCTION MATERIALS AND METHODS THE IMAGOES of most holometabolous insects are Activity levels morphologically and ecologically very different from We have found the following to be a simple and repeat- their respective larvae. The transition from larva to able procedure for measuring levels of enzyme activity in adult requires a unique pupal stage during which lar- individual organisms. A fly or aphid is placed in a small val structures are histolysed and reorganized into mortar with 0-02 ml of deionized water and is thoroughly adult tissues. "The metamorphosis of the higher in- homogenized with a glass rod. Another 0.13 ml of water sects embodies the most profound reorganization of is added and stirred. From this homogenate, 0.10ml is pipetted through gauze to a spectrophotometer microcu- a grown animal that is known" (Wyatt, 1968). In con- vette containing appropriate buffer, substrate, ions, and trast, far fewer morphological and ecological differ- coenzymes. Since each of the catalyzed reactions involves ences are observed between life stages of some hemi- formation of either NADH or NADPH, enzyme activity metabolous insects. Development proceeds through a is measured by the rate of change in absorbance at 340 m~t series of molts and culminates in adults which may over a 3-rain period. Assays were made" at room temp on closely resemble the instars except in size and repro- a Zeiss PMQII spectrophotometer. ductive capacity. Although these gross developmental Stock solutions of the following were prepared: Buf- differences between holo- and hemi-metabolous in- fers--(A) 0"05 M Tris-HC1, pH 8"5; (B) 0-25 M Tris-HC1, sects have long been recognized, relatively few com- pH 7.1; (C) 0.10M Tris-HC1, pH 7.1. Substrates--(D) 370mM 19L-ct-glycerophosphate; (E) 173 mM OL-isocitric parative studies have been made of the underlying acid; (F) 500 mM ~o(+) glucose; (G) 6'6 mM o-fructose-6- biochemical and genetic bases of these differences, In phosphate; (H) 64 mM L-malic acid; (I) 330 mM ~D glu- this study, we quantify activity levels of the isozymic cose-l-phosphate; (J) isopropanol. Cofactors--(K) 53 mM forms of several carbohydrate-metabolizing enzymes ethylenediamine tetraacetic acid; (L) 3"8 mM nicotinamide during life stages of insects exhibiting holometabolic adenine dinucleotide; (M) 1.3mM nicotinamide adenine and hemimetabolic development. Our objective is to dinucleotide phosphate; (N) 227 mM manganese chloride; quantify changes in components of central metabolic (O) 50 mM magnesium chloride; (P) 8.5 mM adenosine 5'- pathways, and to associate these changes with distinct diphosphate; (Q) 1000 units glucose-6-phosphate dehydro- patterns of insect ontogeny. genase per 100ml water; (R) 1400 units hexokinase per Since higher Diptera exhibit the most pronounced 50ml water; (S) 1% histidine. Assay mixtures for the various enzymes were: (1) alcohol metamorphosis among insects, we have examined dehydrogenase (ADH): 0.75 ml of (A), 0.05 ml of (J), and Drosophila pseudoobscura as an extreme representative 0.10ml of (L); (2) ~t-glycerophosphate dehydroyenase of holometabolic development. Larvae are fusiform (ctGPD): 0'6ml of (A), 0.1ml of [D), 0.1ml of (K), and grubs passing through three instars prior to pupation. 0"1 ml of (L); (3) isocitric dehydrogenase (IDH): 0'58 ml of The winged adults bear little morphological or eco- (A), 0.1ml of (E), 0.2ml of (M), and 0-02ml of (N); (4) logical resemblance to the larvae. Hemimetabolic de- phosphoglucose isomerase (PGI): 0.50 ml of (C), 0-10 ml of velopment of the pea aphid, Acyrthosiphon pisum, pro- (G), 0.1 ml of (K), 0.l ml of (M), 0"02 ml of (O), and 0-08 ml ceeds through four instars to adult. All stages feed of (Q); (5) phosphoolucomutase (PGM): 0"63ml of (C), in phloem of legume plants, and, except for size differ- 0.06 ml of (I), 0.1 ml of (M), 0.02 ml of (O), 0.08 ml of (Q), and 0'01 ml of (S); (6) malic enzyme (ME): 0.64 ml of (A), ences, are morphologically nearly indistinguishable. 0.05 ml of (H), 0.20 ml of (M), and 0'01 ml of (O); (7) adeny- Thus Drosophila and Acyrthosiphon provide appro- late kinase (ADKIN): 0.26 ml of (B), 0-2 ml of (F), 0.25 ml priate material for studying the genetic basis of con- of (M), 0.01 ml of (O), 0'05 ml of (P), 0-08 ml of (Q), and trasting developmental strategies. 0.05 ml of (R). In each case, addition of 0-10ml of tissue 393 394 JOHN C. Awst~ AND JOHN F. McDONALI) THIRD FOURTH THIRD FOURTH INSTAR INSTAR ADULT NSTAR INSTAR ADULT I I | I I I 0 I00 4O0 PGM IDH d (5 uJ 300 (/) o o 75 e*3 eo ,.,; 200 (5 c~ 100 I- ..... I ...... i 50 I I L I I I I I LARVA EARLY LATE ADULT LARVA EARLY LATE ADULT PUPA PUPA PUPA PUPA Fig. 1. Levels of phosphoglucomutase activity in third and Fig. 3. Levels of isocitrate dehydrogenase activity during fourth instars and adult Acyrthosiphon, and in larvae, early development of Acyrthosiphon and Drosophila. Explanation and late pupae, and adult Drosophila. Activities expressed as in legend to Fig. 1. as initial change in optical density (x 1000) per 30 see/rag wet body wt. Mean + one S.E. is indicated. RES U LTS homogenate to the microcuvette brought the final assay Holometaholic development vol to I ml. Activity levels of seven enzymes during develop- Flies and aphids were weighed in groups of ten to the ment of Drosophila pseudoohscura are presented in nearest 0-01 mg, and each individual assigned a wt equal Figs. I 6. Since overall mean wt of larvae, pupae, to the mean value for the group. Thus in the graphs that and adults were nearly identical (l.38mg, 1.32rag, follow, the variance about the mean enzyme activity/unit and 1"31 mg per individual, respectively), the curves wt of insect represents a function of the total interorganism variance, a component of which may be due to variability represent both mean activity/organism and mean acti- in weight among organisms. vity/unit wt. Two basic patterns are evident. Follow- ing a significant drop in pupae, enzyme levels either lsozyme analysis recover to near larval levels or higher (PGM, IDH, Horizontal starch gel electrophoresis was carried out PG1, :(GPD, ADKIN) or else continue to drop in according to standard techniques described by Ayala et adults (ADH, ME). al. (1972). Enzymes were localized with staining mixtures Developmental levels of some of these enzymes comparable to those given above (for exact recipes, see have previously been reported in Drosophila. In Ayala et al., 1972). accord with our results, 1DH shows a U-shaped curve In both the spectrophotometric and electrophoretic (pupae lower than late third instar larvae and adults) studies, four developmental stages of Drosophila pseudoohs- in Drosophila melanoqaster (Fox, 1971), as does :~GPD cura were used third instar larvae, early pupae, late (Rechsteiner, 1970; Karlson & Sekeris, 1964). We did pupae, and adults. For our purposes, early pupae are not observe U-shaped variation in alcohol dehydro- defined as those prior to visible development of wings. Adults include only sexually mature flies, older than 12 hr genase in Drosophila pseudoobscura although U- post eclosion. For each life stage in Drosophila and shaped curves have been reported for ADH in D. ,4cyrthosiphon, a minimum of ten individuals were assayed mehmo~laster (Hewitt eta/., 1974) and in D. hydei (lm- individually for each enzyme. berski & Strommen, 1972). In order to confirm our THIRD FOURTH THIRD FOURTH INSTAR INSTAR ADULT INSTAR INSTAR ADULT I I I [ I I O 40( • " 400 IE d (5 ADKIN LU ua(/) 30( 300 2--.. Z01 t-~ Z00 d I- ..... l--- 101 100 "- "I I I L I I I I I LARVA EARLY LATE ADULT LARVA EARLY LATE ADULT PUPA PUPA PUPA PUPA Fig. 2. Levels of phosphoglucose isomerase activity during Fig. 4. Levels of adenylate kinasc activity during develop- development of Acyrthosiphon and Drosophila. Explanation ment of Acyrthosiphon and Drosophila. Explanation as in as in legend to Fig. I. legend to Fig. 1. Development of holo- and hemi-metabolic insects 395 THIRD FOURTH for the enzymes electrophoretically examined, we INSTAR INSTAR ADULT have no evidence of different loci being completely I I I turned on or off in Drosophila pseudoobscura. Two t6 120 sharp bands of very different mobility appear on gels IE stained for PGM. The furthest anodal band is darker "~. |oo M E I.I in larvae and early pupae than in adults, but in all LI.I stages the less anodal band is the most intense.