Enzyme Changes During Development of Holo- and Hemi-Metabolic Insects

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 development 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. A ~,0 o sharp band appears for ME in all life stages, and •~. 60 is most intense in larvae. The locus encoding this zone ,,..,, (ME-I) is known to be polymorphic in D. pseudoobs- c~ 6o ,c3 cura (Ayala & Powell, 1972), as is a very much lighter product of a possible second ME locus (ME-2) on 20 their gels. We did not observe this second light zone I 1 I I of activity. Isocitric dehydrogenase exhibits a single LARVA EARLY LATE ADULT sharp band in all life stages. PUPA PUPA We could not resolve PGI into sharp bands, Fig. 5. Levels of malic enzyme activity during development although a wide blur consistently migrated slightly of Acyrthosiphon and Drosophila. Explanation as in legend toward the anode in larvae, pupae and adults. A to Fig. 1. single sharp band, darkest in adults, appears on gels stained for ~GPD. Using very different electrophore- results, we assayed ADH in D. persimilis (a very close tic techniques, Hubby & Lewontin (1966) show two relative of D. pseudoobscura) and D. melanogaster. close zones of ~GPD activity in D. pseudoobscura. Although D. persimilis shows a nearly identical deve- Since no zymogram variability was observed between lopmental pattern to D. pseudoobscura, D. melanogas- individuals or strains (Lewontin & Hubby, 1966), ter does show a strong U-shaped curve. ADH deve- these bands may simply represent post-translational lopmental patterns thus appear to be variable changes in products of a single locus acting through- between (and perhaps within) Drosophila species and out development. we are presently investigating this phenomenon. The zymogram of ADH shows three sharp bands, The activity of ct-glycerophosphate dehydrogenase two of which migrate cathodally under our condi- is much higher in adults than in larvae and early tions, and one which migrates slightly anodally. All pupae (by at least 100 and 400~o, respectively). A great bands are darkest in larvae and early pupae, and light- deal of attention has been paid to the high levels of est in adults. Although a multiple locus model could ~GPD in winged insects, and it is now known that be proposed for this zymogram pattern, two of the this enzyme plays a critical role in the metabolic pro- bands very likely represent subbands of the primary cesses of energy requiring flight muscle (Wyatt, 1968; gene product (Jacobson et al., 1972). At any rate, there Sacktor, 1965). ct-Glycerophosphate dehydrogenase is presently no conclusive evidence for different ADH forms part of a cytosol-mitoehondrial shuttle system loci being active during the life stages of Drosophila which allows oxidation of extramitochondrial NADH pseudoobscura. and hence continuance of the glycolytic cycle as well The adenylate kinase zymogram is more complex. as transfer of electrons to the oxidative phosphoryla- There are five major zones of activity, each present tion chain (Hochachka & Somero, 1973). This enzyme in all life stages. Lack of variability in the banding thus represents changing levels which are closely tied pattern of the individuals we surveyed makes it pre- to the physiological requirements of an organism. sently impossible to know whether these bands repre- In most cases, the striking changes in activity levels sent products of the same or different ADKIN loci. during development appear to be the result of chang- In summary, the ontogeny of D. pseudoobscura is ing quantities of the same isozymic products. In fact, characterized by dramatic changes in levels of glucose metabolizing enzymes. Whole body enzyme levels in- variably drop significantly in pupae, and usually, although not always, recover to larval levels or higher 60 in adults. In most cases, changing levels of enzyme activity appear not to be a consequence of the com- d 5O /DH tLl plete turning on and off of different genes during de- ~ 40 velopment, but rather appear to represent changing concentrations or activations of molecular products "" 30 of the same loci.

° 2O Hemimetabolic development Activity levels of five enzymes during development IO of Acyrthosiphon are presented in Figs. /-5. The I I I I weight of individual pea aphids increases greatly dur- LARVA EARLY LATE ADULT ing development (about 0.95 mg, 2,60 mg, and 3.85 mg PUPA PUPA apiece for third instars, fourth instars, and adults, res- Fig. 6. Levels of alcohol dehydrogenase and ~-glycero- pectively). Thus we have corrected for weight, and phosphate dehydrogenase activity during development of the graphs represent activities per unit wt of Drosophila pseudoobscura. Explanation as in legend to Fig. organism. (Expressed on a per organism basis, activity 1. of each enzyme increased through development.)

(At.P.(It) ~ 3B I 396 JOHN C. AVlSE AND JOHN F. McDONALD

Again, two patterns are evident, although the pat- decreasing enzyme activity could simply reflect pro- terns are quite different from those in Drosophila. The tein destruction during histolysis. However, histolysis enzymes examined either maintain a relatively con- probably does not result in a depression in whole stant level of activity per unit wt during development body levels of all proteins. Proteinase activity (mea- (PGM, IDH), or else decrease somewhat in later life sured at constant pH) shows very little variation dur- stages (ME), particularly in the adult (PGI, ADKIN). ing development (Agrell, 1964). Also, histolysis and The zymogram patterns appear very similar in the histogenesis are not consecutive but rather concur- three life stages. A single sharp band is expressed for rent, and thus total cellular tissue may not change PGM. A single band also appears on gels stained much (Wyatt, 1968). for ME and for IDH. Phosphoglucose isomerase exhi- In summary, biochemical, physiological, and mor- bits one darkly staining band in all life stages exam- phological changes all parallel one another during incd. In addition, a much slower migrating, very faint Drosophila metamorphosis. Enzyme levels decrease, band appears in fourth instars and adults. This band respiration decreases, and tissues histolize. Although may represent the product of a separate locus, these processes clearly comprise an integrated and although its faint appearance suggests that it contri- adaptive developmental strategy, it is less clear which butes very little to the ,activit3 levels shown in Fig. 2. of the processes are causally related. (Note: In both Acyrthosiphol~ and Drosophila, the The changes in levels of enzyme activity during de- relative intensities of the bands appearing on the gels velopment are much less pronounced in Acyrthosi- support in a general sense the curves depicted in Fig. phon, Enzyme levels per unit body wt either remain 1 through 6. Thus, for example, the c~GPD band in constant or else decrease somewhat in later life stages. Drosophila zymograms appears very intense in adults, There is no evidence of U-shaped developmental pat- less intense in larvae, and faintest in early and late terns of enzyme levels. Again, in most cases, the same pupae. Nonetheless enzyme levels are not readily or isozymic forms of each enzyme are present in third accurately quantified from starch gels alone.) and fourth instars and adults. ~GPD, an enzyme As in Drosophila, the adenylate kinase zymogram which is known to be essential to flying insects, was is complex. Three major zones of activity appear on not detected in our assays of the flightless pea aphid. gels in adults, and the least anodal of the zones can- The decrease in levels of ADKIN, ME, and PGI not be seen in third and fourth instars. The appear- in later life stages was not expected. This pattern may ance of the bands and their wide separation on the reflect changes in body composition. For example, gels suggests that they represent products of different much of the body cavity of adults is occupied by loci, one of which may be turncd on primarily in developing embryos, and, after reproductive age is adults. However, until genetic variants are observed passed, the cavity becomes filled with fat deposits and this interpretation must remain tentative. water (Marv Kinsey, pers. comm.). However, if this is the cause of the decrease in some enzymes per unit body wt, the decrease should be expressed in all func- DISCUSSION tionally related enzymes. Furthermore, the drop from All of the enzymes we have examined in Drosophila third instars to fourth instars is not explained. At any pupae show a significant decrease in activity from rate, the magnitude of the activity changes are not third instar larvae. Although protein molecules are nearly as pronounced as in Drosophila for most several steps removed from the genes, and need not enzymes examined. directly reflect patterns of gene induction or repres- Wyatt (1968) argues that the major metabolic path- sion, they do generally reflect changing metabolic ways during pupation differ quantitatively but not capabilities of organisms. One of the best known qualitatively from those of normal tissues. Our results metabolic observations on metamorphosing insects is agree with this conclusion. Not only are the same the depression of oxygen utilization during pupation enzymes present in late larvae, pupae and adult Dro- (Agrell, 1964; Wigglesworth. 1965). Accompanying sophila, but usually the same isozymic form is repre- this decrease in respiratory rate is a corresponding sented, though in different concentrations. Decreasing decrease in the terminal oxidases of the cytochrome enzyme levels in pupae may reflect gene repression, system (Agrell, 1964), and previously reported de- increased degradation (or inactivation) of existing creases in certain dehydrogenases in pupating meal- enzyme molecules, or both, but the rise in activity worms and houseflies (Ludwig & Barsa, 1958, 1959). levels in adults must largely represent renewed Nonetheless, the rate of metabolism may not be cnzyme synthesis. This renewed synthesis in most limited by levels of respiratory enzymes but rather cases appears to result from induction of the same by the energy requirements of protein and tissue syn- genes rather than a completely different cohort of loci. thesis in the pupa (Wyatt, 1968). The stimulation of Two of the enzymes we have examined in Droso- oxygen uptake by certain chemicals or by mechanical phila did not recover to larval levels in adults. Malic injury of silk moth pupae indicates that respiratory enzyme catalyses the interconversion of pyruvic acid capacity normally exceeds demand. and malic acid, and is thought to be significant for Among the higher Diptera, metamorphosis entails its capacity to generate NADPH for biosynthetic a major degree of histolysis of larval tissues (Whitten, pathways. It is not clear why levels are low in adults 1968). We have examined only whole body enzyme and late pupae. Alcohol dehydrogenase is unique levels and do not know how much activity is contri- among the enzymes examined because its substrates buted by various tissues. Muscles, fat body, and intes- are probably of external origin. ADH may play a tine make up much of the larval weight, and all of critical role in allowing flies to tolerate and/or utilize these organs and tissues apparently completely histo- alcohols in the environment. Thus we might expect lize during metamorphosis (Bodenstein, 1965). Thus ontogenic patterns of ADH to differ among species Development of holo- and hemi-metabolic insects 397 or genotypes depending on ecological requirements. HOCHACHKA P. W. & SOMERO G. N. (1973) Strategies of We are currently investigating ADH levels in a var- Biochemical Adaptation. W. B. Saunders, Philadel- iety of Drosophila species. If the major physiological phia. function of ADH is indeed environmentally related, HuBav J. L. & LEWONTIN R. C. 0966) A molecular approach to the study of genic heterozygosity in natural the lack of significant quantities of alcohol in the pea populations--I. The number of alleles at different loci aphid environment may explain our inability to detect in Drosophila pseudoobscura. Genetics 54, 577-594. ADH activity in those individuals we examined. IMBERSKI R. B. & STROMMEN C. (1972) Developmental Some other enzymes (for example, lactate dehydro- changes in alcohol dehydrogenase activity in Drosophila genase--Rechsteiner, 1970) also decrease in activity hydei. Drosoph. Inf. Serv. 48, 74. in adult Drosophila. The increase in enzyme synthesis JACOBSON K. B., MURPHY J. B. & HARTMAN F. C. (1972) in adults thus does not extend to all loci, but as Isoenzymes of Drosophila alcohol dehydrogenase. J. Biol. expected, reflects selective control. Chem. 245, 1075-1083. KARLSON P. & SEK~ERISC. E. (1964) Biochemistry of insect metamorphosis. In Comparative Biochemistry~VI Acknowledgements--Drs. F. J. Ayala, G. J. Edlin, and (Edited by FLO~KIN M. & MASON H. S.). Academic C. L. Judson were all very kind in allowing us free use Press, New York. of their laboratory facilities. We also wish to thank Mary LEWONTIN R. C. & HUBBY J. L. (1966) A molecular Kinsey for supplying the pea aphids used in this study. approach to the study of genic heterozygosity in natural Research was supported by NIH training grants in gene- populations--II. Amount of variation and degree of tics. heterozygosity in natural populations of Drosophila pseudoobscura. Genetics 51, 595--609. REFERENCES LUDWm D. & BARSA M. C. (1958) Activity of dehydro- AGRELL I. (1964) Physiological and biochemical changes genase enzymes during the metamorphosis of the meal- during insect development. In The Physiology of Insects worm. Tenebrio molitor Linnaeus. Ann. Ent. Soc. Am. 51, (Edited by ROCHSTEIr~M.), VO1. 1, pp. 91-148. Academic 311-314. Press, New York. LUDWIG D. & BARSAM. C. (1959) Activities of respiratory AYALA F. J. & POWELL J. R. (1972) Allozymes as diagnostic enzymes during the metamorphosis of the housefly, characters of sibling species of Drosophila. Proc. Natn. Musca domestica Linnaeus. JI N. Y. Ent. Soc. 67, 151-156. Acad. 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