Quantitative immunochemical and electrophoretic comparisons of glycerophosphate dehydrogenases in several insects

J. Insect Physiol., 11967, Vol. 13, pp. 1757 to 1767. Pergamon Press Ltd. Printed in Great Britain

QUANTITATIVE IMMUNOCHEMICAL AND ELECTROPHORETIC COMPARISONS OF GLYCEROPHOSPHATE DEHYDROGENASES IN SEVERAL INSECTS RONALD

W. BROSEMER, DAVID S. GROSSO, CHARLES W. CARLSON

Department

of Chemistry, Washington (Received

State University, 2.5 July

GLEN

ESTES,

Pullman, Washington

and

99163

1967)

Abstract-An immunochemical method, micro-complement fixation, has been used to compare quantitatively DPN+-dependent glycerophosphate dehydrogenases (IX 1.1.1.8) of various insects. Rabbit antibodies directed against pure honeybee glycerophosphate dehydrogenase were prepared, and the cross-reaction with extracts of other insects measured. The order of decreasing cross-reaction is: honeybee, bumblebee, robust mining bee, leaf-cutting bee, sphecoid wasp, yellow jacket, syrphid, and flesh fly; this is the same as the classical taxonomic order. Several other insects apart from the Hymenoptera show no detectable cross-reaction; the enzymes from six honeybee races and the three honeybee social orders possess identical immunological properties. The specific activity of glycerophosphate dehydrogenase in honeybee thoracic muscle increases fivefold during the week following adult emergence. The immunological properties of the enzyme are identical in the newly emerged and 6-day-old bees. Thus the change in enzyme activity is apparently due to an increase in the number of enzyme molecules. Electrophoretograms show that most of the insects studied here have more than one band with glycerophosphate dehydrogenase activity. There is, however, no obvious taxonomic relationship between the electrophoretic patterns of these insects. INTRODUCTION

STUDIESin recent years on the phylogenetic relationships of individual macromolecules have been focused mainly on vertebrates and micro-organisms (FITCH and MARGOIJASH, 1967). Since pure extramitochondrial glycerophosphate dehydrogenase (L-glycerol-3-phosphate : DPN+ oxidoreductase, EC 1.1.1.8) has been crystallized from honeybees (Apzk mellifera ; MARQUARDTand BROSEMER, 1966), it was possible to initiate studies on the comparative structure of this enzyme in various insects. An immunochemical method, micro-complement fixation, was used for these investigations. This technique was developed by WASSERMANand LEVINE(1961) and applied in the laboratories of KAPLAN (1965) and Wilson @RICH and WILSON, 1966) to studies on the comparative structure of proteins from vertebrates, micro-organisms, and some arthropods. In these types of comparative studies, immunological methods have the major advantage II2

1757

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RONALD W. BROSEMER, DAVIDS. GROSSO, GLENESTES,ANDCHARLES W. CARLSON

that only one protein (the antigen) must be highly purified; the specific advantages of micro-complement fixation are discussed below. The present report describes the application of micro-complement fixation to an investigation of insect phylogeny and ontogeny. In addition, parallel studies of electrophoretic enzyme patterns are reported. METHODS

AND

MATERIALS

Many of the methods and materials have been described (ACQUIT and BROSEMER,1966). The micro-complement fixation technique was described by WASSERMAN and LEVINE(1961) and by SARICHand WILSON (1966). The injection schedule for rabbit I has been described (MARQUARDTand BROSEMER,1966). The injection schedule for rabbit II was essentially the same, except that the honeybee glyeero-P dehydrogenase was complexed with methylated bovine serum albumin before injections (PLESCIAet a!., 1964) and the antigen dosage was about one-quarter of that for rabbit I. The 7s immunoglobulins were isolated from antiserum I (BAUMSTARK et al., 1964) and used in all cases; straight antiserum II was used without further purification. Cellulose acetate electrophoresis was performed at 2°C on 2q.5x 17 cm Sepraphore III strips in a Gelman Deluxe Ele~rophoresis Chamber. The buffer was 13 mM phosphate, 10 mM /3-mercaptoethanol (pH 6.8); the runs were 2 hr at 400 V and about 2 mA initially per strip. Immediately after the run the strips were placed upside down on agar gel plates, as described by PENHOETet al. (1966). The staining solutions for dehydrogenase activities are described by FINE and COSTELLO(1963) ; the glycero-P dehydrogenase staining solution was the same as that for lactate dehydrogenase except that glycero-P was substituted for lactate. Italian honeybee workers were purchased from C. G. Wenner and Sons, Glenn, California. Caucasian, Carniolan, Anatolian, Greek, and Hastings honeybees were kindly donated by M. V. Smith, Guelph, Canada; Dadant and Sons, Hamilton, Illinois, kindly donated specimens of the first three races. Leaf-cutting bees were kindly supplied by C. A. Johansen, Pullman, W~hington. Locusts were supplied by the Anti-Locust Research Centre, London. Other insects were collected in the field. Guinea-pig complement and anti-sheep haemolysin were obtained from Baltimore Biological Laboratory; bovine serum albumin (fraction V) from Armour Pharmaceutical Co. RESULTS

Immunoglobulins I and antiserum II were first checked for homogeneity. In both the antibody preparation reacted with honeybee thoracic extracts and with pure honeybee glycero-P dehydrogenase in identical fashion; that is, a single micro-complement fixation peak was obtained with both the extract and the pure antigen, and these peaks had the same position, shape, and height. The data for immunoglobulins I are shown in Fig. 1; the results obtained with antiserum II

GLYCEROPHOSPHATE DEHYDROGENASES IN SEVERAL INSECTS

1759

were similar. The antibody dilution giving 50 per cent complement tixation was about 1 : 10,CiOOwith immunoglobulins I and 1 : 14,000 with antiserum II.

0

Glycerophosphate

dehydrogenase

(mpg)

FIG. 1. M:icro-complement fixation of immunoglobulins I with honeybee thoracic extract ( 0) and with crystalline honeybee glycero-P dehydrogenase (0).

Antiserum I, immunoglobulins I, and antiserum II were also checked with immunodiffusion. Using cellulose acetate as substrate, sharp single bands were

obtained with antiserum I (MARQUARDT and BROSEMER,1966). However, no consistent success was observed with any of the antibody preparations using agar gel diffusion. Faiint single bands could be observed, but they were always quite diffuse. This puzzling phenomenon may be related to the aggregation of honeybee glycero-P dehydrogenase at moderate concentrations (MARQUARDT and BROSEMER, 1966). The consistent, reproducible results of the micro-complement fixation test demonstrated that normal antigen-antibody reaction was indeed occurring. Cross-reaction in the micro-complement jixation test Cross-reaction of heterologous antigens with antibodies as measured by the micro-complement fixation method is expressed in terms of the immunological distance (I.D.., SARICHand WILSON, 1966). The amount of antibody required to obtain a certain percentage of complement fixation in the peak tube with the homologous system (i.e. antibodies plus honeybee extract) is first determined; the per cent complement fixed must be between 20 and 90 (SARICHand WILSON. 1966).

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RONALDW. BROSEMER,DAVIDS. GRosso, GLEN ESTES, ANDCHARLESW. CARLSON

Then the antibody titre required to fix the same amount of complement in the heterologous system (i.e. antibodies plus extract of another insect) is measured. The ratio of the latter titre to the former titre (heterologous~homologous) is the I.D.; the lower the cross-reaction, the greater is the I.D. The cross-reaction of insect extracts with antibodies directed against honeybee glycero-P dehydrogenase is shown in Table 1. The cross-reaction is completely consistent with the classification of these insects based on anatomical, morphological, and behavioral characteristics. This is true even in the sphecoid and vespoid wasps. Sphecoid wasps are closely related to bees; in fact, bees are probably a branch of the sphecoid wasp line (MICHENER, 1944). The I.D. of sphecoid wasp glycero-P dehydrogenase (4.5) is indeed much lower than that of the yellow jacket enzyme (12). The I.D. values for the syrphid and flesh flies (17 and 18, respectively) do not differ significantly from one another. TABLE ~-CROSS-REACTION OF INSECTTHORAX EXTRACTSWITH ANTI-HONEYBEEGLYCEROPHOSPHATE DEHYDROGENASR Immunological distance (I.D.)

Insect Honeybee (Apis mellifera) Bumblebee (Bombus fervidzcs) Robust mining bee (Tetralonia sp.) Leaf-cutting bee (Megachile rotund&a) Sphecoid wasp (~~~e~e) Yellow jacliet (Vespula ~cu~~~o~s~ Syrphid Ay (Syrphus sp.) Flesh fly (Sarcophaga bullata)

Lowest taxon common with honeybee *

Immunoglobulins I

Antiserum II

Subfamily Family

1.0 2.7 3.1

1.0 2.0 -

Superfamily Suborder

4.2 4.5

2.6 -

-

Suborder Class

12 17

Class

18

5.5 14

* The classification is described by BORRORand DELONG (1964).

The differences in I.D. values between immunoglobulins I and antiserum II may be related to individual variation of the two injected rabbits. However, SARICB and WILSON (1966) reported that I.D. values for primate serum albumins varied only slightly when antisera from different rabbits were used. More likely, the different I.D. values in this study reflect the fact that rabbit I received injections of pure glycero-P dehydrogenase, while rabbit II received glycero-P dehydrogenase complexed with methylated bovine serum albumin, Some of the antigenic determinants of the bee enzyme were probably altered or masked through binding to the carrier protein, thereby changing the cross-reaction pattern of the antiserum with the free enzyme in insect extracts. A value of 25 is about the highest I.D. that can be accurately determined with these antisera, No measurable cross-reaction could be detected with extracts from moths (Arstheruea polyphemus), beetles (Leptinotarsa dece~zine~t~), locusts (Schisto-

GLYCEROPHOSPHATE DEHYDROGENASES IN SEvERAL INSECTS

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cenx greguria), and cockroaches

(Per~pl~~et~ ~~~~u~a). Most of these insects have relatively low glycero-P dehydrogenase values, which is consistent with the fuels utilized by these insects for flight (WEE-F•GH, 1961); the low enzyme levels contribute to the lack of detectable cross-reaction. In addition, six races of Apis mellifera were compared. The glycero-P dehydrogenase used for preparing the two antisera had been isolated from workers of the Italian line. Extracts of Caucasian, Carniolan, ~natolian, Greek, and Hastings (Caucasian) workers all have I.D. values of 1.0 with immunoglobulins I. Therefore, there are no detectable immunological differences of the glycero-P dehydrogenase proteins in these six races. The micro-complement fixation technique was also used to compare Italian workers, drones, and queens. There are no detectable immunological differences in the glycero-P dehydrogenases of these three social classes; that is, the I.D. value in each case is within experimental error of 1.0. Ontogeny of honeybee glycerophosphate dehydrogenase

The micro-complement fixation technique was also used in a study of insect ontogeny, G-lycero-P dehydrogenase activity in the flight muscle of different insects increases severalfold during the period immediately following the imaginal ecdysis; this phenomenon is related to the acquisition of flight ability at this stage of development (ROCKSTEINand BFUNDT, 1963; BROSEMER,1965a). Experiments with mixed extracts and with specific inhibitors of protein synthesis indicated that this enzymic activity increase is due to de noeto synthesis of glycero-P dehydrogenase and not to activation of pre-existing apo-protein (BROSEMER,1965b). Further evidence supporting this conclusion could be obtained by using the antibodies directed against the glycero-P dehydrogenase of adult honeybees. Extracts were made of thoracic muscles of newly emerged bees and of 6-day-old bees. The glycero-P dehydrogenase activity in each was 0.3 and 1.6 pmoles substrate disapp~~aring~min per mg tissue protein, respectively. Both extracts gave identical reactions in the micro-complement fixation test; that is, the position, shape, and height of the peaks were the same. Since the antigenic properties of glycero-P dehydrogenase in newly emerged bees are the same as those of the enzyme in older adults, the fivefold increase in enzymic activity is apparently due to an increase in number of glycero-P dehydrogenase molecules, i.e. due to de no~lo protein synthesis. This is consistent with the prior evidence (BROS~ER, 1965b). E;Eectrophoretic comparison of glycerophosphate dehydrogenases

Cellulose acetate electrophoresis in a phosphate buffer (pH 6.8) was used to compare glyc:ero-P dehydrogenase activities of thoracic extracts from various insects. The data are presented in Table 2. The most striking feature of most of the extracts is. the presence of more than one electrophoretic band with glycero-P dehydrogenase activity. Bumblebee and flesh fly may have four bands, yellow jacket three, and honeybee two. Some of the lighter bands are not always observed, so that great significance should not be assigned to them. The electrophoretic

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RONALDW. BROSEMER,DAVIDS. GROSSO,GLEN ESTES, ANDCHARLESW. CARLSON

TABLE ~-ELE~TR~~HoRETIc PATTERNSOF OLY~ER~PH~S~HATE DEHYDROOENASE IN INSECT THORAXEXTRACTS

Insect *

Per cent migration relative to major honeybee bandt

Relative intensity of band:

Honeybee

100 67

++++ +

Bumblebee

112 96 76 62

++ ++++ + +++

Robust mining bee

93

++++

Leaf-cutting

97 60

++++ +

144 129 107

+ ++++ ++++

94

++++

119 105 85 68

+ ++ +++ ++++

83

++++

Yellow jacket

Syrphid fly Flesh fly

Rabbits

bee

* The systematic names of these insects are given in Table 1. t Migration in each case was toward the anode. $ The major band for each insect is given the value + + + + . Bands indicated with + are faint and not always observed. 3 The purified rabbit enzyme was used. rates vary greatly for the different bands; there is no obvious taxonomic relationship in the electrophoretic patterns. It has been suggested that multiple electrophoretic bands with dehydrogenase activity may be due to a carrier protein which binds more than one type of dehydrogenase enzyme or to enzymes with more than one type of dehydrogenase activity (AGRELL and KJELLBERG, 1965). This was postulated, because certain electrophoretic bands of extracts may stain for more than one dehydrogenase activity. We therefore checked the insect thoracic extract electrophoretograms for activities of glyceraldehyde-P dehydrogenase (EC 1.2.1.12), malate dehydrogenase (EC 1 .l .1.37), and lactate dehydrogenase (EC 1 .l .1.27). As was expected, there is no detectable lactate dehydrogenase activity on any of the electrophoretograms. The various supernatant and mitochondrial malate dehydrogenases are detected, but none of these bands overlap with glycero-P dehydrogenase bands. In most cases, glyceraldehyde-P dehydrogenase and glycero-P migration

GLYCEROPHOSPHATR

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dehydrogenase bands do not overlap either. The two fastest glycero-P dehydrogenase bands of flesh fly do exhibit glyceraldehyde-P dehydrogenase activity, but this probably reflects simply the identical migration rates of these separate proteins. In order to eliminate the possibility that some bands are due to the mito[L-glycerol-3-phosphate: (acceptor)oxidochondrial glycero-P dehydrogenase reductase, EC 1.1.99.51, DPNf was left out of the staining mixture; the mitochondrial enzyme does not require DPN+ in order to reduce a dye. No staining activity is observed in the absence of DPN+. Therefore, all the glycero-P dehydrogenase bands arise from a DPN+-dependent enzyme. When glycero-P is left out of the: staining solution, no bands are observed. DISCUSSION

Immunological comparisons

Serological techniques have been used for insect classification; however, the antibodies used in these studies have almost always been directed against several antigens, e.g. against extracts. This makes interpretation of results most difficult, because the reactions depend not only on formation of the individual antigenantibody complexes but also on the relative amounts of each antigen and of each specific antibody and on interactions between different antigen-antibody systems. The present studies utilize as antigen a highly purified protein with a wellcharacterized biochemical function. Since this function is mainly limited to the specific metabolism associated with insect flight, glycero-P dehydrogenase is an especially appropriate protein for studying insect phylogeny. In addition, many physical, chemical, and enzymic properties of this protein have been reported (MARQUARDT and BROSEMER,1966 ; BROSEMER and MARQUARDT,1966). Once a single antigen-antibody system has been obtained, the immunological methods for measuring cross-reaction must be chosen. The advantages of microcomplement fixation over other standard serological techniques have been described (SARICH and WILSON, 1966). In brief, this method requires very low antigen and a:ntibody titres and is quite sensitive to slight alterations in antigenic structure. These advantages are especially critical in the study of insects. There are often only limited available quantities of the pure antigen for injection and of the desired insect specimens for cross-reaction. Many insect species are so closely related that only micro-complement fixation may have the requisite sensitivity to significantly detect differences in antigenic properties. Micro-complement fixation results obtained with the anti-honeybee glycero-P dehydrogenase are such that cross-reaction studies with these antisera must be limited largely to the advanced Hymenoptera. Slight cross-reaction was obtained with two Diptera, but no reaction could be observed with other insects outside the order Hymenoptera. Even the yellow jacket, which is a fairly advanced hymenopteran, shows relatively low cross-reaction with immunoglobulins I. This may seem to bse a narrow field of investigation, especially to those mainly interested in vertebrate phylogeny. However, the 100,000 hymenopteran species outnumber

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RONALD W. BROSEM~R, DAVIDS. GROSSO, GLENESTES,ANDCHARLES W. CARLSON

the 50,000 species in the whole vertebrate subphylum. Even the number of bee species alone (about 20,000) is greater than that of the m~malian (5000) or avian (9000) species. Hymenoptera also constitute a broad field in terms of the evolutionary time scale; this order appeared before or at the same time as mammals or birds (CARPENTER,1953). Just as classical taxonomy cannot depend upon only one character for classification, molecular taxonomy must compare as many different proteins as possible. Honeybee glyceraldehyde-P dehydrogenase has been crystallized and antibodies directed against this protein obtained; micro-complement fixation tests are presently underway using this system. Studies with antibodies directed against several other pure proteins from honeybees must be undertaken before a comprehensive picture of the relative relationship between proteins of the higher Hymenoptera can begin to emerge. In order to compare closely related species using this immunochemical technique, it is necessary to utilize antibodies to proteins of one of the members of the same or similar taxon (KAPLAN, 1965). For example, if Diptera are being compared, antibodies to a honeybee protein are not very useful. A dipterous protein must be purified and antibodies to it prepared before the taxonomic relationship can be measured. Studies on the molecular phylogeny of insects have lagged behind those of vertebrates and micro-organisms. This is emphasized by the fact that only about five insect enzymes have been crystallized and the amino acid composition of only two of these has been reported. Certainly the ultimate technique for investigating insect molecular phylogeny is the determination of the complete primary structure of several insect proteins. Until such time that this goal can be achieved, microcomplement fixation will remain one of the most rapid and reliable methods for investigating insect phylogeny at the level of comparative protein structure. Since the rate of speciation is by far the greatest among insects, it appears possible that the average rate of change of protein primary structure may be higher in insects than in other animals. The immunological data reported here can be compared with similar data obtained with various vertebrate proteins in order to obtain at least an indication whether insect glycero-P dehydrogenase is altering at a greater rate than corresponding vertebrate enzymes. KAPLAN(1965) has reported I.D. values in the micro-complement fixation test for four separate DPN+-dependent dehydrogenases ; glycero-P dehydrogenase was not included in this study. The I.D. values are, to a first approximation, similar for these four enzymes; that is, when an extract of a species is compared with the corresponding anti-chicken dehydrogenase, the I.D. value is about the same for all four antisera. The following assumptions can be made: (1) the I.D. values for vertebrate glycero-P dehydrogenase would be similar to those for the other enzymes, (2) I.D. values are a valid approximation of primary structural differences (KAPLAN, 19654, (3) a change in primary structure of an insect dehydrogenase results in the same relative I.D. as in a corresponding vertebrate dehydrogenase. In addition, the times of divergence of the lines leading to the various species used in the vertebrate

GLYCEROPHOSPHATE

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study and in the present study must be estimated. For the species mentioned in Table 1, the following times of divergence in millions of years have been estimated (these figures, are subject to large error) : honeybee-fly: 300 (Ross, 1965) ; honeybeeyellow jacket: 200; honeybee-sphecoid wasp: 150 (MICHENER, 1944); honeybee-all other bees: 50 to 150. Using these assumptions and estimates, the insect glycero-P dehydrogenase The change in I.D. data can be compared with those of vertebrate dehydrogenases. value for a given evolutionary time increment is roughly similar in both studies. Therefore, it appears that insect glycero-P dehydrogenase may not be changing at a significantly faster rate than vertebrate dehydrogenases. It must be emphasized that the above assumptions are very tenuous; a definite conclusion on the rate of change of insect glycero-P dehydrogenase must await comparative primary structure studies. In the only investigation to date of the complete primary structure of an insect protein, FITC:H and MARGOLIASH (1967) have shown that cytochrome c changes at approximately the same rate in insects as in other species. Cytochrome c activity is required by all insects; high levels of glycero-P dehydrogenase are present in many if not all flying insects. Therefore, the rate of change of these two proteins may not reflect the high rate of speciation in insects. Possibly those proteins concerned with characteristics not shared by most insects would vary at a fundamentally faster rate; such proteins might be those involved in coloration, feeding habits, specialized anatomical features, and the like. It will probably, however, be very difficult to identify and isolate such proteins, let alone determine their primary structures. Electrophoret ic comparisons The electrophoretograms of glycero-P dehydrogenase activity in the various insects studied here are complex. Most of the insect thoracic extracts show more than one band with glycero-P dehydrogenase activity, but there is no consistent pattern in the number or position or relative intensity of these bands. In the limited sampling of insects tested, there appears to be almost a random distribution of enzyme patterns. For example, the honeybee shows one major and one very minor band ; the closely related bumblebee shows three major and possibly one minor band. The bumblebee pattern more closely resembles that of the flesh fly than of any other insect tested. This emphasizes the fact that electrophoretic comparisons may have limited taxonomic significance; there are cases, however, where such comparisons do give meaningful information (KITTO and WILSON, 1966). The maximum number of bands found in any one species gives an indication of the subu:nit structure of the enzyme. Rabbit glycero-P dehydrogenase is probably a dimer (VAN EYS et al., 1964). Since the molecular weight of the honeybee enzyme is only slightly less than that of the rabbit enzyme (BROSEMER and MARQUARDT, 1966) and since most dehydrogenases are oligomers of subunits with about 35,000 molecular weight (KAPLAN, 1965), the honeybee enzyme may also be

1766

RONALDW. BROSEMER,DAVIDS. GROSSO,GLEN ESTES, ANDCHARLES W. CARLSON

a dimer. If a dimer can be composed of any combination of two different subunits (A and B), there are three possible combinations (A,, AB, B,); if it is composed of any combination of three different subunits, there are six possible combinations (AZ, AB, AC, B,, BC, C,). A trimer composed of two different subunits may have up to four possible combinations (A3, A,B, AB,, B3). Of course, not all combinations may exist, so that the actual number of molecular forms may be less than the theoretical maximum. Bumblebee and flesh fly thoracic extracts show three and possibly four bands with glycero-P dehydrogenase activity (Table 2). In each the fourth band is very light, so that it is of questionable significance. However, if there are indeed four bands, several explanations are possible. Two of the more likely explanations are that the enzyme in these species is : (1) a trimer with two types of subunits or (2) a dimer with three types of subunits but not all possible combinations occurring. Thus the number of subunits in a glycero-P dehydrogenase molecule or the number of different kinds of subunit structures in the molecule may vary in insects, including the closely related honeybee and bumblebee. Such a phenomenon could have phylogenetic and functional significance. The only conclusive method for comparing the subunit structure is to isolate the enzyme from several species. It is, for example, possible that the multiple electrophoretic bands do not represent different primary structures but rather different conformations of the enzyme molecule (KITTO et al., 1966). Acknowledgements-We wish to acknowledge the technical assistance of LOIS BARNES. We wish to thank Dr. ALLAN C. WILSON for introducing one of us (R. W. BROSEMER)to the micro-complement fixation technique. This work was supported in part by grant GB-4863 from the National Science Foundation and by a development award to R. W. BROSEMER of the Public Health Service research career program (number lK03 GM1 1073-01 GMK) from the Institute of General Medical Sciences, and by funds provided for biological and medical research by the State of Washington Initiative Measure No. 171. G. ESTES and D. GROSSOwere National Science Foundation Undergraduate Research Participants (Grant JY-313).

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CARPENTERF. M. (1953) The geological history and evolution of insects. Am. Scient. 41, 256-270. FINE I. H. and COSTELLO L. A. (1963) The use of starch electrophoresis in dehydrogenase studies. In Methods in Enzymology (Ed. by COLOWICK S. P. and KAPLAN N. 0.) 6, 958-972. Academic Press, New York. FITCH W. M. and MARGOLIASHE. (1967) Construction of phylogenetic trees. S&ence, N. Y. 155, 279-284. KAPLAN N. 0. (1965) Evolution of dehydrogenases. ~vo~v~~g Genes and Prote-im (Ed. by BRYSON V. and VOGEL II. J.) 243-277. Academic Press, New York. KITTO G. B., WASSAFXMAN P. M., and KAPLANN. 0. (1966) Enzymatically active conformers of mitochondrial malate dehydrogenase. Proc. nut. Acad. Sci. U.S.A. 56, 578-585. KITTO G. B. .and WILSON A. C. (1966) Evolution of malate dehydrogenases in birds. Science, N. Y. 153, 1408-1410. MARQUARDTR. R. and BROSEMR R. W. (1966) Insect extramitochondrial glycerophosphate dehydrogenase-I. Crystallization and physical properties of the enzyme from honeybee (Apis melE$era) thoraces. Biochim. ~ophys. Actu 128, 454-463. MICHENER C. 11. (1944) Comparative external morphology-, phylogeny, and a classification of the bees (Hymenoptera). Bull. Am. Mu.s. nat. Hist. 82, 151-326. PENHOETE., R~~JKUMAR T., and RUTTER W. J. (1966) Multiple forms of fructose diphosphate aldolase in :mammalian tissues. Proc. nut. Acad. Sci. U.S.A. 56, 1275-1282. PLESCIA 0. J., BRAUNW., and PALCZUKN, C. (1964) Production of antibodies to denatured deoxyribonucleic acid (DNA). Proc. nat. Acad. Sci. U.S.A. 52, 279-285. ROCK~TEINM. and BRANDT K. F. (1963) Enzyme changes in flight muscle correlated with aging and flight ability in the male housefly. Science, N.Y. 139, 1049-1050. Ross H. H. (15165) A Textbook of Entomology. John Wiley, New York. SARICH V. M. iand WILSON A. C. (1966) Quantitative immunochemistry and the evolution of primate albumins: micro-complement fixation. Science, N.Y. 154, 1563-1566. VANEYS J., JUDD J., Form J., and WOMACKW. B. (1964) On the chemistry of rabbit muscle a-gly~erophosphate dehydrogenase. ~~c~~try 3, 1755-1763. WASSERMANE. and LEVINE L. (1961) Q uantitative micro-complement fixation and its use in the study o:f antigenic structure by specific antigen-antibody inhibition. J. Immun. 87, 290-29s. WEIS-FOGH ‘I?. (1961) Power in flapping flight. The Cell and the Organism (Ed. by RAMSAY J. A. and WIGGLESWORTHV. B.) 283-300. Cambridge University Press, London.