Changes in the nucleic acid content and structure of thoracic mitochondria during development of the blowfly, Lucilia cuprina

J. InsectPhysiol.,1967,Vol. 13,pp. 1745to 1756. PergamonPressLtd. Printedin Great Britain

CHANGES IN THE NUCLEIC ACID CONTENT AND STRUCTURE OF THORACIC MITOCHONDRIA DURING DEVELOPMENT OF THE BLOWFLY, LUCILIA CUPRINA R. W. LENNIE,”

D. W. GREGORY,?

and L. M. BIRT

Department of Biochemistry, University of Sheffield (Received 12 April 1967) Abstract-The distribution of nucleic acids during the development of the blowfly Lucilia cuprina has been studied. The DNA content varied from 12.8 pg to 22.0 pg/insect and its distribution suggests that some of the DNA may be extra-nuclear. The RNA content varied from 206 to 402pglinsect; in early pupal life it was concentrated in a fraction containing particles greater than 3 ,u in diameter. The nucleic acid content of purified preparations of mitochondria from the flight muscles has been determined during adult development from 1 day before to 7 days after emergence. The amount of mitochondrial DNA varied between 0.53 and 1.41 pgjinsect with values for the specific content from 1.0 to 5.3 pg DNA/mg protein. The mitochondrial RNA varied between 3.6 and 9.5 pg/ insect, while the values for the specific content were between 4 and 48 pg RNA/mg protein. Electron micrographs of mitochondria, both in tissue preparations and after isolation, showed a marked increase in internal structure, organization, and overall size during the period from 19 days before to 1 day after emergence. The data presented are in agreement with a model previously proposed for the development of flight muscle mitochondria. INTRODUCTION

A CONSIDERABLE amount of data is accumulating about the morphogenesis of mitochondria in a wide variety of tissues (ANDRE?,1962; AVERS et al., 1965; CHRISPEELS et al., 1966). The most detailed reports using insects are given for changes during the diapause and subsequent redevelopment of the flight muscles of the beetle, Leptizotarsa decemlineata (STEGWEE et al., 1963); for the locust, Locusta migratoria (BROSEMERet al., 1963); for the developing moth, Hyalophora cemopia (MICHEJDA, 1964); for the bee, Apis meZZifera (HEROLD, 1965); and for the blowfly, Lucilia cuprina (LENNIE and BIRT, 1967). The last two studies can be compared most readily as they deal with the changes in holometabolous insects developing without diapause. HEROLD(1965) h as reported variations in the amounts of cytochromes and in the structure of mitochondria during pupal and adult development. At an early stage, the mitochondria were found to be ‘vesicular’ * Present address: Victorian Plant Research Institute, Burnley Gardens, E.l, Victoria, Australia. t Medical Research Council Research Group for the Study of the Biochemistry and Physiology of Intracellular Organelles. 1745

1746

R. W. LENNIE,D. W. GREGORY,ANDL. M. BIRT

and almost devoid of ‘internal tubules’; at ecdysis the increase in mitochondrial size and the number of these ‘tubules’ was associated with the appearance of the cytochromes. As no specific activities were quoted, it is not possible to correlate precisely the development of protein and cytochromes. LENNIE and BIRT (1967) have examined the correlation between the dehydrogenases and dehydrogenasefree (‘structural’) protein for Luciliu and have suggested that the nucleic acid detected in these mitochondria was concerned with the synthesis of structural protein. A growing body of evidence points to the existence of a specific mitochondrial DNA (TEWARI et al., 1965 ; BORST and RUTTENBERG,1966; KROON, 1966) differing in both physical properties and chemical composition from nuclear DNA. A number of workers have suggested that this extra-nuclear DNA is concerned with the control of the synthesis of the structural protein of the mitochondrion (KALF, 1964; N~ss et al., 1965; RABINOWITZ et al., 1965). Mitochondria also contain RNA (see FREEMAN, 1965). This paper-presents information about variations in the amount of mitochondrial nucleic acids from 1 day before to 7 days after emergence of the adult fly. Changes in mitochondrial structure from about 19 days before to 1 day after emergence of the adult have also been examined as this period appears to be a critical one in the development of the mitochondria of the flight muscles (LENNIE and BIRT, 1967). The present data are interpreted as supporting the model previously proposed for the morphogenesis of the flight muscle mitochondria of Lucilia . MATERIALS

AND METHODS

Chemicals Orcinol and DNA (from salmon testes) were obtained from British Drug Houses, Poole, Dorset; ATP from Sigma St. Louis Missouri. The 3,5-diaminobenzoic acid, from Koch-Light Laboratories, Bucks., was recrystallized as described by KISSANE and ROBINS (1958) and stored as the dihydrochloride. Preparation of fractions Insects were kept at 30°C throughout the entire life cycle (for details of culture see LENNIE and BIRT, 1967); the larval stage lasted 5 days and the ‘pupal’ stage, covering the entire period in the puparium, 6 days. During this time, the insect is a pupa for the first 2 to 3 days; thereafter, for the next 3 to 4 days, it is a pharate adult. The preparation and fractionation of tissue homogenates were carried out as described previously (LENNIE and BIRT, 1967). The homogenates were separated into four fractions using their Method I ; the ‘soluble’ fraction contained soluble cytoplasmic compounds, ribosomes, and other particles less than 0.2 p in diameter ; the ‘small particle’ fraction (0.2-l p in diameter) contained mainly mitochondria; the ‘intermediate particle’ fraction (l-3 p in diameter) contained a few larger somatic mitochondria and in the adult, flight muscle mitochondria; and the ‘large particle’ fraction (greater than 3 ,U in diameter) contained nuclei, and especially in

DEVELoPiWPNT OFBLOWFLY MITOCHONDRIA

1747

the adult, muscle fragments. All the particulate fractions contained ‘albumenoid bodies’ especially during the pupal and pharate adult stages. The limits for the particle sizes in the various fractions were chosen arbitrarily, having in mind the separation of the subcellular organelles mentioned above. The conditions for sedimentation were calculated on the assumption that all particles were spheres and had a specific gravity of 1.20 (i.e. close to the values reported for mitochondria). Puri$cation of mitochondrial fractions ‘Large’ and ‘small’ mitochondrial fractions were separated according to Method III of LENNIEand BIRT (1967). The material in the large mitochondrial fraction at about 1Q days before emergence was concentrated for electron microscopy by resuspension in 0.25 M sucrose and centrifugation at 70,000 g for 30 min in small conical tubes. This procedure appears to have caused extensive disruption of these mitochondria (see Fig. 3). Estimation of nucleic acids in insects and fractions from tissue homogenates The RNA content was estimated with orcinol by the method of HELE and FINCH (1960). The DNA content of single insects and of fractions from tissue homogenates was estimated by the diphenylamine method of BURTON(1956). A coloured substance was released during the hydrolysis of the materials precipitated by perchloric acid; washing the precipitate twice with cold perchloric acid did not remove it completely. The substance did not react with the diphenylamine reagent and its extinction at 700 rnp was almost identical to that at 600 rnp, the wavelength used in the estimation of DNA. Therefore the difference between the optical densities at 600 rnp and 700 rnp was used to determine the amount of DNA in the extracts. Estimations of the nucleic acids in the fractions were done in duplicate while those on individual insects were done in triplicate. Estimation of nucleic acids in mitochondrial fractions The modification of the Schmidt-Thannhauser extraction as described by HUTCHISONand MUNRO (1961) was used to prepare lipid-free precipitates from the mitochondrial fractions. These were hydrolysed in 1-O ml of O-5 M perchloric acid for 20 min at 70°C centrifuged, and the supernatant fluid removed. This treatment was repeated and the combined supernatant fluids used for the estimations of both DNA and RNA. For the estimation of DNA the perchloric acid was removed by precipitation as potassium perchlorate. The DNA was estimated by the fluorimetric method of KISSANEand ROBINS(1958) ; fluorescence was measured in a Farrand A-2 fluorimeter (Farrand Optical Co., New York) as described by POLAKISand BARTLEY(1966). RNA was estimated with orcinol (HELE and FINCH, 1960).

1748

R.

W. LENNIE,D. W. GREGORY, ANDL. M. BIRT

Electron microscopy (a) Flight muscles. Insects were cooled to 0°C; the heads and abdomens were carefully removed and discarded. The thoraces were bisected dorsoventrally and the two halves were fixed in 1% (w/v) 0~0, in veronal-acetate buffer, pH 7.3 (PALADE, 1952) for 1 hr at 0°C. The fixed tissue was rinsed with water, and whilst under water the longitudinal dorsal muscles were dissected out. (b) Mitochondria. After centrifugation the supernatant sucrose solution was decanted from the mitochondrial pellet. ‘Two methods of fixation and embedding were employed. The first method was to add ice-cold buffered 1% (w/v) 0~0, to the undisturbed pellet for 1 hr. After being washed with water, pieces of the fixed pellet were dehydrated and embedded in Araldite. The second method was to resuspend the mitochondria in buffered l”/” (w/v) 0~0, at 0°C for 1 hr. The suspension was then centrifuged at 2500g for 5 min, and the supernatant fixative was decanted. The pellet of tixed mitochondria was washed with water by resuspension and recentrifuging. The fixed mitochondria were resuspended in a small volume of warm 2% (w/v) agar, and the suspension was poured on to a cold glass plate where it solidified. Small cubes (about 1 mm side) of the agar gel were dehydrated and embedded in Araldite. This method of dispersing mitochondria has been used previously (Dr. H. W. WOOLHOUSE,personal communication) and is essentially similar to that used by KELLENBERGERet al. (1958) for dispersing bacteria. Sections were stained with lead citrate (REYNOLDS, 1963). RESULTS

Variations in the content and distribution of RNA in whob insects during the lif cycle The amount of RNA/insect (Table 1) at the larval stage increased by 18 per cent to a maximum value in l-day-old pupae, declined appreciably (by 50 per cent) during ‘pupal’ life (3-day-old pharate adult) and then rose slightly (by 20 TABLE1 Amount

of nucleic

acid/insect

(w) Larvae (3-day-old) DNA RNA

19.1 340

Pupae (l-day-old) 12.8 402

Pharate adults (3-day-old) 19.3 206

Flies (7-day-old) 22.0 240

per cent) as the fly matured (7-day-old fly). Losses occurred during the preparation of the homogenates, the average recovery being 82 per cent (range 72 to 95 per cent). The total amount of RNA present in all the fractions isolated at each stage averaged 99 per cent (84 to 108 per cent) of the RNA in the homogenate. The

DEVELOPMENT OF BLOWFLY MITOCHONDRIA

1749

distribution of RNA between various subcellular fractions, separated on the basis of apparent size (LENNIE and BIRT, 1967), has been expressed as a percentage of the sum of the material in all the fractions (Table 2) ; the nature of the fractions is described in Materials and Methods. TABLE 2 Nucleic acid (as y0 of total) Larvae (3-day-old) Fraction

DNA

‘Large particles’ (greaterthan 3 I_L 35 in diameter) ‘Intermediateparticles’ (l-3 p in diameter) 4 ‘Small particles’ (0.2-l p in diameter) 17 ‘Soluble’ (less than 0.2~ in diameter) 44

Pupae (1 -day-old)

Pharateadults (3-day-old)

Flies (7-day-old)

RNA

DNA

RNA

DNA

RNA

DNA

RNA

30

100

60

49

22

30

13

4

0

0

21

9

45

13

32

0

23

21

22

18

20

34

0.

17

9

47

7

54

The larval RNA was distributed almost equally between the ‘soluble’, ‘small particle’ and ‘large particle’ fractions, which together accounted for 96 per cent of the total. A marked change in the distribution occurred during pupation especially in the ‘large particle’ fraction in which there was a considerable accumulation of RNA (about 14Opg/ insect); in the ‘soluble’ and ‘small particle’ fractions there was a slight loss of RNA. Further changes in the distribution took place during ‘pupal’ development and in the first 7 days of adult life, at the end of which most (54 per cent) of the RNA was in the ‘soluble’ fraction. There was a decrease of 30 ,ug/insect in the ‘large particle’ fraction; RNA began to appear in the ‘intermediate’ fraction during pharate adult development and continued to accumulate in these particles as the fly matured. The proportion of RNA in the ‘small particle’ fraction was relatively constant during the entire period. The twofold increase in RNA in the ‘large particle’ fraction detected at pupation may be related to development of the ‘albumenoid bodies’ (WIGGLESWORTH, 1942; NAIR and GEORGE, 1964), which are known to contain enzymes for the carboxyl activation of amino acids, especially of tyrosine (LENNIE and BIRT, 1965), and for the incorporation of amino acids into protein. Variations in the content and distribution of DNA in whole insects during the life cycle There was a decrease of about 30 per cent in the DNA content (Table 1) during pupation from the larval value of 19 ,ug/insect followed by a return to the initial value prior to emergence (3-day-old pharate adult). There was a further

1750

R. W. LENNIE,D. W. GREGORY,ANDL. M. BIRT

small rise (15 per cent) as the fly matured (7-day-old fly). These values agree with those previously reported for the same insect (HOWELLS and BIRT, 1964). The amounts of DNA recovered in the homogenates were consistently lower than those for the whole insect at the same stage of development. The average recovery was 74 per cent (range 68-82 per cent), the losses being greater than those of RNA. As separate tests showed that the DNAse activity of the homogenate was low (accounting for not more than 5 per cent of the observed loss), the most likely explanation for the discrepancy was the loss of nuclei during filtration. With larvae, an additional problem was the difficulty of disrupting the epidermal cells (LENNIE and BIRT, 1967) which may contain polyploid nuclei, rich in nucleic acid (AGRELL, 1964; RICHARDS, 1953). The distribution of the DNA between various subcellular fractions has been expressed as a percentage of the sum of the material in all the fractions (Table 2) ; the average recovery was 103 per cent (range 97-107 per cent) of the DNA in the homogenate. Most of the DNA was found in those fractions containing particles greater than 1 ,u in diameter except for the unusually high value (44 per cent) in the ‘soluble’ fraction of the larvae. In the larval stage most of the particulate DNA was in structures greater than 1 I.L in diameter. In the pupal stage all of the DNA was in the ‘large particle’ fraction, suggesting that all the material was nuclear. In the 3-day-old pharate adults, most (70 per cent) of the DNA was detected in the greater than 1 p fractions but with some 21 per cent in the ‘small particle’ fraction. The distribution in the adult (7-day-old) fly was similar, except that some redistribution has taken place in the two fractions greater than 1 p, resulting in 45 per cent of the DNA being in the ‘intermediate’ fraction (l-3 p in diameter). The very high value for DNA in the ‘soluble’ fraction (which will contain particles less than O-2 p in diameter) may be due in part to a release of DNA from the larger particles: it occurred to an appreciable extent only in the larvae. It has been suggested that the non-mitochondrial cytoplasmic DNA found in a variety of animal (mainly egg) and plant tissue represents a store of deoxyribonucleotides (see AGRELL and PERSON, 1956). Although this interpretation has been questioned (DAWID, 1965), such a role would be consistent with the general accumulation of nutrient reserves by larvae. The distribution of DNA amongst particles from the larvae is consistent with reports of a very wide range in the diameter of nuclei from various larval organs of Diptera (AGRELL and PERSSON, 1956; RICHARDS, 1953), but could also be due to However, at the beginning of pupal life the presence of nuclear fragmentation. DNA in only one fraction suggests a more restricted range in the size of nuclei. By the end of pupal life the distribution again points either to a considerable variation in the size of nuclei or to their fragmentation. The ‘large’ and ‘intermediate particle’ fractions contained relatively large numbers of intact nuclei, as detected by phase-contrast microscopy. However, as the ‘small particle’ fraction was free from intact nuclei, the DNA must be in either fragments of nuclei or the mitochondria which were present in large numbers.

DEVELOPMENT

OF BLOWFLY

MITOCHONDRIA

1751

Changes in the content and distribution of nucleic acids of flight muscle mitochondria during development The distribution of RNA and DNA in various fractions isolated from L. cuprina indicated that the mitochondria of this insect might also contain nucleic acids. As information was already available for the changes in protein content and the activity of some respiratory enzymes during the development of the flight muscle mitochondria of this insect, a more detailed investigation of the nucleic acid content of these organelles has been undertaken. As previously discussed (LENNIE and BIRT, 1967) the method used to fractionate the tissue homogenates does not permit complete recovery of mitochondria greater than 1 p in diameter. Therefore values for the concentration of the total nucleic acids (as pg/mg protein) in all the mitochondria from the thorax at a given stage of development have been estimated from the amount of mitochondrial protein/insect (Fig. 3, LENNIE and BIRT, 1967) and the experimentally determined values for the concentrations of nucleic acids in the isolated, purified fractions. In the day before emergence the mitochondrial DNA increased almost threefold to a value of 1.41 pg/insect and then declined (Table 3). At each stage of development the amount in the large mitochondrial fraction was greater than that in the corresponding small mitochondrial fraction, though the difference at emergence and 1 day after emergence was very small. The average concentration of mitochondrial DNA doubled to 4.7 pg/mg protein in the last day of pharate adult life due mainly to the fourfold increase in DNA in the small mitochondrial fraction. At later stages the concentrations were similar in both fractions and were considerably lower than the values at emergence, the decline being due mainly to the continuing increase in mitochondrial protein. The range of values for the specific content of DNA (1.0 to 5.3 pg/mg protein) may be compared with values obtained for other cells. For example, yeast mitochondria have been found by different workers to contain from 0.4 to 10 pg DNA/mg protein (SCHATZ et al., 1964; NASS et al., 1965 ; TEWARI et al., 1965) and values of 3.4 to 5.6 pg DNA/mg protein have been reported for lamb heart mitochondria (KALF, 1964). The amount of mitochondrial RNA rose slightly in the last day of pharate adult life to a value of 9.5 pg/insect then declined until in the mature 7-day-old fly less than 40 per cent of the maximum value was recorded. Thus although the pattern of change was similar to that for DNA the increase in RNA was much less and its decline after emergence was more rapid. Initially more of the RNA was in the small mitochondrial fraction (57 per cent), but with development the pattern slowly changed until in the 7-day-old adult there was slightly more in the large mitochondrial fraction (58 per cent). As a consequence of these changes, the range of specific contents of RNA was from a maximum value of 48 pg/mg protein (in the small mitochondrial fraction isolated 1 day before ecdysis) to 4 pg/mg protein (in the large mitochondrial fraction isolated 7 days after ecdysis). There was little difference in the specific content of the two fractions until the fly had matured, when the specific content of the larger mitochondria dropped to a very low value (about 8 per cent of the maximum). The range of values (4 to 48 pg/mg protein)

100 0.10

4.8 48

(c) pg Protein/insect (talc.) (d) pg DNA/insect (talc.)

(e) ,ug RNA/insect (talc.) RNA/DNA

42* 200 0.53 8.4 16.0*

100 0.43 3.6 8.4

2.6”

36

4.3

4.9 8.8

4.6 5.5

160 085

29

5.3

9.5 6.7*

300 I.41

32*

4=7*

At emergence > 1 ,u Total

140 0.56

35

4.0


4.0 7.8

190 0.51

21

2.7

3.0 5.2

190 0.59

16

3.1

I.5 6.9

140 0.22

380 I.10

I.6 11

7.0 6.4”

7 days

2.1 2.9

520 0.73

4

1.4

3.6 3.8*

660 0.95

5*

1.4”

after emergence > 1 p Total
18”

2*9*

after emergence > 1 p Total


1 day

The values for the concentrations of nucleic~ acids in the mitochondrial fractions were determined experimentally (lines (a) and (b)). The amount of mitochondrial protein (line (c)) in the fractions was derived from Fig. 3 of LENNIE and BIRT (1967). The values for the amount (pg) of nucleic acids/insect (lines (d) and (e)) were determined from the values in lines (a), (b), and (c). * Values were calculated from the total amount (pg) of mitochondrial nucleic acid/insect (lines (d) and (e)) and the total amount (mg) of mitochondrial protein/insect (line (c)) and therefore represent the average values for the whole mitochondrial population.

48

I.0

(b) pg RNA/mgprotein

(a) pg DNA/mg protein


before emergence > 1 p Total

1 day

TABLE 3-MITOCHONDRIAL NUCLEICACIDS

5 F 1

g r

.JJ

i

9

p

“[

9

FiG. 1. Longitudinal section of indirect flight muscle from a fly about 1½ days before emergence, The mitochondria vary greatly in size, often have an irregular outline, and have areas which contain no cristae.

FIGS. 2 and 3. Mitochondrial fractions isolated from the thoraces of flies about 1½ days before emergence, i.e. at the same age as the tissue shown in Fig. 1. Fig. 2: Small ( < 1/z) mitochondrial fraction. Fig. 3 : Large ( > 1/z) mitochondrial fraction, Extensive disruption has occurred in this preparation which was resuspended and recentrifuged (see Materials and Methods),

FIG. 4. L o n g i t u d i n a l section of i n d i r e c t flight muscle f r o m a fly 1 day after emergence. T h e m i t o c h o n d r i a vary greatly in size a n d often have a n irregular outline. I n m o s t of t h e r n i t o c h o n d r i a at this stage the cristae are seen t h r o u g h o u t the entire m i t o c h o n d r i a l space a n d are a r r a n g e d in a regular m a n n e r w i t h a characteristic p a t t e r n of fenestrations (cf. SMITIJ, 1963). FIG. 5. T w o m i t o c h o n d r i a w i t h a m a r k e d l y different a p p e a r a n c e in t h e same tissue as s h o w n in Fig. 4. T h e less organized type is a very small p r o p o r t i o n of the total.

FIGS. 6 and 7. Mitochondrial fractions isolated from the thoraces of flies 1 day after emergence, i.e. at the same age as the tissue shown in Figs. 4 and 5. Fig. 6: Small ( < 1/z) mitochondrial fraction. Fig. 7 : Large ( > 1/~) mitochondrial fraction,

DEVBLOPMBNT

OF BLOWFLY

MITOCHONDRIA

1753

for specific content of RNA is considerably greater than those found in mitochondria from other cells, e.g. from yeast, 18 to 40 pg/mg protein (TEWARI et al., 1965) and 7 to 14 pg in mitochondria from beef heart (KROON, 1966). It has been proposed that when the flight muscle mitochondria reach a size of about 1 p (which for the majority of the particles occurs close to the time of emergence) there is a period when the rate of synthesis of ‘structural’ protein is greater than that of the dehydrogenases (LENNIE and BIRT, 1967). It seemed likely that this development would be preceded by the multiplication of the nucleic acids required for the synthesis of the ‘structural’ protein and the results in Table 3 give support to this proposal. Thus immediately before emergence the growth of the mitochondria to a size greater than 1 EL.in diameter involved a fourfold increase in the specific content of DNA to about 4 pg/mg protein. The concentration of RNA at the earliest stage tested was already at a maximum, and about the same value in both fractions. At emergence, when the population of mitochondria has a mean diameter of about 1 p, the specific content of DNA in both fractions was similar and this was the period when the rapid synthesis of ‘structural’ protein was occurring. During later development the amounts of both DNA and RNA in the mitochondria declined. The RNA was lost more rapidly than the DNA, particularly from the larger mitochondria, in which the rate of protein synthesis was much reduced. It might be expected that during the period when ‘structural’ protein is being formed relatively rapidly a considerable development of intramitochondrial membranes might be detected. Therefore the electron microscope was used to study the structure of mitochondria both in the intact muscle and after isolation. Samples were taken from flies 1Q days before and 1 day after emergence. Structure of mitochondria in tissue sections Figs. 1 and 4 show the appearance of mitochondria in thoracic muscle tissue about 14 days before and 1 day after emergence respectively. The structure of this tissue at intervals from 3 days before to 8 days after emergence of the adult fly will be described in detail elsewhere (GREGORY et al., 1967). The most obvious change during the period 18 days before to 1 day after emergence is the difference in the number of cristae per mitochondrion which increases from about 14 to 321~ of mitochondrial length; these values were obtained from counts of cristae in fifty profiles, selected randomly. The mitochondria in the younger tissue have relatively large areas in which no cristae are present, but at the later stage cristae are packed uniformly throughout the entire profile. In the older tissue the cristae are more regularly ordered, so that the characteristic pattern of fenestrations (SMITH, 1963) is now well defined. The area of mitochondrial profiles seen in the sections, which is probably proportional to the volume of the mitochondria, increases about two and a half times over the 24 day period. Thus the number of cristae per mitochondrion, the estimated mitochondrial volume, and the total mitochondrial protein (LENNIE and BIRT, 1967) all increase two- to threefold during this period.

175’4

R. W. LENNIE,D. W. GREGORY,ANDL. M. BIRT

While it is apparent that most of the mitochondria in the older tissue are densely packed with cristae, it is still possible to find two juxtaposed mitochondria of roughly equal size but of markedly different internal morphology (Fig. 5). Such a difference is unlikely to be a fixation artifact since the two types of mitochondria are found side by side; it may indicate either the existence of mitochondria at different stages of development or degradation in one area of the muscle or a functional diversity of mitochondrial types. SMITH (1963) observed two types of mitochondria in the flight muscle of the mature blowfly Calliphora. He suggested that the less organized type might be degenerating. However, this explanation seems less likely to apply for the present work with tissue at only 1 day post emergence. Structural heterogeneity of mitochondria has also been reported in a number of other cell types (OGAWAand BARRNETT, 1964, 1965; AVERS et al., 1965). Structure

of isolated mitochondria

Electron micrographs of small and large mitochondria isolated in 0.25 M sucrose (Figs. 2, 3, 6, and 7) confirm the adequacy of the method of preparation in providing mitochondrial samples virtually free of contamination by nuclei and albumenoid bodies, as previously reported on the basis of phase contrast microscopy (LENNIE and BIRT, 1967). Considerable difficulty was experienced in isolating from pharate adults sufficient of the large mitochondrial fraction for electron microscopic examination, and the relatively high g value used to sediment the material has caused extensive damage to the mitochondria with the release of the inner membranes. The pellets also contain some small granules which may be ribosomes, possibly of non-mitochondrial origin. Nevertheless, the pattern of changes for mitochondrial RNA described previously (Table 3) would be little affected by some contamination of this fraction. Most of the mitochondria tend to become spherical during the separation procedure though some of the smaller ones isolated from flies not yet emerged are still elongated. Before emergence (Figs. 2 and 3) all the mitochondria are relatively small, those in the large mitochondrial fraction being only slightly greater than 1 p in apparent diameter. After emergence (Figs. 6 and 7) there is a considerable difference in the mean size of the mitochondria in the two fractions ; the larger mitochondria may be up to 4 p in apparent diameter, while most of those in the other fraction are approaching 1 I”. It is also clear that by the later stage the density and complexity of cristae have increased markedly in mitochondria in both fractions. In general the small mitochondria have retained their internal organization more completely, whereas many of the large mitochondria are swollen; they are similar in appearance to the mitochondria of the housefly isolated in O-154 M KC1 (CARNEY, 1966). The mitochondria in the tissue section resemble more closely housefly mitochondria isolated in 0.25 M sucrose, which are supposed by Carney to be shrunken. In contrast to this supposition it seems likely that for blowfly mitochondria the sucrose medium preserves the mitochondrial structure more adequately. Thus the electron micrographs of isolated mitochondria confirm the conclusion drawn from those of the tissue that in the period from 18 days before to 1 day

DEVELOPMENT OF BLOWFLYMITOCHONDRIA

17.55

after emergence of the fly, there is a marked increase in the average size of the thoracic mitochondria and a considerable increase in the number and organization of their cristae. All the data reported in this paper are consistent with the model for mitochondrial development previously proposed (LENNIEand BIRT, 1967). Acknowledgements--R. W. LENNIE was a recipient of a post-graduate award from the Commonwealth Office of Education, Australia, and was on study leave from the Victorian Department of Agriculture. Financial support was provided by the Medical Research Council, (D. W. GREGORY)and the Science Research Council (L. M. BIRT). Facilities for electron microscopy were provided by the Nuflield Foundation and were made available by Professor R. BARERof the Department of Human Biology and Anatomy, University of Sheffield. We also wish to thank Professor BARTLEYfor his interest and encouragement, Miss M. DOWSON for technical assistance in specimen preparation, and Mr. P. GARLICK for taking the electron micrographs. REFERENCES AGRELL I. (1964) Physiological and biochemical changes during insect development. In The Physiology of Insecta (Ed. by ROCK~TEIN M.) 1,91-149. Academic Press, New York. AGRELLI. and PERSSONH. (1956) Changes in the amount of nucleic acids and free nucleotides during early embryonic development of sea urchins. Nature, Lond. 178,1398-1399. ANDREJ. (1962) Modifications ultrastructurales du chondriome. r. Ultrastruct. Res. (Suppl.) 3, 7-185. AVERSC. J., PFEFFER C. R., and RANCOURT N. W. J. (1965) Acriflavine induction of different kinds of ‘petite’ mitochondrial populations in Saccharomyces cerevisiae. J. Bact. 90, 481494. BORSTP. and RUTTENBERG C. J. C. M. (1966) On the presence of DNA in mitochondria of animal tissues. In Regulation of Metabolic Processes in Mitochondria (Ed. by TAGER J. M., PAPA S., QUAGLIARIELLO E., and SLATERE. C.), pp. 4544.58. Elsevier, Amsterdam. BROSEMER R. W., VOGELLW., and BOCHERT. (1963) Morphologische und enzymatische Muster bei der Entwicklung indirekter Flugmuskeln von Locusta migratoria. Biochem. 2. 338, 854-910. BURTONK. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. CARNEY G. C. (1966) The effects of different isolation media on the respiration and morphology of housefly sarcosomes. J. Insect Physiol. 12, 1093-1103. CHRISPEELS M. J., VATTERA. E., and HANSONJ. B. (1966) Morphological development of mitochondria during cell elongation in the roots of Zea Mays seedlings. Quart. J. micv. Sot. 85, 2944. DAWID I. B. (1965) Deoxyribonucleic acid in amphibian eggs. y. molec. Biol. 12, 581-599. FREEMANK. B. (1965) Protein synthesis in mitochondria. Bi0chem.J. 94, 494-501. GREGORYD. W., LENNIER. W., and BIRT L. M. (1967) An electronmicroscopic study of flight muscle development in the blowfly Lucilia cuprina. J. R. microsc. Sot. In press. HELEP. and FINCH L. R. (1960) Amino acid-activating systems from pig liver. Bi0chem.J. 75, 352-363. HEROLDR. C. (1965) Development and ultrastructural changes of sarcosomes during honey bee flight muscle development. Devel. Biol. 12, 269-286. HOWELLSA. J. and BIRT L. M. (1964) Amino acid-dependent pyrophosphate exchange during the life cycle of the blowfly Lucilia cup&a. Comp. Biochem. Physiol. 11, 61-83. HUTCHISONW. C. and MUNROH. N. (1961) The determination of nucleic acids in biological materials. Analyst 86, 768-813.

1756

R. W. LENNIE, D. W. GREGORY, ANDL. M. BIRT

G. F. (1964) Deoxyribose nucleic acid in mitochondria and its role in protein synthesis. Biochemistry, N. Y. 3, 1702-1706. KELLENBERGERE., RYTER A., and SECHAUDJ. (1958) Electron microscope study of DNAcontaining plasms-II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different states. y. biophys. biochem. Cytol. 4, 671-678. KISSANE J. M. and ROBINS E. (1958) Fluorimetric measurement of DNA in animal tissues with specific reference to the central nervous system. r. biol. Chem. 233, 184-188. KROON A. M. (1966) Amino acid incorporation by isolated mitochondria. In Regulation of Metabolic Processes in Mitochondria (Ed. by TAGER J. M., PAPA S., QUAGLIAR~ELLO E., and SLATER E. C.), pp. 397413. Elsevier, Amsterdam. LENNIE R. W. and BIRT L. M. (1965) The localization of a particle-bound tyrosine-activating enzyme in Lucilia cuprina and the distribution of free amino acids during the life cycle. r. Insect Physiol. 11, 1213-1224. LENNIE R. W. and BIRT L. M. (1967) Aspects of the development of flight muscle sarcosomes in the sheep blowfly, Lucilia cuprina, in relation to changes in the distribution of protein and some respiratory enzymes during metamorphosis. Biochem. J. 102, 338-350. MICHEJDA J. (1964) Physiology and structure of flight muscle sarcosomes in silkworm, Hyalophora cecropia L. Bull. Sot. Amis Sci. Lett. Poznan (D) 4, 61-84. NAIR K. S. S. and GEORGE J. C. (1964) A histological and histochemical study of larval fat body of Anthrenus vorax Waterhouse (Dermestidae, Coleoptera). y. Insect Physiol. 10, 509-517. NASS S., NASS M. M. K., and HERRIX U. (1965) DNA in isolated rat liver mitochondria. Biochim. biophys. Acta 95, 426-435. OGAWAK. and BARRNETTR. J. (1964) The histochemical examination of oxidative enzymes and mitochondria. Nature, Lond. 203, 724-726. OGAWAK. and BARRNETTR. J. (1965) The cytochemical studies of succinic dehydrogenase and dihydronicotinamide adenine nucleotide diaphorase activity. J. Ultrastruct. Res. 12,488-508. PALADEG. E. (1952) A study of fixation for electron microscopy. g. exp. Med. 95, 285-298. POLAKIS E. S. and BARTLEY W. (1966) Changes in the intracellular concentrations of adenosine phosphates and nicotinamide nucleotides during the aerobic growth cycle of yeast on different carbon sources. Biochem. J. 99, 521-533. RABINOWITZM., SINCLAIRJ., DE SALLE L., HASELKORNR., and SWIFT H. H. (1965) Isolation of DNA from mitochondria of chick embryo heart and liver. Proc. nat. Acad. Sci. U.S.A. 53, 1126-1133. REYNOLDS E; S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. RICHARDSA. G. (1953) Structure and development of the integument. In Insect Physiology (Ed. by ROEDER K. D.), Chapter 1. John Wiley, New York. SCHATZC., HASLBRUNNERE., and TUPPY H. (1964) DNA associated with yeast mitochondria. Biochem. biophys. Res. Commun. 15, 127-138. SMITH D. S. (1963) The structure of flight muscle sarcosomes in the blowfly Calliphora erythrocephala (Diptera). y. Cell Biol. 19, 115-138. STEGWEE D., KIMMEL E. C., DE BOER J. A., and HENSTRA S. (1963) Hormonal control of reversible degeneration of flight muscle in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera). J. Cell Biol. 19, 519-527. TEWARI K. K., JAYARAMANJ., and MAHLER H. R. (1965) Separation and characterization of mitochondrial DNA from yeast. Biochem. biophys. Res. Commun. 21, 141-148. WIGGLE~WORTHV. B. (1942) The storage of protein, fat, glycogen, and uric acid in the fat body and other tissues of mosquito larvae. J. exp. Biol. 19, 56-77. KALF