Changes in the amounts of carbohydrates, phosphagen, and related compounds during the metamorphosis of the blowfly, Lucilia cuprina

J. l.mect Physical., 1967, Vol. 13, pp. 1575 to 1592.

Pergamon Press Ltd.

Printed in Great Britain

CHANGES IN THE AMOUNTS OF CARBOHYDRATES, PHOSPHAGEN, AND RELATED COMPOUNDS DURING THE METAMORPHOSIS OF THE BLOWFLY, L UCILIA CUPRINA M. CROMPTON Department

and

of Biochemistry,

L. M. BIRT

University of Sheffield

(Receded 29 March 1967)

Abstract-The total amount of carbohydrate (as glucose) decreased during the larval-pupal transformation by about 1 pmole/insect (about 20 per cent), and thereafter remained almost constant until emergence. Thus, during pharate adult development, the increase in chitin was balanced by a corresponding decrease in glycogen (these are the two principal carbohydrates; together accounting for about 94 per cent of the total). Glucose-oligosaccharides, trehalose, and a number of monosaccharides were also estimated. There was little evidence for the accumulation, at any stage, of L-lactate, L-glycerol-l-phosphate, cy-alanine, glycerol, and/or D-sorbitol. ‘High-energy’ phosphate compounds decreased during the larval-pupal transformation and increased during pharate adult life. The R.Q. value increased from 0.72 in prepupae to 0.93O-99 in late pupal and early pharate adult life, and then decreased to 0.78 in late pharate adult life. These data are discussed in relation to the information available on the changes in lipids, proteins, and amino acids in Lucilia. It is concluded that the energy requirements are provided principally by utilization of protein and fat (during pupal and late pharate adult development) and that the contribution of carbohydrate is very small. INTRODUCTION

Irg THE Diptera carbohydrate appears to serve as the major fuel for flight (SACKTOR, 1965). In the pupae, however, the consensus of opinion is that the main source of energy is fat together with a smaller amount of carbohydrate; this conclusion has been based on studies of the R.Q. (respiratory quotient) and on changes in the total amounts of fat and carbohydrate (review by AGRELL, 1964). It has been inferred by some workers that insects convert fat to carbohydrate during pupal development (e.g. HAUB and HITCHCOCK, 1941; AGRELL, 1953; LUDWIG et al., 1964). Chitin, besides being one of the principal structural elements of the exoskeleton, may also serve as a source of reserve carbohydrate in some insects (BADE and WYATT, 1962). Thus during the pupal moult of Hyalophora, some of the chitin from the old cuticle is reabsorbed and converted into glycogen and sugars; also, it has been suggested that there is a specific re-use of chitin from the old cuticle in the formation of the new one, although the intermediates in this transfer have not been identified. It is now established that haemolymph glucose is incorporated 1575

1576

M. CROMPTON AND L. M. BIRT

into the chitin of the growing cuticle (BADEand WYATT, 1962; CANDYand KILBY, 1962; LIPKE et al., 1965). During the larval moulting cycles of Peripknata, LIPKE et al. (1965) h ave shown that the chitin is continually being degraded and resynthesized, although the relative rates of the two processes varied according to the stage in the cycle. Thus, chitin may give rise to, and be derived from, soluble sugars during metamorphosis. A fuller comprehension of the provision of energy during metamorphosis of any one insect, and the relative importance of carbohydrates in this respect, is dependent on detailed information about changes in the amount of proteins, amino acids, fats, and also phosphagen, besides carbohydrates. This paper describes the changes occuring in the amounts of structural and soluble carbohydrate and of phosphagen during the metamorphosis of Lucilia, and relates these to the data available on changes in lipids, proteins, and amino acids in the same insect. It is concluded that the contribution of carbohydrate to the energy requirements of metamorphosis is very small. MATERIALS Chemicals

Trehalase was purified from Lucilia adults (22 g) by the method of KALF and REIDER (1958). The final protein precipitate was dissolved in 2.5 ml of 0.05 M maleate buffer, pH 6.5. A polyol dehydrogenase was prepared from rat liver according to WOLFSONand WILLIAMS-ASHMAN(1958) ; the pH of the initial extract was adjusted to pH 4.7 as described and the protein fraction used was that which precipitated between ethanol concentrations of 40 and 50 per cent. L-Arginine phosphokinase was prepared from the muscle of lobster by the method of MORRISONet al. (1957). Diazyme is an amyloglucosidase obtained from Miles Chemical, Clifton, New Jersey; it hydrolyses a: 1 -+4 linkages and also 011 -+6 linkages, but at a very much slower rate (see PAZURand KLEPPE, 1962). Chitinase was obtained from Calbiochem, Los Angeles. Invertase concentrate and catalase were supplied by British Drug Houses, Poole, Dorset. All other enzymes were obtained from C. F. Boehringer & Soehne, Mannheim. NAD, NADP, and ADP were purchased from Sigma, St. Louis, Missouri. Arginine-phosphate was kindly supplied by J. P. Heslop, Pest Infestation Laboratory, Slough, Bucks. All other reagents were of A.R. grade or of the highest purity commercially available. Insect culture

The insects (Lucilia cup&a Wied) were maintained at 30°C throughout the entire life cycle. Larvae were fed on ox-liver and adults on sucrose and water. All insects chosen for studies of pupal and adult development had started pupation within a single period of 3 to 4 hr. The term ‘prepupa’ is used to describe an organism which has stopped feeding and has become motionless, but has not yet

CARBOHYDRATES,

PHOSPHAGEN

AND

RELATED

COMPOUNDS

IN

changed from the larval colour. The term ‘pupa’ covers the from the larval-pupal moult to the emergence of the fly from Lucilicz the pupal-adult moult occurs 2 to 3 days after pupation; the next 34 days of ‘pupal’ life) the insect is a pharate adult. designated L, the prepupal Pp, the ‘pupal’ on successive days the adult stage half day after emergence F.

LUCILIA

1577

entire development the puparium. In thereafter (i.e. for The larval stage is P,, P,, P,, etc., and

METHODS

Extraction of water-soluble carbohydrates and related compounds Preliminary experiments showed that extractions (for 10 min) by cold (O’C) water (followed by heating in a boiling-water bath), boiling water, cold (0°C) O-3 N HClO,, and hot (100°C) 20% KOH were equally efficient in extracting total soluble carbohydrate. However, extraction in cold water followed immediately by heating in a boiling-water bath for 10 min led to losses of glycogen amounting to about 15 per cent of the total in larvae, about 50 per cent in pupae, and about 25 per cent in newly emerged flies; there was no loss, however, of total carbohydrate. A possible explanation is the presence of a carbohydrase degrading glycogen into units not precipitated by ethanol. Analyses, therefore, were carried out on HCIOl extracts in which these changes did not occur. Insects (twenty to thirty) were washed in water, dried on filter paper, and weighed. After a further washing to ensure complete removal of filter paper fibres, they were homogenized in O-3 N HClO, (5 ml) at 0°C for 1 min in a glass homogenizer with a Teflon pestle. The homogenate was kept in ice for a further 10 min. Insoluble matter was removed by centrifuging for 3 min at 2000g in a bench centrifuge, and washed twice in ice-cold HClO* (5 ml) by redispersion and centrifugation. The three supernatant fluids were combined into an acid extract. When necessary the extract was neutralized by addition of solid KHCO,. The neutralized extract was kept in ice for about 30 min and the KClO, precipitate sedimented b-\’ centrifugation. Both acid and neutralized extracts were stored at - 15°C and with this precaution there was no significant decrease in the total carbohydrate (of either acid or neutralized extracts) or the free glucose content (of neutralized extracts) over the whole period (about 3 months) in which they were used for analysis. Estimation of water-soluble carbohydrates and related compounds Total carbohydrate was determined in acid extracts by the phenol-sulphuric acid reaction (DUBOIS et al., 1956) with 1.5 ml of sample, 0.5 ml of phenol (20 per cent w/v) and 5.0 ml of H,SO, (98%). Absorbancies were measured at 490 rnp and referred to glucose standards. Glycogen was precipitated from the acid extract (O-5 ml) by addition of saturated Na,SO, solution (0.25 ml) and then ethanol to 70% (v/v) concentration. The mixture was kept at - 15°C overnight. The precipitate was sedimented by centrifugation, redispersed in fresh ethanol (4.0 ml), and resedimented. The

1578

M. CROMPTONANDL. M. BIRT

washed pellet was dried at 105°C and then dispersed in water. The glycogen fractions were assayed by the phenol-sulphuric method and also by the diazymeglucose oxidase method (KREBS et al., 1963). Glucose and glucose-6-phosphate were determined with hexokinase (for glucose) and glucose-6-phosphate dehydrogenase (SLEIN, 1965). The glucoseoxidase method was not used because of interference by some component (possibly a polyphenol) of the extracts, which caused a considerable underestimate of the glucose content. The component was absent from the glycogen fraction. Trehalose was determined as glucose after hydrolysis by trehalase. The preparation was contaminated with maltase activity, but this did not interfere with the trehalose estimation since samples were freed from oi 1 +4 linked glucosides by pretreatment with diazyme which has a negligible hydrolytic activity towards trehalose (PAZUR and KLEPPE, 1962). The sample (containing 100-700 pg glycogen) of neutralized extract was treated with diazyme (as for the estimation of glycogen) and then heated in a boiling-water bath for 1.5 min. The solution (2-3 ml) was neutralized with 2 N KOH, incubated at 37°C for 1 hr with 0.2 ml of glucoseoxidase-catalase reagent (glucose oxidase, 25 mg; catalase, 0.5 ml; 1 M phosphate buffer, pH 5.8, 4-O ml), and then heated again in a boiling-water bath for 15 min ; this procedure lowered the control glucose value (to about 12 per cent of the test glucose value) and the H,O, released was completely destroyed by the catalase and the heating. A sample (1.2 ml containing up to 0.4 pmoles trehalose) of this solution was incubated with trehalase (0.1 ml, sufficient to hydrolyse at least 1 pmole trehalose to 2 pmoles glucose) at 35°C for 12 hr, heated for 15 min in a boilingwater bath, and neutralized with O-1 M NaOH. The control was treated similarly but without trehalase. The glucose content of both solutions was determined with hexokinase and glucoseQ-phosphate dehydrogenase, and the amount of trehalose obtained by difference. The fraction designated ‘01 1 +4 linked oligosaccharides’ refers to all those compounds soluble in 70% ethanol and hydrolysed by diazyme to glucose. The supernatant fluid resulting from glycogen precipitation was evaporated at room temperature under a stream of air until all the ethanol had been removed. The glucose content of the solution remaining was determined (with hexokinase and glucose-6-phosphate dehydrogenase) before and after hydrolysis with diazyme, the difference being the ‘oligosaccharide’ content. Sucrose was determined as glucose and fructose after hydrolysis with invertase (BERGMEYER and KLOTZSCH, 1965). L-Glycerol-l-phosphate was determined with L-glycerol-l-phosphate dehydrogenase as described by HOHORST (1965a). L-Lactic acid was determined using L-lactic dehydrogenase as described by HOHORST (196513). D-Sorbitol was determined with polyol dehydrogenase. The assay system used was that described by WILLIAMS-ASHMAN (1965). With this system there was no reduction of NADf by glucose-6-phosphate, glycerol-l-phosphate, isocitrate, or L-lactate but inclusion of L-malate did cause a reduction of NAD+.

CARBOHYDRATES,

PHOSPHAGEN,

AND

RELATED

COMPOUNDS

IN

LUCILIA

1579

Glycerol was determined with glycerokinase and glycerol-l -phosphate dehydrogenase (WIELAND, 1965). N-Acetylhexosamines were determined by the method of REISSIG et al. (1955). Blank solutions were prepared in the same way as test solutions, but were not boiled. Absorbancies were read at 58.5 rnp. Hexosamines were estimated as the corresponding N-acetylhexosamines b>, the method of GHOSH et al. (1960). The amounts of hexosamines were determined by subtracting the values obtained for N-acetylhexosamines from those after acetylation of the extracts. E,xtraction of phosphagen, adenosine phosphates and inorganic orthophosphate Insects (about twenty) were crushed between two stainless steel blocks, previously cooled in liquid nitrogen. The tissue was quickly scraped into a mortar chilled with liquid nitrogen, and then ground to a fine powder under liquid nitrogen. The liquid nitrogen was allowed to evaporate and the tissue powder shaken on to a sheet of smooth paper (also chilled with liquid nitrogen) from which it could easily be transferred to a weighing bottle containing a magnetic flea and ice-cold HClO, (0.4 N, 2-O ml). The bottle was capped as soon as the tissue had been transferred. The whole operation was carried out in a room at -20°C and as quickly as possible. The bottle was packed in ice and the contents stirred for 15 min. The weight of tissue extracted was obtained by weighing the capped bottle and contents before and after the addition of the tissue. Insoluble matter was removed by centrifugation at 0°C. A sample (1.40 ml) of the supernatant fluid was mixed with ice-cold triethanolamine-buffer (O-01 ml, pH 7.4) and the pH adjusted to 7.4 by the very slow addition, with constant stirring, of ice-cold 3 N KOH ; this addition was carried out in ice. The extract, pH 7.4, was stored at - 15°C. The procedure was tested for the efficiency of recovery in two ways: (a) By determining the recoveries of known amounts of arginine phosphate and ATP introduced into the HCOl, immediately prior to the addition of tissue powder; the recoveries were quantitative. (b) By prolonging fourfold the time for which the frozen tissue powder remained in the mortar and on the paper. The ATP content/ mg wet wt. tissue was 90 per cent of that in another sample of the tissue extracted by the standard procedure; this indicated that any errors in these stages of the procedure amounted to no more than about 3 per cent. Estimation of phosphagen, adenosine phosphates, and inorganic orthophosphate Both arginine-phosphate and ATP were estimated within a few hours of extraction; ADP and AMP within a week; labile phosphate (i.e. that hydrolysed by 3 N H,SO, for 10 min) and inorganic phosphate within a fortnight, and the values were corrected for the hydrolysis of arginine phosphate that had occurred during storage of the extracts. ATP was determined with hexokinase and glucose-6-phosphate dehydrogenase (LAMPRECHT and TRATJTSCHOLD, 1965).

1580

M. CROMPTONAND

L. M. BIRT

L-Arginine phosphate was estimated by measuring the ATP formed when arginine phosphokinase was added to the ATP assay system on completion of the ATP determination. ADP and AMP were determined with pyruvate kinase, lactic dehydrogenase, and myokinase (for AMP) as described by ADAM (1965). Inorganic orthophosphate was determined by the method of MARTIN and DOTY (1949). Labile phosphate was determined by measurement of the inorganic phosphate released by hydrolysis in 1 N H&SO, at 100°C for 10 min. Determination of chitin Insects (twenty) were crushed in N NaOH (5 ml) and the mixture heated in a boiling-water bath for 24 hr. During this period, the alkali was changed once, since the first portion became very viscous and difficult to filter subsequently. This change was made by sedimenting (2OOOg, 10 min) the solid matter from the solution (which had been diluted with an equal volume of ethanol) and resuspending it in fresh alkali. After the digestion period, the undissolved material (chitin) was collected in a tared, dried filter stick (porosity No. l), washed with water (to remove alkali) and then with ethanol followed by ether, and dried at 110°C to constant weight. Estimations were duplicated. The chitin samples from larvae and from ‘pupae’ in the first 4 days of development were almost pure white; samples from ‘pupae’ half a day before emergence and from newly emerged flies contained a small amount of dark material, possibly derived from the adult cuticle. Further heating (in a boiling-water bath) of these samples with either 10 N KOH for 24 hr or 1 N KOH + 5% SnCI, for 24 hr did not extract any coloured material and decreased the dry weight by less than 2 per cent. Attempts to dissolve the samples with chitinase according to the method of JEUNIAUX (1965) were unsuccessful. The nitrogen contents of chitin samples were determined by the Kjeldahl method as described by CHIBNALL et al. (1943), except that the digestion mixtures were cleared by vigorous boiling for 2 hr ; less vigorous boiling for longer periods produced lower (by as much as 25 per cent) nitrogen yields. The digested samples were made alkaline and the ammonia distilled into 2% boric acid solution in the Markham apparatus and estimated by titration. The nitrogen contents of the samples were 4.6 per cent for larvae, 5.2 per cent for prepupae and ‘pupae’ and 2.3 per cent for flies; the theoretical value for (CSH1a05N), is 6.9 per cent. These low nitrogen contents may indicate nitrogen losses during purification, although HACKMANN (1960), using isolated puparia from L. cup&a was able to obtain almost pure chitin, nitrogen content 7.0 per cent by the same purification procedure. Determination of the R.Q. This was carried out by the direct method for the calculations of R.Q. values were those

of Warburg. taken during

The readings the first hour

used after

CARBOHYDRATES,

equilibration (see Results); period were constant.

PHOSPHAGEN,

1581

AND RELATED COMPOUNDS IN LUCILIA

the rates of CO, evolution

and 0, uptake

during

this

RESULTS

Changes in the amounts of chitin and total carbohydrate The values for the amounts of chitin calculated from the dry weights and with the assumption that the molecule is a polymer of N-acetylglucosamine (M.W. 221), are shown in Fig. l(a). If the average chitin contents are calculated from the nrtrogen contents of the weighed samples, the values given in Table 1 are obtained.

1

Pupo

IPhorote Age.

adult

1

days

FIG. 1. (a) Variations in the amounts of total carbohydrate and chitin during metamorphosis. The amounts of total carbohydrate were obtained by summing those for total soluble carbohydrate and for chitin. Chitin was determined by weighing the dried residue left after alkali digestion of whole insects. Total carbohydrate, O----- q ; chitin, O0. (b) Variations in the amounts of total soluble carbohydrate, glycogen, and trehalose during metamorphosis. Total soluble carbohydrate was estimated after precipitation by ethanol by the phenol sulphuric acid reaction and also by the diazyme-glucose-oxidase method. Trehalose was estimated as glucose after hydrolysis with trehalose. Total soluble carbohydrate, q-0; glycogen by phenol sulphuric acid, An ; glycogen by diazyme-glucose-oxidase, 0 -0 ; trehalose, O-O.

1582

M. CROMPTONAND L. M. BIRT TABLE ~-CHANGES IN THE AMOUNTOF CHITINDURINGMETAMORPHOSIS OF Lucilia Pupal L

Chitin (as pmoles N-acetyl glucosamine)/ 2~43 insect

Pharate adult

P,

PI

Pa

P,

P,

F

2.38

1.88

2.04

2.09

2.60

1.24

L refers to the larval stage, P, to the prepupal, PI, P,, etc., to the period within the puparium (i.e. ‘pupal’) on successive days of development, and F to the adult half a day after emergence. The amounts of chitin were calculated from the N contents of alkaliinsoluble material derived from whole insects (see Methods).

Although there is a discrepancy between the two series of values calculated, the patterns of change indicated by them are similar, i.e. there is a decrease (31 per cent from dry weights, 23 per cent from N contents) in the amount of chitin during the larval-pupal transformation, followed by a small increase (9 per cent) in pupal life (P, +P,); for the following 2 days the amount was almost constant, but then increased (by 25 per cent) during pharate adult development (P, +PJ. The values after emergence (F) do not include the chitin of the shed puparium. The general pattern of change is similar to that found in a number of other insects (e.g. Bomb~~x, ZALUSKA, 1959; Hyalophora, BADE and WYATT, 1962). The decrease in the amount of chitin at the onset of pupation must represent a partial degradation of the larval cuticle. The slight increase in pupal life (P, +P,) may be due to the formation of the pupal cuticle which is only an extremely fine structure, and therefore would account for little of the total chitin. A partial breakdown of the pupal cuticle may occur during the pharate adult stage, and thus account for the increase in the amount of free (IV-acetyl)hexosamines at this stage (Fig. 2a) as shown for Hyalophora also by PASSONEAU and WILLIAMS (1953); nevertheless, the concurrent synthesis of the much more massive adult cuticle would cause a net increase in the amount of chitin, as observed. The amounts of total carbohydrate (Fig. la) were obtained by summing the values for chitin (from the dry weights) and for the total soluble carbohydrate; the values for soluble carbohydrate were corrected at each stage of development for the differences in fresh weight between the two batches of insects. There was a decrease (O-9 pmole/insect) in the amount of total carbohydrate during the larvalpupal transformation, an increase (O-25 pmole/insect) in pupal life (P, +P,), but very little change (< 0.1 pmole/insect) thereafter until emergence. Changes in the amounts of soluble carbohydrates and related compounds The total amount of soluble carbohydrate (Fig. lb) decreased slightly immediately before pupation. During and after pupation the amount increased, attaining a maximum in the latter half of pupal life, after which it decreased steadily. Extracts of newly emerged insects contained sucrose which was probably present in the gut

CARBOHYDRATES,

PHOSPHAGEN,

AND RELATED COMPOUNDS

IN

LUCILIA

1583

after ingestion, or was incompletely removed from the outside of the flies before extraction; the value after emergence, therefore, has been corrected for sucrose content (about 50 mpmoles/insect). A similar pattern of changes was obtained from extracts of a number of different populations of insects, although the absolute values varied. Thus, in four such series of extractions the average content (as pmoles glucose/insect) in prepupae was 1.6, 24 days after pupation it was 2.1, and in insects half a day before emergence it was 1.3. Glycogen changes (Fig. lb) reflected those of the total soluble carbohydrate, the difference between them (the ethanol-soluble fraction) remaining almost constant (approx. O-3 pmole/insect). These changes in the amounts of glycogen are somewhat similar to those occuring during metamorphosis of the silkworms Bombyx (ZLLUSKA, 1959) and HyaZophora (BADE and WYATT, 1962) but are unlike those described for Calliphora by AGRELL(1953). Of the remaining constituents assayed, the following were sensitive to phenolsulphuric acid : trehalose, glucose-6-phosphate, fructose plus fructose-6-phosphate, ami a: 1 -t4 linked ‘ oligosaccharides’. During ‘pupal ’ life, quantitatively the most important of these was trehalose. Trehalose (Fig. lb) was undetectable at the larval stage but thereafter the amount increased steadily reaching about 100 mpmoles/ insect 14 days after pupation, a value which was maintained for the next 2 days; the amount decreased slightly during development of the pharate adult. There are other reports of the absence of trehalose from blowfly larvae (Phormia, EVANSand DFPHIER, 1957; Caliiphora, DUTRIEUX, 1961). The amount of glucose (Fig. 2a) decreased greatly during the larval-pupal transformation from about 86 mpmoles/ insect to about 2 m,umoles/insect in late pupal life; the value then rose steadily to about 50 mpmoleslinsect in newly emerged flies. Similar changes occured in the amounts of fructose plus fructose-6-phosphate (about 5 1 mpmoles/insect in larvae, < .i m~moles/insect in ‘mid-pupal’ life, about 21 mpmoles/insect in flies). The amount of glucose-6-phosphate was very low at all times, being highest in larvae (about 11 mpmoles/insect) and flies (about 6 mpmoles/insect). These changes in glucose, glucose-6-phosphate, and fructose plus fructose-6-phosphate are similar to those of the reducing sugars of Calliphora (AGRELL, 1953) in which the changes are much less pronounced. The LX1 --4 linked glucose oligosaccharides (Fig. 2a) varied between 25 and 32 mpmoles (as glucose)/insect during the first 4 days of ‘pupal’ life, declining slightly to between 20 and 24 mpmoles/insect in the latter half of pharate adult life and after emergence. V-acetylhexosamines (Fig. 2a) were undetectable (< 1 mpmole/insect) in larvae and prepupae, but appeared on pupation in low amounts (between 2 and 5 mpmoles/ insect). During the pharate adult stage the amount increased rapidly to about 28 mpmoles/insect, and then decreased so that none was detectable on emergence. The pattern of changes in hexosamines (Fig. 2a) was similar but the values were lower; thus they were undetectable before the pharate adult stage (P4), after which they increased to about 9 mpmoIes/insect and then decreased to an undetectable value in newly emerged flies. The considerable increase in (N-acetyl)hexosamines just before adult emergence is similar to that occuring in Hyalophora (PASSONEAU

1584

M. CROMPTON AND L. M. BIRT

and WILLIAMS, 1953). Passoneau and Williams concluded that the increase was a result of the partial degradation of the pupal cuticle by the moulting fluid; this explanation would appear to be feasible for Luciliu as well. The appearance of (iV-acetyl)hexosamines at pupation may be related to the degradation of some of the larval cuticle (see Fig. la); the appearance, however, of only very low amounts may imply a rapid transfer of products released from the larval cuticle to the forming pupal cuticle as suggested by BADE and WYATT (1962) for Hyalophora.

SO

(bf

60

Age.

days

FIG. 2. (a) Variations in the amounts of glucose, ‘oligosaccharides’, and (N-acetyl)hexosamines during metamorphosis. Glucose was estimated using hexokinase and glucose-6-phosphate dehydrogenase. ‘Oligosaccharides’ refer to glucose released by diazytne treatment of supernatant fluids after precipitation of glycogen. N-acetylhexosamines were estimated calorimetrically using p-dimethyl aminobenzaldehyde. Hexosamines were estimated as N-acetylhexosamines after acetylaGlucose, q --17 ; ‘oligosaccharides’, tion. O@ ; N-acetylhexosamines, A; hexosamines, l0; (b) Variations in the amounts of glycerolAl-phosphate and lactate during metamorphosis. Glycerol-l-phosphate was estimated using glycerol-l-phosphate dehydrogenase and lactate using lactic dehydrogenase. Glycerol-l -phosphate, A ; lactate, U-0. A--

The amount of L-glycerol-l-phosphate (Fig. 2b) decreased from a maximum value of about 41 mpmoles/insect in larvae to about 13 mpmoles/insect in prepupae,

CARBOHYDRATES,

PHOSPHAGEN,

Ah-D

RELATED

COMPOUNDS

IN

LUCILIA

1585

and then increased to about 34 mpmoles/insect half a day after pupation; thereafter the value decreased steadily to a minimum of about 10 mpmoles/insect in flies. Glycerol was undetectable (< 2 mpmoles/insect) at any stage. The amount of L-lactate (Fig. 2b) decreased sharply immediately before pupation from about 80 mpmoleslinsect in larvae to about 10 mpmoles/insect in prepupae; the amount decreased still further during ‘pupal’ life and emergence to about 2 mpmoles/insect. Sorbitol was present only in very low concentrations (< 13 mpmoles/insect), or was absent, throughout the whole period (since the enzyme preparation used in this assay was not absolutely specific, this value must be maximal). C%rnges iz the amounts of phosphagen, adenosine phosphates, and inorganic phosphate These compounds were determined in a single series of extracts prepared from one population of insects. L-Arginine phosphate (Fig. 3a) decreased (by 50 per cent) from about 280 mp.moles/insect in larvae to about 139 mpmoles/insect in prepupae, and then further to about 100 mpmoles/insect (about 80 mg/lOO g fresh weight) in pupal life (P2). The amount rose to a maximum ‘pupal’ value of about 170 mpmoles/insect 1 day before emergence; the emerged insect contained somewhat less, i.e. about 115 mpmoles/insect (90 mg/lOO g fresh weight). In Calliphora (LEVENBOOK, 1953) the amounts of arginine phosphate per unit wet weight decrease steadily during pupal life and are much smaller at all stages than in Lucilia. The amount of ATP (Fig. 3a) present during the larval and ‘pupal’ periods was about half that of arginine phosphate. ATP decreased steadily from about 142 mpmoles/insect in larvae to about 50 mpmoles/insect (i.e. about 6.5 pg adenine/ insect) in early pupal life; this level was maintained until early in the pharate adult stage (PJ. Later in pharate adult life and on emergence the amount increased to about 128 mpmoleslinsect, which exceeded the amount of arginine phosphate. The pattern of ADP changes (Fig. 3a) was also U-shaped. The amount decreased steadily from about 34 mpmoles/insect in larvae to about 20 mpmoles/insect (i.e. about 2.5 pg adenine/insect) immediately after pupation and then further to about 9 mpmoles/insect in early pharate adult life; thereafter it increased to about 16 mp,moles/insect (I). In contrast to the changes in the amounts of arginine phosphate, ATP, and _4DP, the amount of AMP (Fig. 3a) increased during the larval pupal transformation, from about 8 mpmoles/insect in larvae to a maximum of about 37 mpmoleslinsect l+ days after pupation, and then it decreased to about 18 mpmoles/insect in flies. There is no resemblance between these values for the amounts of ATP and AMP in Lucilia and those described for Calliphora by ACRELL (1953). However, the absolute amounts per unit weight and the pattern of changes in the sum of ATP and ADP do agree fairly closely (the comparative values may be obtained by taking the weight of Calliphora pupae as 70 mg/insect (AGRELL, 1953) and of Lucilia pupae as 35 mg/insect). Labile phosphates (Fig. 3b) decreased by two-thirds during the larval-pupal transformation to about 200 mpmoles/insect (i.e. about 200 mg P/l00 g fresh weight); this value was maintained until early pharate adult life (Pa), after which

1586

M. CROMPTON AND

L. M.

BIRT

there was a steady increase to about 391 mpmoles/insect on emergence. These values agree well with the summed ‘high-energy’ phosphate values of arginine phosphate, ATP, and ADP ( average disparity 5 per cent; see Fig. 3b). Inorganic orthophosphate (Fig. 3b) increased steadily from about 200 mpmoles/insect in

600

I Pupa Age,

IPharate

adult

1

days

FIG. 3. (a) Variations in the amounts of arginine phosphate and adenosine phosphates during metamorphosis. Arginine phosphate was estimated as ATP formed in the arginine phosphokinase reaction. ATP was estimated using hexokinase and ADP using pyruvate kinase and lactic glucose-6-phosphate dehydrogenase, and AMP as ADP after phosphorylation using myokinase. dehydrogenase, i, . Arginine phosphate, qq I; ATP, c. -c; ADP, .0; AMP, A(b) Variations in the amounts of inorganic phosphate and ‘labile phosphate’ during metamorphosis. Inorganic phosphate was estimated calorimetrically using ammonium molybdate. ‘Labile phosphate’ is the phosphate released by hydrolysis by N H,SOI in a boiling water bath for 10 min. Inorganic phosphate, A-A ; ‘labile phosphate’, C--c! ; the total ‘high-energy’ phosphate present in arginine phosphate, ATP and ADP, 0-O.

CARBOHYDRATES, PHOSPHAGEN, ANDRELATED COMPOUNDS IN LUCILIA

1587

larvae to about 560 mpmoles/insect l+ days after pupation (i.e. about 19 pg P/insect or 57 mg/lOO g fresh weight), and then decreased to a post-emergence value of about 190 mpmoles/insect. Values obtained by AGRELL(1953) and LEVENBOOK(1953) with Calliphora for the total labile P and inorganic phosphate agree reasonably with those for Lucilia. Respiratory quotients The amounts of CO, evolved and 0, taken up atdifferent stages of metamorphosis, and the resultant R.Q. values are given in Table 2. The curves of the 0, consumption and CO, evolution have the familiar U-shaped form. These values T‘IBLE ~-THE

AMOUNTSOF COa EVOLVED,0,

CONSUMED, AND THE R.Q. VALUEDURIKG OF ikCi&l

METAMORPHOSIS

Pharate adult

Pupal

pl:rng wet wt. { COP evolved tissue per hr O2 consumed R.Q.

P,

PI

Pa

Pa

P,

P5

Ps

0.780 1.070 0.72

0.309 0.401 0.77

0.262 0.283 0.93

0.234 0.252 0.93

0.262 0.267 0.99

0.370 0.456 0.81

0.475 0.610 0.78

P, refers to the prepupal stage and P,, P,, etc., to the period within the puparium (i.e. ‘pupal’) on successive days of development. Gas exchange was determined manometrically by the direct method of Warburg using ten insects in a gas volume of about 2’: ml incubated for 1 hr at 30°C.

are calculated from the gaseous exchanges occuring during the first hour after equilibration. After this period, with insects in the first 4 days of ‘pupal’ life at least, the R.Q. decreased with time to a final value of 0.71 to 0.74; the initial R.Q. value was restored by regassing the flasks with air. Furthermore, this decrease occured sooner (about 30 min) when the volume of the gas phase was reduced by about 75 per cent (i.e. from about 25 ml to about 6 ml). It is possible that the decrease in the value of the R.Q. is due to the accumulation of CO,. These results emphasize that for the estimation of R.Q. values for organisms of this type in an enclosed system, factors such as the duration of the incubation and the volume of gas available to the insects must be considered. DISCUSSION The

utilization of energy reserves during metamorphosis

The information available for Lucilia permits an attempt to integrate the data on th.e changes in the amounts of total carbohydrate, total fatty acids (D’COSTA and BIRT, 1966), protein plus free amino acids (viz. total amino acid), CO, output, and the R.Q. during the prepupal and ‘pupal’ periods. All these changes have been corrected to a standard fresh weight/insect of 40 mg. Total amino acid catabolism during the period P,, +P, has been obtained from the measured decrease in protein

1588

M. CROMPTONANDL. M. BIRT

and free amino acids during this period (LENNIE and BIRT, 1967); BIRT and CHRISTIAN, unpublished); during the period P, +P, it has been calculated from the amounts of uric acid present at each stage of development (BIRT and CHRISTIAN, unpublished) with the assumption that all the uric acid appearing has resulted from amino acid catabolism. These calculations were based on an average content of 4.6 atoms C/amino acid mol. (calculated from data on the amino acid composition of the protein present at different stages of development; BIRT and CHRISTIAN, unpublished). For discussion, it is convenient to divide the period into three stages, viz. P, -+P, (pupation and early pupal life), P, +P, (late pupal and early pharate adult life), and P, +P, (the remainder of pharate adult life until half a day before emergence). Stage P, -+P,: During pupation (P, +P,) there was disappearance of total amino acid (about 37 pg atoms C/insect), fatty acids (about 32 pg atoms C/insect) and carbohydrate (l-2 pg atoms C/insect) and an R.Q. value of 0.72 to 0.77. These data suggest oxidation of total amino acid, fatty acids, and a very small amount of carbohydrate. During early pupal life (P, +Pz) there was a further disappearance of total amino acid (about 23 pg atoms C/insect), but an appearance of fatty acids (about 28 pg atoms C/insect) and carbohydrate (about 1 pg atom C/insect) and an R.Q. value of 0.79 to 0.93. These data suggest that total amino acid is being oxidized and also being converted to fatty acids and carbohydrate. The apparent deficiency in C made available from total amino acid catabolism for the syntheses of fatty acids and carbohydrate and for the CO, evolution (14 pmoles/insect) may be due to the difficulty in making a precise calculation for the net change in fatty acids during the period, owing to the rapidity of the change from a net degradation to a net synthesis of fatty acids. However, the values obtained could be explained by assuming that some of the amino acids deaminated during the period P, +P, are not oxidized immediately (perhaps they are stored temporarily as organic acids), but subsequently, during the period P, G-P,. Thus, during the whole period (P, -+Pz), there is suffiicient C released by total amino acid catabolism (about 60 pg atoms C/insect) to account for the synthesis of both fatty acids and carbohydrate (a total of about 29 r_Lgatoms C/insect). The remaining C released by total amino acid catabolism plus that from fatty acids and carbohydrates (altogether, a total of about 63 pg atoms C/insect) is sufficient to account for the CO, evolved (about 38 pmoles/insect). In summary, therefore, the main source of energy during pupation (P, -+P,) would seem to be the oxidation of total amino acid and fatty acids, while during early pupal life (P, +PJ oxidation of total amino acid alone appears to provide the There have been a number of reports of a net conversion of fat to energy required. carbohydrate occuring during the pupal period of a variety of insects (e.g. in Phormia, HAUB and HITCHCOCK, 1941; in Popillia, LUDWIG and ROTHSTEIN, 1949; in Calliphora, AGRELL, 1953; in Musca, LUDWIG et al., 1964). In Lucilia, the only increase in total carbohydrate during the prepupal and pupal periods takes place during the period P, +P,, when the amount of fat is also increasing; it seems

CARBOHYDRATES,

PHOSPHAGEN,

AND

RELATED

COMPOUNDS

IN LUCILIA

1589

likely that these syntheses occur at the expense of total amino acid (gluconeogenesis from amino acids at this time has also been suggested for Hyalophora by BADE and WYATT, 1962). Stage P, +P,: During this period there was a decrease in the amount of total amino acid (about 11 pg atoms C/insect) and of fatty acid (about 3 pg atoms C/ was very small (about 1 pg atom C/insect). insect) ; any decreas e in carbohydrate ‘I’hus almost all the energy required would seem to be provided by oxidation of total amino acids, although this is not accurately reflected in the rather high R.Q. value (0.93-0.99). The measured CO, evolution over the whole period was 23 pmoles/insect. Stage P, +P,: During this period there was a decrease in the amounts of fatty acid (about 32 ,ug atoms C/insect) and total amino acid (about 5 pg atoms C/insect), but the amount of carbohydrate was almost constant (changing by < 1 pg atom C/ insect); the amount of CO, evolved was about 35 pmoles/insect. In the transition to the latter half of pharate adult development, therefore, it would seem that fatty acids become the principal source of energy, and this is reflected in the decrease in the R.Q. value to 0.81-0.78. Accumulation of glycolytic end-products A number of compounds have been established as end-products of insect anaerobic glycolysis, e.g. L-lactate, L-glycerol-l-phosphate, and ol-alanine (PRICE, 1961); in certain diapausing insects the accumulation of glycerol (in Hyalophora: CHINO, 1958; WYATT and MEYER, 1959; WILHELM et al., 1961) and D-sorbitol (in Bombyx: CHINO, 1960) has been shown to allow anaerobic energy production. As none of these compounds accumulated to any great extent in Lucilia (ol-alanine, I~IRT and CHRISTIAN, unpublished), it appears likely that anaerobic energy production utilizing a terminal H acceptor other than 0, was quantitatively unimportant (the relatively large amount of lactate despite the high 0,uptake present in the larvae suggests that this was much more active immediately before this stage, i.e. in the feeding larvae). Indeed, the lack of any pronounced decrease in the total amount of carbohydrate indicates only a low activity of the glycolytic pathway. Thus, the glycogen disappearing during pharate adult life would be utilized almost completely for the formation of the chitin of the adult. From this it would follow that the U-shaped respiratory curve during metamorphosis is a good index of the energy metabolism. The relatively low rate of respiration during the period Pa-P,, when furthermore there is no net utilization of high-energy phosphate compounds (see Fig. 3b), indicates that energy demands during this period are lowest. Variation in ‘high-energy’ phosphate compounds During amount of part, to the hydrolysis,

the transformation of larva to pupa there was a decrease in the total ‘high-energy’ phosphate compounds. This may be due, at least in histolysis of larval tissues releasing such compounds, followed by their perhaps by a haemolymph phosphatase as suggested for Calliphora by

1590

M. CROMPTONAND

L. M. BIRT

LEVENBOOK (1953); the almost proportional increase in inorganic phosphate is consistent with this suggestion. The comparatively small accumulation of AMP during this period indicates that the breakdown of adenine polyphosphates goes further than AMP, perhaps to IMP (see RAY and HESLOP, 1963) for which no analysis was made. The increase in ‘high-energy’ phosphate compounds beginning about 2i days before emergence can be correlated with the development of the thoracic muscles which also begins at this time (BIRT et al., unpublished); this, also, has been suggested by LEVENBOOK (1953) for Calliphora. During the period P, -+Pq, although the total amount of ‘high-energy’ phosphate is nearly constant, the amount of arginine phosphate increases. This suggests that the rate of ATP production (even though the respiration rate is minimal) exceeds the demand, and this is in line with the previous conclusion that energy demands during this period are minimal; this would seem to be inconsistent with a period of histogenesis in ‘mid-pupal’ life, in contrast to that proposed for CalZiphora by AGRELL (1953). The decrease in the amount of arginine-phosphate beginning about 1 day before emergence suggests a sharp increase in the demand for ATP at this time which cannot be met by its rate of synthesis. Acknowledgements-We wish to thank Professor BARTLEYfor his assistance in the preparation of the manuscript. One of us (M. C.) acknowledges receipt of an award from the Medical Research Council for training in research methods ; the work was also supported by a grant from the Science Research Council. REFERENCES

ADAM H. (1965) Adenosine-5’-diphosphate and adenosine-5’-monophosphate. of Enzymatic Analysis (Ed. by BERGMEYER H.), pp. 573-577. Academic York.

In Methods Press, New

AGRELL I. (1953) The aerobic and anaerobic utilization of metabolic energy during insect metamorphosis. Actu physiol. sca?zd. 28, 306-335.

AGRELL I. (1964) Physiological The Physiology ofInsecta

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BADEM. L. and WYATT G. R. (1962) Metabolic conversions during pupation of the Cecropia silkworm-I. Deposition and utilization of nutrient reserves. Biochem. J. 83, 470477. BERCMEYER H. and KLOTZSCHH. (1965) Sucrose. In Methods of Enzymatic Analysis (Ed. b! BERGMEYER H.), pp. 99-102. Academic Press, New York. CAXDY D. J. and KILBY B. A. (1962) Studies on chitin synthesis in the desert locust. J. exp. Biol. 39, 129-140. CHIBNALLA. C., REES M. W., and WILLIAMS E. F. (1943) The total nitrogen content of egg albumin and other proteins. Biochem. J. 37, 354-359. CHINO H. (1958) Carbohydrate metabolism in the diapause egg of the silkworm, Bombyx mori-II. Conversion of glycogen into sorbitol and glycerol during diapause. J. Insect Physiol. 2, 1-l 2. CHINO H. (1960) Enzymatic pathways in the formation of sorbitol and glycerol in the diapausing egg of the silkworm, Bombyx mori-I. On the polyol dehydrogenases. J. Insect Physiol. 5, l-1 5. D’COSTA M. A. and BIRT L. M. (1966) Changes in the lipid content during the metamorphosis of the blowfly, Lucilia. J. Insect Physiol. 12, 1377-1394. DUBOISM., GILLES K. A., HAMILTONJ. K., REBER~P. A., and SMITHF. (1956) Calorimetric method for determination of sugars and related substances. Analyt. Chem. 28, 350-356.

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DUTRIEUXJ. (1961) Variations in the trehalose content during embryonic development of Bombyx mori and metamorphosis of Calliphora erythrocephala. C. R. Acad. Sci., Paris 252, 347-349. EVANSD. R. and DETHIER V. G. (1957) The regulation of taste thresholds for sugars in the blowfly. J. Insect Physiol. 1, 3-17. GHOSH S., BLUMENTHALH. J., DAVIDSONE., and ROSEMANS. (1960) Glucosamine metabolism-v. Enzymatic synthesis of glucosamine-6-phosphate. J. biol. Chem. 235, 1265-1273. IHAUB J. C. and HITCHCOCKF. A. (1941) The interconversion of foodstuffs in the blowfly, Phormia regina, during metamorphosis-III. Chemical composition of larvae, pupae and adults. Ann. ent. Sot. Am. 34, 32-37. ~HAC~MANN R. H. (1960) Studies on chitin-IV. The occurrence of complexes in which chitin and protein are covalently linked. Aust. J. biol. Sci. 13, 569-577. Determination with glycerol-l-phosphate HOHORST H. (1965a) Glycerol-l-phosphate. dehydrogenase. In Methods of Enzymatic Analysis (Ed. by BERGMEYERH.), pp. 215-219. Academic Press, New York. HOHORST H. (1965b) Lactate. Determination with lactic dehydrogenase and DPN. In &&hods of Enzymatic Analysis (Ed. by BERGMEYERH.), pp. 266-270. Academic Press, New York. ,JEUNIAUXC. (1965) Chitine et phylogenie: application d’une methode enzymatique de dosage de la chitine. Bull. Sot. chim. biol. 47, 2267-2278. KALF G. F. and REIDER S. V. (1958) The purification and properties of trehalase. J. biol. Chem. 230, 691-698. KREBS H. A., BENNETT D. A. H., DE GASQUETP., GASCOYNET., and YOSHIDA T. (1963) Renal gluconeogenesis. The effect of diet on the gluconeogenetic capacity of rat-kidneycortex slices. Biochem. J, 86, 22-27. LANIPRECHT W. and TRAUTSCHOLDI (1965) Adenosine-5’-triphosphate. Determination with hexokinase and glucose-6-phosphate dehydrogenase. In Methods of Enzymatic Analysis (Ed. by BERGMEYERH.), pp. 543-551. Academic Press, New York. 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. LEVENBOOI~L. (1953) The variation in phosphorus compounds during metamorphosis of the blowfly, Calliphora erythrocephala Meig. J. cell. camp. Physiol. 41, 313-334. LIPKE H., GRAVESB., and LETO S. (1965) Polysaccharide and glycoprotein formation in the cockroach-II. Incorporation of n-glucose-rlC into bound carbohydrate, J. biol. Chem. 240, 601-608. LUDWIG D., CROWEP. A., and HASSEMERM. M. (1964) Free fat and glycogen during metamorphosis of Musca domestica L. J. N. Y. ent. Sot. 72, 23-28. I,uD~I(; D. and ROTHSTEIN F. (1949) Changes in the carbohydrate and fat content of the Japanese beetle (Popillia japonica Newnam) during metamorphosis. Physiol. 2061. 22, 308-317. MARTIN J. B. and DOTY D. M. (1949) Determination of inorganic phosphate. Analvt. Chem. 21, 965-967. MORRISONJ. F., GRIFFITHS D. E., and ENNORA. H. (1957) The purification and properties of arginine phosphokinase. Biochem. J. 65, 143-153. PASSONEA~J. V. and WILLIAMS C. M. (1953) The moulting fluid of the Cecropia silkworm. J. esp. Biol. 30, 545-566. PAZURH. J. and KLEPPE K. (1962) The hydrolysis of ar-o-glucosides by an amyloglucosidase from Aspergillus niger. J. biol. Chem. 237, 1002-1006. PRICE G. M. (1961) The accumulation of a-alanine in the housefly, Musca oicina. Bi0chem.J. 81, 15P-16P.

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