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J. Exp. Biol. (1962), 39, 129-140

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STUDIES ON CHITIN SYNTHESIS IN THE DESERT LOCUST

BY D. J. CANDY AND B. A. KILBY

Department of Biochemistry, University of Leeds

(Received 7 November 1961)

INTRODUCTION

Chitin is an important constituent of the exoskeleton of insects since about a thirdof the dry weight of the entire cuticle is composed of this polysaccharide, taking anaverage value from a wide range of insect species (Wigglesworth, 1953). Someknowledge of the mechanism of biosynthesis of this compound would appear to bedesirable for a better understanding of the biochemistry of the insect cuticle.

Chitin is built up of long unbranched chains composed of iV-acetyl-D-glucosamineresidues joined in 1-4-^-Unkages; it is not found in the pure state in the cuticle butalways in association with protein. It can, however, be separated from the othercomponents of the cuticle by alkaline digestion, and this method was used by Zaluska(1959) to study the amounts of chitin in silkworms at different ages. He found thatchitin was formed throughout the period of larval development, but the rate of forma-tion was highest on the first day of feeding after the moults leading to the fourth andfifth stages of growth. A part of the present investigation was concerned with a studyof chitin formation during the growth of the desert locust, Schistocerca gregariaForskal, and of the incorporation into the polysaccharide of radioactivity derived fromlabelled glucose which had been injected into the insect.

Little has been published on the biochemistry of chitin synthesis in insects, but thelast stage of the process has been studied in the mould, Neurospora crassa by Glaser &Brown (1957). They obtained from extracts a particulate enzyme which wouldcatalyse the synthesis of a chitin-like polysaccharide from 14C-labelled uridine di-phosphate iV-acetylglucosamine, provided that a chitodextrin was present. It wassuggested that chitin formation occurred by the stagewise transfer of iV-acetyl-glucosamine groups from the nucleotide to the end of the chain of the chitodextrinprimer.

Uridine diphosphate glucose is known to participate in the synthesis of the gluco-sidic bond of the disaccharide trehalose (Candy & Kilby, 1961) and of glycogen(Trivelloni, i960) in locust tissues. It would not be surprising, therefore, if ananalogous mechanism involving uridine diphosphate iV-acetylglucosamine was in-volved in chitin biosynthesis, and it is interesting to note that this nucleotide hasactually been shown to be present in the haemolymph of Cecropia moths by Carey &Wyatt (i960). In the second part of the investigation it is shown that cuticular tissuefrom the locust is able to make this nucleotide by a pathway which has been elucidatedin detail, and an effort has been made to demonstrate that chitin may be made from it.

Exp. Biol. 39, 1

130 D. J. CANDY AND B. A. KILBY

MATERIALS AND METHODS

Special chemicals

Adenosine triphosphate (ATP), fructose-6-phosphate, glucose-6-phosphate, co-enzyme-A, alkaline phosphatase and hexokinase were purchased from L. Light andCo. Ltd.; uridine triphosphate (UTP) and uridine diphosphate iV-acetylglucosamine(UDPAG) were purchased from the Sigma Chemical Co.; and glucosamine-6-phosphate was purchased from the Nutritional Biochemicals Corporation. 14C-acetatelabelled in the 1-position, and generally labelled 14C-glucose were obtained from theRadiochemical Centre, Amersham, Bucks.

Acetyl coenzyme-A was prepared by the method of Ochoa (1955); N-acetyl-glucosamine-6-phosphate was prepared by the method of Roseman (1954) and purifiedby gradient elution with hydrochloric acid from a column of Amberlite CG-400(chloride form); 14C-acetylglucosamine-6-phosphate (labelled in the acetate group)was prepared enzymically by the U3e of a yeast extract (Brown, 1955); "C-UDPAG(labelled in the acetate group) was prepared by the method of Glaser & Brown (1955);uC-glucose-6-phosphate was prepared by phosphorylation of 14C-glucose with ATPin the presence of hexokinase; chitodextrins were prepared by the method of Zech-meister &Toth(i93i); and chitinase was prepared from puff balls (Lycoperdon sp.) bythe method described by Carlisle (i960).

Experimental animals

Fourth and 5th instar hoppers of the desert locust (Schistocerca gregaria Forskal)were obtained weekly from the Anti-Locust Research Centre, and were fed on anartificial diet (Howden & Hunter-Jones, 1958). For the in vivo experiments on chitinformation, a temperature of 340 C. was maintained throughout, but for other experi-ments heat and light were switched off in the cages for a night-time period of 8 hr.

Measurements of chitin dry weight and radioactivity

For measurements of chitin in a sample of cuticle the chitin was first separated fromother material by digestion with sodium hydroxide solution according to the methodof Tsao & Richards (1952). After repeated washing in ethanol and water, the chitinwas dried in an oven at 1 io° C. and the dry weight was determined.

For measurement of radioactivity unlabelled chitin was added to the sample so thatthe final total weight was: thorax, 15 mg.; wings, 15 mg.; abdomen, 8 mg.; and hindleg, 6 mg. The chitin was then dissolved by heating in concentrated hydrochloric acidsolution. The hydrochloric acid was removed by evaporation, and the residue was dis-solved in a known volume of water. A portion of the solution was spotted on to astandard 3 cm. diameter disk of filter-paper for which fitting planchets were available,and the radioactivity of the sample was then measured using a thin-windowedGeiger-Muller tube.

Preparation of wing extracts

For enzyme studies extracts were prepared of wings removed from decapitated adultlocusts (2-3 days old). The extraction was carried out by grinding the wings in a mortarand pestle with fine glass beads and a buffer solution appropriate to the enzyme beinai

Studies on chitin synthesis in the desert locust 131

Btudied. The homogenate was centrifuged for a few min. at 1000 g and the sedimentwas discarded. The supernatant fraction was then centrifuged at io,ooog for 30 min.,and the resultant clear solution was freeze-dried.

Paper chromatography

The following developing solvents were used for paper chromatography: solvent 1,propan-i-ol-ethyl acetate-water (7:1:2, by vol.) (Baar & Bull, 1953); solvent 2, 95 %ethanol-M-ammonium acetate (pH 7*5)(is:6, v/v) (Paladini & Leloir, 1952); sol vent 3,95% ethanol-M-ammonium acetate buffer (pH 3-8) (15:6, v/v) (Paladini & Leloir,1952); solvent 4, propan-i-ol-water-pyridine-acetic acid (8:4:8:1, by vol.) (Gordon,Thornburg & Werum, 1956); solvent 5, isobutyric acid-aq. NH3 soln. (sp.gr. o-88)-water (66:1:33, by vol.) (Pabst Laboratories, 1956); solvent 6, ethyl acetate-pyridine-aq. NH3 soln. (sp.gr. o-88)-water (10:5:3:3, by vol.) (Cahib, Leloir & Cardini, 1953).

Sugars were detected on paper chromatograms by the silver nitrate method ofTrevelyan, Procter & Harrison (1950); glucosamine and iV-acetylglucosamine by themethod of Partridge (1948); sugar phosphates by the method of Hanes and Isher-wood (1949); nucleotides by contact printing of the chromatogram in ultra-violetlight as described by Markham & Smith (1951); and radioactive compounds by radio-autography with Kodirex X-ray film.

Radioactive compounds were frequently identified by the technique of co-chroma-tography. A trace amount of the labelled material was mixed with authentic un-labelled material and the mixture subjected to paper chromatography. If theradioactive compound and the unlabelled compounds were chemically identical, theradioactive spot on the chromatogram (as found by radioautography) would corre-spond exactly in size and position with the chemically identified spot. Additionalconfirmation was afforded by repeating with different developing solvents.

Other analytical methods

Glucosamine was estimated by the method of Blix (1948); iV-acetylglucosamine bythe method of Reissig, Strominger & Leloir (1955); and fructose-6-phosphateestimated as fructose by the method of Roe (1934).

RESULTS

Chitin formation by the locust in vivo

The preliminary investigation was designed to study the formation of chitin bydifferent parts of locusts of different ages, and also to follow the incorporation ofuC-glucose into chitin. For the latter, female locusts only were used, and at a knownage each was injected with a solution containing "C-glucose (0-5 pinole, 2-5 /xc.).24 hr. later, the insect was killed with chloroform, and the thorax, abdomen, hind legsand wings were removed. The chitin of each sample was purified, weighed and theradioactivity counted.

Fig. 1 shows the variation in the total chitin content in the rear leg (expressed asmg. dry chitin per leg) taken from locusts of different ages from the beginning of the

9-2


5th instar. It is seen that there is a rapid increase during the earlier part of the instar,but this slows down and there is a slight fall as the period of moulting approaches andthis is presumably due to the hydrolysis of some of the chitin by the chitinase of themoulting fluid (Jeuniaux, 1955). There is a fall when the old cuticle is shed and thena fairly steady increase during the first 150 hr. or so of the adult stage. Very similarpatterns were found when assays were made using locust thorax or abdomen. Some

Adult

60 120 0 60 120

Age (hr.)

180 240 300

Fig. 1. Chitin content in locust leg, expressed as mg. dry wt. of chitin per hind legin insects of different ages.

400r°

Adult

Average age

Fig. a. The incorporation of radioactivity from l*C-glucose into locust leg chitin in a 24 hr.period after injection of 14C-glucose. The age of each locust was taken as mid-way through the24 hr. period.

measure of the amount of chitin laid down during particular 24 hr. periods wasobtained by measuring the radioactivity incorporated into the chitin and derived fromthe labelled glucose which had been injected 24 hr. before the insect was killed andthe chitin isolated. The results obtained for locust leg are shown in Fig. 2. During theearlier part of the 5th instar new chitin is being formed as there is appreciable


incorporation of radioactivity, but this falls almost to zero about two-thirds of the waythrough the instar, followed by a slight rise before moulting. As expected from thecurve of Fig. 1, there is active incorporation of label during the first 120-180 hr. ofadult life, but this then almost ceases, showing that the adult cuticle has reached itsfinal chitin content. The periods of miximum incorporation of radioactivity were thesame as the periods of maximum increase in chitin content.

20 r

16 -

12 -

8 -

4 -

_

5th

i

inscar

r

O

/

o

7

o

/<•

o /

Adult

1

Or>

^~

1

w O

I I

60 120 180 240 30060 120

Age (hr.)

Fig- 3- The chitin content of locust wing, expressed as mg. dry wt. of chitin per set of fourwings for insects of different ages.

400r

60 120 60 120 180 240Average age (hr.)

Fig. 4. The incorporation of radioactivity from "C-glucose into locust wing chitin in a 34 hr.period after injection of "C-glucose. The age of each locust was taken mid-way through the24 hr. period.

The results for chitin formation in the wing are shown in Figs. 3 and 4. In the adultstage the whole wing was used, but with 5th-instar insects, the estimations were madeon the vestigial adult wing which had been freed from the cuticle of the 5th-instarwing. Chitin formation is seen to begin towards the end of the 5th instar and continuesduring the first few days of adult life. These results suggested that the epidermis,whether from the leg, thorax, abdomen or wing, was active in the formation of chitin


for the first few days after a moult. In the wing, the cuticle forms a high proportionof the total tissue and it appeared that it might therefore provide the most usefulsource of chitin-forming tissue for in vitro studies. Before proceeding further theidentity of the labelled chitin was confirmed by hydrolsing a sample with chitinase.It was found that the principal radioactive product of hydrolysis was iV-acetyl-glucosamine, this being identified by co-chromatography with the authentic com-pound using solvents i, 4 and 6.

A cell-free extract from adult wings was then examined for the presence of variousenzyme systems which might be expected to occur if assumptions were made abouta possible pathway for chitin biosynthesis.

Heocokinase

A wing extract was made using a buffer solution containing histidine (0-02 M),tris-(2-amino-2-hydroxymethylpropane-i,3-diol) (0-02 M) ethylenediamine-tetra-acetic acid (EDTA, 0-002 M) and magnesium chloride (0*002 M) and which had beenadjusted to pH 7-0 by addition of hydrochloric acid. A portion of this enzyme extract(0-08 ml.) was mixed with 14C-glucose (0-02 /xmole, 0-5 /xc.), ATP (3.0 /xmole) andmagnesium chloride (6-0 (imole) in a total volume of 0-26 ml. and incubated for10 min. at 370 C. The products were separated on a paper chromatogram developedwith solvent 1. Most of the radioactivity ran as sugar phosphate, and only 6-6 % ofthe total was present in the glucose spot. When, however, ATP was omitted from theincubation mixture it was found that 76 % of the total radioactivity remained in theglucose spot. The labelled product from the complete incubation mixure which wasassumed to be sugar phosphate, was shown to be hydrolysable by alkaline phosphataseto give a product which behaved as glucose on chromatograms using solvent 1. Theenzyme hexokinase, which is required for the initial phosphorylation of free sugar byATP was thus shown to be present in the extract.

Formation of glucosamine-6-phosphate

Normally, a sodium phosphate buffer (o-i M, pH 6-8) containing EDTA (0-0025 M)was used for the preparation of the wing extract. It was found that the addition ofglutamine (o-oi M) to the solution would protect the rather unstable enzyme frominactivation, and such addition was made whenever the conditions of the experimentallowed. The formation of glucosamine-6-phosphate from simple sugar phosphateswas demonstrated as follows: Glutamine (10 /xmole), fructose-6-phosphate (io^mole)and enzyme extract (o-8 ml.) in a total volume of i-o ml. was incubated for 240 min.at 30° C. and then the reaction stopped by heating to ioo° C. for 3 min. Precipitatedprotein was removed by centrifugation and a 0-5 ml. sample of supernatant was assayedfor glucosamine-6-phosphate. An appropriate control incubation mixture which hadbeen inactivated at zero time was run in parallel in this and the other experiments, andthe difference between the two values for glucosamine-6-phosphate was taken as ameasure of the amount of this compound formed in the active system. In a typicalexperiment of the type above it was found that 54 fig. of glucosamine-6-phosphate wassynthesized and that a similar amount appeared when glucose-6-phosphate was substi-tuted for fructose-6-phosphate, but glucose-1-phosphate, free glucose or fructose wereinactive as the sugar substrate. Various possible nitrogen donors were tried in place of

Studies on cJritin synthesis in the desert locust 135

^lutamine (such as asparagine or a mixture of ammonium chloride, ATP and gluta-mate) but no significant activity was found with any of them. It is concluded that thereaction takes place by the transfer of an amino group from glutamine to eitherfructose-6-phosphate or glucose-6-phosphate to give glutamine-6-phosphate.

When MC-glucose-6-phosphate was used as substrate in this system a radioactiveproduct was obtained which was found, by co-chromatography, to be identical withglucosamine-6-phosphate in solvents 1, 2, 3 and 4. After hydrolysis with alkalinephosphatase, the labelled product was co-chromatographically identical with glucos-amine in solvents 1, 4 and 6.

Phosphohexose isomerase

The similar rates of glucosamine-6-phosphate formation from glucose-6-phosphateand fructose-6-phosphate suggested either than the enzyme was non-specific, or moreprobably, that an active isomerase was present in the extracts. 4 mg. enzyme (pre-pared in o-i M-tris buffer, pH 9-0) and 10 /xmole fructose-6-phosphate in 10 ml. trisbuffer, pH 9-0, were incubated at 370 C , and 1 ml. samples were taken at intervalsduring the incubation. A similar reaction was also carried out in which the fructose-6-phosphate was replaced by an equimolecular amount of glucose-6-phosphate. Each1 ml. aliquot was analysed for fructose-6-phosphate. In both incubation mixtures, anequilibrium position was reached after about 20 min., in which the fructose-6-phosphate content corresponded to 32% of the total substrate originally present.This indicates equilibration of glucose-6-phosphate and fructose-6-phosphate byphosphohexose isomerase, and the value for the equilibrium position is similar to thatobtained by Lohmann (1933) for muscle extracts, where fructose-6-phosphate formed30 % of the total.

The formation of N-acetyIglucosamine-6-phosphate

In these experiments the enzyme solution used was prepared by extraction of adultwing with 0-02 M sodium phosphate buffer, pH 7-0. A typical reaction mixture con-tained: 2-5 /xmole reduced glutathione, o-i fimole coenzyme A, 5*0 /imole ATP,1 /xmole magnesium chloride, 15 ^rnole sodium acetate buffer, pH 7-0, and 5-0 mg.enzyme (freeze-dried) in a total volume of 0-5 ml. After incubation for 120 min. at370 C. the reaction was stopped by immersion in boiling water for 3 min., the mixturewas diluted with i-o ml. water, the precipitated protein spun off, and N-acetyl-glucosamine was estimated in i-o ml. of the supernatant. The value obtained witha boiled enzyme control was subtracted. Table 1 shows the results obtained with thecomplete reaction mixture described above, and the effect of omission of variousconstituents.

The identity of the product was established by replacing the unlabelled acetate with14C-acetate and by separation of the products of reaction by paper chromatographywith solvent 3. The principal radioactive spot was found to correspond to iV-acetyl-glucosamine-6-phosphate, and the identity was confirmed by co-chromatography withauthentic material in solvents 1, 2 and 3.

When the mixture of acetate, glutathione, coenzyme-A, ATP and magnesiumchloride in the incubation mixture was replaced by acetyl coenzyme-A, approximatelyfour times as much JV-acetylglucosamine-6-phosphate was formed, indicating thatacetyl coenzyme A is the active acetate donor.


Table 1. Formation of N-acetylglucosatmne-6-phosphate

N-acetylglucowmine-6-phosphate formed

Reaction mixture (jwaole)

Complete 0-132Glucosamine-6-phosphate replaced by glucosamine 0-036No glucosamine-6-phosphate 0*012No sodium acetate 0-087No glutathione 0-063No coenzyme-A 0-015No ATP 0-024No magnesium chloride 0-086

Formation of uridine diphosphate N-acetylglucosamine {UDPAG)o*i M-sodium phosphate buffer, pH yo, was used for extraction of the enzyme.

0-045/xmole of 14C-A^-acetylglucosamine-6-phosphate (o-i8/xc.), 0*4/xmole UTP,i-o fimote cysteine, 3-0 /xmole sodium phosphate buffer, pH 7-0, and i-o mg. enzymein a total vol. of 0-06 ml. were incubated at 370 C. for 60 min., and the reactionproducts were separated on a paper chromatogram developed with solvent 3. Radio-activity was found in two areas on the chromatogram—one corresponding to iV-acetyl-glucosamine-6-phosphate, the other to UDPAG, and these were eluted from thechromatogram and counted. Table 2 shows the results, together with those obtainedfrom control reactions in which components were omitted. UDPAG formation wasmaximal in the presence of both UTP and cysteine.

The identity of the UDPAG was confirmed by co-chromatography with theauthentic material in solvents 2, 3 and 5. After hydrolysis by heating for 10 min. atioo° C. in c i N hydrochloric acid a radioactive compound was formed which wasco-chromatographically identical with iV-acetylglucosamine in solvents 1, 4 and 6.

Table 2. Conversion ofltC-N-acetylglucosamine-6-phosphateinto labelled UDPAG by locust wing extract

After incubation, the starting material and the product were separated by chromatography.The figures show the percentage of the radioactivity originally spotted on to the chromato-gram which wa» found in each of the two spots after development.

Incubation iV-acetylglucosaminemixture 6-phosphate UDPAG

Complete 39 68Boiled enzyme 97 oNo UTP 91 8No cygteine 58 43

(Totals differ from 100 due to experimental error.)

CMtin synthesis

A number of attempts were made to show the formation of chitin from 14C-UDPAGin locust wing extracts, but no indication of such a process could be obtained. In thisseries of experiments a number of factors were varied in attempts to find the rightconditions to show chitin formation. The enzyme was prepared using a number ofdifferent techniques for extraction, and three different buffers were tried in the


extraction process. The extracts were fortified with different chitodextrin preparations,iV-acetylglucosamine, unlabelled carrier UDPAG, ATP, magnesium sulphate, EDTA,cysteine, glutathione, and boiled yeast extract. It should be noted, however, that all ofthese conditions were not varied independently.

Typically, the reaction was stopped by the addition of perchlorate (Glaser & Brown,1957), the insoluble portion was washed and counted, and the soluble fraction was runon a paper chromatogram developed with solvent 3. In none of these experiments didthe insoluble portion have a significant amount of the radioactivity, nor was a signifi-cant radioactive area of low Rf found on the chromatogram (chitodextxins have lowRf values, and would be expected to remain near to the origin of the chromatograms).In many cases much of the radioactivity corresponded to UDPAG.

DISCUSSION

The in vivo experiments with the desert locust showed that a high rate of chitinformation occurred during, and shortly after, the moult and that this was paralleledby a high rate of incorporation of radioactivity from "C-glucose. This is of interestsince it is known that the insect does not feed during the first 24 hr. after moultinginto the 5th instar (Howden & Kilby, i960), and a similar fasting period occurs afterthe moult into the adult form. The chitin must, therefore, be formed from reservefood substances during this period. Howden & Kilby (i960) have shown that theconcentration of trehalose (which occurs in high concentrations in the haemolymphof insects) falls during the first 24hr. after the moult, and it is possible that at least partof this is used for chitin synthesis. Glycogen could also be an important source ofreserve material for chitin formation, but Zaluska (1959) has followed the metabolismof glycogen and chitin during the development of the silkworm, and found that thetotal amount of glycogen utilized during the post-diapause period was not sufficientto account for the amount of chitin formed. He suggested that lipids might take partin chitin synthesis as the amounts of such reserves descrease considerably at this time.During the moulting period large quantities of glucosamine and ./V-acetylglucosamineappear in the moulting fluid of the silkmoth (Zielinska & Laskowska, 1958) andZaluska suggested that these two might be used in the formation of the new cuticle.

The results obtained from the enzymic studies on locust wing extracts are consistentwith the scheme shown in Fig. 5 for the formation of UDPAG. In Neurospora crassathe biosynthesis of chitin takes place by transfer of iV-acetylglucosamine units fromUDPAG to a chitodextrin chain. Although such a reaction could not be shownexperimentally in locust wing extracts, it is suggested that this was possibly due to afailure to obtain the correct experimental conditions, rather than to a lack of theenzyme or enzymes involved. It is difficult to understand why there is an activesystem for the formation of UDPAG if this is not used for chitin synthesis. For thesereasons, the final reaction from UDPAG to chitin is tentatively included in the schemeshown in Fig. 5.

Carey & Wyatt (i960) reported that UDPAG and other UDP-sugars were presentin the haemolymph of cecropia moths, and that the concentration of UDP-sugars inthe wing epidermis increased from o-8 ^.mole/g. in diapause (when little cuticle forma-tion takes place) to about 4 fimole/g. in the young adult (when cuticle formation is


most active). It was not stated which of the UDP-sugars made up this total in thewing, but it is tempting to speculate that UDPAG makes up at least a part of thisfraction and that it is involved in the formation of chitin.

Glucose

(J>) __ Fructose-6-Glucose-6- 1. phosphatephosphate

Acetyi Glucosamine-6- <* *• Glutamic acidcoenzyme A ^N / phosphate

ATP + acetate Coenzyme A x Acetylglucosamine-6-phosphate

IhUTP Acetylglucosamine-

1-phosphate

(*)

Pyrophosphate ' ^ UDPAG

Chitin

Fig. 5. Postulated scheme for chitin biosynthesis. The enzymes involved are: a, hexokinase;b, phosphohexose isomerase; c, glutamine transaminase; d, phosphoglucosamine trans-acetylase; e, acetyi coenzyme-A synthetase;/, phosphoacetylglucosamine mutase; g, UDPAGpyrophosphorylase.

In Fig. 3, fructose-6-phosphate is shown as reacting with glutamine, rather thanglucose-6-phosphate, although no clear experimental evidence was obtained for thisdistinction in wing extracts. However, this enzyme (glutamine transaminase) has beenpurified from iV. crassa, Escherischia coli and rat liver; in each case it was found to bespecific for fructose-6-phosphate (Ghosh, Blumenthal, Davidson & Roseman, i960).It seems more likely than not, therefore, that the locust enzyme is also specific forfructose-6-phosphate rather than for glucose-6-phosphate. The phosphohexose iso-merase shown to be present in wing extracts presumably equilibrated the two sugarphosphates.

The formation of acetyi coenzyme-A from ATP, acetate and coenzyme-A was notdirectly shown in the wing extracts, but it was clearly implied by the fact that in thesystem which acetylated glucosamine-6-phosphate, acetyi coenzyme-A could be re-placed by a mixture of acetate, coenzyme-A and ATP. Similarly, although thephosphoglucosamine mutase reaction was not shown directly, it was suggested by thefact that UDPAG was formed from UTP and iV-acetylglucosamine-6-phosphate.Thus the generally accepted mechanism for the formation of a UDP-sugar is byreaction of UTP with the sugar-1 -phosphate.


SUMMARY

1. In vivo studies on 5th instar and adult locusts revealed that chitin formation inthorax, abdomen, hind leg and wing was greatest during the first few days aftermoulting.

2. Wing extracts were used for in vitro experiments, and evidence was obtained forthe presence of the following enzymes: hexokinase, phosphohexose isomerase,glutamine transaminase, phosphoglucosamine transacetylase, acetyl coenzyme-Asynthetase, phosphoacetylglucosamine mutase and uridine diphosphate iV-acetyl-glucosamine pyrophosphorylase.

3. The results are discussed, and a tentative scheme is presented for the bio-synthesis of chitin in the wing of Schistocerca gregaria.

We are indebted to the Anti-Locust Research Centre for supplies of locusts andfor a research studentship for one of us (D. J. C) .

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