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Changes in acid mucopolysaccharides during the development of the blowfly, Calliphora erythrocephala
J. Insect Physiol., 1976, Vol. 22, pp. 917 to 924. Pergamon Press. Printed in Great Britain.
DURING CHANGES IN ACID MUCOPOLYSACCHARIDES THE DEVELOPMENT OF THE BLOWFLY,
CALLIPHORA
ERYTHROCEPHALA LARS H&LUND
Department of Zoophysiology, University of UmeB, S-901 87 UmeB. Sweden (Received 16 July 1975; revised 27 January 1976) Abstract-Acid mucopolysaccharides have been isolated from different developmental stages of Calliphora. Hyaluronic acid and a ‘larval AMPS were identified during all developmental stages. During the later part of the development chondroitin and a poorly-sulphated keratin sulphate-like compound were also present. Chondroitin sulphate and heparan sulphate could be detected at all development stages and during the latter part possibly keratin sulphate. The variation of acid mucopolysaccharides during development is discussed.
INTRODUCTION As ACID mucopolysaccharides (AMPS) are believed to play an essential r81e in developmental processes (IMMERS,1961; SUGIYAMA,1972; TOOLE, 1972; KOSHER and SEARLS, 1973; LOIVTRUP,1974), it may be of interest to try to characterize the AMPS occurring during ontogenesis in various animal taxa. Consequently, I have undertaken to study the development of the blowfly, Calliphora erythrocephula, partly in an
attempt to characterize the different AMPS present during the ontogenesis of the fly, and partly to see if there are any alterations in their amount. Few investigations have been published regarding AMPS in developing insects. Hyaluronic acid has been found in isolated tissues, e.g. the peritrophic membrane of the silkworm, Bombyx mori (NBIZAWA et al., 1963) and the midgut of larvae of the greater wax moth, Galleriu mellonella (ESTES and FAUST, 1964). SHARIEF et al. (1973) have shown that there
are unsulphated and sulphated compounds present during larval development of Phormia regim. From my own investigations on the adult fly C. erythrocephala (H&LUND, 1976). I have found that both unsulphated and sulphated AMPS occur. The
presence of chondroitin, hyaluronic acid, chondroitin sulphate, and possibly keratin sulphate was demonstrated together with two more unsulphated compounds. The latter are a poorly-sulphated keratin sulphate-like substance and a compound rich in amino sugars and galactose but containing also hexuronic acid. MATERIAL
AND METHODS
The stock culture of C. erythrocephala has been maintained continuously in our laboratory for several years, housed in wire cages and supplied with sucrose and water. For propagation young adults are fed on
fresh ox liver for 4 days. After another 7 days a new fresh piece of liver is supplied and the eggs are laid. The eggs are transferred to a humid chamber and reared at room temperature (20°C). At certain time intervals samples of eggs, enough for about 3g of dry powder, were collected and homogenized in cold acetone. At room temperature hatching occurs after about 22 hr. The larvae, fed on ox heart, form the white puparium on the seventh day, and the adults emerge after another 10 days. Larvae and pupae were collected at intervals of 24 to 48 hr and homogenized in cold acetone, giving samples of about 20g of dry powder. The dry powder was digested with papain and the AMPS precipitated with cetylpyridinium chloride (CPC). The isolation procedure resulted in two main fractions, namely, neutral polysaccharides in the CPCsupernatant and AMPS in the CPC-precipitation. The CPC-supernatant was collected through precipitation with 4 volumes of 95% ethanol. This precipitate was dissolved in 3 ml 2 M NaCl and applied to a molecular sieve column, Bio-Gel P-100. Eluation was performed with 0.1 M NaCl and 3-ml fractions were collected and assayed for hexoses, hexuronic acid, and protein. Fractionation of AMPS was achieved by running the polysaccharides through an anion exchange column (Dowex 1 x 10 Cl- form, 2WOO mesh). The column was washed with water and eluated stepwise with 0.2, 0.4. 0.6, 0.8, 1.2 and 4.0M NaCl (H&LUND, 1975). The methods
employed for the enzyme degradation of AMPS and for the chemical determinations of hexuranic acid, hexosamine, neutral sugars, sulphate, protein, phosphate, and sialic acid have been reported earlier (RAHEMTULLAand LWTRUP, 1974a ; H&LUND, 1976). 917
LARSHOGLUND
The amount of neutral polysaccharides isolated from each developmental stage is presented in Fig. 1. During the first hours of development there is no apparent loss of glycogen. but after 8 hr the amount decreases steadily until hatching. Subsequently, during larval development. the glycogen content rises, reaching a peak on day 6 and thereafter declining very rapidly.
600
400
200
I 0
Acid mucopolysaccharides
I
16 (hours)
I
I
5
I
9 (days)
13
TIME
I
17
Fig. I. Amount of glycogen during different developmental stages of Calliphorci.
Fractionation of the extracted AMPS on a column of Dowex-1 (Cl-) resin gave at each developmental stage rise to six subfractions (Fl-F6), of which the two first are unsulphated, the four last sulphated. The chemical analyses of the individual fractions are below discussed separately for the embryonic and the other developmental stages. AMPS isolated during thrjirst 24 hours of developmrnt
RESULTS Neutral polysaccharidrs
The gel chromatography of the neutral polysaccharides resulted in one peak rich in hexose and poor in protein. The subsequent tubes contained much protein but little hexose and were not further analyzed. Glucose is the only monosaccharide detectable in any fraction and glycogen thus the sole polysaccharide present. The protein content in the glycogen is less than 0.055; and no phosphate is present.
The unsulphated fractions FI and F2 contain hexosamine and hexuronic acid in the ratio of about 1:2 (Table 1). The ratio glucosamine/galactosamine is about 1:3 in F2 at 1 hr. Galactose is found in all fractions and sometimes trace amounts of glucose and mannose. The high glucose value in Fl probably implies contamination by glycogen. The presence of both glucosamine and galactosamine indicates a mixture of AMPS. To get an idea of the number of different AMPS present, FI, F2,
Table I. Composition of acid mucopolysaccharides
from embryos of C. rrythrocrphala
6.Y
u.5 I
U.36
0 1”
Hyaluronic acld ‘Larval AMPS
C.Y Hyaluromc
,lilCC
‘Larval 0.?11
I, 44
0 15
fr:IcL!
5x
actd AMPS
Hyaluromc aad ‘Larval AMPS Chondroitin sulphate
1~4
I II
Chondromn sulphatc Hrparm sulphatr
F5
U YJ
Chondrottln sulphaw Heparin sulphafe
Fh
,I 9
Chondroltm sulphatc Hcparm rulphate
0.67
lJ.85
OS17
65
Hyaluromc actd ‘Larval AMPS
0.36
0 93
0 10
0 Ub
5.5
Hysluromc acld ‘Larval AMPS
031
11.7s
4.u
Hyaluromc acid ‘Larval AMPS Chondralm sulphatr
tract
0,114
,I 35
0 17
Chondroltm sulphate Heparm sulphale
0.46
0.16
Chondromn sulphate Hcpann sulphate
(I.3 1
U.12
Chondrottm sulphatc Heparm wlphate
919
Changes in acid mucopolysaccharides
B
A
*
C-p
F2
H+A HS
T2 Em
F5
*
--ST-
c-y3 HA
F5
H’s
i&O:
F5
C-h
F6
r.i+Q
Fig. 2. Electrophoretic patterns of AMPS from Calliphora, 1hr of development. (A) In barium acetate, before and after treatment of fraction 2 with hyaluronidase (enz). (B) In barium acetate, before and after treatment of fraction 5 with nitrous acid. (C) In cupric acetate. Standards. Chondroitin~-sulphate (C 4-S); Chondroitin-6sulphate (C 6-S); Heparin sulphate (HS). The arrow indicates the point of origin.
and F3 were submitted to cellulose acetate electrophoresis in barium acetate buffer. As shown by the results obtained on F2_ two spots were visualized (Fig. 2). one moving like, and the other slower than, the hyaluronic acid standard. This fraction was also subjected to cupric acetate electrophoresis which separates chondroitin and hyaluronic acid. Two spots could be detected, moving closely together, opposite the hyaluronic acid standard. No spot with a mobility comparable to chondroitin could be detected, indicating that this substance is absent during embryogenesis. On treatment of F2 with testicular hyaluronidase about 12% of the material was degraded. When comparing the electrophoretic patterns before and after the enzyme treatment it was found that the compound moving like the hyaluronic acid standard is susceptible to hyaluronidase (Fig. 2). Similar results were obtained with Fl and F3. On these data it is justified to suggest the presence of hyaluronic acid and the absence of chondroitin during the first day of development. The non-susceptible part, comprising most of the unsulphated fractions, contains galactosamine, glucosamine, hexuronic acid, and galactose in the molar ratios 1:0.2: 1:O.i. A fraction of undegradable material with a composition similar to this is found in the adult fly (HCKTLUND, 1976). The proportion of this AMPS is higher during development than in the adult and may be characterized as a ‘larval AMPS. The only record of a substance of similar composition (the molar ratios being 0.2:0.8 :O.l : 1 plus 0.5 mole sulphate) concerns the Lorenzan sulphate found in ~q~u~~s ~cu~f~jus (DOYLE, 1967).The presence of molecules with a mixture of amino sugars and neutral sugars has also been reported from Clitellates (RAHEMTULLAand LQVTRUP.1975). The possibility of ‘larval AMPS containing more than one compound cannot be ruled out, in spite of the chro~tographic and electrophoretic results. It is
difficult, however, to account for the presence of the various monosaccharides on the basis of any mixture of known substances, especially since the results with hyaluronidase exclude the presence of chondroitin which might account for the large amounts of galactosamine and hexuronic acid in this material. The sulphated fractions contain both glucosamine and galactosamine (Table l), indicating a mixture of sulphated components. The sulphated fractions reveal two spots on barium acetate electrophoresis, one moving like the chondroitin sulphaie, and the other like heparin sulphate, standard (Fig. 2). On cupric acetate electrophoresis of F.5 and F6, two spots could be detected in the chondroitin sulphate area, the main spot moving like chondroitin-6-sulphate and a faint spot moving like chondroitin-4-sulphate. Apart from these two spots a compound with the same mobility as the heparin sulphate standard could be visualized (Fig. 2). The sulphate in F3 probably is due to the presence of undersulphated chondroitin sulphate. When the sulphated fractions were subjected to hyaluronidase treatment, it was found that the spot moving like chondroitin sulphate in barium acetate buffer disappears after hyaluronidase digestion. About 407; of the fractions was susceptible to this enzyme. Treatment of F5 with nitrous acid led to the disappearance, in barium acetate electrophoresis, of the compound with the mobility of heparin sulphate (Fig. 2). Nitrous acid is known to attack N-sulphated hexosamines and, hence, to degrade heparin slphate (LAGUNOFFand WARREN,1962; CIFONJZLLI, 1968). The presence of N-sulphate in this fraction (Table 1) corroborates the identification of the hyaluroni~se-resistant substance in FS as heparin sulphate. The occurrence of galactose indicates the presence of a substance other than chondroitin sulphate or heparin sulphate. This is not shown on the electrophoretical strips, but it is possible that the amount is too small or that the separation from the other
LARS HOGLUND
920
substances is incomplete. The presence of a sulphated ‘larval AMPS molecule cannot be excluded. AMPS
isolated from day 2 and onwards
On cellulose acetate electrophoresis of the unsulphated fractions Fl and F2 two spots were visualized during all developmental stages. In Verona1 buffer one moves like. and the other slower than, hyaluronic acid. In barium acetate the presence of a hyaluronic acid-like substance is confirmed. The other component migrates slower than heparin sulphate. F2 was also subjected to cupric acetate electrophoresis, giving a resolution into three, rather than two, spots, the fastest of which has a mobility similar to that of chondroitin. as reported by HATA and NAGAI (1972). This spot can clearly be visualized in F2 from day 12 and onward. A very faint spot can still be detected in strips from day 9. but not at and before day 7, indicating that chondroitin is synthesized only during the latter part of development. The remaining two spots are situated closely together, almost opposite the hyaluronic acid standard. It may be presumed that they represent hyaluronic acid plus the second component in the barium acetate electrophoresis. The latter has the same mobility as the ‘larval AMPS described above. F3 has a electrophoretic pattern that is similar to Fl and F2, but an elongation of the spots in front
of hyaluronic acid can be seen in Verona1 and hydrochloric acid buffer. This indicates the presence of a sulphated compound, as confirmed by the chemical analyses (Table 2). The concomitant appearance in barium acetate of a faint spot with the mobility of heparin sulphate probably means that it is an undersulphated heparin sulphate. On treatment of F2 and F3 with hyaluronidase only part of the fractions was degraded. When comparing the electrophoretic patterns before and after treatment with the enzyme, it was found that the component moving like the hyaluranic acid in Verona1 and barium acetate buffer is susceptible to hyaluronidase, in contrast to the slowmoving component. The fraction of degradable unsulphated material varies only slightly during the first 24 hr of development but, as appears from Table 3, there is a distinct increase during the subsequent stage. The chemical composition of the unsulphated fractions is characterized by a ratio of amino acid sugars to hexuronic acid of between 1.1 to 1.5 (Table 2). All hexuronic acid values are corrected for interference from the neutral sugars. Galactose and glucose are present and trace amounts of mannose. Fucose and sialic acid could not be detected, nor could phosphate. The large amounts of galactose might indicate the presence of a keratin-like substance, but this proposal does not account for all the galactose present.
Table 2. Composition of acid mucopolysaccharides from larvae and pupae of C. erythrocephala
,
duw FI
u, dPtYlog,rw!,r 0.40
0 22
03x
0.74
021
006
55
Hyaluromc aad ‘Larval AMPS
F?
1.01
0.54
0.57
0.34
0.37
004
32
Hyaluromc aad ‘Larval AMPS
F3
0.911
0.45
0.4x
0 IS
I).
3.4
Hvaluromc acid ‘L&al AMPS Chondromn sulphate
F4
I ?b
0.62
0 57
0. I b
0 20
0.50
0 30
3.2
Chondroitm sulphate Heparin sulphatc
F5
I 17
0.70
0.4Y
005
0 I6
IO5
045
35
Chondrmtm sulphatu Heparin sulphatc
Fb
I.1
u.10
0.1
I.04
0.2x
3Y
Chondrodm sulphate Hepann sulphale
I
I.oi
IX
O 03
I
0.10
i 2duyr0,ilw‘4opnl‘wr FI
11.36
0.27
0. I Y
04h
0.28
012
b5
Hyaluromc acld ‘Larval AMPS
F?
0.96
0.66
0.53
0.24
043
0.0x
4.6
Hyaluromc aad ‘Larval AMPS Chondrolt,n
F?
I U3
0 50
0.47
0.09
O 34
009
F4
09s
0.46
0.50
0.09
F5
0.99
0.46
0.46
0 IO
Fb
0.84
040
UJh
0.
I0
010
trace
41
Hyaluromc actd ‘Larval AMPS Chondroitin solphatc
0.20
0.45
I, 22
3.x
Chondroitm sulphafe Heparm sulphate
0 20
0.X5
032
3.2
Chondromn &hate Heparm sulphiitc
0.24
0.93
0.20
3.2
Chondromn sulphaw Heparm sulphate Keratin sulphatc [‘I
921
Changes in acid mucopolysaccharides Table 3. Hyaluronidase-sensitive material in F2 in per cent of the whole fraction Day
%
1 2 4 7 9 12 15 Adult
15 19 23 25 26 30 35 45*
--q--
F4
H+A F? HS
* (H~JGLuND, 1976)
Some of this is probably found in ‘larval AMPS. On day 7 about 25% of the material is hyaluronidase sensitive, and on the assumption that all of this is hyaluronic acid, an evaluation of the composition of the enzyme-resistant material can be made (Table 4). The sulphated fractions contain both galactosamine and glucosamine, indicating the presence of chondroitin sulphate and of either heparin sulphate or keratin sulphate. As N-sulphate is found, some of the glucosamine must occur in a heparin sulphate-like compound, and the galactose makes it possible that a keratin sulphate-like molecule is present. The results of barium acetate electrophoresis suggests the presence in F4 of chondroitin sulphate and heparin sulphate, and the result in Verona1 and hydrochloric acid buffers that the degree of sulphatation is low. F5 and F6 also contain chondroitin sulphate and heparin sulphate, but in these fractions the substances are more sulphated. When submitting F4, FS and F6 to hyaluronidase treatment it was found that only part of the fractions was degraded. The proportion of enzyme-susceptible material varies during development, increasing from about 32% at day 2 to 65% in the adult fly. When the non-degradable material of the sulphated fractions was subjected to electrophoresis it was found that the spot moving like chondroitin sulphate had disappeared. To test for the presence of amido sulphate, F4 and FS from day 9 were treated with nitrous acid and applied to Bio-Gel P-100 columns. They contained
7 days
1 0.25 0.75
1
F5
H’S H1;02
substances interfering with the hexuronic acid determinations, but some hexuronic acid preceded the oxidizing agent, and it was thus possible to establish the susceptibility to nitrous acid. When, after this treatment, the nondegradable material was subjected to electrophoresis, the spots moving like heparin sulphate had disappeared (Fig. 3).
DISCUSSION Glycogen is the sole neutral polysaccharide recorded in the present investigation. Besides its role in energy production (LUDWIG et al., 1965), glycogen is thought to be converted to other sugars that are used for synthetic purposes, such as ribose for RNA production (SASSE,1968), deoxyribose, chitin, and AMPS (VAN DER STARRE-VANDER MOLEN, 1972a). By histochemical methods the latter author could find no drastic alteration in the amount of glycogen during the first part of embryonic development and only small changes during the subsequent segmentation phase. This observation is confirmed by the analyses reported above.
Glucosamine
fraction of F2
Galactosamine
Neutral sugars
of development
Before digestion Susceptible Non-susceptible Non-susceptible ( x I .33)
HA
Fig. 3. Barium acetate electrophoresis of AMPS from Calliphora, 9 days of development. Electrophoretic patterns of fractions 4 and 5 after treatment with nitrous acid. Standards, Chondroitin sulphate (CS); Hyaluronic acid (HA); Heparin sulphate (HS). The arrow indicates the point of origin.
Table 4. Relative molar composition of the hyaluronidase-resistant Hexuronic acid
F5
HN02
0.7 0.25 0.45 0.6
0.6
0.9
0.6 0.8
0.9 1.2
The calculations are based on the premises that (1) the hyaluronidasesensitive fraction amounts to 25 per cent and (2) that it is made up of hyaluronic acid.
LARSH~~GLUND
922 Cl BI
TS
Midgut
Hatch
8 .-o-e-*
=
z 26
5 c <4 P
_
I z
I
/ t?,--r-’
z2
--I.,
/
ll .-*---w--.2
l/.-•
0 ----b---.3
&.-.j*
0
0
I 5
I 10
I 15 TIME (hours)
e-0
4
I 20
1 25
Fig. 4. Graphic representation of AMPS present during embryonic development of Calliphora. (1) ‘Larval AMPS, (2) Heparin sulphate, (3) Chondroitin sulphate, (4) Hyaluronic acid. Abbreviations, segmentation
blastula (BL); cleavage (TS); hatching (Hatch).
(CL); true
Glycogen is accumulated in Calliphora during larval development (Fig, l), most of which is utilized during metamorphosis. Its principal use is for the formation of chitin, only minor amounts being used as an energy source (CROMPTONand BIRT. 1967; STAFFORD, 1973). AGRELL(1952) has shown that most of the energy supply is derived from fat decomposition. Before the discussion of the results on AMPS, it may be pointed out that the distinction between AMPS and glycoproteins is not very sharp but rather the current classification of carbohydrate-protein compounds should be regarded as an operational one (GOTTSCHALK,1972). The main characteristics of AMPS are their large mol. wt and their polyelectric properties, which probably are responsible for the contribution of these substances to various morpho-
5
10
15
TIME (days) Fig. 5. Graphic representation of unsulphated AMPS present during post-embryonic development of Calliphora. (1) ‘Larval AMPS. (2) Hyaluronic acid. (3) Chondroitin. (4) Keratin-like substance (?).
5
10 TIME (days)
15
Fig. 6. Graphic representation of sulphated AMPS during development of Cdiphoru. (1) Heparin sulphate. (2) Chon-
droitin sulphate. genetic processes. ‘Glycoproteins are best defined as conjugated proteins containing as prosthetic groups one or more heterosaccharides, usually branched, with a relatively low number of sugar residues, lacking a serially repeating unit’ (GOTTSCHALK,1972). They are devoid of hexuronic acid and usually of sulphate. Keratin sulphate combines the characteristic of both classes. It possesses a repeating N-acetyl-lactosamine, and may contain mannose, fucose, and sialic acid, but it lacks hexuronic acid (GW~SCHALK,1972). The ‘larval AMPS from Calliphora has characters in common with both glycoproteins and AMPS, but as it shows the same characteristics as AMPS uis b uis polyanion detergents it has been classed as an AMPS in this investigation. During the first 24 hr of development four different compounds are present. The unsulphated ones are hyaluronic acid and ‘larval AMPS. The monosaccharides present suggest that the latter may be a mixture of at least two compounds. The amount of hyaluranic acid does not change considerably during the embryonic stages, whereas ‘larval AMPS shows a distinct rise for the first 3 hr (Fig. 4). This period coincides with cleavage, nuclear migration, and the beginning of the blastoderm formation (VAN DERSTARREVAN DER MOLEN, 1972b). The two sulphated embryonic fractions are chondroitin sulphate and heparin sulphate. The variations of these compounds are moderate, heparin sulphate exhibiting the most obvious change between 3 to 5 hr of development. i.e. during blastoderm formation and pseudo-segmentation in the embryo. ‘Larval AMPS increases rapidly until day 7. The amount of hyaluronic acid also increases but to a lesser degree (Fig. 5). Chondroitin sulphate increases during the first 4 days of larval development and then remains almost constant. The variation in heparin sulphate is similar, except that a slight peak occurs around day 8 (Fig. 6). SHARIEFet al. (1973) have found that there is an increase of sulphated compounds and an incorporation of 35SO:- during the first 2 days of larval development of P. regina, followed by a gradual reduc-
923
Changes in acid mucopolysaccharides
tion in the total amount of sulphated AMPS, and especially in the rate of sulphate binding. If these results are recalculated on an individual basis they agree with those observed by me. About the time of the formation of the white puparium there is marked histolysis and reorganization. Some of the tissues are broken down, whereas the imaginal discs remain uneffected and serve as centres for reorganization (PEREZ. 1910; AGRELL, 1964; DE PRIESTER,1972). During this time a noticeable decrease of unsulphated material is taking place. This is most pronounced for the ‘larval AMPS, and it continues after hatching. Hyaluronic acid, however, seems to increase in the adult. From day 9 and onwards chondroitin is synthesized and from day 13 a keratin-like substance can be detected (Fig. 5). The sulphated AMPS comprises two components which, by electrophoretical, enzymatical and chemical data, are demonstrated to be chondroitin sulphate and heparin sulphate (or possibly keratin sulphate). The proportion of hyaluronidase-sensitive material in the sulphated fractions is constant during the first 7 days of development but increases thereafter. This phenomenon reflects a reduction in the amount of heparin sulphate (Fig. 6). and supported also by chemical analyses which show that the proportion of galactosamine increases in the sulphated fractions from 40% at day 2 to 73% in the pharate adult during development. A similar decrease in heparin sulphate has also been reported in the development of the frog (KOSHERand SEARLS.1973) and the chicken (MEIER and HAY, 1973). The relatively low mobility of the AMPS in hydrochloric acid buffer suggests the presence of molecules with a low sulphate content in F4 and F5. A similar phenomenon has been reported from investigations on chickens (FIUNCO-BROWDERet al., 1963; KWST and FINNEGAN,1970) and frogs (HATA and NAGAI, 1973). It appears that under-sulphation is characteristic of AMPS in developing tissues. It is a striking feature that during the development of such widely different animals as the fly, the frog and the chicken, heparin sulphate is an important AMPS. In all three chondroitin sulphate is one of the substances which take its place in the adult. The same holds for ‘larval AMPS in the fly, which is abundant during development but is replaced in the emerged adult by hyaluronic acid and chondroitin.
I. (1952) The aerobic and anaerobic utilization of
metabolic
energy during insect metamorphosis.
Churchill,
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HATAR. and NAGAIY. (1972) A rapid and micro method for separation of acidic glycosaminoglycans by twodimensional electrophoresis. Anal. Biochem. 45. 462-468. H&LUND L. (I 976) The comparative biochemistry of invertebrate mucopolysaccharides. V. Insecta (Calliphora erythrocephala). Comp. Biochem. Physiol. 53(B), %14.
IMMERS J. (1961) Incornoration of 35SOa- in the sea urchin egg and‘larvae. Ark: Zool. 13. 561-564. KOSHERR. and SEARLSR. (1973) Sulfated mucopolysaccharide synthesis during the development of Rana pipiens. Develop. Biol. 32. 5&68.
KVISTT. N. and FINNEGANC. V. (1970) The distribution of glycosaminoglycans in the axial region of the developing chick embryo II. Biochemical analysis. J. exp. Zool. 175. 241-258. LAGUNOFFD. and WARRENG. (1962) Determination of 2-deoxy-2-sulfoaminohexose content of mucopolysaccharides. Arch. Biochem. Biophys. 99. 396400. LQVTRUPS. (1974) Epigenrtics-A treatise on theoretical biology. Wiley, London. LUDWIG D.. CROWEP. A., and HA~~EMYRM. M. (1965) Energy sources during embryogenesis of the yellow mealworm, Tenehrio moflitor. Ann. ent. Sot. Am. 58. 543-546.
MEIERS. and HAY E. (1973)Synthesis of sulphated glycosa-
minoglycans by embryonic cornea1 epithelium. Develop. Biol. 35. 318-331. NISIZAWAK., YAMAGUCHI T., HAUDA N., MAEDAM.. and YAMAZAKIH. (1963) Chemical nature of a uranic acidcontaining polysaccharide in the peritrophic membrane of the silkworm. Biochem. J. 54. 419-426. PEREZC. (1910) Recherches histologiques sur la metamorphose des muscides Calliphora erythrocephala. Meig. Arch. Zool. exp. gin. (5) 4. l-274.
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RAHEMTULLA F. and L~VTRLJPS. (1974) The comparative biochemistry of invertebrate mucopolysaccharides I. Methods. Platyhelminthes. Comp. Biochem. Physiol. 48. B, 631-637. RAHEMTULLA F. and L~VTRUPS. (1975) The comparative biochemistry of invertebrate mucopolysaccharides III. Oligochaeta and Hirudinea. Comp. Biochem. Physiol. SO. B, 627-629. SASSE D. (1968) Glycogen in der Ontogenese des Verdauungstrakts. Ergehn. Anat. Entw Gesh. 40. 2-66.
924
LARSH~GLLJND
SHARIEFF.. PERDUEJ., and JXBROG~SZ W. (1973) Biochemical. histochemical and autoradiographic evidence for the existence and formation of acid mucopolysaccharide in larvae of Phormia regina. Insect Biochem. 3. 243-262. STAFFORDW. (1973) Changes in the content of glycogen. sugars. nucleic acids, protein and free amino acids in the pupa of GIossina morsitarts during its development. Camp. Biochem. Physiol. 45(B). 763-168. SUCIYAMAK. (1972)Occurrence of mucopolysaccharides in the early development of the sea urchin embryo and its role in gastrulation. Develop. Growth DifSer. 14. 63-73.
T~OLEB. P. (1972) Hyaluronate turnover during chondrogenesis in the developing chick limb and axial skeleton. Duelop. Biol. 29. 321-329. VAN DER STARRE-VAN DER MOLENL.
G. (1972a) Embryogenesis of Cdiphorn erythrocephda Meigen II. Glycogen pattern. Neth. J. Zool. 22. 183-206. VAN DERSTARRE-VANDERMOLENL. G. (1972b) Embryogenesis of Cdiphora erythrocephda Meigen I. Morphology. Neth. J. Zool. 22. 119-182.
DURING CHANGES IN ACID MUCOPOLYSACCHARIDES THE DEVELOPMENT OF THE BLOWFLY,
CALLIPHORA
ERYTHROCEPHALA LARS H&LUND
Department of Zoophysiology, University of UmeB, S-901 87 UmeB. Sweden (Received 16 July 1975; revised 27 January 1976) Abstract-Acid mucopolysaccharides have been isolated from different developmental stages of Calliphora. Hyaluronic acid and a ‘larval AMPS were identified during all developmental stages. During the later part of the development chondroitin and a poorly-sulphated keratin sulphate-like compound were also present. Chondroitin sulphate and heparan sulphate could be detected at all development stages and during the latter part possibly keratin sulphate. The variation of acid mucopolysaccharides during development is discussed.
INTRODUCTION As ACID mucopolysaccharides (AMPS) are believed to play an essential r81e in developmental processes (IMMERS,1961; SUGIYAMA,1972; TOOLE, 1972; KOSHER and SEARLS, 1973; LOIVTRUP,1974), it may be of interest to try to characterize the AMPS occurring during ontogenesis in various animal taxa. Consequently, I have undertaken to study the development of the blowfly, Calliphora erythrocephula, partly in an
attempt to characterize the different AMPS present during the ontogenesis of the fly, and partly to see if there are any alterations in their amount. Few investigations have been published regarding AMPS in developing insects. Hyaluronic acid has been found in isolated tissues, e.g. the peritrophic membrane of the silkworm, Bombyx mori (NBIZAWA et al., 1963) and the midgut of larvae of the greater wax moth, Galleriu mellonella (ESTES and FAUST, 1964). SHARIEF et al. (1973) have shown that there
are unsulphated and sulphated compounds present during larval development of Phormia regim. From my own investigations on the adult fly C. erythrocephala (H&LUND, 1976). I have found that both unsulphated and sulphated AMPS occur. The
presence of chondroitin, hyaluronic acid, chondroitin sulphate, and possibly keratin sulphate was demonstrated together with two more unsulphated compounds. The latter are a poorly-sulphated keratin sulphate-like substance and a compound rich in amino sugars and galactose but containing also hexuronic acid. MATERIAL
AND METHODS
The stock culture of C. erythrocephala has been maintained continuously in our laboratory for several years, housed in wire cages and supplied with sucrose and water. For propagation young adults are fed on
fresh ox liver for 4 days. After another 7 days a new fresh piece of liver is supplied and the eggs are laid. The eggs are transferred to a humid chamber and reared at room temperature (20°C). At certain time intervals samples of eggs, enough for about 3g of dry powder, were collected and homogenized in cold acetone. At room temperature hatching occurs after about 22 hr. The larvae, fed on ox heart, form the white puparium on the seventh day, and the adults emerge after another 10 days. Larvae and pupae were collected at intervals of 24 to 48 hr and homogenized in cold acetone, giving samples of about 20g of dry powder. The dry powder was digested with papain and the AMPS precipitated with cetylpyridinium chloride (CPC). The isolation procedure resulted in two main fractions, namely, neutral polysaccharides in the CPCsupernatant and AMPS in the CPC-precipitation. The CPC-supernatant was collected through precipitation with 4 volumes of 95% ethanol. This precipitate was dissolved in 3 ml 2 M NaCl and applied to a molecular sieve column, Bio-Gel P-100. Eluation was performed with 0.1 M NaCl and 3-ml fractions were collected and assayed for hexoses, hexuronic acid, and protein. Fractionation of AMPS was achieved by running the polysaccharides through an anion exchange column (Dowex 1 x 10 Cl- form, 2WOO mesh). The column was washed with water and eluated stepwise with 0.2, 0.4. 0.6, 0.8, 1.2 and 4.0M NaCl (H&LUND, 1975). The methods
employed for the enzyme degradation of AMPS and for the chemical determinations of hexuranic acid, hexosamine, neutral sugars, sulphate, protein, phosphate, and sialic acid have been reported earlier (RAHEMTULLAand LWTRUP, 1974a ; H&LUND, 1976). 917
LARSHOGLUND
The amount of neutral polysaccharides isolated from each developmental stage is presented in Fig. 1. During the first hours of development there is no apparent loss of glycogen. but after 8 hr the amount decreases steadily until hatching. Subsequently, during larval development. the glycogen content rises, reaching a peak on day 6 and thereafter declining very rapidly.
600
400
200
I 0
Acid mucopolysaccharides
I
16 (hours)
I
I
5
I
9 (days)
13
TIME
I
17
Fig. I. Amount of glycogen during different developmental stages of Calliphorci.
Fractionation of the extracted AMPS on a column of Dowex-1 (Cl-) resin gave at each developmental stage rise to six subfractions (Fl-F6), of which the two first are unsulphated, the four last sulphated. The chemical analyses of the individual fractions are below discussed separately for the embryonic and the other developmental stages. AMPS isolated during thrjirst 24 hours of developmrnt
RESULTS Neutral polysaccharidrs
The gel chromatography of the neutral polysaccharides resulted in one peak rich in hexose and poor in protein. The subsequent tubes contained much protein but little hexose and were not further analyzed. Glucose is the only monosaccharide detectable in any fraction and glycogen thus the sole polysaccharide present. The protein content in the glycogen is less than 0.055; and no phosphate is present.
The unsulphated fractions FI and F2 contain hexosamine and hexuronic acid in the ratio of about 1:2 (Table 1). The ratio glucosamine/galactosamine is about 1:3 in F2 at 1 hr. Galactose is found in all fractions and sometimes trace amounts of glucose and mannose. The high glucose value in Fl probably implies contamination by glycogen. The presence of both glucosamine and galactosamine indicates a mixture of AMPS. To get an idea of the number of different AMPS present, FI, F2,
Table I. Composition of acid mucopolysaccharides
from embryos of C. rrythrocrphala
6.Y
u.5 I
U.36
0 1”
Hyaluronic acld ‘Larval AMPS
C.Y Hyaluromc
,lilCC
‘Larval 0.?11
I, 44
0 15
fr:IcL!
5x
actd AMPS
Hyaluromc aad ‘Larval AMPS Chondroitin sulphate
1~4
I II
Chondromn sulphatc Hrparm sulphatr
F5
U YJ
Chondrottln sulphaw Heparin sulphafe
Fh
,I 9
Chondroltm sulphatc Hcparm rulphate
0.67
lJ.85
OS17
65
Hyaluromc actd ‘Larval AMPS
0.36
0 93
0 10
0 Ub
5.5
Hysluromc acld ‘Larval AMPS
031
11.7s
4.u
Hyaluromc acid ‘Larval AMPS Chondralm sulphatr
tract
0,114
,I 35
0 17
Chondroltm sulphate Heparm sulphale
0.46
0.16
Chondromn sulphate Hcpann sulphate
(I.3 1
U.12
Chondrottm sulphatc Heparm wlphate
919
Changes in acid mucopolysaccharides
B
A
*
C-p
F2
H+A HS
T2 Em
F5
*
--ST-
c-y3 HA
F5
H’s
i&O:
F5
C-h
F6
r.i+Q
Fig. 2. Electrophoretic patterns of AMPS from Calliphora, 1hr of development. (A) In barium acetate, before and after treatment of fraction 2 with hyaluronidase (enz). (B) In barium acetate, before and after treatment of fraction 5 with nitrous acid. (C) In cupric acetate. Standards. Chondroitin~-sulphate (C 4-S); Chondroitin-6sulphate (C 6-S); Heparin sulphate (HS). The arrow indicates the point of origin.
and F3 were submitted to cellulose acetate electrophoresis in barium acetate buffer. As shown by the results obtained on F2_ two spots were visualized (Fig. 2). one moving like, and the other slower than, the hyaluronic acid standard. This fraction was also subjected to cupric acetate electrophoresis which separates chondroitin and hyaluronic acid. Two spots could be detected, moving closely together, opposite the hyaluronic acid standard. No spot with a mobility comparable to chondroitin could be detected, indicating that this substance is absent during embryogenesis. On treatment of F2 with testicular hyaluronidase about 12% of the material was degraded. When comparing the electrophoretic patterns before and after the enzyme treatment it was found that the compound moving like the hyaluronic acid standard is susceptible to hyaluronidase (Fig. 2). Similar results were obtained with Fl and F3. On these data it is justified to suggest the presence of hyaluronic acid and the absence of chondroitin during the first day of development. The non-susceptible part, comprising most of the unsulphated fractions, contains galactosamine, glucosamine, hexuronic acid, and galactose in the molar ratios 1:0.2: 1:O.i. A fraction of undegradable material with a composition similar to this is found in the adult fly (HCKTLUND, 1976). The proportion of this AMPS is higher during development than in the adult and may be characterized as a ‘larval AMPS. The only record of a substance of similar composition (the molar ratios being 0.2:0.8 :O.l : 1 plus 0.5 mole sulphate) concerns the Lorenzan sulphate found in ~q~u~~s ~cu~f~jus (DOYLE, 1967).The presence of molecules with a mixture of amino sugars and neutral sugars has also been reported from Clitellates (RAHEMTULLAand LQVTRUP.1975). The possibility of ‘larval AMPS containing more than one compound cannot be ruled out, in spite of the chro~tographic and electrophoretic results. It is
difficult, however, to account for the presence of the various monosaccharides on the basis of any mixture of known substances, especially since the results with hyaluronidase exclude the presence of chondroitin which might account for the large amounts of galactosamine and hexuronic acid in this material. The sulphated fractions contain both glucosamine and galactosamine (Table l), indicating a mixture of sulphated components. The sulphated fractions reveal two spots on barium acetate electrophoresis, one moving like the chondroitin sulphaie, and the other like heparin sulphate, standard (Fig. 2). On cupric acetate electrophoresis of F.5 and F6, two spots could be detected in the chondroitin sulphate area, the main spot moving like chondroitin-6-sulphate and a faint spot moving like chondroitin-4-sulphate. Apart from these two spots a compound with the same mobility as the heparin sulphate standard could be visualized (Fig. 2). The sulphate in F3 probably is due to the presence of undersulphated chondroitin sulphate. When the sulphated fractions were subjected to hyaluronidase treatment, it was found that the spot moving like chondroitin sulphate in barium acetate buffer disappears after hyaluronidase digestion. About 407; of the fractions was susceptible to this enzyme. Treatment of F5 with nitrous acid led to the disappearance, in barium acetate electrophoresis, of the compound with the mobility of heparin sulphate (Fig. 2). Nitrous acid is known to attack N-sulphated hexosamines and, hence, to degrade heparin slphate (LAGUNOFFand WARREN,1962; CIFONJZLLI, 1968). The presence of N-sulphate in this fraction (Table 1) corroborates the identification of the hyaluroni~se-resistant substance in FS as heparin sulphate. The occurrence of galactose indicates the presence of a substance other than chondroitin sulphate or heparin sulphate. This is not shown on the electrophoretical strips, but it is possible that the amount is too small or that the separation from the other
LARS HOGLUND
920
substances is incomplete. The presence of a sulphated ‘larval AMPS molecule cannot be excluded. AMPS
isolated from day 2 and onwards
On cellulose acetate electrophoresis of the unsulphated fractions Fl and F2 two spots were visualized during all developmental stages. In Verona1 buffer one moves like. and the other slower than, hyaluronic acid. In barium acetate the presence of a hyaluronic acid-like substance is confirmed. The other component migrates slower than heparin sulphate. F2 was also subjected to cupric acetate electrophoresis, giving a resolution into three, rather than two, spots, the fastest of which has a mobility similar to that of chondroitin. as reported by HATA and NAGAI (1972). This spot can clearly be visualized in F2 from day 12 and onward. A very faint spot can still be detected in strips from day 9. but not at and before day 7, indicating that chondroitin is synthesized only during the latter part of development. The remaining two spots are situated closely together, almost opposite the hyaluronic acid standard. It may be presumed that they represent hyaluronic acid plus the second component in the barium acetate electrophoresis. The latter has the same mobility as the ‘larval AMPS described above. F3 has a electrophoretic pattern that is similar to Fl and F2, but an elongation of the spots in front
of hyaluronic acid can be seen in Verona1 and hydrochloric acid buffer. This indicates the presence of a sulphated compound, as confirmed by the chemical analyses (Table 2). The concomitant appearance in barium acetate of a faint spot with the mobility of heparin sulphate probably means that it is an undersulphated heparin sulphate. On treatment of F2 and F3 with hyaluronidase only part of the fractions was degraded. When comparing the electrophoretic patterns before and after treatment with the enzyme, it was found that the component moving like the hyaluranic acid in Verona1 and barium acetate buffer is susceptible to hyaluronidase, in contrast to the slowmoving component. The fraction of degradable unsulphated material varies only slightly during the first 24 hr of development but, as appears from Table 3, there is a distinct increase during the subsequent stage. The chemical composition of the unsulphated fractions is characterized by a ratio of amino acid sugars to hexuronic acid of between 1.1 to 1.5 (Table 2). All hexuronic acid values are corrected for interference from the neutral sugars. Galactose and glucose are present and trace amounts of mannose. Fucose and sialic acid could not be detected, nor could phosphate. The large amounts of galactose might indicate the presence of a keratin-like substance, but this proposal does not account for all the galactose present.
Table 2. Composition of acid mucopolysaccharides from larvae and pupae of C. erythrocephala
,
duw FI
u, dPtYlog,rw!,r 0.40
0 22
03x
0.74
021
006
55
Hyaluromc aad ‘Larval AMPS
F?
1.01
0.54
0.57
0.34
0.37
004
32
Hyaluromc aad ‘Larval AMPS
F3
0.911
0.45
0.4x
0 IS
I).
3.4
Hvaluromc acid ‘L&al AMPS Chondromn sulphate
F4
I ?b
0.62
0 57
0. I b
0 20
0.50
0 30
3.2
Chondroitm sulphate Heparin sulphatc
F5
I 17
0.70
0.4Y
005
0 I6
IO5
045
35
Chondrmtm sulphatu Heparin sulphatc
Fb
I.1
u.10
0.1
I.04
0.2x
3Y
Chondrodm sulphate Hepann sulphale
I
I.oi
IX
O 03
I
0.10
i 2duyr0,ilw‘4opnl‘wr FI
11.36
0.27
0. I Y
04h
0.28
012
b5
Hyaluromc acld ‘Larval AMPS
F?
0.96
0.66
0.53
0.24
043
0.0x
4.6
Hyaluromc aad ‘Larval AMPS Chondrolt,n
F?
I U3
0 50
0.47
0.09
O 34
009
F4
09s
0.46
0.50
0.09
F5
0.99
0.46
0.46
0 IO
Fb
0.84
040
UJh
0.
I0
010
trace
41
Hyaluromc actd ‘Larval AMPS Chondroitin solphatc
0.20
0.45
I, 22
3.x
Chondroitm sulphafe Heparm sulphate
0 20
0.X5
032
3.2
Chondromn &hate Heparm sulphiitc
0.24
0.93
0.20
3.2
Chondromn sulphaw Heparm sulphate Keratin sulphatc [‘I
921
Changes in acid mucopolysaccharides Table 3. Hyaluronidase-sensitive material in F2 in per cent of the whole fraction Day
%
1 2 4 7 9 12 15 Adult
15 19 23 25 26 30 35 45*
--q--
F4
H+A F? HS
* (H~JGLuND, 1976)
Some of this is probably found in ‘larval AMPS. On day 7 about 25% of the material is hyaluronidase sensitive, and on the assumption that all of this is hyaluronic acid, an evaluation of the composition of the enzyme-resistant material can be made (Table 4). The sulphated fractions contain both galactosamine and glucosamine, indicating the presence of chondroitin sulphate and of either heparin sulphate or keratin sulphate. As N-sulphate is found, some of the glucosamine must occur in a heparin sulphate-like compound, and the galactose makes it possible that a keratin sulphate-like molecule is present. The results of barium acetate electrophoresis suggests the presence in F4 of chondroitin sulphate and heparin sulphate, and the result in Verona1 and hydrochloric acid buffers that the degree of sulphatation is low. F5 and F6 also contain chondroitin sulphate and heparin sulphate, but in these fractions the substances are more sulphated. When submitting F4, FS and F6 to hyaluronidase treatment it was found that only part of the fractions was degraded. The proportion of enzyme-susceptible material varies during development, increasing from about 32% at day 2 to 65% in the adult fly. When the non-degradable material of the sulphated fractions was subjected to electrophoresis it was found that the spot moving like chondroitin sulphate had disappeared. To test for the presence of amido sulphate, F4 and FS from day 9 were treated with nitrous acid and applied to Bio-Gel P-100 columns. They contained
7 days
1 0.25 0.75
1
F5
H’S H1;02
substances interfering with the hexuronic acid determinations, but some hexuronic acid preceded the oxidizing agent, and it was thus possible to establish the susceptibility to nitrous acid. When, after this treatment, the nondegradable material was subjected to electrophoresis, the spots moving like heparin sulphate had disappeared (Fig. 3).
DISCUSSION Glycogen is the sole neutral polysaccharide recorded in the present investigation. Besides its role in energy production (LUDWIG et al., 1965), glycogen is thought to be converted to other sugars that are used for synthetic purposes, such as ribose for RNA production (SASSE,1968), deoxyribose, chitin, and AMPS (VAN DER STARRE-VANDER MOLEN, 1972a). By histochemical methods the latter author could find no drastic alteration in the amount of glycogen during the first part of embryonic development and only small changes during the subsequent segmentation phase. This observation is confirmed by the analyses reported above.
Glucosamine
fraction of F2
Galactosamine
Neutral sugars
of development
Before digestion Susceptible Non-susceptible Non-susceptible ( x I .33)
HA
Fig. 3. Barium acetate electrophoresis of AMPS from Calliphora, 9 days of development. Electrophoretic patterns of fractions 4 and 5 after treatment with nitrous acid. Standards, Chondroitin sulphate (CS); Hyaluronic acid (HA); Heparin sulphate (HS). The arrow indicates the point of origin.
Table 4. Relative molar composition of the hyaluronidase-resistant Hexuronic acid
F5
HN02
0.7 0.25 0.45 0.6
0.6
0.9
0.6 0.8
0.9 1.2
The calculations are based on the premises that (1) the hyaluronidasesensitive fraction amounts to 25 per cent and (2) that it is made up of hyaluronic acid.
LARSH~~GLUND
922 Cl BI
TS
Midgut
Hatch
8 .-o-e-*
=
z 26
5 c <4 P
_
I z
I
/ t?,--r-’
z2
--I.,
/
ll .-*---w--.2
l/.-•
0 ----b---.3
&.-.j*
0
0
I 5
I 10
I 15 TIME (hours)
e-0
4
I 20
1 25
Fig. 4. Graphic representation of AMPS present during embryonic development of Calliphora. (1) ‘Larval AMPS, (2) Heparin sulphate, (3) Chondroitin sulphate, (4) Hyaluronic acid. Abbreviations, segmentation
blastula (BL); cleavage (TS); hatching (Hatch).
(CL); true
Glycogen is accumulated in Calliphora during larval development (Fig, l), most of which is utilized during metamorphosis. Its principal use is for the formation of chitin, only minor amounts being used as an energy source (CROMPTONand BIRT. 1967; STAFFORD, 1973). AGRELL(1952) has shown that most of the energy supply is derived from fat decomposition. Before the discussion of the results on AMPS, it may be pointed out that the distinction between AMPS and glycoproteins is not very sharp but rather the current classification of carbohydrate-protein compounds should be regarded as an operational one (GOTTSCHALK,1972). The main characteristics of AMPS are their large mol. wt and their polyelectric properties, which probably are responsible for the contribution of these substances to various morpho-
5
10
15
TIME (days) Fig. 5. Graphic representation of unsulphated AMPS present during post-embryonic development of Calliphora. (1) ‘Larval AMPS. (2) Hyaluronic acid. (3) Chondroitin. (4) Keratin-like substance (?).
5
10 TIME (days)
15
Fig. 6. Graphic representation of sulphated AMPS during development of Cdiphoru. (1) Heparin sulphate. (2) Chon-
droitin sulphate. genetic processes. ‘Glycoproteins are best defined as conjugated proteins containing as prosthetic groups one or more heterosaccharides, usually branched, with a relatively low number of sugar residues, lacking a serially repeating unit’ (GOTTSCHALK,1972). They are devoid of hexuronic acid and usually of sulphate. Keratin sulphate combines the characteristic of both classes. It possesses a repeating N-acetyl-lactosamine, and may contain mannose, fucose, and sialic acid, but it lacks hexuronic acid (GW~SCHALK,1972). The ‘larval AMPS from Calliphora has characters in common with both glycoproteins and AMPS, but as it shows the same characteristics as AMPS uis b uis polyanion detergents it has been classed as an AMPS in this investigation. During the first 24 hr of development four different compounds are present. The unsulphated ones are hyaluronic acid and ‘larval AMPS. The monosaccharides present suggest that the latter may be a mixture of at least two compounds. The amount of hyaluranic acid does not change considerably during the embryonic stages, whereas ‘larval AMPS shows a distinct rise for the first 3 hr (Fig. 4). This period coincides with cleavage, nuclear migration, and the beginning of the blastoderm formation (VAN DERSTARREVAN DER MOLEN, 1972b). The two sulphated embryonic fractions are chondroitin sulphate and heparin sulphate. The variations of these compounds are moderate, heparin sulphate exhibiting the most obvious change between 3 to 5 hr of development. i.e. during blastoderm formation and pseudo-segmentation in the embryo. ‘Larval AMPS increases rapidly until day 7. The amount of hyaluronic acid also increases but to a lesser degree (Fig. 5). Chondroitin sulphate increases during the first 4 days of larval development and then remains almost constant. The variation in heparin sulphate is similar, except that a slight peak occurs around day 8 (Fig. 6). SHARIEFet al. (1973) have found that there is an increase of sulphated compounds and an incorporation of 35SO:- during the first 2 days of larval development of P. regina, followed by a gradual reduc-
923
Changes in acid mucopolysaccharides
tion in the total amount of sulphated AMPS, and especially in the rate of sulphate binding. If these results are recalculated on an individual basis they agree with those observed by me. About the time of the formation of the white puparium there is marked histolysis and reorganization. Some of the tissues are broken down, whereas the imaginal discs remain uneffected and serve as centres for reorganization (PEREZ. 1910; AGRELL, 1964; DE PRIESTER,1972). During this time a noticeable decrease of unsulphated material is taking place. This is most pronounced for the ‘larval AMPS, and it continues after hatching. Hyaluronic acid, however, seems to increase in the adult. From day 9 and onwards chondroitin is synthesized and from day 13 a keratin-like substance can be detected (Fig. 5). The sulphated AMPS comprises two components which, by electrophoretical, enzymatical and chemical data, are demonstrated to be chondroitin sulphate and heparin sulphate (or possibly keratin sulphate). The proportion of hyaluronidase-sensitive material in the sulphated fractions is constant during the first 7 days of development but increases thereafter. This phenomenon reflects a reduction in the amount of heparin sulphate (Fig. 6). and supported also by chemical analyses which show that the proportion of galactosamine increases in the sulphated fractions from 40% at day 2 to 73% in the pharate adult during development. A similar decrease in heparin sulphate has also been reported in the development of the frog (KOSHERand SEARLS.1973) and the chicken (MEIER and HAY, 1973). The relatively low mobility of the AMPS in hydrochloric acid buffer suggests the presence of molecules with a low sulphate content in F4 and F5. A similar phenomenon has been reported from investigations on chickens (FIUNCO-BROWDERet al., 1963; KWST and FINNEGAN,1970) and frogs (HATA and NAGAI, 1973). It appears that under-sulphation is characteristic of AMPS in developing tissues. It is a striking feature that during the development of such widely different animals as the fly, the frog and the chicken, heparin sulphate is an important AMPS. In all three chondroitin sulphate is one of the substances which take its place in the adult. The same holds for ‘larval AMPS in the fly, which is abundant during development but is replaced in the emerged adult by hyaluronic acid and chondroitin.
I. (1952) The aerobic and anaerobic utilization of
metabolic
energy during insect metamorphosis.
Churchill,
London. M. and BIRTL. (1967) Changes in the amounts of carbohydrates, phosphagen, and related compounds during the metamorphosis of the blowfly Lucilia cuprina.
CROMFTON
J. Insect Physiol. 13. 1575-1592.
DE PRIESTERW. (1972) Ultrastructural changes in developing Midgut epithelium of Calliphora erythrocephala Meigen. Z. Zellforsch. Mikrosk. Anat. 129. 278-289. kIYLE J. (1967) The ‘Lorenzan sulphates’. A new group of vertebrate mucopolysaccharides. Biochem. J. 103. 325-330.
ESTESZ. and FAUSTR. (1964) Studies on the mucopolysaccharides of the greater wax moth, Galleria mellonella (Linnaeus). Comp. Biochem. Physiol. 13. 443452. GOTTSCHALKA. (1972) Definition of glycoproteins and their delineation from other carbohydrate-protein complexes. In Glycoproteins (Ed. by GOTTSCHALK A.), BBA Library, 5. 2430. Elsevier. Amsterdam. FRANC+BROWDERS.. DE RYDTJ.. and DORFMANA. (1963) The identification of a sulphated mucopolysaccharide in chick embryos stages 1 l-23. Proc. nat. Acad. Sci. U.S.A. 49. 643-647.
HATAR. and NAGAIY. (1972) A rapid and micro method for separation of acidic glycosaminoglycans by twodimensional electrophoresis. Anal. Biochem. 45. 462-468. H&LUND L. (I 976) The comparative biochemistry of invertebrate mucopolysaccharides. V. Insecta (Calliphora erythrocephala). Comp. Biochem. Physiol. 53(B), %14.
IMMERS J. (1961) Incornoration of 35SOa- in the sea urchin egg and‘larvae. Ark: Zool. 13. 561-564. KOSHERR. and SEARLSR. (1973) Sulfated mucopolysaccharide synthesis during the development of Rana pipiens. Develop. Biol. 32. 5&68.
KVISTT. N. and FINNEGANC. V. (1970) The distribution of glycosaminoglycans in the axial region of the developing chick embryo II. Biochemical analysis. J. exp. Zool. 175. 241-258. LAGUNOFFD. and WARRENG. (1962) Determination of 2-deoxy-2-sulfoaminohexose content of mucopolysaccharides. Arch. Biochem. Biophys. 99. 396400. LQVTRUPS. (1974) Epigenrtics-A treatise on theoretical biology. Wiley, London. LUDWIG D.. CROWEP. A., and HA~~EMYRM. M. (1965) Energy sources during embryogenesis of the yellow mealworm, Tenehrio moflitor. Ann. ent. Sot. Am. 58. 543-546.
MEIERS. and HAY E. (1973)Synthesis of sulphated glycosa-
minoglycans by embryonic cornea1 epithelium. Develop. Biol. 35. 318-331. NISIZAWAK., YAMAGUCHI T., HAUDA N., MAEDAM.. and YAMAZAKIH. (1963) Chemical nature of a uranic acidcontaining polysaccharide in the peritrophic membrane of the silkworm. Biochem. J. 54. 419-426. PEREZC. (1910) Recherches histologiques sur la metamorphose des muscides Calliphora erythrocephala. Meig. Arch. Zool. exp. gin. (5) 4. l-274.
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charides (Ed. by QUINTARELLI G.),pp. 9 l-94.
Acta
physiol. Stand. 28. 30&335. AGRELL I.(1964) Physiological
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