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Developmental changes in cuticular proteins of Ascaris suum
Comp. Biochem. Physiol. Vol. 90B, No. 2, pp. 321-327, 1988
0305-0491/88 $3.00+ 0.00 © 1988 Pergamon Press pie
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DEVELOPMENTAL CHANGES IN CUTICULAR PROTEINS OF ASCARIS S U U M R. H. FETTERER* and J. F. URBAN,JR Helminthic Diseases Laboratory, Animal Parasitology Institute, Agricultural Research Service, USDA, BARC-East Beltsville, MD 20705, USA (Received I0 August 1987)
Abstract--l. Cuticles were isolated from developmental stages of the swine nematode Ascaris suum by a combination of mechanical disruption and detergent treatment of larvae or by surgical removal of cuticle from adults. Proteins from the isolated cuticles were solubilized with 2-mercaptoethanol (2ME) and analyzed by SDS-PAGE. 2.2ME soluble, cuticular proteins from adults consisted of 5 to 6 bands with 80% of proteins in 2 bands with tool. wts of 106,000 and 93,000. Cuticular proteins from the third and fourth larval stages (L3 and L4) were comparable to adult, but differences in the number of bands were observed. The soluble proteins from the adult, L3 and L4 were readily degraded by a bacterial collagenase suggesting that these proteins are collagen-like structural elements of the cuticle. 3. The soluble proteins from the second stage (L2) differed from the adult and other larval stages in both the number and tool. wt of protein bands and their lack of degradation by bacterial collagenase. Amino acid composition of soluble cuticular proteins were similar for adult and L4, but glycine and proline were present in lower amounts in the L2. 4. These results support a hypothesis that there are stage specific differences in cuticular proteins from A. suum and that the greatest differences appear to exist between L2 and other stages.
INTRODUCTION The cuticle, the thin, flexible outer covering of nematodes is composed primarily of protein with trace amounts of lipid and carbohydrate. The cuticle varies widely in morphology among both nematode species and the developmental stage within a species but, in general, the cuticle can be considered to consist of an outer epicuticle, a cortical zone, a medial zone and an inner basal layer (Bird and Deutsch, 1957; Bird, 1971, 1980). Biochemical studies of composition of the cuticle have centered on the adult stage of the free-living nematode ( Caenorhabditis elegans (Cox et al., 1981a; Ouanza and Herbage, 1981) and the swine parasitic nematode Ascaris suum (Winkfein et al., 1985; Fujimoto and Kanaya, 1973). These studies indicate that the nematode cuticle consists of two general protein classes: (I) Collagen-like proteins that are extracted from the cuticle with reducing agents such as 2-mereaptoethanol (2ME), and (2) proteins that are insoluble in reducing agents. Electrophoretic characterization of the 2ME soluble cuticular proteins from adults have demonstrated a variety of tool. wt species. In the cuticle of both C. elegans (Cox et al., 1981a) and the plant parasitic nematode, Meloidogyne incognita (Reddigari et al., 1986), 9 or 10 proteins of various tool. wt species were resolved. In another free-living nematode, Panagrellus sillusiae (Leushner et al., 1979), 18 or more proteins were observed. The cuticle of A. suum, however, appears to contain only 5 or 6 proteins (Winkfein et al., 1985). Developmental changes in the composition of the cuticle observed in C. elegans suggest stage specific synthesis of cuticular proteins (Cox et al., 1981b). *To whom all correspondence should be addressed.
This concept was extended to plant parasitic nematodes because collagen-like cuticular proteins differed between the adult female and second stage larvae of M. incognita (Reddigari et al., 1986). In contrast, there have been no reports of developmental changes in the biochemistry of the cuticle in an animal parasitic nematode. An objective of the present study was to obtain data on the developmental biochemistry of the cuticle of the swine roundworm A. suum. Distinct developmental stages o f A. suum were obtained from in vivo sources as well as from recently developed in vitro culture systems, and cuticular proteins from different developmental stages were characterized using polyacrylamide gel electrophoresis and other biochemical methods. Comparison of these data with that of studies of cuticles from other free-living nematodes may give a more complete understanding of the role of the cuticle in adapting the parasitic nematode to the unique environment in which it lives. MATERIALS AND
METHODS
Parasites
Adults of Ascaris suum were obtained from a commercial abattoir, maintained in the laboratory (Fleming and Fetterer, 1984) and used for experiments within 48 hr after removal from swine. Developmental stages of A. suum were obtained using several in vivo and in vitro methods that have been previously reported and are briefly described below. Second stage larvae (L2) were obtained by mechanical hatching of embryonated eggs (Urban et al., 1981). The isolated L2 were placed in a defined culture medium (Dulbecco's Modified Eagles Medium; DMEM) (Urban and Tromba, 1982; Rew et al., 1986) and after 14 days in culture (DIC) 60-70% of the larvae had developed to the third stage (L3). The larvae from this culture system are referred
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R. H. FETTERERand J. F. URBAN,JR
to as L2/3. Larvae in mid- to late-third stage (L3) (Douvres et al., 1969) were obtained from lungs of pigs 7 days after inoculation with infective eggs. The L3 recovered from lungs were placed in culture medium DMEM supplemented with 10% fetal bovine serum and after 7 DIC greater than 90°/. of the larvae had developed to the fourth stage (L4) (Urban and Douvres, 1981). This culture system was also used to harvest third stage cuticles that were released into the culture media during the ecdysis of L4. Young adults (YA) of A. suum were obtained by incubating the L3 obtained from pig lung in a roller bottle culture system for 28 days (Douvres and Urban, 1986). This culture system was also used to collect the fourth stage cuticles that were released during the ecdysis of YA. Isolation o f cuticles
Adult female A. suum were dissected along the lateral line and the intestine and reproductive tract removed. The muscle and hypodermis were then removed from the cuticle by scraping with a scalpel blade (Fetterer and Wasiuta, 1987). Cuticle was isolated from intact A. suum larvae by treatment with detergents (Cox et al., 1981a; Fetterer and Urban, 1985). Briefly, larvae were broken by gently grinding with a glass homogenizer (L4) or by stirring 3-6 hr with 5 mm diameter glass beads (L2 and L2/3). Larval pieces were suspended in ST buffer (2% SDS, 100mM Tris, pH 6.8) and heated 5 min at 100°C. After cooling to room temperature the suspension was stirred for 2-3 hr. The suspension was centrifuged (10 min, 2000g) and the pellet resuspended in ST buffer and reheated as above. After centrifugation the pellet was examined microscopically and if tissue was observed to adhere to the cuticles, the pellet was re-extracted with ST buffer. Third stage cuticles released during the ecdysis of L4 in vitro were concentrated by centrifugation of culture media (10min, 2000g) and then separated from the larvae by centrifugation (20 min, 2000 g) on an aqueous Percoll (30%; Sigma St Louis, MO) solution. The fourth stage cuticles released during the ecdysis of YA in vitro were large enough to be picked from culture media by aspiration with a small diameter pipette. After isolation, all cuticles were washed 3 times with water and frozen at 20°C. -
Isolation o f cuticular proteins
Cuticles were suspended in ST buffer containing 5% 2-mercaptoethanol (2ME), heated at 100°C for 5min, cooled to room temperature and stirred overnight at 4°C. The preparation was then reheated, cooled and centrifuged. The supernatant containing solubilized cuticular proteins was dialyzed overnight against 100 mM Tris containing 1% 2ME (pH 8.0). The resulting pellet containing the insoluble cuticular proteins was washed 5 times with water, dried under vacuum and frozen at -20°C until analyzed for amino acid content. Electrophoresis
Sodium dodecylsulfate polyacrylamide (SDS-PAGE) gel (6-15%) linear gradients were prepared and run with only minor modifications according to the methods of Laemmli and Favre (1973). Proteins were visualized by staining overnight with 0.125% Coomassie Brilliant Blue R-250 in 35% methanol, 10% acetic acid and then destained by diffusion in 35% methanol, 10% acetic acid. The susceptibility of isolated cuticles to enzymatic degradation were tested by suspending cuticles in appropriate buffer containing enzymes (1000 cuticles/ml) followed by incubation at 37°C for periods of 6-18 hr. The enzymes and incubation conditions were as follows: collagenase (Sigma, Type VIII, 0.4 mg/ml) in 50 mM Tris, 150 mM NaC1, 5 mM CaCI at pH 7.4; protease (Sigma Type IV 1.5 mg/ml) in 100 mM Tris, 5 mM CaC1, pH 7.5; elastase (Sigma Type IV 0.8 mg/ml) in 200 mM Tris, pH 8.8; lipase (Sigma Type VII,
1.7 mg/ml) in 50 mM Tris, pH 7.5. Degradation of cuticles was verified using the light microscope. The sensitivity of the 2ME soluble cuticular proteins to collagenase digestion was evaluated by SDS-PAGE. Samples of 2ME soluble proteins were precipitated from solution by addition of cold acetone (10:1 v/v), dried under vacuum and resuspended in 50raM Tris, 150mM NaCI, 5 mM CaC1, pH 7.4 with or without 10 units of bacterial collagenase (Sigma, Type VIII). The samples were incubated for 4 or 18 hr at 37°C and digestions terminated by heating at 100°C for 5 rain in electrophoresis sample buffer. Samples were analyzed on SDS-PAGE gels as described above. Amino acid and carbohydrate analysis
Soluble cuticular proteins were precipitated from solution with cold acetone (10:1 v/v) and dried under vacuum. Samples of 2ME insoluble cuticular proteins were washed 5 times with water, twice with cold acetone and dried under vacuum. All samples were hydrolyzed for 20hr at 115°C. One crystal of phenol was added before hydrolysis and samples were analyzed on a Beckman Amino Acid Analyzer. Following overnight hydrolysis of cuticles at 100°C in 2N HCI, amino sugars were assayed by method of Gatt and Berman (1966) and neutral sugars were measured by the method of Trevelyan and Harrison (1952). Unless otherwise noted all chemicals were of reagent grade and were obtained from local sources. Protein concentrations were determined using the method of Bradford (1976).
RESULTS Treatment of broken larvae with detergent produces segments of the cuticle that, upon examination by the light microscope, appear free of adhering tissue (Fig. 1). Analysis by S D S - P A G E of soluble larval proteins released into solution by detergent treatment reveal a complex protein mixture with mol. wts from 200,000 to less than 30,000 (Fig. 2). In contrast, solubilization of proteins from isolated cuticle of various developmental stages by treatment with 2ME yields a relatively small number of protein bands on S D S - P A G E that have a similar mol. wt range (Figs 2 and 3). Proteins from adult cuticle separated into 5-6 protein bands on S D S - P A G E with the greatest amount of protein localized in 2 bands with mol. wts of 106,000 and 93,000. Lesser amounts o f protein were in 2 bands with mol. wts near 67,000, and a single band with mol. wt of 35,000. A minor band with tool. wt of 45,000 was also observed. The 2 M E soluble cuticular proteins from adult male and female A . suum appeared to be identical (not shown). Cuticular proteins isolated from L4 and L3 and third stage cuticle released during ecdysis in vitro were similar to proteins from adult cuticle (Fig. 3). Prominent protein bands with mol. wts from 90,000 to 106,000 were observed for cuticular proteins from these stages. Other minor bands were present at 67,000, 45,000 and 35,000. The bands observed at 67,000 were generally more prominent in proteins from the third stage cuticle released during ecdysis than from L4, L3 or adult. Cuticular proteins from L2/3 demonstrated prominent bands at 92,000, 106,000, 67,000 and 35,000 but unlike the other stages examined prominent bands were also observed at tool. wts between 200,000-116,000; a large number of minor bands were also present throughout the tool. wt range examined. Cuticular proteins solubilized from L2 cuticle contained 3 to 4 prominent bands
Developmental changes in Ascaris cuticle proteins
Fig. I. Photomicrograph of fragments of cuticle isolated from Ascaris suum L4 by mechanical disruption and detergent treatment as described in Materials and Methods. Horizontal bar indicates 500/am.
A
B
C
D
E
STD
---200KD
116KD 92KD ~
67KD
.......
45KD
31KD Fig. 2. SDS-PAGE gels (5-15%) of detergent soluble proteins released from Ascaris suum I A (lane C), L3, (lane D) and L2 (lane E) following the mechanical disruption and SDS treatment. 2ME soluble proteins from adult cuticle lane A, and an example of a mammalian collagen preparation (calf skin) is shown in lane B.
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R. H. FETTERERand J. F. URBAN,JR
324
2
3
4
5
~200KD
2 0 0 K D ....
~116KD 92KD 116KD''" ~ 6 7 K D
92KD~ 45KD
67KD
,,
45KD
• 31KD
31KD~
"O <
fq ..a
co
O)
Fig. 3. SDS-PAGE (5-15%) analysis of 2ME soluble cuticular proteins from adult and larvae stages of 25-30 gg of larval proteins were applied to each lane, while 50 #g of adult proteins were used to reveal the presence of minor bands in the preparation.
Ascaris suum:
with mol. wts between 116,000 and 200,000 and with at least one prominent band greater than 200,000. A major band with mol. wt of 72,000 was also observed along with a number of minor bands at the lower mol. wt which were often poorly resolved. The soluble cuticular proteins from adult, IA and L3 were degraded by a highly purified bacterial collagenase (Fig. 4). Non-collagenous protein standards are relatively resistant to collagen digestion, while a calfskin collagen is readily degraded by
bacterial collagenase. In contrast, 2ME soluble cuticular proteins from L2 appeared to be relatively resistant to digestion by bacterial collagenase, even after incubation for more than 12 hr. The amino acid, neutral and amino sugar content of 2ME soluble and 2ME insoluble cuticular proteins were determined (Table 1). The amino acid content of the 2ME soluble cuticular proteins from all stages examined had relatively high percentages of glycine, proline and hydroxyproline, characteristic of
Fig. 4. The effect of bacterial collagenase on 2ME soluble cuticular proteins from adult and larval stages of Ascaris suum. Proteins were analyzed on 5-15% SDS-PAGE after incubation with (+) or without ( - ) 10 units of collagenase. Non-collagenous proteins (std) served as negative controls and calf skin collagen (CSC) was used as a positive control. C-indicates position of collagenase.
D e v e l o p m e n t a l c h a n g e s in A s c a r i s cuticle p r o t e i n s
325
Table 1. The amino acid composition of 2-mereaptoethanol soluble and insoluble cuticular proteins from adult and larvae of Ascaris suum
Amino acid
Soluble cutieular proteins Stage L4 L2/3
Adult
G.lycine Small* Basict Aeidie~ Proline Hydroxyproline Amino sugar Neutral sugar
271 346 105 143 251 20 0.44% 0.5%
Glycine Small* Basict Acidic:~ Proline Hydroxyproline Amino sugar Neutral sugar
182 350 45 134 292 ND ND ND
239 289 52 141 191 17 1.04% 4.6%
181 256 111 186 119 25 1.96% 2.9%
Insoluble cuticular proteins 183 342 364 486 21 42 321 141 169 137 ND ND 0,09% 0.6% 2,25% 1.2%
L2 212 302 106 232 141 29 -
306 416 58 140 123 14 0.99% 0.06%
Values for amino acids are residues per thousand. Sugars are given as % dry wt, ND, not detected. - Assay not performed. *Glycine + alanine tLysine + histidine + arginine :~Glutamic + aspartic
collagen-like proteins. The largest amount of glycine was measured in the adult (27%) and L4 (24%) with lesser amounts in L2/3 (18%) and L2 (21%). Proline residues were most numerous in adult cuticular proteins (25%) compared to L4 (19.1%), L2/3 (12%) and L2 (14%). Hydroxyproline was higher in L2 (2.9%) and L2/3 (2.5%) than adult (2.0) or L4 (1.7). Both neutral and amino sugars of 2ME soluble proteins were lowest in adult and highest in both of the larval stages examined (L4 and L2/3). The amino acid analysis of the 2ME insoluble cuticular proteins indicated that adult and L4 were lower in glycine content, contained similar amounts of proline and hydroxyproline compared to 2ME soluble proteins from these 2 stages. The 2ME insoluble cuticular proteins from L2/3 and L2 had greater glycine content than insoluble cuticular proteins from adult and L4, but had lower proline content. Hydroxyproline, although absent in 2ME insoluble cuticular proteins from L2/3, was present in L2 (1.4%).
Table 2. The effect of various enzymes and 2-mercaptoethanol on structure of cuticles isolated from adult and larvae stages of Ascaris suum
Stage
COL
Adult Fourth cuticle* L4 Third cuticlet L2
+++ +++ +++ + + +
ELAST + + + -
Treatment LIP CHIT -
-
PRO
2ME
+++ +++ +++ + + +
+++ +++ +++ + + +
COL, collagenase; ELAST, elastase; LIP, lipase; CHIT, chitinase; PRO, bacterial protease; 2ME, 2-mercaptoethanol. Enzyme concentrations and incubation conditions given in Materials and Methods. Alteration of microscopically observed cuticular structure: + + + , large; + + , moderate; + , slight; - , negligible. *Fourth stage cuticle released during eedysis of young adult. tThird stage cuticle released during ecdysis of L4.
The susceptibility of isolated fragments of cuticles from L4 and from 4th stage cuticle released/n vitro during ecdysis of YA to collagenase, bacterial protease and 2ME was greater than fragments of third stage cuticle released in vitro during ecdysis of L4 and from L2 (Table 2). The most prominent effect of both coUagenase and 2ME appeared to be on the basal region of the cuticle while the protease caused a general loss of integrity in all layers of the cuticle. Of the other enzymes tested only elastase had a relatively weak effect on cuticular structure of fragments from adult, fourth stage cuticle released during ecdysis of YA and L4; lipase and chitinase were without significant effect. The amount of cuticular protein solubilized by 2ME varied considerably between stages. Cuticles from adult stage had 80% soluble protein while L4, L3 and L2 had 70, 50 and 20%, respectively. DISCUSSION
The present data demonstrate that mechanical disruption combined with detergent solubilization is an effective method for the isolation of cuticle from larval stages of the animal parasitic nematode, A s earls suum. Three lines of evidence suggest that the above method is suitable for preparation of isolated cuticle: (1) Ascaris suum cuticle prepared by this method appeared to be free of adhering tissue when observed by light microscope; (2) the large amount of protein released by detergent solubilization had a wide range of mol. wts while cuticular proteins solubilized by 2ME consisted of relatively few protein bands; (3) solubilized cuticular proteins derived either from dissected adults or from cuticle released by larvae during ecdysis in vitro are qualitatively similar to cuticular proteins from mechanically disrupted and detergent treated preparations. Although detergent treatment may be suitable for isolation of
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structural proteins from the cuticle, many other important proteins, particularly those from the surface of the cuticle, may be removed by this treatment. The results of the current study indicate that the A. suum cuticular proteins have properties similar to cuticular proteins from other nematode species. Cuticles from A. suum larvae and adults consist of both 2ME soluble and 2ME insoluble proteins like cuticles from C. elegans and M . incognita (Cox et al., 1981a; Ouanza and Herbage, 1981; Reddigari et al., 1986). In addition, the 2ME soluble proteins from A. suum are collagen-like because they are sensitive to collagenase, and their amino acid composition is rich in glycine and proline. Although the 2ME soluble cuticular proteins from A. suum L2 were not degraded by a bacterial collagenase it should be noted that this is also a property of collagen from other invertebrate sources (Kimura and Tanzer, 1977). The cuticle of A. suum differs from some other nematode cuticle in at least two ways: (1) The ratio of hydroxyproline to proline (H/P) observed in the 2ME soluble cuticular proteins of A. suum was relatively high (1:13, adult; 1: 5, larvae) compared to C. elegans (1:1), M . incognita (1:1, adult; 1 : 3 larvae) or mammalian collagen (1:1) (Cox et al., 1981a, b; Reddigari et al., 1986; Miller and Undenfriend, 1970). It is possible that the relatively low amount of hydroxylation of proline observed in A. suum is the result of the low oxygen tensions found in the intestinal environment. However, the free-living nematode Panagrellus sillusiae has a H/P ratio (1:7) similar to that of A. suum (Leushner et al., 1979) suggesting that there is not a clear dichotomy in H/P ratio between A. suum and other nematode species. (2) The metachromatic staining by Coomassie Brilliant Blue of the collagen-like cuticular proteins separated by SDSPAGE observed by other authors (Cox et al., 1981a; Winkfein et aL, 1985) was only occasionally noted in the present investigations. Previous studies of cuticular structure have demonstrated stage specific changes in morphology of the nematode cuticle during development from larvae to adult (Samoiloff and Pasternak, 1986; Bird, 1971; Cox et al., 1981b). In A. suum there is a particularly marked increase in the thickness of the medial and basal layers of the cuticle as the parasite grows to the adult stage. This increase in the medial layer may represent an adaptation to a parasitic environment (Bird, 1971; Watson, 1965). Two observations from the current studies are consistent with the hypothesis that there is a progressive increase in the amount of collagen-like, 2ME soluble proteins in the basal and medial layers of the cuticle of A. suum L2 developing to adult: (1) treatment with 2ME and bacterial collagenase was most disruptive of the structure of the basal and medial layers of the cuticle of the adult, young adult and fourth stage larvae, and (2) the amount of 2ME soluble cuticular protein markedly increased from the L2 through the adult stage. This increase may represent an adaptation to a changing parasitic environment, but this phenomenon is not unique to parasitic worms. Adults of the free-living nematode C. elegans also have larger amounts of 2ME soluble cuticular proteins compared to their larvae (Cox et al., 1981a). Studies of C. elegans cuticular proteins and their
genes have led to the hypothesis that nematodes have genes that are developmentally regulated resulting in stage specific cuticular proteins (Cox et al., 1981b; Kramer et al., 1982, 1985). A parasitic plant nematode, M . incognita, has recently been shown to have cuticular proteins that differ between the adult and the second stage juvenile (Reddigari et al., 1986). This developmental change in cuticular proteins of the animal parasitic nematode A. suum are in general agreement with the above hypothesis. The 2ME soluble cuticular proteins from L2 were clearly different from other stages not only in pattern and numbers of protein bands on SDS-PAGE, but in the lack of sensitivity to digestion by a bacterial collagenase. The L2 differs biologically from other parasitic stages of A. suum examined because it is found naturally within the egg shell in the environment. It is not clear, however, if the differences in cuticular proteins observed are related to differences in the environment in which the stages are found. The cuticular proteins from L2/3 also appear to be different from adult, L4 and L3. The interpretation of these results are complicated by the fact that there are mixtures of stages derived from this culture system and second stage cuticles released during ecdysis of L3 are also present. Cuticular proteins from adult, L3 and L4 were readily degraded by collagenase, and had a similar protein banding pattern by SDS-PAGE analysis. However, differences in the number of protein bands in each stage were noted, suggesting that A. suum cuticular proteins differ between stages. Analysis of amino acid composition of cuticles from different stages also support this concept. The amino acid composition of 2ME soluble cuticular proteins from the adult was similar to that reported by Winkfein et al. (1985). Nearly one-third of the residues were glycine and there was a relatively high proportion of proline. This composition is characteristic of collagen-like proteins. The soluble cuticular proteins from L4 were similar to adult by SDS-PAGE analysis but differed slightly in amino acid composition. The L4 had less glycine and slightly less proline than adults. Amino acid composition of L2 and L2/3 were most different from adult. They had about 21% glycine compared to 27% for adults and had markedly lower amounts of proline than adults. Stage differences in amino acid composition of the 2ME insoluble cuticular proteins were also observed. The 2ME insoluble cuticular proteins of IA were similar in composition to adult, but those from L2/3 and L2 were considerably different, especially in the greater amount of glycine found in those early larval stages. The amounts of both amino and neutral sugars were generally greater in larval stages than adult. In summary, the results of the present study show that there are stage differences in cuticular proteins of developing A. suum, and that the differences between adult and L2 are most prominent. There is also a quantitative increase in the amount of 2ME soluble proteins in the cuticle during development. However, it is not possible to determine from the current study if there are multiple stage specific genes responsible for the various cuticular proteins as has been suggested for C. elegans. Since recent experiments indi-
Developmental changes in Ascaris cuticle proteins cate that C. elegans collagen genes may be useful probes for identification o f genes o f cuticular protein in A. suum (Zarlenga and Fetterer, unpublished observation), it may be possible in future studies to more precisely determine the mechanisms o f developmental regulation o f A. suum cuticular proteins. Acknowledgements--We are indebted to J. Blythe for expert technical assistance. We thank Dr R. S. Rew and Dr P. C. Allen for critical reading of this manuscript. Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee of warranty by U.S. Department of Agriculture and does not imply its approval to exclusion of other products that may be suitable.
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Bird A. F. (1971) The Structure o f Nematodes. Academic Press, New York. Bird A. F. (1980) The nematode cuticle and its surface. In Nematodes as Biological Models (Edited by Zuckerman B. M.), pp. 213-235. Academic Press, New York. Bird A. F. and Deutsch K. (1957) The structure of the cuticle of Ascaris lumbricoides var. suis. Parasitology 47, 319-328. Bradford M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Bioehem. 50, 573-585. Cox G. N., Kusch M. and Edgar R. S. (1981a) Cuticle of Caenorhabditis elegans: Isolation and partial characterization. J. Cell BioL 90, 7-17. Cox G. N., Straprans S. and Edgar R. S. (1981b) The cuticle of Caenorhabditis elegans--II. Stage specific changes in ultrastructure and protein composition during postembryonic development. Devl BioL 86, 456-470. Douvres F. W., Malakatis G. M. and Tromba F. G. (1969) Morphogenesis and migration of Ascaris suum larvae developing to fourth stage in swine. J. Parasit. 55, 689-712. Douvres F. W. and Urban J. F. Jr (1986) Development of Ascaris suum from /n vivo-derived third stage larvae to egg-laying adults in vitro. Proc. Helm. Soc. Wash. 53, 256-262. Fetterer R. H. and Wasiuta M. (1987) Ascaris suum: Partial isolation and characterization of hypodermis from the adult female. Exp. Parasit. 63, 312-318. Fetterer R. H. and Urban J. F. Jr (1985) The inhibition of growth and development of Ascaris suum larvae in vitro by proline analogs. Comp. Biochem. PhysioL $2C, 331-335. Fleming M. W. and Fetterer R. H. (1984) Ascaris suum: Continuous perfusion of pseudocoelom and nutrient absorption. Exp. Parasit. 576, 142-148. Fujimoto D. and Kanaya S. (1973) Cuticilin: A noncollagen structural protein from Ascaris cuticle. Archs Biochem. Biophys. 157, 1-6.
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Gatt R. and Berman E, R. (1966) A rapid method for estimation of amino sugars on a micro scale. Analyt, Bioehem. 15, 167-171. Kimura S. and Tanzer M. L. (1977) Nereis cuticle collagen--isolation and properties of a large fragment resistant to proteolysis by bacterial collagenase. J. biol. Chem. 252, 8018-8022. Kramer J. M., Cox G. N. and Hirsch D. (1982) Comparison of the complete sequence of two collagen genes from Caenorhabditis elegans. Cell 30, 599-606. Kramer J. M., Cox G. N. and Hirsch D. (1985) Expression of Caenorhabditis elegans collagen genes col-I and col-2 is developmentally regulated. J. biol. Chem. 260, 1945-1951. Laemmli U. K. and Favre M. (1973) Maturation of the head of bacteriophage T4--I. DNA packaging events, J. Molec. Biol. 80, 575-599. Leushner J. R., Semple N. E. and Pasternak J. P. (1979) Isolation and characterization of cuticle from the freeliving nematode Panagrellus sillusiae. Archs Biochem. Biophys. 139, 104-113. Miller R. L. and Undenfriend S. (1970) Hydroxylation of proline residues in collagen nascent chains. Archs Biochem. Biophys. 1939, 104-113. Ouanza R. and Herbage D. (1981) Biochemical characterization of cuticle collagen of the nematode (Caenorhabditis elegans). Biochim. biophys. Acta 669, 236-243. Reddigari S. R., Jansma P. L., Premachandran D. and Hussey R. S. (1986) Cuticular collagenous proteins of second-stage juveniles and adult females of Meloidogyne incognita--Isolation and partial characterization. J. Nemat. 18, 294-302. Pew R. S., Urban J. F., Jr and Douvres F. W. (1986) Screen for anthelmintics using larvae of Ascaris suum. Am. J. Vet. Res. 47, 869-873. Samoiloff M. R. and Pasternak J. (1986) Nematode morphogenesis--Fine structure of the cuticle of each stage of the nematode Panagrellas sillusiae. Can. J. ZooL 46, 1019-1022. Trevelyan W. E. and Harrison J. S. (1952) Studies on yeast metabolism--I. Bioehem. J. 50, 298-302. Urban J. F., Jr and Douvres F. W. 0981) In vitro development of dscaris suum from third to fourth stage larvae and detection of metabolic antigens in a multiwell culture system. J. Parasit. 67, 800-806. Urban J. F., Jr and Tromba F. G. (1982) Development of immune responsiveness to Ascaris suum antigens in pigs vaccinated with ultraviolet attenuated eggs. Vet. Immun. Immunopath. 3, 339-402. Urban J. F., Jr, Douvres F. W. and Tromba F, G. (1981) A rapid method for hatching of Ascaris eggs in vitro. Proc. Helm. Soc. Wash. 48, 241-243. Watson B. D. (1965) The fine structure of the body wall and growth of the cuticle in the adult nematode Ascaris lumbricoides: Quart. J. Microscop. Soc. 106, 89-91. Winkfein R. J., Pasternak J. P., Mudry T. and Martin L. H. (1985) Ascaris lumbricoides--Charaeterization of collagenous compounds of adult cuticle. Exp. Parasit. 59, 197-203.
0305-0491/88 $3.00+ 0.00 © 1988 Pergamon Press pie
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DEVELOPMENTAL CHANGES IN CUTICULAR PROTEINS OF ASCARIS S U U M R. H. FETTERER* and J. F. URBAN,JR Helminthic Diseases Laboratory, Animal Parasitology Institute, Agricultural Research Service, USDA, BARC-East Beltsville, MD 20705, USA (Received I0 August 1987)
Abstract--l. Cuticles were isolated from developmental stages of the swine nematode Ascaris suum by a combination of mechanical disruption and detergent treatment of larvae or by surgical removal of cuticle from adults. Proteins from the isolated cuticles were solubilized with 2-mercaptoethanol (2ME) and analyzed by SDS-PAGE. 2.2ME soluble, cuticular proteins from adults consisted of 5 to 6 bands with 80% of proteins in 2 bands with tool. wts of 106,000 and 93,000. Cuticular proteins from the third and fourth larval stages (L3 and L4) were comparable to adult, but differences in the number of bands were observed. The soluble proteins from the adult, L3 and L4 were readily degraded by a bacterial collagenase suggesting that these proteins are collagen-like structural elements of the cuticle. 3. The soluble proteins from the second stage (L2) differed from the adult and other larval stages in both the number and tool. wt of protein bands and their lack of degradation by bacterial collagenase. Amino acid composition of soluble cuticular proteins were similar for adult and L4, but glycine and proline were present in lower amounts in the L2. 4. These results support a hypothesis that there are stage specific differences in cuticular proteins from A. suum and that the greatest differences appear to exist between L2 and other stages.
INTRODUCTION The cuticle, the thin, flexible outer covering of nematodes is composed primarily of protein with trace amounts of lipid and carbohydrate. The cuticle varies widely in morphology among both nematode species and the developmental stage within a species but, in general, the cuticle can be considered to consist of an outer epicuticle, a cortical zone, a medial zone and an inner basal layer (Bird and Deutsch, 1957; Bird, 1971, 1980). Biochemical studies of composition of the cuticle have centered on the adult stage of the free-living nematode ( Caenorhabditis elegans (Cox et al., 1981a; Ouanza and Herbage, 1981) and the swine parasitic nematode Ascaris suum (Winkfein et al., 1985; Fujimoto and Kanaya, 1973). These studies indicate that the nematode cuticle consists of two general protein classes: (I) Collagen-like proteins that are extracted from the cuticle with reducing agents such as 2-mereaptoethanol (2ME), and (2) proteins that are insoluble in reducing agents. Electrophoretic characterization of the 2ME soluble cuticular proteins from adults have demonstrated a variety of tool. wt species. In the cuticle of both C. elegans (Cox et al., 1981a) and the plant parasitic nematode, Meloidogyne incognita (Reddigari et al., 1986), 9 or 10 proteins of various tool. wt species were resolved. In another free-living nematode, Panagrellus sillusiae (Leushner et al., 1979), 18 or more proteins were observed. The cuticle of A. suum, however, appears to contain only 5 or 6 proteins (Winkfein et al., 1985). Developmental changes in the composition of the cuticle observed in C. elegans suggest stage specific synthesis of cuticular proteins (Cox et al., 1981b). *To whom all correspondence should be addressed.
This concept was extended to plant parasitic nematodes because collagen-like cuticular proteins differed between the adult female and second stage larvae of M. incognita (Reddigari et al., 1986). In contrast, there have been no reports of developmental changes in the biochemistry of the cuticle in an animal parasitic nematode. An objective of the present study was to obtain data on the developmental biochemistry of the cuticle of the swine roundworm A. suum. Distinct developmental stages o f A. suum were obtained from in vivo sources as well as from recently developed in vitro culture systems, and cuticular proteins from different developmental stages were characterized using polyacrylamide gel electrophoresis and other biochemical methods. Comparison of these data with that of studies of cuticles from other free-living nematodes may give a more complete understanding of the role of the cuticle in adapting the parasitic nematode to the unique environment in which it lives. MATERIALS AND
METHODS
Parasites
Adults of Ascaris suum were obtained from a commercial abattoir, maintained in the laboratory (Fleming and Fetterer, 1984) and used for experiments within 48 hr after removal from swine. Developmental stages of A. suum were obtained using several in vivo and in vitro methods that have been previously reported and are briefly described below. Second stage larvae (L2) were obtained by mechanical hatching of embryonated eggs (Urban et al., 1981). The isolated L2 were placed in a defined culture medium (Dulbecco's Modified Eagles Medium; DMEM) (Urban and Tromba, 1982; Rew et al., 1986) and after 14 days in culture (DIC) 60-70% of the larvae had developed to the third stage (L3). The larvae from this culture system are referred
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R. H. FETTERERand J. F. URBAN,JR
to as L2/3. Larvae in mid- to late-third stage (L3) (Douvres et al., 1969) were obtained from lungs of pigs 7 days after inoculation with infective eggs. The L3 recovered from lungs were placed in culture medium DMEM supplemented with 10% fetal bovine serum and after 7 DIC greater than 90°/. of the larvae had developed to the fourth stage (L4) (Urban and Douvres, 1981). This culture system was also used to harvest third stage cuticles that were released into the culture media during the ecdysis of L4. Young adults (YA) of A. suum were obtained by incubating the L3 obtained from pig lung in a roller bottle culture system for 28 days (Douvres and Urban, 1986). This culture system was also used to collect the fourth stage cuticles that were released during the ecdysis of YA. Isolation o f cuticles
Adult female A. suum were dissected along the lateral line and the intestine and reproductive tract removed. The muscle and hypodermis were then removed from the cuticle by scraping with a scalpel blade (Fetterer and Wasiuta, 1987). Cuticle was isolated from intact A. suum larvae by treatment with detergents (Cox et al., 1981a; Fetterer and Urban, 1985). Briefly, larvae were broken by gently grinding with a glass homogenizer (L4) or by stirring 3-6 hr with 5 mm diameter glass beads (L2 and L2/3). Larval pieces were suspended in ST buffer (2% SDS, 100mM Tris, pH 6.8) and heated 5 min at 100°C. After cooling to room temperature the suspension was stirred for 2-3 hr. The suspension was centrifuged (10 min, 2000g) and the pellet resuspended in ST buffer and reheated as above. After centrifugation the pellet was examined microscopically and if tissue was observed to adhere to the cuticles, the pellet was re-extracted with ST buffer. Third stage cuticles released during the ecdysis of L4 in vitro were concentrated by centrifugation of culture media (10min, 2000g) and then separated from the larvae by centrifugation (20 min, 2000 g) on an aqueous Percoll (30%; Sigma St Louis, MO) solution. The fourth stage cuticles released during the ecdysis of YA in vitro were large enough to be picked from culture media by aspiration with a small diameter pipette. After isolation, all cuticles were washed 3 times with water and frozen at 20°C. -
Isolation o f cuticular proteins
Cuticles were suspended in ST buffer containing 5% 2-mercaptoethanol (2ME), heated at 100°C for 5min, cooled to room temperature and stirred overnight at 4°C. The preparation was then reheated, cooled and centrifuged. The supernatant containing solubilized cuticular proteins was dialyzed overnight against 100 mM Tris containing 1% 2ME (pH 8.0). The resulting pellet containing the insoluble cuticular proteins was washed 5 times with water, dried under vacuum and frozen at -20°C until analyzed for amino acid content. Electrophoresis
Sodium dodecylsulfate polyacrylamide (SDS-PAGE) gel (6-15%) linear gradients were prepared and run with only minor modifications according to the methods of Laemmli and Favre (1973). Proteins were visualized by staining overnight with 0.125% Coomassie Brilliant Blue R-250 in 35% methanol, 10% acetic acid and then destained by diffusion in 35% methanol, 10% acetic acid. The susceptibility of isolated cuticles to enzymatic degradation were tested by suspending cuticles in appropriate buffer containing enzymes (1000 cuticles/ml) followed by incubation at 37°C for periods of 6-18 hr. The enzymes and incubation conditions were as follows: collagenase (Sigma, Type VIII, 0.4 mg/ml) in 50 mM Tris, 150 mM NaC1, 5 mM CaCI at pH 7.4; protease (Sigma Type IV 1.5 mg/ml) in 100 mM Tris, 5 mM CaC1, pH 7.5; elastase (Sigma Type IV 0.8 mg/ml) in 200 mM Tris, pH 8.8; lipase (Sigma Type VII,
1.7 mg/ml) in 50 mM Tris, pH 7.5. Degradation of cuticles was verified using the light microscope. The sensitivity of the 2ME soluble cuticular proteins to collagenase digestion was evaluated by SDS-PAGE. Samples of 2ME soluble proteins were precipitated from solution by addition of cold acetone (10:1 v/v), dried under vacuum and resuspended in 50raM Tris, 150mM NaCI, 5 mM CaC1, pH 7.4 with or without 10 units of bacterial collagenase (Sigma, Type VIII). The samples were incubated for 4 or 18 hr at 37°C and digestions terminated by heating at 100°C for 5 rain in electrophoresis sample buffer. Samples were analyzed on SDS-PAGE gels as described above. Amino acid and carbohydrate analysis
Soluble cuticular proteins were precipitated from solution with cold acetone (10:1 v/v) and dried under vacuum. Samples of 2ME insoluble cuticular proteins were washed 5 times with water, twice with cold acetone and dried under vacuum. All samples were hydrolyzed for 20hr at 115°C. One crystal of phenol was added before hydrolysis and samples were analyzed on a Beckman Amino Acid Analyzer. Following overnight hydrolysis of cuticles at 100°C in 2N HCI, amino sugars were assayed by method of Gatt and Berman (1966) and neutral sugars were measured by the method of Trevelyan and Harrison (1952). Unless otherwise noted all chemicals were of reagent grade and were obtained from local sources. Protein concentrations were determined using the method of Bradford (1976).
RESULTS Treatment of broken larvae with detergent produces segments of the cuticle that, upon examination by the light microscope, appear free of adhering tissue (Fig. 1). Analysis by S D S - P A G E of soluble larval proteins released into solution by detergent treatment reveal a complex protein mixture with mol. wts from 200,000 to less than 30,000 (Fig. 2). In contrast, solubilization of proteins from isolated cuticle of various developmental stages by treatment with 2ME yields a relatively small number of protein bands on S D S - P A G E that have a similar mol. wt range (Figs 2 and 3). Proteins from adult cuticle separated into 5-6 protein bands on S D S - P A G E with the greatest amount of protein localized in 2 bands with mol. wts of 106,000 and 93,000. Lesser amounts o f protein were in 2 bands with mol. wts near 67,000, and a single band with mol. wt of 35,000. A minor band with tool. wt of 45,000 was also observed. The 2 M E soluble cuticular proteins from adult male and female A . suum appeared to be identical (not shown). Cuticular proteins isolated from L4 and L3 and third stage cuticle released during ecdysis in vitro were similar to proteins from adult cuticle (Fig. 3). Prominent protein bands with mol. wts from 90,000 to 106,000 were observed for cuticular proteins from these stages. Other minor bands were present at 67,000, 45,000 and 35,000. The bands observed at 67,000 were generally more prominent in proteins from the third stage cuticle released during ecdysis than from L4, L3 or adult. Cuticular proteins from L2/3 demonstrated prominent bands at 92,000, 106,000, 67,000 and 35,000 but unlike the other stages examined prominent bands were also observed at tool. wts between 200,000-116,000; a large number of minor bands were also present throughout the tool. wt range examined. Cuticular proteins solubilized from L2 cuticle contained 3 to 4 prominent bands
Developmental changes in Ascaris cuticle proteins
Fig. I. Photomicrograph of fragments of cuticle isolated from Ascaris suum L4 by mechanical disruption and detergent treatment as described in Materials and Methods. Horizontal bar indicates 500/am.
A
B
C
D
E
STD
---200KD
116KD 92KD ~
67KD
.......
45KD
31KD Fig. 2. SDS-PAGE gels (5-15%) of detergent soluble proteins released from Ascaris suum I A (lane C), L3, (lane D) and L2 (lane E) following the mechanical disruption and SDS treatment. 2ME soluble proteins from adult cuticle lane A, and an example of a mammalian collagen preparation (calf skin) is shown in lane B.
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R. H. FETTERERand J. F. URBAN,JR
324
2
3
4
5
~200KD
2 0 0 K D ....
~116KD 92KD 116KD''" ~ 6 7 K D
92KD~ 45KD
67KD
,,
45KD
• 31KD
31KD~
"O <
fq ..a
co
O)
Fig. 3. SDS-PAGE (5-15%) analysis of 2ME soluble cuticular proteins from adult and larvae stages of 25-30 gg of larval proteins were applied to each lane, while 50 #g of adult proteins were used to reveal the presence of minor bands in the preparation.
Ascaris suum:
with mol. wts between 116,000 and 200,000 and with at least one prominent band greater than 200,000. A major band with mol. wt of 72,000 was also observed along with a number of minor bands at the lower mol. wt which were often poorly resolved. The soluble cuticular proteins from adult, IA and L3 were degraded by a highly purified bacterial collagenase (Fig. 4). Non-collagenous protein standards are relatively resistant to collagen digestion, while a calfskin collagen is readily degraded by
bacterial collagenase. In contrast, 2ME soluble cuticular proteins from L2 appeared to be relatively resistant to digestion by bacterial collagenase, even after incubation for more than 12 hr. The amino acid, neutral and amino sugar content of 2ME soluble and 2ME insoluble cuticular proteins were determined (Table 1). The amino acid content of the 2ME soluble cuticular proteins from all stages examined had relatively high percentages of glycine, proline and hydroxyproline, characteristic of
Fig. 4. The effect of bacterial collagenase on 2ME soluble cuticular proteins from adult and larval stages of Ascaris suum. Proteins were analyzed on 5-15% SDS-PAGE after incubation with (+) or without ( - ) 10 units of collagenase. Non-collagenous proteins (std) served as negative controls and calf skin collagen (CSC) was used as a positive control. C-indicates position of collagenase.
D e v e l o p m e n t a l c h a n g e s in A s c a r i s cuticle p r o t e i n s
325
Table 1. The amino acid composition of 2-mereaptoethanol soluble and insoluble cuticular proteins from adult and larvae of Ascaris suum
Amino acid
Soluble cutieular proteins Stage L4 L2/3
Adult
G.lycine Small* Basict Aeidie~ Proline Hydroxyproline Amino sugar Neutral sugar
271 346 105 143 251 20 0.44% 0.5%
Glycine Small* Basict Acidic:~ Proline Hydroxyproline Amino sugar Neutral sugar
182 350 45 134 292 ND ND ND
239 289 52 141 191 17 1.04% 4.6%
181 256 111 186 119 25 1.96% 2.9%
Insoluble cuticular proteins 183 342 364 486 21 42 321 141 169 137 ND ND 0,09% 0.6% 2,25% 1.2%
L2 212 302 106 232 141 29 -
306 416 58 140 123 14 0.99% 0.06%
Values for amino acids are residues per thousand. Sugars are given as % dry wt, ND, not detected. - Assay not performed. *Glycine + alanine tLysine + histidine + arginine :~Glutamic + aspartic
collagen-like proteins. The largest amount of glycine was measured in the adult (27%) and L4 (24%) with lesser amounts in L2/3 (18%) and L2 (21%). Proline residues were most numerous in adult cuticular proteins (25%) compared to L4 (19.1%), L2/3 (12%) and L2 (14%). Hydroxyproline was higher in L2 (2.9%) and L2/3 (2.5%) than adult (2.0) or L4 (1.7). Both neutral and amino sugars of 2ME soluble proteins were lowest in adult and highest in both of the larval stages examined (L4 and L2/3). The amino acid analysis of the 2ME insoluble cuticular proteins indicated that adult and L4 were lower in glycine content, contained similar amounts of proline and hydroxyproline compared to 2ME soluble proteins from these 2 stages. The 2ME insoluble cuticular proteins from L2/3 and L2 had greater glycine content than insoluble cuticular proteins from adult and L4, but had lower proline content. Hydroxyproline, although absent in 2ME insoluble cuticular proteins from L2/3, was present in L2 (1.4%).
Table 2. The effect of various enzymes and 2-mercaptoethanol on structure of cuticles isolated from adult and larvae stages of Ascaris suum
Stage
COL
Adult Fourth cuticle* L4 Third cuticlet L2
+++ +++ +++ + + +
ELAST + + + -
Treatment LIP CHIT -
-
PRO
2ME
+++ +++ +++ + + +
+++ +++ +++ + + +
COL, collagenase; ELAST, elastase; LIP, lipase; CHIT, chitinase; PRO, bacterial protease; 2ME, 2-mercaptoethanol. Enzyme concentrations and incubation conditions given in Materials and Methods. Alteration of microscopically observed cuticular structure: + + + , large; + + , moderate; + , slight; - , negligible. *Fourth stage cuticle released during eedysis of young adult. tThird stage cuticle released during ecdysis of L4.
The susceptibility of isolated fragments of cuticles from L4 and from 4th stage cuticle released/n vitro during ecdysis of YA to collagenase, bacterial protease and 2ME was greater than fragments of third stage cuticle released in vitro during ecdysis of L4 and from L2 (Table 2). The most prominent effect of both coUagenase and 2ME appeared to be on the basal region of the cuticle while the protease caused a general loss of integrity in all layers of the cuticle. Of the other enzymes tested only elastase had a relatively weak effect on cuticular structure of fragments from adult, fourth stage cuticle released during ecdysis of YA and L4; lipase and chitinase were without significant effect. The amount of cuticular protein solubilized by 2ME varied considerably between stages. Cuticles from adult stage had 80% soluble protein while L4, L3 and L2 had 70, 50 and 20%, respectively. DISCUSSION
The present data demonstrate that mechanical disruption combined with detergent solubilization is an effective method for the isolation of cuticle from larval stages of the animal parasitic nematode, A s earls suum. Three lines of evidence suggest that the above method is suitable for preparation of isolated cuticle: (1) Ascaris suum cuticle prepared by this method appeared to be free of adhering tissue when observed by light microscope; (2) the large amount of protein released by detergent solubilization had a wide range of mol. wts while cuticular proteins solubilized by 2ME consisted of relatively few protein bands; (3) solubilized cuticular proteins derived either from dissected adults or from cuticle released by larvae during ecdysis in vitro are qualitatively similar to cuticular proteins from mechanically disrupted and detergent treated preparations. Although detergent treatment may be suitable for isolation of
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structural proteins from the cuticle, many other important proteins, particularly those from the surface of the cuticle, may be removed by this treatment. The results of the current study indicate that the A. suum cuticular proteins have properties similar to cuticular proteins from other nematode species. Cuticles from A. suum larvae and adults consist of both 2ME soluble and 2ME insoluble proteins like cuticles from C. elegans and M . incognita (Cox et al., 1981a; Ouanza and Herbage, 1981; Reddigari et al., 1986). In addition, the 2ME soluble proteins from A. suum are collagen-like because they are sensitive to collagenase, and their amino acid composition is rich in glycine and proline. Although the 2ME soluble cuticular proteins from A. suum L2 were not degraded by a bacterial collagenase it should be noted that this is also a property of collagen from other invertebrate sources (Kimura and Tanzer, 1977). The cuticle of A. suum differs from some other nematode cuticle in at least two ways: (1) The ratio of hydroxyproline to proline (H/P) observed in the 2ME soluble cuticular proteins of A. suum was relatively high (1:13, adult; 1: 5, larvae) compared to C. elegans (1:1), M . incognita (1:1, adult; 1 : 3 larvae) or mammalian collagen (1:1) (Cox et al., 1981a, b; Reddigari et al., 1986; Miller and Undenfriend, 1970). It is possible that the relatively low amount of hydroxylation of proline observed in A. suum is the result of the low oxygen tensions found in the intestinal environment. However, the free-living nematode Panagrellus sillusiae has a H/P ratio (1:7) similar to that of A. suum (Leushner et al., 1979) suggesting that there is not a clear dichotomy in H/P ratio between A. suum and other nematode species. (2) The metachromatic staining by Coomassie Brilliant Blue of the collagen-like cuticular proteins separated by SDSPAGE observed by other authors (Cox et al., 1981a; Winkfein et aL, 1985) was only occasionally noted in the present investigations. Previous studies of cuticular structure have demonstrated stage specific changes in morphology of the nematode cuticle during development from larvae to adult (Samoiloff and Pasternak, 1986; Bird, 1971; Cox et al., 1981b). In A. suum there is a particularly marked increase in the thickness of the medial and basal layers of the cuticle as the parasite grows to the adult stage. This increase in the medial layer may represent an adaptation to a parasitic environment (Bird, 1971; Watson, 1965). Two observations from the current studies are consistent with the hypothesis that there is a progressive increase in the amount of collagen-like, 2ME soluble proteins in the basal and medial layers of the cuticle of A. suum L2 developing to adult: (1) treatment with 2ME and bacterial collagenase was most disruptive of the structure of the basal and medial layers of the cuticle of the adult, young adult and fourth stage larvae, and (2) the amount of 2ME soluble cuticular protein markedly increased from the L2 through the adult stage. This increase may represent an adaptation to a changing parasitic environment, but this phenomenon is not unique to parasitic worms. Adults of the free-living nematode C. elegans also have larger amounts of 2ME soluble cuticular proteins compared to their larvae (Cox et al., 1981a). Studies of C. elegans cuticular proteins and their
genes have led to the hypothesis that nematodes have genes that are developmentally regulated resulting in stage specific cuticular proteins (Cox et al., 1981b; Kramer et al., 1982, 1985). A parasitic plant nematode, M . incognita, has recently been shown to have cuticular proteins that differ between the adult and the second stage juvenile (Reddigari et al., 1986). This developmental change in cuticular proteins of the animal parasitic nematode A. suum are in general agreement with the above hypothesis. The 2ME soluble cuticular proteins from L2 were clearly different from other stages not only in pattern and numbers of protein bands on SDS-PAGE, but in the lack of sensitivity to digestion by a bacterial collagenase. The L2 differs biologically from other parasitic stages of A. suum examined because it is found naturally within the egg shell in the environment. It is not clear, however, if the differences in cuticular proteins observed are related to differences in the environment in which the stages are found. The cuticular proteins from L2/3 also appear to be different from adult, L4 and L3. The interpretation of these results are complicated by the fact that there are mixtures of stages derived from this culture system and second stage cuticles released during ecdysis of L3 are also present. Cuticular proteins from adult, L3 and L4 were readily degraded by collagenase, and had a similar protein banding pattern by SDS-PAGE analysis. However, differences in the number of protein bands in each stage were noted, suggesting that A. suum cuticular proteins differ between stages. Analysis of amino acid composition of cuticles from different stages also support this concept. The amino acid composition of 2ME soluble cuticular proteins from the adult was similar to that reported by Winkfein et al. (1985). Nearly one-third of the residues were glycine and there was a relatively high proportion of proline. This composition is characteristic of collagen-like proteins. The soluble cuticular proteins from L4 were similar to adult by SDS-PAGE analysis but differed slightly in amino acid composition. The L4 had less glycine and slightly less proline than adults. Amino acid composition of L2 and L2/3 were most different from adult. They had about 21% glycine compared to 27% for adults and had markedly lower amounts of proline than adults. Stage differences in amino acid composition of the 2ME insoluble cuticular proteins were also observed. The 2ME insoluble cuticular proteins of IA were similar in composition to adult, but those from L2/3 and L2 were considerably different, especially in the greater amount of glycine found in those early larval stages. The amounts of both amino and neutral sugars were generally greater in larval stages than adult. In summary, the results of the present study show that there are stage differences in cuticular proteins of developing A. suum, and that the differences between adult and L2 are most prominent. There is also a quantitative increase in the amount of 2ME soluble proteins in the cuticle during development. However, it is not possible to determine from the current study if there are multiple stage specific genes responsible for the various cuticular proteins as has been suggested for C. elegans. Since recent experiments indi-
Developmental changes in Ascaris cuticle proteins cate that C. elegans collagen genes may be useful probes for identification o f genes o f cuticular protein in A. suum (Zarlenga and Fetterer, unpublished observation), it may be possible in future studies to more precisely determine the mechanisms o f developmental regulation o f A. suum cuticular proteins. Acknowledgements--We are indebted to J. Blythe for expert technical assistance. We thank Dr R. S. Rew and Dr P. C. Allen for critical reading of this manuscript. Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee of warranty by U.S. Department of Agriculture and does not imply its approval to exclusion of other products that may be suitable.
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