Comp. Biochem. Physiol. Vol. 103B,No. 3, pp. 681--686, 1992
0305-0491/92 $5.00 + 0.00 Pergamon Press Ltd
Printed in Great Britain
CHARACTERIZATION OF THE EGGSHELL OF HAEMONCHUS CONTORTUS--I. STRUCTURAL COMPONENTS L. S. MANSFIELD,H. R. GAMBLE and R. H. FETTERER United States Department of Agriculture, Agricultural Research Service, Livestock and Poultry Sciences Institute, Helminthic Diseases Laboratory, Beltsville, MD 20705-2350, U.S.A. [Tel: (301) 504-8774; Fax: (301) 504-5306] (Received 27 March 1992; accepted I May 1992)
Abstract--1. By transmission electron microscopy, the eggshell of Haemonchus contortus was seen to be similar to previously studied nematodes, with an outer vitelline layer bounded by a trilaminate membrane, a broad medial region, containing chitin, and an electron dense basal region, containing lipid and protein. 2. Exposure of Haemonchus contortus eggs to proteases resulted in disruption of the shell with removal of components of the outer, medial and basal regions. Exposure to chitinase depleted fibrillar components of the medial region of the shell, while collagenase had no effect. 3. Chloroform/methanol extraction of fresh eggshells caused a minor condensation of the outer, vitelline layer and some depletion of the basal layer. 4. After normal hatching, shells appeared similar to those treated with protease and chitinase, but also lacked the basal, lipid layer. 5. Extracts of isolated unhatched eggshells and hatched eggshells, and extracts of biotin-labelled whole fresh eggs showed three major protein bands when run on sodium dodecyl sulphate--polyacrylamide gels indicating that these three proteins are most likely structural in nature and do not participate in the release of the larva from the eggshell. 6. Biotin-labelled protein bands were degraded by proteases and chitinase, but not collagenase or lipase.
INTRODUCTION The nematode egg is an important stage of the parasite's life cycle both from the perspective of development of the parasite and as a potential target for control strategies. Adult female worms of Haeraonehus contortus reside in the abomasum of ruminants and pass unembryonated eggs (8-16 cells) which serve as a relatively resistant dispersal stage. Eggs hatch following a 24-48-hr embryonation period releasing a first-stage larva. Hatching and subsequent release of the first-stage larva are thought to result from a combination of mechanical and enzymatic events which are not completely characterized (Bird, 1971; Croll, 1976; Rogers and Brooks, 1977; Bone and Parish, 1988; Perry, 1989). The target sites of these putative hatching enzymes are, at this time, undefined (Perry, 1989). Electron microscopy has been used to study the synthesis of the nematode eggshell (Bird, 1971; Croll, 1976). Up to 30% of the total investment in energy utilization is devoted to eggshell production in some helminth species (Wharton, 1983). The eggshell is thought to consist of three basic layers that are secreted by the embryo. These include an inner lipid layer, a middle chitinous layer and an outer vitelline layer. In some nematodes such as Ascaris, a fourth outermost layer termed the uterine layer (Croll, 1976) is found which is secreted by the uterus. These layers begin to form immediately after fertilization of the egg. The outer vitelline membrane is thought to originate at fertilization when a second membrane 681
forms beneath the oolemma. The medial, cortical layer forms between these two membranes, pushing the vitelline membrane to the outside where it hardens (Bird, 1971). During this time, the cytoplasm of the egg becomes very active, with rough endoplasmic reticulum and refringent granules developing in the cortical area (Croll, 1976). Within hours after fertilization the chitinous, proteinaceous cortical area thickens and develops (Croll, 1976). In several nematode species chitin and protein complexes occur as 2.8 nm chitin microfibrils embedded in a protein matrix (Neville, 1975). In Triehuris suis the chitinous layer is composed of this size fibril surrounded by a protein coat, suggesting this as the basic unit of this layer (Wharton, 1980). The basal or lipid layer is formed by material secreted at the surface of the egg cytoplasm or by the extrusion of refringent granules from the egg (Wharton, 1979). As the lipid layer develops the egg takes on its characteristic impermeability. Despite this detailed knowledge of the formation of the eggshell, little is known about the mechanisms of breakdown of the shell coincident with the release of the first-stage larva. It is important to have a clear understanding of the composition and organization of the eggshell, to form a basis for understanding the nature of the environmental resistance afforded by the eggshell and to understand the action of enzymes on the shell structure. In the following studies we have examined the eggshell of H. contortus, both pre- and posthatching, using several techniques including electron
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microscopy, enzymatic digestion, biotin labelling, a n d s o d i u m dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) in a n effort to determine the basic structure. MATERIALS AND METHODS Egg production
Parasite-naive polled Dorset lambs were infected with the Beltsville strain of H. contortus. Faeces from lambs with patent infections were collected and cultured for 10 days to obtain infective third-stage larvae for inoculation. Unembryonated eggs (8-16 cells) were collected by obtaining fresh faeces from infected lambs over a 3-hr interval. Faeces were homogenized in tap water using a blender, pressed through a 300 p m mesh screen, mixed with an equal volume of 150% sucrose and centrifuged for 10min at 1400g. Following centrifugation, eggs were aspirated from the surface, mixed with 100 volumes of tap water and allowed to sediment for 30 rain. The supernatant was aspirated and the sediment floated again by mixing with an equal volume of 150% sucrose for 10min at 1400g. After a final sedimentation through 100 volumes of tap water, eggs were collected, inspected for extraneous faecal debris and concentrated by centrifugation for use in experimental studies. Eggshells from either unembryonated or hatched eggs were prepared as described by Gamble and Mansfield (1992). Briefly, freshly collected eggs were layered on the surface of a step gradient made up of 30 and 45% Percoll, centrifuged at 600 g for I0 min and the eggs, free of faecal debris, were collected from the interface between the 30 and 45% layers. Purified unembryonated eggs were chopped repeatedly with razor blades, until the majority of the eggs appeared nicked when examined microscopically. Nicked eggs were resuspended in sterile double distilled water (sdd H20) and disrupted further by sonication. Following sonication, pure eggshells, free of embryonic material, were collected by layering the mixture on 20% Percoll and centrifuging as above. Pure shells were collected from the surface, washed three times in sdd H20 and concentrated by centrifugation for biochemical studies or electron microscopy. To collect hatched shells, unembryonated eggs were purified as described above and incubated in sterile tap water supplemented with antibiotics at room temperature until more than 90% of the eggs had developed and hatched (approximately 4 2 4 8 hr). Sterility of hatching eggs was assessed by streaking supernatants from hatching preparations on nutrient agar. Cultures were discarded if bacterial contamination was present. Hatched eggshells were separated from first stage larvae and debris by centrifugation on 10% Percoll, washing in sdd H20 and extraction in sodium dodecyl sulphate (SDS) and 2-mercaptoethanol (2-ME) as described below. Electron microscopy Samples of unembryonated eggs (treated as described below, or untreated) were pelleted in Eppendorf tubes at 1000g and fixed for transmission electron microscopy (TEM) in 20 volumes of osmium tetroxide for 1 hr with gentle agitation. Three 10-min washes in Sorenson's buffer (Barta et al., 1987) were followed by three, 5 min washes in sdd H20. Samples were then incubated in 0.5% uranyl acetate overnight, washed in sdd H20 and dehydrated in several changes of ethanol (30, 50, 70, 85, 95%). Following dehydration, the samples were infiltrated by passing through increasing concentrations of Spurr's medium (3:1, 1:1, 1:3, 100% ethanol to Spurr's) (Barta et al., 1987). Specimens were embedded in Spurr's, hardened by baking in a 70°C oven for 8 hr, cut in sections from 60 to 90/~m on a Sorvall microtome and mounted on HX 200 copper grids. Grids were examined using a Phillips EM 200 electron microscope.
Eggs examined by TEM included the following treatment groups: (1) untreated unembryonated eggs, (2) proteasetreated unembryonated eggs, (3) collagenase-treated unembryonated eggs, (4) chitinase-treated unembryonated eggs, (5) chloroform/methanol extracted unembryonated eggs and (6) hatched eggshells. Enzymatic treatments were carried out for 1 hr at 37°C followed by fixation in Spurr's medium. All enzymes were used at a concentration of 10 U enzyme/25/~l egg extract. In some cases, chitinase was mixed with a protease inhibitor cocktail including 10 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulphonyl fluoride (PMSF) (Sigma, St. Louis, Missouri), 1/~M pepstatin, 100#M antipain and 10mM aprotinin prior to addition to eggs or extracts. Lipid extraction was performed by adding 500/~1 of an equal volume mixture of chloroform and methanol to the whole egg pellet in an Eppendorf tube, vortexing for 1 min and centrifuging at 8000g for 1 min. The organic phase was removed, and the egg pellet remaining was washed three times in phosphate buffered saline (PBS) and fixed for TEM as described above. Unembryonated eggs treated with N-hydroxysuccinimide biotin (NHS-biotin) and sulphosuccinimido biotin (sulphoNHS-biotin) (Pierce, Rockford, Illinois) (Hill et al., 1990) were also fixed according to the above protocol and examined by TEM. SDS PAGE and blotting o f egg extracts
For extraction, 5% sodium dodecyl sulphate (SDS) and 5% 2-mercaptoethanol (2-ME) were added to 0.5ml of biotin-labelled whole eggs or 0.25 ml of purified eggshells, vortexed for I min, boiled for 1 min and the process repeated. Soluble extracts prepared by this treatment were run on denaturing 12% SDS-PAGE gels to determine the presence of and size of any detergent soluble eggshell proteins. Soluble extracts run on similar 12% SDS-PAGE gels were stained with periodic acid Schiff (PAS) stain by the method of Kapitany and Zebrowski (1973) to test for the presence of glycoproteins. Extracts of biotinylated eggs from SDS-PAGE gels were electrophoretically transferred to nitrocellulose membranes, washed sequentially with wash buffer I [WB I, 5% nonfat dry milk in 50 mM Tris (pH 7.5) and 150 mM NaC1], wash buffer II (WB II, containing in addition to the components of WB I, 1.0% Triton X-100 and 0.1% SDS), and WB I and incubated at room temperature with streptavidin-horseradish peroxidase (Bethesda Research Laboratories, Gaithersburg, Maryland) in WB I overnight. After incubation, membranes were washed by the same procedure then developed using 0.18% 4-chloronaphthol and 0.012% H202 in Tris buffered saline (50 mM Tris, 150mM NaC1, pH 7.5). Biotin labelling Intact, unembryonated eggs were surface labelled by incubating with sulpho-NHS-biotin or NHS-biotin (Hill et al., 1990). Sulpho-NHS-biotin and NHS-biotin were prepared in the dark at I0 mg/ml, the former in 10 mM PBS and the latter in dimethylsulphoxide (DMSO), and diluted 1/20 in 10 mM PBS, pH 7.4. Five hundred microlitres of packed fresh eggs were washed three times in PBS, suspended in 2.5 ml of PBS and mixed with 25 ~1 of either sulpho-NHS-biotin or NHS-biotin. This mixture was incubated at room temperature for I0 min. Labelled eggs were pelleted in an Eppendorf centrifuge at 8000 g and washed three times in sterile PBS. In one experiment, NHS-biotintreated eggs were fixed for TEM by techniques described previously, sectioned and the sections incubated with strepavidin-immunogold. These sections were then observed for gold foci. Enzymatic digestion Extracts of biotinylated eggs were tested for sensitivity to chitinase (Sigma, St. Louis, Missouri), lipase, collagenase and proteinase K. NHS-biotinylated extracts were dialysed
Haemonchus contortus eggshell characterization against sdd H20 to remove SDS and 2-ME and divided into four groups for exposure to the enzymes. Extracts were incubated with enzymes for I hr at 37°C. All enzymes were used at a concentration of 10 U enzyme/25/d of extracted material. Digested extracts were analysed by SDS--PAGE, blotted to nitrocellulose, incobated with streptavidin-HRP and developed as above to determine the effects of enzyme treatment. In control experiments, proteolytic activity present in the ehitinase enzyme was found to be inhibited by a cocktail of protease inhibitors including 10mM EDTA, I mM PMSF, I/~M pepstatin, 100/~M antipain and I0 mM aprotinin. The inhibitor cocktail was used in subsequent chitinase digests to block nonspecific proteolytic activity in ehitinase enzyme preparations. RESULTS
Electron microscopy and enzymatic digestion Unembryonated eggs, recovered from the faeces of infected source sheep were purified, exposed to a
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series of treatments and examined using the electron microscope. Shells of intact, unembryonated eggs had three layers including a basal electron dense layer, an extensive medial amorphous cortical layer and an outer dense layer bounded by a thin trilaminate membrane (Fig. 1). The basal layer was the most dense of the three layers with little discernible structure. This layer varied considerably in thickness. The medial layer had distinctive electron dense fibrillar components interspersed throughout an area that, although occupying the greatest volume of the three layers, was the least dense. The outer layer was bounded by a trilaminate membrane and was closely adherent to the cortical layer (Fig. 2a). Enzyme treatments selectively affected the three layers of the eggshell (Fig. 2). Protease treatment affected all three layers. The vitelline membrane was disrupted, and the trilaminate layers obliterated, indicating that this layer was largely composed of
Fig. 1. Transmission electron micrograph of the eggshell of Haemonchus contortus showing the basic layers including the outer trilaminate vitelline layer (v), the medial chitinous layer (ch), the basal lipid/protein layer (1), the embryonic membrane (em) and a cross-section of the embryo.
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protein. Some components of the medial and basal areas were removed, causing a decrease in density. The cortical area appeared less dense with clumping of the electron dense fibrous strands. The cortical stroma or matrix seemed to be the missing element. The basal layer appeared somewhat thinner and condensed and in some cases was absent (Fig. 2b). Collagenase treatment had little detectable effect on the eggshell (Fig. 2c). Chitinase treatment depleted components of the medial layer, selectively removing the electron dense fibrous strands present in the cortical area (Fig. 2d). Chloroform/methanol extraction had little or no effect on the cortical layer of the eggshell, but caused a minor condensation of the vitelline layer and some depletion of the basal layer (Fig. 2e). Eggshells examined following hatching had more pronounced changes in structure than any of the
ii ~ ii
iii
I
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3
4
2 0 0 K --,,,-
116K-,-92K--~ 66K---~
45K-~
Fig. 3. SDS-PAGE separation of detergent (5% SDS, 5% 2-ME) soluble extracts of eggshells of fresh eggs of Haemonchus contortus. Low molecular weight standards (Lane I), high molecular weight standards (Lane 2), 12.25/z g of detergent soluble extract of fresh eggshells (Lane 3), 6.12/z g of detergent soluble extract of hatched eggshells (Lane 4). enzyme-treated groups. Hatched eggshells showed similarities to shells digested with both protease and chitinase. There was extensive depletion of the chitin/protein complexes of the medial layer leaving a uniform amorphous matrix and disruption of the bounding trilaminate membrane. Additionally, the basal lipid layer was largely removed by the normal hatching process (Fig. 2f). Transmission electron micrographs of biotinlabelled eggs showed that sulpho-NHS-biotin or NHS-biotin bound to the eggshell at low levels. When streptavidin-immunogold was used to stain biotintreated eggs of the parasite, few gold foci were found, and these were dispersed randomly throughout the three layers of the eggshell (data not shown). S D S - P A G E o f eggshell proteins Three major protein bands were seen on Coomassie Blue and silver stained S D S - P A G E gels of extracts of whole unembryonated eggs. These bands migrated at approximate molecular weights (MW) of 186, 132 and 89 kDa; many minor protein bands were also evident (Fig. 3). Biotinylated eggshell extracts also showed three major bands and some minor bands which corresponded in MW to Coomassie Blue stained bands (Fig. 4). Extracts of eggshells of H. contortus following hatching showed the same three major protein bands seen in extracts of unembryonated eggs (Fig. 3). Only a single high M W band ( > 2 0 0 k D a ) stained with PAS (Fig. 4).
Fig. 2. Transmission electron micrographs of the eggshell of Haemonchus contortus after various treatment regimens. (a) Untreated control eggshells. (b) Protease-treated eggshells. (c) Collagenase-treated eggshells. (d) Chitinase-treated eggshells. (e) Chloroform/methanol-treated eggshells. (f) Naturally hatched eggshells.
Biotinylation and enzymatic digestion o f eggs Sulpho-NHS-biotin and NHS-biotin treatment resuited in low level labelling of H. contortus eggshells. NHS-biotin penetrated the eggshell more effectively than sulpho-NHS-biotin, as judged by heavier banding patterns on S D S - P A G E gels (data not shown)
Haemonchua contortus eggshell characterization
I
2
3
4
5
200K 116K 66K 45K
31K
Fig. 4. SDS-PAGE analysis of detergent (5% SDS, 5% 2-ME) soluble protein extracts of NHS-biotin-labelled whole eggs of Haemonchus contortus. High molecular weight standards (Lane 1), low molecular weight standards (Lane 2), Coomassie stained soluble extract of biotin-labelled whole fresh eggs (Lane 3), PAS stained soluble extract of biotin-labelled whole fresh eggs (Lane 4), streptavidin-horse radish peroxidase stained blot of SDS-PAGE of soluble extract of biotin-labelled whole fresh eggs. and a greater number of gold particles on strepavidin-immunogold stained transmission electron micrographs. The three major protein species seen on these blots ran at the same size as those seen in Coomassie/silver stained gels. Proteinase K and chitinas¢ digestion completely removed the three major and all the minor eggshell protein bands (Fig. 5). Addition of proteolytic inhibitor cocktail to chitinase digests of eggshell extracts only partially inhibited this digestion. Lipase and collagenase had no apparent effect on the biotinylated eggshell protein bands (Fig. 5).
I
2
3
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5
6
200K --~ 116K
Fig. 5. SDS-PAGE analysis of detergent (5% SDS, 5% 2-ME) soluble protein extracts of NHS-biotin-labelled eggshells of Haemonchus contortus. Control--untreated whole eggs (Lane 1), chitinase-digested whole eggs (Lane 2), chitinase-digested whole eggs with inhibitor cocktail (Lane 3), llpase-digested whole eggs (Lane 4), eollagenase-digested whole eggs (Lane 5), and proteinase K-digested whole eggs (Lane 6).
685 DISCUSSION
Based on these studies, the structure of the eggshell of H. contortus appears to be similar to that described for other nematodes. Electron microscopy revealed the presence of three basic structural layers described previously (Foor, 1967; Kaulenaus and Fairbairn, 1968; Bird, 1968) including an inner electron dense layer, an extensive amorphous middle layer and an outer thin trilaminate bounding membrane. No uterine layer, as described for Ascaris spp. (Croll, 1976), was seen in H. contortus. The outermost layer of the eggshell appeared as a thin trilaminate membrane reminiscent of, but thicker than, a cell membrane. Based on the enzyme degradation and lipid extraction studies, it was composed largely of protein and lipid. The cortical or medial layer of the eggshell of H. contortus was the thickest layer and appeared to be composed of electron dense fibrils in a randomly oriented, regular distribution. These fibrillar complexes were surrounded by a less dense matrix. Subtraction studies, using enzymes to selectively remove elements of the cortical area, suggested that this layer may derive its uniform matrix appearance from a random association of chitinous fibrils surrounded by protein. This protein may coat the chitin fibrils in an arrangement similar to Trichuris spp. (Wharton, 1980). This arrangement of chitin, in some type of covalent linkage, may provide a degree of resistance to chemical attack, as suggested by Wharton (1980). The basal layer of the eggshell is a matrix made up of lipid in some association with protein, similar to the findings of Wharton (1983). This layer varied in thickness along the length of the shell and was electron dense with a randomly oriented, regularly distributed matrix. Lipid extraction procedures merely depleted, but did not entirely remove, the basal layer of the eggshell. This layer was the only area noticeably affected by this treatment, and the major effect was to cause a condensation, like a collapse of a proteinaceous matrix. Proteinase K treatment also affected this layer causing a similar condensation without removing the entire structure. Natural hatching more completely depleted this layer suggesting that both lipid and protein components are reduced during the hatching process. Isolated components of the eggshell of H. eontortus were also sensitive to the action of enzymes. Three major proteins were detected in extracts of the eggshell with relative masses of 186, 132 and 89 kDa, as determined by Coomassie staining of SDS-PAGE gels and biotin labelling. These major bands were degraded by either proteinase K or chitinase digestion but not by lipase or collagenase treatment of extracts of biotinylated eggshells. Similarly, collagenase had no apparent effect on in situ components of the eggshell as determined by TEM. Based on these results, it appears that collagenous proteins play little if any role in maintaining the structure of the eggshell in H. contortus. This is unlike the adult nematode cuticle in which collagen comprises 80% of the cuticular proteins (Fetterer and Urban, 1988; Kingston, 1991). When observed using TEM, the natural hatching process produced structural changes in the eggshell
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similar to those produced by enzyme treatments (chitinase/proteinase K). Hatching depleted structural components of all three layers of the eggshell as observed at the TEM level. A difference in the density of the eggshells, another measure of enzyme depletion, was also observed after hatching. Hatched eggshells floated on 10% Percoll while fresh eggshells floated on 20% Percoll (Gamble and Mansfield, 1992). Another interesting finding of these studies is that the eggshell of H. contortus may be impermeable to biotin or may have few available binding sites for the biotin molecule. Both forms of biotin used in this study react with primary amines to form amide bonds. Neither sulpho-NHS-biotin, which is soluble in aqueous environments, or NHS-biotin, which is soluble in organic solvents, bound well to the eggshell when viewed by TEM analysis and staining with avidin-gold particles. There were a few sites of labelling in the eggshell, but gold foci were in low quantity and were dispersed randomly throughout the three layers of the eggshell. In contrast, the cuticles of Ascaris suum larvae (Hill et al., 1990) and Onchocerca linealis microfilariae (Hill et aL, 1992) are strongly labelled by NHS-biotin. The minimal binding of biotin to eggshell proteins in H. contortus may also reflect structural differences between the eggshell and the cuticle. The studies presented here indicate that the eggshell of Haemonchus contortus is structurally similar to eggs of other nematodes. Electron microscopic and enzymatic degradation studies of the eggshell also support claims in H. contortus (Rogers and Brooks, 1977) and in other nematode species (Bone and Parish, 1988; Perry, 1989) that the hatching process may occur as a result of enzymatic events. Further work is needed to relate the structural findings in this study to the hatching process. Acknowledgements--The authors would like to acknowledge the expert technical assistance of Ms Jean Corbin, Ms Eleanor E. Moore and Mr James L. McCrary.
REFERENCES
Barta J. R., Boulard Y. and Desser S. S. (1987) Ultrastructural observations on secondary merogony and
gametogony of Dactylosoma ranarum Labbe, 1894 (Eucoccidiida; Apicomplexa).J. Parasitol. 73, 1019-1029. Bird A. F. (1968) Changes associated with parasitism in nematodes. III. Ultrastructure of the eggshell, larval cuticle, and contents of the subventral oesophageal glands in Meloidogyne javanica, with some observations on hatching. J. Parasitol. 54, 699-719. Bird A. F. (1971) The Structure of Nematodes, pp. 114-137. Academic Press, New York. Bone L. W. and Parish E. J. (1988) Egg enzymes of the ruminant nematode Trichostrongylus colubriformis. Invert. Reprod. Develop. 14, 299-302. Croll N. A. (1976) The Organization of Nematodes, pp. 279-300. Academic Press, New York. Fetterer R. H. and Urban J. F., Jr (1988) Developmental changes in cuticular proteins of Ascaris suum. Comp. Biochem. Physiol. 90B, 321-327. Foor W. E. (1967) Ultrastructural aspects of oocyte development and shell formation in Ascaris lumbricoides. J. ParasitoL 53, 1245-1261. Gamble H. R. and Mansfield L. S. (1992) Purification of egg shells from Haemonchus contortus. J. Helminthol. Soc. Wash. 59, 234-236. Hill D. E., Fetterer R. H. and Urban J. F., Jr (1990) Biotin as a probe of the surface of Ascaris suum developmental stages. Molec. Biochem. Parasitol. 41, 45-52. Hill D. E., Mahin Khapami, Lok J. B. and Rocky J. H. (1992) Onchocerca linealis: Surface glycolipid masking cuticular antigens of Onchocerca microfilariae. Exp. Parasitol. (In press). Kapitany R. A. and Zebrowski E. J. (1973) High resolution PAS Stain for polyacrylamide gel electrophoresis. Analyt. Biochem. 56, 361-369. Kaulenaus M. S. and Fairbairn D. (1968) RNA metabolism of fertilized Ascaris lumbricoides eggs during uterine development. Exp. Cell Res. 52, 233. Kingston I. B. (1991) Nematode collagen genes. Parasitol. Today 7(1), 11-14. Neville A. C. (1975) Biology of the Arthropod Cuticle, pp. 1-250. Springer, Berlin, Germany. Perry R. N. (1989) Dormancy and hatching of nematode eggs. Parasitol. Today 5(12), 377-383. Rogers W. P. and Brooks F. (1977) The mechanism of hatching of eggs of Haemonchus contortus. Int. J. Parasitol. 7, 61 65. Wharton D. A. (1979) Oogenesis and eggshell formation in Aspiculuris tetraptera Schulz (Nematoda: Oxyuroidea). Parasitology 78, 131-143. Wharton D. A. (1980) Nematode egg-shells. Parasitology 81, 447-463. Wharton D. A. (1983) The production and functional morphology of helminth egg-shells. Parasitology 86, 85-97.
0305-0491/92 $5.00 + 0.00 Pergamon Press Ltd
Printed in Great Britain
CHARACTERIZATION OF THE EGGSHELL OF HAEMONCHUS CONTORTUS--I. STRUCTURAL COMPONENTS L. S. MANSFIELD,H. R. GAMBLE and R. H. FETTERER United States Department of Agriculture, Agricultural Research Service, Livestock and Poultry Sciences Institute, Helminthic Diseases Laboratory, Beltsville, MD 20705-2350, U.S.A. [Tel: (301) 504-8774; Fax: (301) 504-5306] (Received 27 March 1992; accepted I May 1992)
Abstract--1. By transmission electron microscopy, the eggshell of Haemonchus contortus was seen to be similar to previously studied nematodes, with an outer vitelline layer bounded by a trilaminate membrane, a broad medial region, containing chitin, and an electron dense basal region, containing lipid and protein. 2. Exposure of Haemonchus contortus eggs to proteases resulted in disruption of the shell with removal of components of the outer, medial and basal regions. Exposure to chitinase depleted fibrillar components of the medial region of the shell, while collagenase had no effect. 3. Chloroform/methanol extraction of fresh eggshells caused a minor condensation of the outer, vitelline layer and some depletion of the basal layer. 4. After normal hatching, shells appeared similar to those treated with protease and chitinase, but also lacked the basal, lipid layer. 5. Extracts of isolated unhatched eggshells and hatched eggshells, and extracts of biotin-labelled whole fresh eggs showed three major protein bands when run on sodium dodecyl sulphate--polyacrylamide gels indicating that these three proteins are most likely structural in nature and do not participate in the release of the larva from the eggshell. 6. Biotin-labelled protein bands were degraded by proteases and chitinase, but not collagenase or lipase.
INTRODUCTION The nematode egg is an important stage of the parasite's life cycle both from the perspective of development of the parasite and as a potential target for control strategies. Adult female worms of Haeraonehus contortus reside in the abomasum of ruminants and pass unembryonated eggs (8-16 cells) which serve as a relatively resistant dispersal stage. Eggs hatch following a 24-48-hr embryonation period releasing a first-stage larva. Hatching and subsequent release of the first-stage larva are thought to result from a combination of mechanical and enzymatic events which are not completely characterized (Bird, 1971; Croll, 1976; Rogers and Brooks, 1977; Bone and Parish, 1988; Perry, 1989). The target sites of these putative hatching enzymes are, at this time, undefined (Perry, 1989). Electron microscopy has been used to study the synthesis of the nematode eggshell (Bird, 1971; Croll, 1976). Up to 30% of the total investment in energy utilization is devoted to eggshell production in some helminth species (Wharton, 1983). The eggshell is thought to consist of three basic layers that are secreted by the embryo. These include an inner lipid layer, a middle chitinous layer and an outer vitelline layer. In some nematodes such as Ascaris, a fourth outermost layer termed the uterine layer (Croll, 1976) is found which is secreted by the uterus. These layers begin to form immediately after fertilization of the egg. The outer vitelline membrane is thought to originate at fertilization when a second membrane 681
forms beneath the oolemma. The medial, cortical layer forms between these two membranes, pushing the vitelline membrane to the outside where it hardens (Bird, 1971). During this time, the cytoplasm of the egg becomes very active, with rough endoplasmic reticulum and refringent granules developing in the cortical area (Croll, 1976). Within hours after fertilization the chitinous, proteinaceous cortical area thickens and develops (Croll, 1976). In several nematode species chitin and protein complexes occur as 2.8 nm chitin microfibrils embedded in a protein matrix (Neville, 1975). In Triehuris suis the chitinous layer is composed of this size fibril surrounded by a protein coat, suggesting this as the basic unit of this layer (Wharton, 1980). The basal or lipid layer is formed by material secreted at the surface of the egg cytoplasm or by the extrusion of refringent granules from the egg (Wharton, 1979). As the lipid layer develops the egg takes on its characteristic impermeability. Despite this detailed knowledge of the formation of the eggshell, little is known about the mechanisms of breakdown of the shell coincident with the release of the first-stage larva. It is important to have a clear understanding of the composition and organization of the eggshell, to form a basis for understanding the nature of the environmental resistance afforded by the eggshell and to understand the action of enzymes on the shell structure. In the following studies we have examined the eggshell of H. contortus, both pre- and posthatching, using several techniques including electron
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microscopy, enzymatic digestion, biotin labelling, a n d s o d i u m dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) in a n effort to determine the basic structure. MATERIALS AND METHODS Egg production
Parasite-naive polled Dorset lambs were infected with the Beltsville strain of H. contortus. Faeces from lambs with patent infections were collected and cultured for 10 days to obtain infective third-stage larvae for inoculation. Unembryonated eggs (8-16 cells) were collected by obtaining fresh faeces from infected lambs over a 3-hr interval. Faeces were homogenized in tap water using a blender, pressed through a 300 p m mesh screen, mixed with an equal volume of 150% sucrose and centrifuged for 10min at 1400g. Following centrifugation, eggs were aspirated from the surface, mixed with 100 volumes of tap water and allowed to sediment for 30 rain. The supernatant was aspirated and the sediment floated again by mixing with an equal volume of 150% sucrose for 10min at 1400g. After a final sedimentation through 100 volumes of tap water, eggs were collected, inspected for extraneous faecal debris and concentrated by centrifugation for use in experimental studies. Eggshells from either unembryonated or hatched eggs were prepared as described by Gamble and Mansfield (1992). Briefly, freshly collected eggs were layered on the surface of a step gradient made up of 30 and 45% Percoll, centrifuged at 600 g for I0 min and the eggs, free of faecal debris, were collected from the interface between the 30 and 45% layers. Purified unembryonated eggs were chopped repeatedly with razor blades, until the majority of the eggs appeared nicked when examined microscopically. Nicked eggs were resuspended in sterile double distilled water (sdd H20) and disrupted further by sonication. Following sonication, pure eggshells, free of embryonic material, were collected by layering the mixture on 20% Percoll and centrifuging as above. Pure shells were collected from the surface, washed three times in sdd H20 and concentrated by centrifugation for biochemical studies or electron microscopy. To collect hatched shells, unembryonated eggs were purified as described above and incubated in sterile tap water supplemented with antibiotics at room temperature until more than 90% of the eggs had developed and hatched (approximately 4 2 4 8 hr). Sterility of hatching eggs was assessed by streaking supernatants from hatching preparations on nutrient agar. Cultures were discarded if bacterial contamination was present. Hatched eggshells were separated from first stage larvae and debris by centrifugation on 10% Percoll, washing in sdd H20 and extraction in sodium dodecyl sulphate (SDS) and 2-mercaptoethanol (2-ME) as described below. Electron microscopy Samples of unembryonated eggs (treated as described below, or untreated) were pelleted in Eppendorf tubes at 1000g and fixed for transmission electron microscopy (TEM) in 20 volumes of osmium tetroxide for 1 hr with gentle agitation. Three 10-min washes in Sorenson's buffer (Barta et al., 1987) were followed by three, 5 min washes in sdd H20. Samples were then incubated in 0.5% uranyl acetate overnight, washed in sdd H20 and dehydrated in several changes of ethanol (30, 50, 70, 85, 95%). Following dehydration, the samples were infiltrated by passing through increasing concentrations of Spurr's medium (3:1, 1:1, 1:3, 100% ethanol to Spurr's) (Barta et al., 1987). Specimens were embedded in Spurr's, hardened by baking in a 70°C oven for 8 hr, cut in sections from 60 to 90/~m on a Sorvall microtome and mounted on HX 200 copper grids. Grids were examined using a Phillips EM 200 electron microscope.
Eggs examined by TEM included the following treatment groups: (1) untreated unembryonated eggs, (2) proteasetreated unembryonated eggs, (3) collagenase-treated unembryonated eggs, (4) chitinase-treated unembryonated eggs, (5) chloroform/methanol extracted unembryonated eggs and (6) hatched eggshells. Enzymatic treatments were carried out for 1 hr at 37°C followed by fixation in Spurr's medium. All enzymes were used at a concentration of 10 U enzyme/25/~l egg extract. In some cases, chitinase was mixed with a protease inhibitor cocktail including 10 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulphonyl fluoride (PMSF) (Sigma, St. Louis, Missouri), 1/~M pepstatin, 100#M antipain and 10mM aprotinin prior to addition to eggs or extracts. Lipid extraction was performed by adding 500/~1 of an equal volume mixture of chloroform and methanol to the whole egg pellet in an Eppendorf tube, vortexing for 1 min and centrifuging at 8000g for 1 min. The organic phase was removed, and the egg pellet remaining was washed three times in phosphate buffered saline (PBS) and fixed for TEM as described above. Unembryonated eggs treated with N-hydroxysuccinimide biotin (NHS-biotin) and sulphosuccinimido biotin (sulphoNHS-biotin) (Pierce, Rockford, Illinois) (Hill et al., 1990) were also fixed according to the above protocol and examined by TEM. SDS PAGE and blotting o f egg extracts
For extraction, 5% sodium dodecyl sulphate (SDS) and 5% 2-mercaptoethanol (2-ME) were added to 0.5ml of biotin-labelled whole eggs or 0.25 ml of purified eggshells, vortexed for I min, boiled for 1 min and the process repeated. Soluble extracts prepared by this treatment were run on denaturing 12% SDS-PAGE gels to determine the presence of and size of any detergent soluble eggshell proteins. Soluble extracts run on similar 12% SDS-PAGE gels were stained with periodic acid Schiff (PAS) stain by the method of Kapitany and Zebrowski (1973) to test for the presence of glycoproteins. Extracts of biotinylated eggs from SDS-PAGE gels were electrophoretically transferred to nitrocellulose membranes, washed sequentially with wash buffer I [WB I, 5% nonfat dry milk in 50 mM Tris (pH 7.5) and 150 mM NaC1], wash buffer II (WB II, containing in addition to the components of WB I, 1.0% Triton X-100 and 0.1% SDS), and WB I and incubated at room temperature with streptavidin-horseradish peroxidase (Bethesda Research Laboratories, Gaithersburg, Maryland) in WB I overnight. After incubation, membranes were washed by the same procedure then developed using 0.18% 4-chloronaphthol and 0.012% H202 in Tris buffered saline (50 mM Tris, 150mM NaC1, pH 7.5). Biotin labelling Intact, unembryonated eggs were surface labelled by incubating with sulpho-NHS-biotin or NHS-biotin (Hill et al., 1990). Sulpho-NHS-biotin and NHS-biotin were prepared in the dark at I0 mg/ml, the former in 10 mM PBS and the latter in dimethylsulphoxide (DMSO), and diluted 1/20 in 10 mM PBS, pH 7.4. Five hundred microlitres of packed fresh eggs were washed three times in PBS, suspended in 2.5 ml of PBS and mixed with 25 ~1 of either sulpho-NHS-biotin or NHS-biotin. This mixture was incubated at room temperature for I0 min. Labelled eggs were pelleted in an Eppendorf centrifuge at 8000 g and washed three times in sterile PBS. In one experiment, NHS-biotintreated eggs were fixed for TEM by techniques described previously, sectioned and the sections incubated with strepavidin-immunogold. These sections were then observed for gold foci. Enzymatic digestion Extracts of biotinylated eggs were tested for sensitivity to chitinase (Sigma, St. Louis, Missouri), lipase, collagenase and proteinase K. NHS-biotinylated extracts were dialysed
Haemonchus contortus eggshell characterization against sdd H20 to remove SDS and 2-ME and divided into four groups for exposure to the enzymes. Extracts were incubated with enzymes for I hr at 37°C. All enzymes were used at a concentration of 10 U enzyme/25/d of extracted material. Digested extracts were analysed by SDS--PAGE, blotted to nitrocellulose, incobated with streptavidin-HRP and developed as above to determine the effects of enzyme treatment. In control experiments, proteolytic activity present in the ehitinase enzyme was found to be inhibited by a cocktail of protease inhibitors including 10mM EDTA, I mM PMSF, I/~M pepstatin, 100/~M antipain and I0 mM aprotinin. The inhibitor cocktail was used in subsequent chitinase digests to block nonspecific proteolytic activity in ehitinase enzyme preparations. RESULTS
Electron microscopy and enzymatic digestion Unembryonated eggs, recovered from the faeces of infected source sheep were purified, exposed to a
683
series of treatments and examined using the electron microscope. Shells of intact, unembryonated eggs had three layers including a basal electron dense layer, an extensive medial amorphous cortical layer and an outer dense layer bounded by a thin trilaminate membrane (Fig. 1). The basal layer was the most dense of the three layers with little discernible structure. This layer varied considerably in thickness. The medial layer had distinctive electron dense fibrillar components interspersed throughout an area that, although occupying the greatest volume of the three layers, was the least dense. The outer layer was bounded by a trilaminate membrane and was closely adherent to the cortical layer (Fig. 2a). Enzyme treatments selectively affected the three layers of the eggshell (Fig. 2). Protease treatment affected all three layers. The vitelline membrane was disrupted, and the trilaminate layers obliterated, indicating that this layer was largely composed of
Fig. 1. Transmission electron micrograph of the eggshell of Haemonchus contortus showing the basic layers including the outer trilaminate vitelline layer (v), the medial chitinous layer (ch), the basal lipid/protein layer (1), the embryonic membrane (em) and a cross-section of the embryo.
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protein. Some components of the medial and basal areas were removed, causing a decrease in density. The cortical area appeared less dense with clumping of the electron dense fibrous strands. The cortical stroma or matrix seemed to be the missing element. The basal layer appeared somewhat thinner and condensed and in some cases was absent (Fig. 2b). Collagenase treatment had little detectable effect on the eggshell (Fig. 2c). Chitinase treatment depleted components of the medial layer, selectively removing the electron dense fibrous strands present in the cortical area (Fig. 2d). Chloroform/methanol extraction had little or no effect on the cortical layer of the eggshell, but caused a minor condensation of the vitelline layer and some depletion of the basal layer (Fig. 2e). Eggshells examined following hatching had more pronounced changes in structure than any of the
ii ~ ii
iii
I
2
3
4
2 0 0 K --,,,-
116K-,-92K--~ 66K---~
45K-~
Fig. 3. SDS-PAGE separation of detergent (5% SDS, 5% 2-ME) soluble extracts of eggshells of fresh eggs of Haemonchus contortus. Low molecular weight standards (Lane I), high molecular weight standards (Lane 2), 12.25/z g of detergent soluble extract of fresh eggshells (Lane 3), 6.12/z g of detergent soluble extract of hatched eggshells (Lane 4). enzyme-treated groups. Hatched eggshells showed similarities to shells digested with both protease and chitinase. There was extensive depletion of the chitin/protein complexes of the medial layer leaving a uniform amorphous matrix and disruption of the bounding trilaminate membrane. Additionally, the basal lipid layer was largely removed by the normal hatching process (Fig. 2f). Transmission electron micrographs of biotinlabelled eggs showed that sulpho-NHS-biotin or NHS-biotin bound to the eggshell at low levels. When streptavidin-immunogold was used to stain biotintreated eggs of the parasite, few gold foci were found, and these were dispersed randomly throughout the three layers of the eggshell (data not shown). S D S - P A G E o f eggshell proteins Three major protein bands were seen on Coomassie Blue and silver stained S D S - P A G E gels of extracts of whole unembryonated eggs. These bands migrated at approximate molecular weights (MW) of 186, 132 and 89 kDa; many minor protein bands were also evident (Fig. 3). Biotinylated eggshell extracts also showed three major bands and some minor bands which corresponded in MW to Coomassie Blue stained bands (Fig. 4). Extracts of eggshells of H. contortus following hatching showed the same three major protein bands seen in extracts of unembryonated eggs (Fig. 3). Only a single high M W band ( > 2 0 0 k D a ) stained with PAS (Fig. 4).
Fig. 2. Transmission electron micrographs of the eggshell of Haemonchus contortus after various treatment regimens. (a) Untreated control eggshells. (b) Protease-treated eggshells. (c) Collagenase-treated eggshells. (d) Chitinase-treated eggshells. (e) Chloroform/methanol-treated eggshells. (f) Naturally hatched eggshells.
Biotinylation and enzymatic digestion o f eggs Sulpho-NHS-biotin and NHS-biotin treatment resuited in low level labelling of H. contortus eggshells. NHS-biotin penetrated the eggshell more effectively than sulpho-NHS-biotin, as judged by heavier banding patterns on S D S - P A G E gels (data not shown)
Haemonchua contortus eggshell characterization
I
2
3
4
5
200K 116K 66K 45K
31K
Fig. 4. SDS-PAGE analysis of detergent (5% SDS, 5% 2-ME) soluble protein extracts of NHS-biotin-labelled whole eggs of Haemonchus contortus. High molecular weight standards (Lane 1), low molecular weight standards (Lane 2), Coomassie stained soluble extract of biotin-labelled whole fresh eggs (Lane 3), PAS stained soluble extract of biotin-labelled whole fresh eggs (Lane 4), streptavidin-horse radish peroxidase stained blot of SDS-PAGE of soluble extract of biotin-labelled whole fresh eggs. and a greater number of gold particles on strepavidin-immunogold stained transmission electron micrographs. The three major protein species seen on these blots ran at the same size as those seen in Coomassie/silver stained gels. Proteinase K and chitinas¢ digestion completely removed the three major and all the minor eggshell protein bands (Fig. 5). Addition of proteolytic inhibitor cocktail to chitinase digests of eggshell extracts only partially inhibited this digestion. Lipase and collagenase had no apparent effect on the biotinylated eggshell protein bands (Fig. 5).
I
2
3
4
5
6
200K --~ 116K
Fig. 5. SDS-PAGE analysis of detergent (5% SDS, 5% 2-ME) soluble protein extracts of NHS-biotin-labelled eggshells of Haemonchus contortus. Control--untreated whole eggs (Lane 1), chitinase-digested whole eggs (Lane 2), chitinase-digested whole eggs with inhibitor cocktail (Lane 3), llpase-digested whole eggs (Lane 4), eollagenase-digested whole eggs (Lane 5), and proteinase K-digested whole eggs (Lane 6).
685 DISCUSSION
Based on these studies, the structure of the eggshell of H. contortus appears to be similar to that described for other nematodes. Electron microscopy revealed the presence of three basic structural layers described previously (Foor, 1967; Kaulenaus and Fairbairn, 1968; Bird, 1968) including an inner electron dense layer, an extensive amorphous middle layer and an outer thin trilaminate bounding membrane. No uterine layer, as described for Ascaris spp. (Croll, 1976), was seen in H. contortus. The outermost layer of the eggshell appeared as a thin trilaminate membrane reminiscent of, but thicker than, a cell membrane. Based on the enzyme degradation and lipid extraction studies, it was composed largely of protein and lipid. The cortical or medial layer of the eggshell of H. contortus was the thickest layer and appeared to be composed of electron dense fibrils in a randomly oriented, regular distribution. These fibrillar complexes were surrounded by a less dense matrix. Subtraction studies, using enzymes to selectively remove elements of the cortical area, suggested that this layer may derive its uniform matrix appearance from a random association of chitinous fibrils surrounded by protein. This protein may coat the chitin fibrils in an arrangement similar to Trichuris spp. (Wharton, 1980). This arrangement of chitin, in some type of covalent linkage, may provide a degree of resistance to chemical attack, as suggested by Wharton (1980). The basal layer of the eggshell is a matrix made up of lipid in some association with protein, similar to the findings of Wharton (1983). This layer varied in thickness along the length of the shell and was electron dense with a randomly oriented, regularly distributed matrix. Lipid extraction procedures merely depleted, but did not entirely remove, the basal layer of the eggshell. This layer was the only area noticeably affected by this treatment, and the major effect was to cause a condensation, like a collapse of a proteinaceous matrix. Proteinase K treatment also affected this layer causing a similar condensation without removing the entire structure. Natural hatching more completely depleted this layer suggesting that both lipid and protein components are reduced during the hatching process. Isolated components of the eggshell of H. eontortus were also sensitive to the action of enzymes. Three major proteins were detected in extracts of the eggshell with relative masses of 186, 132 and 89 kDa, as determined by Coomassie staining of SDS-PAGE gels and biotin labelling. These major bands were degraded by either proteinase K or chitinase digestion but not by lipase or collagenase treatment of extracts of biotinylated eggshells. Similarly, collagenase had no apparent effect on in situ components of the eggshell as determined by TEM. Based on these results, it appears that collagenous proteins play little if any role in maintaining the structure of the eggshell in H. contortus. This is unlike the adult nematode cuticle in which collagen comprises 80% of the cuticular proteins (Fetterer and Urban, 1988; Kingston, 1991). When observed using TEM, the natural hatching process produced structural changes in the eggshell
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similar to those produced by enzyme treatments (chitinase/proteinase K). Hatching depleted structural components of all three layers of the eggshell as observed at the TEM level. A difference in the density of the eggshells, another measure of enzyme depletion, was also observed after hatching. Hatched eggshells floated on 10% Percoll while fresh eggshells floated on 20% Percoll (Gamble and Mansfield, 1992). Another interesting finding of these studies is that the eggshell of H. contortus may be impermeable to biotin or may have few available binding sites for the biotin molecule. Both forms of biotin used in this study react with primary amines to form amide bonds. Neither sulpho-NHS-biotin, which is soluble in aqueous environments, or NHS-biotin, which is soluble in organic solvents, bound well to the eggshell when viewed by TEM analysis and staining with avidin-gold particles. There were a few sites of labelling in the eggshell, but gold foci were in low quantity and were dispersed randomly throughout the three layers of the eggshell. In contrast, the cuticles of Ascaris suum larvae (Hill et al., 1990) and Onchocerca linealis microfilariae (Hill et aL, 1992) are strongly labelled by NHS-biotin. The minimal binding of biotin to eggshell proteins in H. contortus may also reflect structural differences between the eggshell and the cuticle. The studies presented here indicate that the eggshell of Haemonchus contortus is structurally similar to eggs of other nematodes. Electron microscopic and enzymatic degradation studies of the eggshell also support claims in H. contortus (Rogers and Brooks, 1977) and in other nematode species (Bone and Parish, 1988; Perry, 1989) that the hatching process may occur as a result of enzymatic events. Further work is needed to relate the structural findings in this study to the hatching process. Acknowledgements--The authors would like to acknowledge the expert technical assistance of Ms Jean Corbin, Ms Eleanor E. Moore and Mr James L. McCrary.
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Barta J. R., Boulard Y. and Desser S. S. (1987) Ultrastructural observations on secondary merogony and
gametogony of Dactylosoma ranarum Labbe, 1894 (Eucoccidiida; Apicomplexa).J. Parasitol. 73, 1019-1029. Bird A. F. (1968) Changes associated with parasitism in nematodes. III. Ultrastructure of the eggshell, larval cuticle, and contents of the subventral oesophageal glands in Meloidogyne javanica, with some observations on hatching. J. Parasitol. 54, 699-719. Bird A. F. (1971) The Structure of Nematodes, pp. 114-137. Academic Press, New York. Bone L. W. and Parish E. J. (1988) Egg enzymes of the ruminant nematode Trichostrongylus colubriformis. Invert. Reprod. Develop. 14, 299-302. Croll N. A. (1976) The Organization of Nematodes, pp. 279-300. Academic Press, New York. Fetterer R. H. and Urban J. F., Jr (1988) Developmental changes in cuticular proteins of Ascaris suum. Comp. Biochem. Physiol. 90B, 321-327. Foor W. E. (1967) Ultrastructural aspects of oocyte development and shell formation in Ascaris lumbricoides. J. ParasitoL 53, 1245-1261. Gamble H. R. and Mansfield L. S. (1992) Purification of egg shells from Haemonchus contortus. J. Helminthol. Soc. Wash. 59, 234-236. Hill D. E., Fetterer R. H. and Urban J. F., Jr (1990) Biotin as a probe of the surface of Ascaris suum developmental stages. Molec. Biochem. Parasitol. 41, 45-52. Hill D. E., Mahin Khapami, Lok J. B. and Rocky J. H. (1992) Onchocerca linealis: Surface glycolipid masking cuticular antigens of Onchocerca microfilariae. Exp. Parasitol. (In press). Kapitany R. A. and Zebrowski E. J. (1973) High resolution PAS Stain for polyacrylamide gel electrophoresis. Analyt. Biochem. 56, 361-369. Kaulenaus M. S. and Fairbairn D. (1968) RNA metabolism of fertilized Ascaris lumbricoides eggs during uterine development. Exp. Cell Res. 52, 233. Kingston I. B. (1991) Nematode collagen genes. Parasitol. Today 7(1), 11-14. Neville A. C. (1975) Biology of the Arthropod Cuticle, pp. 1-250. Springer, Berlin, Germany. Perry R. N. (1989) Dormancy and hatching of nematode eggs. Parasitol. Today 5(12), 377-383. Rogers W. P. and Brooks F. (1977) The mechanism of hatching of eggs of Haemonchus contortus. Int. J. Parasitol. 7, 61 65. Wharton D. A. (1979) Oogenesis and eggshell formation in Aspiculuris tetraptera Schulz (Nematoda: Oxyuroidea). Parasitology 78, 131-143. Wharton D. A. (1980) Nematode egg-shells. Parasitology 81, 447-463. Wharton D. A. (1983) The production and functional morphology of helminth egg-shells. Parasitology 86, 85-97.
