- Email: [email protected]
Ascaris lumbricoides suum: Morphological characterization of apparent cuticular pores by ionic permeability and electron microscopy
EWERIMENTALPARASITOLOGY63,
329-336(1987)
Ascaris lumbricoides sum: Morphological Characterization Apparent Cuticular Pores by Ionic Permeability and Electron Microscopy REX
of
E. MARTIN, L. ANN FOSTER, A. STEPHEN KESTER, AND MANUS J. DONAHUE
Department
of Biological
Sciences, North Texas State University,
Denton, Texas 76203, U.S.A
(Accepted for publication 12 November 1986) MARTIN, R. E., FOSTER, L. A., KESTER, A. S., AND DONAHUE, M. J. 1987. Ascaris lumbricoides suum: Morphological characterization of apparent cuticular pores by ionic permeability and electron microscopy. Experimental Parasitology 63, 329-336. The cuticle of the parasitic roundworm Ascaris lumbricoides suum has been found to contain apparent
pores, using scanning and transmission electron microscopy. However, X-ray spectral analysis and dot mapping analysis of diffusing divalent cations in the cuticle found these ions to be randomly distributed on the pseudocoelomic surface of the cuticle. This indicates that the pores seen with the electron microscope were not true structural pores and that the 0 1987 Academic Press, h. cuticle is homogeneously permeable to ions. INDEX DESCRIPTORSAND ABBREVIATIONS: Ascaris lumbricoides suum; Nematode, parasitic; Cuticle; Electron microscopy; Pores; Ionic permeability; Scanning electron microscopy (SEM); Transmission electron microscopy (TEM).
INTRODUCTION
The cuticle of Ascaris lumbricoides suum is divided into three layers, all of which may have subdivisions (Bird 1958). The cuticle of A. 1. suum has been shown to be impermeable to glucose (Castro and Fairbairn 1969) although Fleming and Fetterer (1984) have suggested more recently that the cuticle may be the site of absorption of cholesterol and glucose. Other recent data have suggested that the cuticle may be permeable to amino .acids because an important enzyme in the y-glutamyl cycle (Orlowski and Meister 1970),namely, y-glutamyl transpeptidase (EC 2.3.2.2), has been demonstrated in the cuticle-hypodermis sections of this worm (Dass and Donahue 1986). This y-glutamyl cycle has been linked to amino acid transport in some mammalian systems (Sekura and Meister 1977). Pores have been observed in the cuticle of A. 1. suum (Bird and Bird 1969)but the function of these pores is unknown.
This paper investigates whether or not organized structural pores (Singer and Nicholson 1972) are present in the cuticle of A. 1. suum and whether or not the cuticle is permeable to divalent cations. The data suggest that the cuticle does not have structurally organized pores but is permeable homogeneously to barium, iron, and copper, and may be a possible site for amino acid transport in A. 1. suum. MATERIALS
AND METHODS
Female Ascaris lumbricoides suum were collected at a slaughterhouse and transported to the laboratory in a salt solution (Donahue et al. 1981a).The worms were maintained in the laboratory at 37 C in a buffered salt solution (A. 1. suum saline) as described previously (Donahue et al. 1981b)and constantly gassed with 95% N, and 5% CO,. Adult female parasites that were 25-30 cm in length were used in these experiments. The worms (female) were cut into pieces and incubated overnight in 40% formaldehyde. The pieces were embedded in Histo Prep (frozen tissue embedding medium) and 20 to 25 Frn sections from each piece were cut in a precooled (-20 C) Cryo-cut mi-
329 0014-4894/87$3.00 Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
330
MARTIN ET AL.
crotome (Model 840 from American Optical). The frozen sections were mounted on pregelatinized glass slides treated with alcohol and xylene and stained in 0.5% aqueous solution of crystal violet at 50-60 C for 3 min. Black and white photographs (using 32 ASA panatomic-X-film) were taken (Dury and Wallington 1980). The apparent pores in the cuticle seen in the light micrographs were examined using a JEOL JSM T-300 scanning electron microscope at an accelerating voltage of 20 KeV. The cuticle was prepared as described previously (Donahue et al. 1983). Briefly, female worms were removed from the holding vessel and dissected longitudinally. The reproductive and digestive tracts were removed. The cuticle layer was
separated from the muscle layer by scraping the muscle layer from the underlying cuticle with a glass slide. Cuticle treated for scanning electron microscopy was fixed with 3% glutaraldehyde in A. !. suum saline for 2 hr. Then, the tissue was dehydrated by serial transfer in ethanol:water mixtures (30:70, 50:50, 70:30,90:10, and two changes in 100% anhydrous ethanol). The tissue was allowed to stand in each mixture for 15-20 min before transfer to the next mixture. Dehydrated cuticle was critical point dried with CO, in a Bomar SPC-900/EX ciritical point dryer, mounted with colloidal carbon or conductive silver paint, and coated with gold using a SPI-module sputter coater. Unfixed specimens to be analyzed by X-ray micro-
FIG. 1. Light micrograph of cross section of Ascaris lumbricoides suum taken from midgut region; C is the cuticle and M is the muscle of the worm; magnification is 100x .
Ascaris lumbricoides sum: CUTICULAR PORES analysis were lyophilized and mounted with colloidal carbon and carbon coated in a JEOL, JEE-4X vacuum evaporator. To examine the apparent pores of the SEM at the ultrastructural level, the TEM was used. The cuticle was separated from muscle as described previously and cut into 5 to 10 mm sections under a solution containing paraformaldehyde powder, 1.5 g; distilled water 25 ml; sodium hydroxide 1 M; 5 drops glutaraldehyde (25%) and 25 ml A. 1. suum saline (Harpur 1963). The paraformaldehyde was dissolved in water at 60 C and the solution was cleared with the sodium hydroxide. After cooling, the other ingredients were added; the final pH was 7.2. After dissecting the cuticle in this fixative, it was transferred to another fixation solution containing 5 ml glutaraldehyde (25%) in distilled water, and 20 ml A. 1. suutn saline. The sections were fixed in this solution overnight at 4 C, after which they were fixed in 2% osmium tetroxide in A. 1.SUWI saline for 2 hr. This procedure for fixation was a modification of the one reported by Hayat (1981). After fixation, the specimens were dried in acetone:water mixtures (30:50, 50:50, 70:30, 90:10, and two changes in 100%
331
anhydrous acetone) and flat embedded in Luft’s Epon 812 (Luft 1961). Thin sections (60-80 nm) of embedded specimens were cut with a diamond knife and a Sorvall MT-6000 ultramicrotome, mounted on 50 mesh Formvar coated grids, and stained with uranyl acetate and lead citrate. Stained specimens were observed on a JEOL 1OOCX TEM at an accelerating voltage of 60 KeV. The question of whether or not these apparent pores were real was examined using X-ray spectral analysis. The cuticle was removed and prepared as described under SEM. The cuticle was scraped with a glass slide to assure that no hypodermis or cellular debris adhered to the cuticle. The cuticle was then transported to a chamber (wax chuck) and oriented in the chamber so that the position of the cuticle was known, i.e., the outside of the cuticle faced the investigator. Samples of 100 m.U BaCl,, CuCl,, and FeCl, in double distilled water were placed on the cuticle and allowed to move to the inside of the cuticle. These ions were selected because they demonstrated the least signal overlap using the X-ray spectral analysis technique. The wax chuck was so positioned that no diffusion could occur
FIG. 2. Scanning electron micrograph showing the outside of the cuticle (OC) of Ascaris hmbricoides SUM and a side view showing the middle cuticle (MC) layers of the cuticle. Bar represents 10 w.
FIG. 3. Scanning electron micrograph showing the pseudocoelomic side of the cuticle (PC) from lumbricoides suum. Part of the cuticle has been scraped free of cellular debris exposing apparent pores (p). Note also the cellular debris (D) that remains attached to the cuticle if the surface is not fully scraped. Bar represents 10 pm.
Ascaris
FIG. 4. Transmission electron micrograph of the cuticle (C) from Ascaris iumbricoides suum. The (P) represents the apparent pore which partially transverses the cuticle. Bar represents 5 pm. 332
Ascaris lumbricoides SUU~:CUTICULARPORES around the cuticle. This was checked by using a high molecular weight impermeable dye (Blue Dextran) and the dye was never observed on the opposite side of the chamber even if the preparation was left overnight. At the end of the experiment, the excess diffusing ion was removed with filter paper. After incu-
I
333
bation, the chamber and samples (cuticle) were then dried by lyophilization. The sections were mounted on carbon coated metal stubs and carbon coated in a JEOL Model vacuum evaporator for 7 set at 40 A with a vacuum of 2 x 10m4urn. The samples were then examined with the SEM equipped with a Tracer
S
:
control-A
;
: 5:
: 10 min-B
Emission
j
energy (K ev)
FIG. 5. The X-ray spectral analysis of the pseudocoelomic side of the cuticle from Ascaris lumbric&es suum. The scraped and cleaned cuticle was incubated for various time periods with 100 mM BaCl, on the outside of the cuticle. The cuticle was lyophilized, and the sections were then mounted on carbon coated aluminum stubs and carbon coated in JEOL vacuum evaporator and examined by scanning electron micrograph. (A) Control cuticle; (b), (c), and (d) sections incubated for 10, 30, and 60 min, respectively. The different barium peaks correspond to different emission energy spectra from a barium atom. The total amount of barium diffusing through the cuticle is represented by the total of all the barium energy peaks. Na, sodium; P, phosphorus; S, sulfur; Cl, chloride; K, potassium; and Ba, barium.
MARTIN ET AL.
334
Northern TN 5500 X-ray analyzer. This allows for X-ray spectral analysis of the elemental composition of the specimen detecting any elements with atomic numbers greater than sodium (atomic number 11). For elemental analysis, an accelerating voltage of 20 KeV and a count time of 30 set were used. Dot mapping analysis is a SEM technique that allows for localization of regions in the specimen containing the ions of interest (barium, copper, or iron) which were identified by X-ray spectral analysis; a Tracer Northern Hardware Disc interfaced with the Tracer Computer and SEM performs this analysis. RESULTS
Light microscopic analysis of the cuticle of Ascaris lumbricoides suum revealed an outer covering approximately 2-3 pm thick that is a complex structure made up of several layers. A partial cross-sectional view of A. 1. suum in the midgut of the worm is seen in Fig. 1. Scanning electron microscopy revealed a much convoluted outer surface of the cuticle (Fig. 2). When the cuticle was observed from the side showing the various layers of the cuticle (Fig. 2), a complex structure is apparent. When the cuticle is observed from the pseudocoelomic surface (basal surface of cuticle) after the hypodermis layer and muscle layer were scraped off, numerous structures which appear to be pores are observed. These pore structures may allow passage of nutrients through the cuticle to the hypodermis. Figure 3 is a scanning electron micrograph showing the pseudocoelomic surface of the cuticle. Part of the picture shows the pseudocoelomic surface of cuticle where the hypodermis and cellular debris are partially attached. Transmission electron microscopy was used to examine these structures more closely. The porelike structures appeared to pass from the grooves on the outer surface of the cuticle at least partially through the cuticle and possibly on to the basal side of the cuticle (Fig. 4). These structures did not have a clear channel but appeared to be tilled with a fibrous material. To further examine the nature of these
porelike structures in the cuticle, segments of cuticle were incubated with 100 mA4 BaCl, for various time periods. After incubation, the BaCl, was removed from the outside of the cuticle, and the cuticle was examined on the pseudocoelomic surface using SEM-X-ray analysis to quantitate the amount of barium which crossed through the cuticle (Fig. 5). The barium was found to accumulate on the pseudocoelomic surface of the cuticle at a linear rate (Table I). The earliest time that the cations could be detected on the pseudocoelomic surface of the cuticle was 8 min after the ions were added to the outer surface of the cuticle. Using the SEM-dot mapping technique, it was possible to localize where the barium was penetrating the A. 1. suum cuticle. For example, if the barium was penetrating through structurally organized pores in the cuticle, then the barium would be concentrated in these areas alone. If no structurally organized pores were present, and barium was diffusing through all of the cuticle at random, a homogeneous diffusion patterns for barium should be observed. Figure 6 is an SEM dot map of barium on the pseudocoelomic surface of the cuticle. TABLE 1 Movement of Barium across Cuticle of Ascaris lumbricoides
suum
Time (mitt)
Total barium movement intensity
0 0.5 2 4 6 8 10 30 60
0 0 0 0 0 601 3904 6619 9093
Note. Cuticle samples were incubated with 100 mM BaCI, for various times. Their X-ray spectral analysis was performed using scanning electron micrograph JEOL JSM-T-300 to determine the amount of barium that diffused across cuticle.
Ascaris lumbricoides suum: CUTICULARPORES
335
FIG. 6. A dot map analysis of barium taken from the pseudocoelomic side of a female Ascaris lumbricoides suum after it had been exposed to 100mM BaCl, on the outside surface for 1 hr. The dots
represent individual quanta of barium energy. Note that the barium is distributed homogeneously on the SEM photomicrograph. The bar represents 100 pm.
The barium which diffused through the cuticle was randomly distributed. Similar results were obtained for iron and copper (data not shown). Random diffusion was also observed when the cations were placed on the basal surface of the cuticle and allowed to diffuse from the basal side of the cuticle to the outside surface of the cuticle and also at all time points from 8 to 60 min. DISCUSSION Light micrographs, scanning electron micrographs, and transmission electron micrographs have indicated that the cuticle of Ascaris lumbricoides suum may contain structural pores which could function in absorption or excretion. However, X-ray
analysis of diffusing divalent cations through the cuticle of A. 1. suum clearly demonstrated that ions accumulated on the pseudocoelomic surface of the cuticle at linear rates up to 1 hr. This is good evidence that the cuticle is permeable to ions and that their transport was due to simple diffusion. X-ray mapping allowed for visualization of the location of the diffusing ions in the cuticle. The ions were found to be ubiquitously present throughout the cuticle which indicated that there were no organized structural pores and that the cuticle may act as a selective barrier to diffusion functioning similarly to the basal lamella in the intestinal tract of this worm (Donahue et al. 1983).
MARTIN ET AL.
336
The apparent pores were seen in the electron micrograph (Fig. 3). These same pores would be approximately 10 km apart in the dot map picture seen in Fig. 6. This indicated that the apparent pores of the TEM picture are too few to account for all the ions represented by dots in Fig. 6. Also, if the divalent cations were diffusing through pores and then diffusing laterally on the pseudocoelomic surface of the cuticle, areas of high concentration of dots should be seen at approximately 10 km intervals in Fig. 6. None were observed. To further corroborate that pores were not present, the X-ray map taken at 8 min was very similar to the 10 min X-ray map in Fig. 6. This indicated that even at the earliest possible point of detecting the diffusing ions (8 min), they were homogeneously distributed on the pseudocoelomic surface of the cuticle. Collectively, the data indicate that the cuticle of Ascaris lumbricoides suum is permeable to ions and that the cuticle may be acting as a selective barrier to the transport of ions and other nutrients, possibly cholesterol (Fleming and Fetterer 1984) and amino acids (Dass and Donahue 1986), into this parasite but the pores of the cuticle do not appear to be the transport site. ACKNOWLEDGMENTS This work was supported by Grant AI22479 from the U.S. National Institutes of Health and Organized Research Funds from North Texas State University. We thank Jayanta Chaudhuri for technical assistance. REFERENCES BIRD, ture BIRD, and
A. E 1958. Further observations on the strucof nematode cuticle. Parasitology 48, 32-37. A. F., AND BIRD, .I. 1969. Skeletal structures integument of Acanthocephala and Nematoda.
In “Chemical Zoology” (M. Florkin, and B. .I. Scheer ) 7.Vol. III, pp. 253-288. Academic _ J,Eds_.ITeSS, NeW YOt’K. CASTRO,G. A., AND FAIRBAIRN D. 1969. Comparison of cuticular and intestinal absorption of glucose by adult Ascaris lumbricoides. Journal of Parasitology 55, 14-16. DASS, P. D., AND DONAHUE, M. J. 1986. y-Glutamyl transpeptidase activity in Ascaris suum. Molecular and Biochemical
Parasitology
20, 233-236.
DONAHUE, M. J., BEAMES, C. G., AND BOST, K. S. 1983. Permeability characteristics of the basement membrane (basal lamella) of the intestine of Ascaris suum. Journal of Biological Physics 11, 1l- 13. DONAHUE, M. J., YACOUB, N. J., KAEINI, M. R., AND HARRIS, B. G. 1981a. Activity of enzymes regulating glycogen metabolism in perfused musclecuticle sections of Ascaris suum (Nematoda). Journal
of Parasitology
67, 362-367.
DONAHUE, M. J., YACOUB, N. J., KAEINI, M. R., MASARACCHIA, R. A., AND HARRIS, B. G. 1981b. Glycogen metabolizing enzymes during starvation and feeding of Ascaris suum maintained in a perfusion chamber. Journal of Parasitology 67, 505-510. DURY, R. A. B., AND WALLINGTON, E. A. 1980. “Carleton’s Histological Technique,” pp. 132- 135. Oxford Univ. Press, Fair Lawn, NJ. FLEMING, M. W., AND FETTERER, R. H. 1984. Ascaris suum: Continuous perfusion of the Pseudocoelom and nutrient absorption. Experimental Parasitology 57, 142-148. HARPUR, R. 1963. Maintenance of Ascaris lumbricoides in vitro. III. Changes in the hydrostatic skeleton. Comparative Biochemistry and Physiology 13, 71-85. HAYAT, M. A. 1981. “Fixation for Electron Microscopy,” pp. 402. Academic Press, New York. Lum, H. J. 1961. Improvements in epoxy resin embedding methods. Journal of Biophysical and Biochemical
Cytology
9,409-414.
ORLOWSKI, M., AND MEISTER, A. 1970. The y-glutamyl cycle: A possible transport system for amino acids. Proceedings of the National Academy of Science USA 67, 1248-1255. SEKURA, R., AND MEISTER, A. 1977. y-Glutamylcystein synthetase. Journal of Biological Chemistry 252, 2599-2605. SINGER, S. J., AND NICOLSON, G. L. 1972. The fluid mosaic model of the structure of cell membranes. Science 175, 720-731.
329-336(1987)
Ascaris lumbricoides sum: Morphological Characterization Apparent Cuticular Pores by Ionic Permeability and Electron Microscopy REX
of
E. MARTIN, L. ANN FOSTER, A. STEPHEN KESTER, AND MANUS J. DONAHUE
Department
of Biological
Sciences, North Texas State University,
Denton, Texas 76203, U.S.A
(Accepted for publication 12 November 1986) MARTIN, R. E., FOSTER, L. A., KESTER, A. S., AND DONAHUE, M. J. 1987. Ascaris lumbricoides suum: Morphological characterization of apparent cuticular pores by ionic permeability and electron microscopy. Experimental Parasitology 63, 329-336. The cuticle of the parasitic roundworm Ascaris lumbricoides suum has been found to contain apparent
pores, using scanning and transmission electron microscopy. However, X-ray spectral analysis and dot mapping analysis of diffusing divalent cations in the cuticle found these ions to be randomly distributed on the pseudocoelomic surface of the cuticle. This indicates that the pores seen with the electron microscope were not true structural pores and that the 0 1987 Academic Press, h. cuticle is homogeneously permeable to ions. INDEX DESCRIPTORSAND ABBREVIATIONS: Ascaris lumbricoides suum; Nematode, parasitic; Cuticle; Electron microscopy; Pores; Ionic permeability; Scanning electron microscopy (SEM); Transmission electron microscopy (TEM).
INTRODUCTION
The cuticle of Ascaris lumbricoides suum is divided into three layers, all of which may have subdivisions (Bird 1958). The cuticle of A. 1. suum has been shown to be impermeable to glucose (Castro and Fairbairn 1969) although Fleming and Fetterer (1984) have suggested more recently that the cuticle may be the site of absorption of cholesterol and glucose. Other recent data have suggested that the cuticle may be permeable to amino .acids because an important enzyme in the y-glutamyl cycle (Orlowski and Meister 1970),namely, y-glutamyl transpeptidase (EC 2.3.2.2), has been demonstrated in the cuticle-hypodermis sections of this worm (Dass and Donahue 1986). This y-glutamyl cycle has been linked to amino acid transport in some mammalian systems (Sekura and Meister 1977). Pores have been observed in the cuticle of A. 1. suum (Bird and Bird 1969)but the function of these pores is unknown.
This paper investigates whether or not organized structural pores (Singer and Nicholson 1972) are present in the cuticle of A. 1. suum and whether or not the cuticle is permeable to divalent cations. The data suggest that the cuticle does not have structurally organized pores but is permeable homogeneously to barium, iron, and copper, and may be a possible site for amino acid transport in A. 1. suum. MATERIALS
AND METHODS
Female Ascaris lumbricoides suum were collected at a slaughterhouse and transported to the laboratory in a salt solution (Donahue et al. 1981a).The worms were maintained in the laboratory at 37 C in a buffered salt solution (A. 1. suum saline) as described previously (Donahue et al. 1981b)and constantly gassed with 95% N, and 5% CO,. Adult female parasites that were 25-30 cm in length were used in these experiments. The worms (female) were cut into pieces and incubated overnight in 40% formaldehyde. The pieces were embedded in Histo Prep (frozen tissue embedding medium) and 20 to 25 Frn sections from each piece were cut in a precooled (-20 C) Cryo-cut mi-
329 0014-4894/87$3.00 Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
330
MARTIN ET AL.
crotome (Model 840 from American Optical). The frozen sections were mounted on pregelatinized glass slides treated with alcohol and xylene and stained in 0.5% aqueous solution of crystal violet at 50-60 C for 3 min. Black and white photographs (using 32 ASA panatomic-X-film) were taken (Dury and Wallington 1980). The apparent pores in the cuticle seen in the light micrographs were examined using a JEOL JSM T-300 scanning electron microscope at an accelerating voltage of 20 KeV. The cuticle was prepared as described previously (Donahue et al. 1983). Briefly, female worms were removed from the holding vessel and dissected longitudinally. The reproductive and digestive tracts were removed. The cuticle layer was
separated from the muscle layer by scraping the muscle layer from the underlying cuticle with a glass slide. Cuticle treated for scanning electron microscopy was fixed with 3% glutaraldehyde in A. !. suum saline for 2 hr. Then, the tissue was dehydrated by serial transfer in ethanol:water mixtures (30:70, 50:50, 70:30,90:10, and two changes in 100% anhydrous ethanol). The tissue was allowed to stand in each mixture for 15-20 min before transfer to the next mixture. Dehydrated cuticle was critical point dried with CO, in a Bomar SPC-900/EX ciritical point dryer, mounted with colloidal carbon or conductive silver paint, and coated with gold using a SPI-module sputter coater. Unfixed specimens to be analyzed by X-ray micro-
FIG. 1. Light micrograph of cross section of Ascaris lumbricoides suum taken from midgut region; C is the cuticle and M is the muscle of the worm; magnification is 100x .
Ascaris lumbricoides sum: CUTICULAR PORES analysis were lyophilized and mounted with colloidal carbon and carbon coated in a JEOL, JEE-4X vacuum evaporator. To examine the apparent pores of the SEM at the ultrastructural level, the TEM was used. The cuticle was separated from muscle as described previously and cut into 5 to 10 mm sections under a solution containing paraformaldehyde powder, 1.5 g; distilled water 25 ml; sodium hydroxide 1 M; 5 drops glutaraldehyde (25%) and 25 ml A. 1. suum saline (Harpur 1963). The paraformaldehyde was dissolved in water at 60 C and the solution was cleared with the sodium hydroxide. After cooling, the other ingredients were added; the final pH was 7.2. After dissecting the cuticle in this fixative, it was transferred to another fixation solution containing 5 ml glutaraldehyde (25%) in distilled water, and 20 ml A. 1. suutn saline. The sections were fixed in this solution overnight at 4 C, after which they were fixed in 2% osmium tetroxide in A. 1.SUWI saline for 2 hr. This procedure for fixation was a modification of the one reported by Hayat (1981). After fixation, the specimens were dried in acetone:water mixtures (30:50, 50:50, 70:30, 90:10, and two changes in 100%
331
anhydrous acetone) and flat embedded in Luft’s Epon 812 (Luft 1961). Thin sections (60-80 nm) of embedded specimens were cut with a diamond knife and a Sorvall MT-6000 ultramicrotome, mounted on 50 mesh Formvar coated grids, and stained with uranyl acetate and lead citrate. Stained specimens were observed on a JEOL 1OOCX TEM at an accelerating voltage of 60 KeV. The question of whether or not these apparent pores were real was examined using X-ray spectral analysis. The cuticle was removed and prepared as described under SEM. The cuticle was scraped with a glass slide to assure that no hypodermis or cellular debris adhered to the cuticle. The cuticle was then transported to a chamber (wax chuck) and oriented in the chamber so that the position of the cuticle was known, i.e., the outside of the cuticle faced the investigator. Samples of 100 m.U BaCl,, CuCl,, and FeCl, in double distilled water were placed on the cuticle and allowed to move to the inside of the cuticle. These ions were selected because they demonstrated the least signal overlap using the X-ray spectral analysis technique. The wax chuck was so positioned that no diffusion could occur
FIG. 2. Scanning electron micrograph showing the outside of the cuticle (OC) of Ascaris hmbricoides SUM and a side view showing the middle cuticle (MC) layers of the cuticle. Bar represents 10 w.
FIG. 3. Scanning electron micrograph showing the pseudocoelomic side of the cuticle (PC) from lumbricoides suum. Part of the cuticle has been scraped free of cellular debris exposing apparent pores (p). Note also the cellular debris (D) that remains attached to the cuticle if the surface is not fully scraped. Bar represents 10 pm.
Ascaris
FIG. 4. Transmission electron micrograph of the cuticle (C) from Ascaris iumbricoides suum. The (P) represents the apparent pore which partially transverses the cuticle. Bar represents 5 pm. 332
Ascaris lumbricoides SUU~:CUTICULARPORES around the cuticle. This was checked by using a high molecular weight impermeable dye (Blue Dextran) and the dye was never observed on the opposite side of the chamber even if the preparation was left overnight. At the end of the experiment, the excess diffusing ion was removed with filter paper. After incu-
I
333
bation, the chamber and samples (cuticle) were then dried by lyophilization. The sections were mounted on carbon coated metal stubs and carbon coated in a JEOL Model vacuum evaporator for 7 set at 40 A with a vacuum of 2 x 10m4urn. The samples were then examined with the SEM equipped with a Tracer
S
:
control-A
;
: 5:
: 10 min-B
Emission
j
energy (K ev)
FIG. 5. The X-ray spectral analysis of the pseudocoelomic side of the cuticle from Ascaris lumbric&es suum. The scraped and cleaned cuticle was incubated for various time periods with 100 mM BaCl, on the outside of the cuticle. The cuticle was lyophilized, and the sections were then mounted on carbon coated aluminum stubs and carbon coated in JEOL vacuum evaporator and examined by scanning electron micrograph. (A) Control cuticle; (b), (c), and (d) sections incubated for 10, 30, and 60 min, respectively. The different barium peaks correspond to different emission energy spectra from a barium atom. The total amount of barium diffusing through the cuticle is represented by the total of all the barium energy peaks. Na, sodium; P, phosphorus; S, sulfur; Cl, chloride; K, potassium; and Ba, barium.
MARTIN ET AL.
334
Northern TN 5500 X-ray analyzer. This allows for X-ray spectral analysis of the elemental composition of the specimen detecting any elements with atomic numbers greater than sodium (atomic number 11). For elemental analysis, an accelerating voltage of 20 KeV and a count time of 30 set were used. Dot mapping analysis is a SEM technique that allows for localization of regions in the specimen containing the ions of interest (barium, copper, or iron) which were identified by X-ray spectral analysis; a Tracer Northern Hardware Disc interfaced with the Tracer Computer and SEM performs this analysis. RESULTS
Light microscopic analysis of the cuticle of Ascaris lumbricoides suum revealed an outer covering approximately 2-3 pm thick that is a complex structure made up of several layers. A partial cross-sectional view of A. 1. suum in the midgut of the worm is seen in Fig. 1. Scanning electron microscopy revealed a much convoluted outer surface of the cuticle (Fig. 2). When the cuticle was observed from the side showing the various layers of the cuticle (Fig. 2), a complex structure is apparent. When the cuticle is observed from the pseudocoelomic surface (basal surface of cuticle) after the hypodermis layer and muscle layer were scraped off, numerous structures which appear to be pores are observed. These pore structures may allow passage of nutrients through the cuticle to the hypodermis. Figure 3 is a scanning electron micrograph showing the pseudocoelomic surface of the cuticle. Part of the picture shows the pseudocoelomic surface of cuticle where the hypodermis and cellular debris are partially attached. Transmission electron microscopy was used to examine these structures more closely. The porelike structures appeared to pass from the grooves on the outer surface of the cuticle at least partially through the cuticle and possibly on to the basal side of the cuticle (Fig. 4). These structures did not have a clear channel but appeared to be tilled with a fibrous material. To further examine the nature of these
porelike structures in the cuticle, segments of cuticle were incubated with 100 mA4 BaCl, for various time periods. After incubation, the BaCl, was removed from the outside of the cuticle, and the cuticle was examined on the pseudocoelomic surface using SEM-X-ray analysis to quantitate the amount of barium which crossed through the cuticle (Fig. 5). The barium was found to accumulate on the pseudocoelomic surface of the cuticle at a linear rate (Table I). The earliest time that the cations could be detected on the pseudocoelomic surface of the cuticle was 8 min after the ions were added to the outer surface of the cuticle. Using the SEM-dot mapping technique, it was possible to localize where the barium was penetrating the A. 1. suum cuticle. For example, if the barium was penetrating through structurally organized pores in the cuticle, then the barium would be concentrated in these areas alone. If no structurally organized pores were present, and barium was diffusing through all of the cuticle at random, a homogeneous diffusion patterns for barium should be observed. Figure 6 is an SEM dot map of barium on the pseudocoelomic surface of the cuticle. TABLE 1 Movement of Barium across Cuticle of Ascaris lumbricoides
suum
Time (mitt)
Total barium movement intensity
0 0.5 2 4 6 8 10 30 60
0 0 0 0 0 601 3904 6619 9093
Note. Cuticle samples were incubated with 100 mM BaCI, for various times. Their X-ray spectral analysis was performed using scanning electron micrograph JEOL JSM-T-300 to determine the amount of barium that diffused across cuticle.
Ascaris lumbricoides suum: CUTICULARPORES
335
FIG. 6. A dot map analysis of barium taken from the pseudocoelomic side of a female Ascaris lumbricoides suum after it had been exposed to 100mM BaCl, on the outside surface for 1 hr. The dots
represent individual quanta of barium energy. Note that the barium is distributed homogeneously on the SEM photomicrograph. The bar represents 100 pm.
The barium which diffused through the cuticle was randomly distributed. Similar results were obtained for iron and copper (data not shown). Random diffusion was also observed when the cations were placed on the basal surface of the cuticle and allowed to diffuse from the basal side of the cuticle to the outside surface of the cuticle and also at all time points from 8 to 60 min. DISCUSSION Light micrographs, scanning electron micrographs, and transmission electron micrographs have indicated that the cuticle of Ascaris lumbricoides suum may contain structural pores which could function in absorption or excretion. However, X-ray
analysis of diffusing divalent cations through the cuticle of A. 1. suum clearly demonstrated that ions accumulated on the pseudocoelomic surface of the cuticle at linear rates up to 1 hr. This is good evidence that the cuticle is permeable to ions and that their transport was due to simple diffusion. X-ray mapping allowed for visualization of the location of the diffusing ions in the cuticle. The ions were found to be ubiquitously present throughout the cuticle which indicated that there were no organized structural pores and that the cuticle may act as a selective barrier to diffusion functioning similarly to the basal lamella in the intestinal tract of this worm (Donahue et al. 1983).
MARTIN ET AL.
336
The apparent pores were seen in the electron micrograph (Fig. 3). These same pores would be approximately 10 km apart in the dot map picture seen in Fig. 6. This indicated that the apparent pores of the TEM picture are too few to account for all the ions represented by dots in Fig. 6. Also, if the divalent cations were diffusing through pores and then diffusing laterally on the pseudocoelomic surface of the cuticle, areas of high concentration of dots should be seen at approximately 10 km intervals in Fig. 6. None were observed. To further corroborate that pores were not present, the X-ray map taken at 8 min was very similar to the 10 min X-ray map in Fig. 6. This indicated that even at the earliest possible point of detecting the diffusing ions (8 min), they were homogeneously distributed on the pseudocoelomic surface of the cuticle. Collectively, the data indicate that the cuticle of Ascaris lumbricoides suum is permeable to ions and that the cuticle may be acting as a selective barrier to the transport of ions and other nutrients, possibly cholesterol (Fleming and Fetterer 1984) and amino acids (Dass and Donahue 1986), into this parasite but the pores of the cuticle do not appear to be the transport site. ACKNOWLEDGMENTS This work was supported by Grant AI22479 from the U.S. National Institutes of Health and Organized Research Funds from North Texas State University. We thank Jayanta Chaudhuri for technical assistance. REFERENCES BIRD, ture BIRD, and
A. E 1958. Further observations on the strucof nematode cuticle. Parasitology 48, 32-37. A. F., AND BIRD, .I. 1969. Skeletal structures integument of Acanthocephala and Nematoda.
In “Chemical Zoology” (M. Florkin, and B. .I. Scheer ) 7.Vol. III, pp. 253-288. Academic _ J,Eds_.ITeSS, NeW YOt’K. CASTRO,G. A., AND FAIRBAIRN D. 1969. Comparison of cuticular and intestinal absorption of glucose by adult Ascaris lumbricoides. Journal of Parasitology 55, 14-16. DASS, P. D., AND DONAHUE, M. J. 1986. y-Glutamyl transpeptidase activity in Ascaris suum. Molecular and Biochemical
Parasitology
20, 233-236.
DONAHUE, M. J., BEAMES, C. G., AND BOST, K. S. 1983. Permeability characteristics of the basement membrane (basal lamella) of the intestine of Ascaris suum. Journal of Biological Physics 11, 1l- 13. DONAHUE, M. J., YACOUB, N. J., KAEINI, M. R., AND HARRIS, B. G. 1981a. Activity of enzymes regulating glycogen metabolism in perfused musclecuticle sections of Ascaris suum (Nematoda). Journal
of Parasitology
67, 362-367.
DONAHUE, M. J., YACOUB, N. J., KAEINI, M. R., MASARACCHIA, R. A., AND HARRIS, B. G. 1981b. Glycogen metabolizing enzymes during starvation and feeding of Ascaris suum maintained in a perfusion chamber. Journal of Parasitology 67, 505-510. DURY, R. A. B., AND WALLINGTON, E. A. 1980. “Carleton’s Histological Technique,” pp. 132- 135. Oxford Univ. Press, Fair Lawn, NJ. FLEMING, M. W., AND FETTERER, R. H. 1984. Ascaris suum: Continuous perfusion of the Pseudocoelom and nutrient absorption. Experimental Parasitology 57, 142-148. HARPUR, R. 1963. Maintenance of Ascaris lumbricoides in vitro. III. Changes in the hydrostatic skeleton. Comparative Biochemistry and Physiology 13, 71-85. HAYAT, M. A. 1981. “Fixation for Electron Microscopy,” pp. 402. Academic Press, New York. Lum, H. J. 1961. Improvements in epoxy resin embedding methods. Journal of Biophysical and Biochemical
Cytology
9,409-414.
ORLOWSKI, M., AND MEISTER, A. 1970. The y-glutamyl cycle: A possible transport system for amino acids. Proceedings of the National Academy of Science USA 67, 1248-1255. SEKURA, R., AND MEISTER, A. 1977. y-Glutamylcystein synthetase. Journal of Biological Chemistry 252, 2599-2605. SINGER, S. J., AND NICOLSON, G. L. 1972. The fluid mosaic model of the structure of cell membranes. Science 175, 720-731.