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Mesocestoides corti: Cation concentration in calcareous corpuscles of tetrathyridia grown in vitro
EXPERIMENTAL
PARASITOLOGY
4,
(1978)
190-196
Mesocestoides co&: Cation Concentration in Calcareous Corpuscles of Tetrathyridia Grown in Vitro JANNET L. BALDWIN,* * Oregon
ALLEN K. BERNTZEN,"
AND BRUCE W.
BROWN
Zoological Research Center, Portland, Oregon 97221, and t Department Chemistry, Portland State University, P.O. Box 751, Portland, Oregon 97207, U.S.A. (Accepted
for publication
5 December
+,I
of
1977)
BALDWIN, J. L., BERNTZEN, A. K., AND BROWN, B. W. 1978. Mesocestoides corti: Cation concentration in calcareous corpuscles of tetrathyridia grown in vitro. Experimental Parasitology 44, 190-196. The tetrathyridia larval stage of the cestode Mesocestoides codi has been maintained for 5 days in vitro in the presence of Cr( III), Cu( II), Ga( III), In( III) Tl( I), VO( II), Zn( II), and Zr (IV) cations. With the exception of the VO( II) culture, all tetrathyridia concentrated these cations in the calcareous corpuscles. Vanadium, as the vanadyl ion, appeared to have a toxic effect on the organisms and no vanadium was detected in these corpuscles by emission spectrographic analysis. Incorporation of the other ions has been shown by emission spectrographic analysis and, in the case of Ga, In, and Tl, compounds containing these ions were identified by X-ray powder diffraction. The identification by emission spectrography of cations whose compounds were not identifiable by X-ray diffraction has led to the speculation that these ions may have been incorporated into the organic portion of the calcareous corpuscles rather than the inorganic portion. INDEX DESCRIPTORS: Mesocestoides co&i; Tetrathyridia; Cestode; Tapeworm; Calcareous Corpuscles, organic, inorganic portions; X-ray diffraction; Emission spectrography; fn vz’tro cultivation; Cation concentration; Vanadyl ion toxicity; Peromyscus maniculatrcs; Mice.
Calcareous corpuscles comprise a major portion of the larval cestode tissue. Inorganic substances are localized in these corpuscles along with an organic base containing deoxyribonucleic acid, ribonucleic acid, glycogen, proteins, mucopolysaccharides, and alkaline phosphatase (Chowdhury et al. 1962; von Brand et al. 1960). Variations in the inorganic compositions of calcareous corpuscle has been noted between species of cestodes, the same species from different classes of hosts, and the same 1 Author to whom be addressed.
requests
for reprints
should
species from different geographic locations (von Brand et al. 1965a; von Brand et al. 1967 ) . Total phosphate concentration in the corpuscles may be altered by a change in the nutritional state of the host (von Brand et al. 196513). Experiments with Tuenia taeniaformis indicate the possibility that one of the functions of the calcareous corpuscle is to provide a phosphate reserve for the metabolic needs of the cestode (von Brand et al. 1965b). Chowdhury et al. (1962) have suggested that the calcareous corpuscles may serve as elements of a very rudimentary skeletal system for the cestode, with the alkaline phosphatase taking part in energy transport 190
OOI4-4894/78/0442-0I90$02.60/0 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Mesocestoides
Corti:
CATIONS
during cellular metabolism. Another passible role of the corpuscle may be in storage, transformation, and distribution of carbohydrates, proteins, and other necessary materials, The buffering capacities of the corpuscles may be important in the host-parasite relationship. Kegley et al. (1970) have shown that Mesocestoides corti tetrathyridia concentrate divalent cations in their calcareous corpuscles, both in viva and when exposed to these ions in their in vitro environment. In the present study we have exposed to additional cations, the tetrathyridia maintained the culture for several days, isolated the corpuscles, and analyzed their content. Analysis was carried out by emission spectrography for elemental determination and X-ray diffraction for compound identification. MATERIALS
AND
METHODS
The strain of Mesocestoides corti tetrathyridia used in these studies were originally isolated from the liver of Peromyscus municulatus collected in Coos County, Oregon, U.S.A. Tetrathyridia were propagated in the laboratory by means of interperitoneal injection into Swiss albino mice. Infected mice were maintained on Purina Lab Chow ad lib. Tetrathyridia were recovered at autopsy from mice infected for 5-8 months. Preparation of the tetrathyridia for in vitro culture was accomplished by repeated washings with a sterile saline solution (0.85% NaCl) containing the antibiotics streptomycin sulfate and penicillin G (Sigma Chemical Co., St. Louis, Missouri). Medium 115 developed by Berntzen and Mueller (1964) was used in place of the NCTC 109 (Grand Island Biological Co., Grand Island, New York) developed by Evans (1956) because of the ease of preparation and modification. M. co& tetrathyridia have been maintained equally well for short periods of
IN
CALCAREOUS
191
CORPUSCLES TABLE
I
Compounds Used to Prepare in Vitro &fesocestoides co& Tetrathyridia Ion
added
Compound
used
Cr(II1) cu (II) Ga(II1) In(II1) Tl (1) vo (II) Zn (II) Zr (IV)
Media for
Cation concentration (mM/liter)
Crz03
10
CuSOh. 5HzO Gas (Sod 3
10 20 20 20 20 10 20
Inz(SO.i)3 T&304 VOSOa.7HzO ZnSOc.7Hz0 Zr(S0412
time in both NCTC 109 and M 115 (Voge and Coulombe 1966). To the basic M 115 was added a solution of Ficoll ( Sigma Chemical Co. ) at the 20% level to replace the horse serum used in the previous study (Kegley et al. 1970). Ficoll, a nonionic synthetic polymer of sucrose, was added in order to minimize the addition of other cations to the system. No noticeable changes in the growth or development of the tetrathyridia were noted due to the addition of the Ficoll. The Ficoll solution was prepared by dissolving 8 g of the powdered Ficoll in 100 ml of distilled water. The Ficoll solution was then autoclaved in order to sterilize it before adding to the rest of the media. The cations were added to the system by the incorporation into stock solution A of M I.15 before combining with the rest of the media; see Table I for compounds used to prepare the media. Due to the relative insolubility of copper, chromium, and zinc compounds in the media it was necessary to add these ions at the 10 mM concentration, while all the other ions were incorporated at the 20 mM concentration. The removal of CaClz and NaH2P04 from stock solution A facilitated the addition of the copper, gallium, indium, thallium, and zinc compounds. It was necessary to initially dissolve the Cr203 in a minimum
192
BALDWIN,
BERNTZEN,
quantity of 0.01 M Sigma 7-9 buffer (Sigma Chemical Co.) before the addition to stock solution A in order to prevent precipitation. Once all the cations were dissolved in stock solution A, it was combined according to the method of Berntzen and Mueller (1964). The Ficoll solution was then added and streptomycin sulfate and penicillin G were incorporated. Cultures were grown for 5 days at 37 C under a gas phase of 10% COz, 10% 02, and SOY, Nz. Culture methods were similar to those used by Voge and Coulombe (1966). Upon termination of the culture the tetrathyridia were washed free of the media with distilled water and frozen. Residual worm tissue was removed from the corpuscles by digestion with 1% trypsin, pH 7, as described by Kegley et al. ( 1969). Isolated corpuscles were freed of any residual tissue by alternating washing and centrifugation with saline, distilled water, ethanol, and diethyl ether. Corpuscles were then air dried. Samples of the dried corpuscles were ashed at 800 C for a period of 0.5 hr prior to emission spectrographic analysis and a period of 18 hr prior to X-ray diffraction analysis. Methods of analysis were similar to those used by Kegley et al. ( 1969). RESULTS
Results of emission spectrographic analysis of the calcareous corpuscles from the Mesocestoides corti tetrathyridia grown in vitro in media containing various cations are given in Table II. With the exception of the tetrathyridia grown in media containing vanadyl ions, all corpuscles show elements that were incorporated to at least moderate levels. In some cases extraneous cations which were contained in compounds used to prepare the media were also concentrated in trace amounts. X-ray diffraction analysis, given in Table III, confirms the presence of incorporated ions by the identification of compounds such as CaGa201, InPOa, and TlpOa.
AND
BROWN
Since X-ray diffraction analysis is successful only for crystalline components of the ignited corpuscles it is not surprising that all incorporated ions were not detected. Trace components may have been incorporated into the hydroxyapatite, dolomite, or oxide structures by an isomorphic replacement process such as that proposed for nickel, lead, and tin cations in a previous study (Kegley et al. 1970). The possibility also has arisen that some of the cations, such as copper and chromium, may be incorporated into the organic portion of the corpuscle. Upon ignititon of the corpuscles at high temperatures these organic compounds would be destroyed leaving these cations in the ash as an amorphous powder. No powder pattern would be identifiable from these remains. All the X-ray diffraction patterns contain additional lines which remain as yet unidentified. These lines may be evidence of additional compounds but an acceptable comparison has not been found with published patterns in the JCPDS fiile (International Centre for Diffraction Data 1971). DISCUSSION
It has been shown by Kegley et al. (1970) that divalent cations are incorporated into the structure of the calcareous corpuscles of Mesocestoides corti tetrathyridia. In the present study, the following cations were incorporated into the in vitro culture media; Cu( II), Ga( III), In( III), Tl(I), VO(II), Zn(II), and Zr( IV). With the exception of the VO( II) culture, all organisms in culture remained active and appeared normal. Moderate concentrations of chromium, as shown by emission spectrographic analysis, without the identification of a chromium compound by X-ray diffraction, may be the result of the chromium being incorporated into the organic portion of the corpuscle rather than the inorganic portion. Cestode calcareous corpuscles are known to contain organic and inorganic fractions.
Mesocestoides
TABLE Environmental Emission
incorporated
into medium used to grow
Normalb
Cu(I1)
Cr(II1)
Ga(II1)
Al Ca Cr CU Fe Ga In
+ +++ + + + ___ ___
+ +++ _--
+ +++ ++
+ +++ ---
nfg Mn Na Ni P Si Sn ‘I’1
+++ + + ___
+++ + + _--
V zn &.
++ + _____ --____-
II
Cation Concentration in Mesocestoides corti Tetrathyridia: Spectrographic Data of Isolated Calcareous Corpuscles”
Element
Elements found
+++ + _- _- -
++ ++ --___ __-----
+” + ----+++ + + +” ++ ++ --_- _-$” ---
In(II1)
Tl(U
+ ++ ---
+ +-t+ ---
+ + +++ ---
+ + --+++
-t + -----
+++ + + ---
+++ + + ---
++ ++ --_- -------
++ ++ --___ ----_ ---
The organic portion consists of a protein base with such substances as glycogen, mucopolysaccharides, lipides, and alkaline phosphatase incorporated into it. (Chowdbury et al. 1962; von Brand et al. 1965a).
Identified
by X-Ray
Cation added to medium Normala Chromium (III) Copper (11) Gallium (III) Indium (III) Thallium (I) Vanadium VO (II) Zinc (II) Zirconium (IV)
Diffraction
M. corti larvae VO (II)
Zn (II)
Zr (IV)
+ +++ _--
+ +++ ---
+ +++ __-
$”
+”
+ +” _+ ---
-+ ---
+++ + + ---
+++ + + ---
+++ + + ___
++ ++ -_+++ -------
-t+ ++ ----_-- __ ---
a - - -, Not found; +, trace; + f, moderate; f + +, major component. b From Kegley et al. (1970). Listed here for comparison with tetrathyridia without added cations. c Present in compounds used to build media.
Compounds
193
corti: CATIONS IN CALCAREOUS CORPUXLES
-r_ --+++ + +
++ ++ -__ -----
++; ++ ___ ___ ---
++ ___
--++
grown in NCTC
199 medium
Chromium in man and animals is not easily absorbed an anionic hexavalent chromium. Absorbed hexavalent chromium passes through the red blood cell membrane and becomes bound to the globulin
TABLE
III
Analysis
of Calcareous
Corpuscles
from Mesocestoides corti
Compounds identified (heated to 800 C) Ca,(P04)30H, CaO, Cas(P04)30H, CaO, Cab(POa)30H, MgO, Cas(POd)sOH, CaO, CaO, MgO, InlO Ca,(POS30H, CaO, Ca,(POb)sOH, CaO, Cab(P04)80H,b CaO, Ca,(POa)aOH, CaO,
MgO, CaSOa, FenOa MgO, Fe203 MgSO+ Fe203, CaO,b CaSO2 MgO, CaSOd, Fez03, CaGazOdc MgO, MgO, MgO, MgO,
CaS04, Fez03, T1203 CaSOa, FezOl CaS04, MgS04 CaS04
a See footnote b in Table II. b Due to a sl-ift in d-spacings only a questionable match was achieved. c Identified from d-spacings reported in a phase study of the system CaO-Ga203.
194
BALDWIN,
BERNTZEN,
fraction of hemoglobin. Trivalent chromium, on the other hand, is unable to pass through the membrane and combines with the 8-globulin fraction of plasma proteins (Gray and Sterling 1950). Identification of only a moderate concentration of chromium in the corpuscles may be due to the use of Cr( III) to prepare the media. Copper was found in high concentration in the corpuscIes by emission spectrograp hit analysis. No copper-containing compounds were identified by X-ray diffraction. A change in crystalline components in the ignited corpuscles is noted by the questionable match of CaO and CaSOd and the addition of MgS04 to the mixture. The problems associated with unambiguously identifying trace amounts of CaS04 have been discussed previously (Kegley et aZ. 1969). Copper, like chromium, is also known to be closely associated with proteins and enzymes (Underwood 1971) and may have been incorporated into the organic portion of the corpuscles. Addition of gallium (III) to the growth media resulted in relatively high concentrations of gallium in the corpuscles. X-ray powder diffraction analysis confirms the presence of CaGazOd. CaGaxOJ has been synthesized by the combination of Gaz03 and CaC03 quenched at temperatures over 1200 C (Jeevaratnam and Glasser 1961; Jeevaratnam et al. 1963). Ashing corpuscles at 800 C for 18 hr appears to have produced the same compound. It is thought that this may be possible due to the finegrained crystalline structure of the corpuscles ( Glasser 1972 ) . Trivalent indium produced the most marked changes in the crystalline structure of the calcareous corpuscles. Hydroxyapatite can no longer be identified in the powder pattern In203, CaO, and MgO are the only identified compounds. Although some reaction is known to take place between InzOa and MgO forming Mg(InOz)z, the necessary temperatures (I.300 C) are considerably in excess of the 800 C used
AND
BROWN
in our study (Moeller and Schnizlein 1947). Tetrathyridia grown in media containing thalIium( I ) concentrated the foreign cation in the calcareous corpuscle to a considerable degree as shown by emission spectrographic analysis and by the identification of T&O, in the powder pattern. The tetrathyridia grown in the media containing VO( II) started to balloon similar to the situation described by Voge and Coulombe ( 1966 ) and by the end of the 5 days all were inactive. An additional culture was grown at the 2 mM VO ( II) concentration with the same effect being noted after only a few days in culture, Vanadium was not detected in either sample of corpuscles by emission spectrographic means and no change in the powder pattern was apparent. Zinc and zirconium also produced effects similar to chromium; only a moderate concentration of each ion was found and no zinc or zirconium compounds were identified in the powder pattern of the isolated corpuscles. Zinc is known to be a component of many metaloenzymes such as aIkaline cestode calcareous corpuscles (Chowdhury et al. 1962; von Brand et al. 1960). It is possible that the zinc has been incorporated into the organic portion of the corpuscles, as may also be the situation with both copper and chromium. As observed previously (Kegley et al, 1970) trace elements in compounds used to build the media appear in the calcareous corpuscles and are occasionally identified in moderate concentrations by emission spectrography ( see Table II ) . Research has attempted to find a relation between the position of elements in the periodic table and the essentiality of those elements in plant and animal cells (FreyWyssling 1935; Steinberg 1928). In the tetrathyridia of M. co&, the calcareous corpuscles have been shown to serve as a reservoir for calcium, magnesium, and phosphate ions (von Brand et al. 1956b).
Mesocestoides
corti: CATIONS IN CALCAREOUS CORPUSCLES
Attempts to develop media for phytoplankton culturing (Barber and Wyther 1969) suggest a mechanism for environmental cation accumulation by the tetrathyridia. Regardless of the cation introduced into the medium, the phytoplankton failed to grow well without the addition of a chelating agent. In the case of the tetrathyridia, the constituents of the NCTC 109 medium (Kegley et al. 1970) or the Ml15 medium used here may supply the agents that convert the cations into a chemical form that facilitates the absorption into the interior through the acellular cuticulate membrane ( Rothman 1963). The incorporation of divalent environmental cations in the inorganic layers of the calcareous corpuscles by an isomorphic replacement process (Kegley eZ al. 1970) also explains the efficient concentration of Cu(I1) and Zn(I1) in this study of Mesocestoides corti. The incorporation of other cations, with the exception of vanadyl which was not detected, must proceed by a more complex route. ACKNOWLEDGMENTS The authors wish to acknowledge the Oregon Zoological Research Center and Portland State University for providing the research space and equipment necessary for the completion of this project. This work was supported in part by the U.S. National Institutes of Health through Grant No. ROl-AI-09860-03 TMP. A. K. Berntzen died prior to completion of this work. REFERENCES BARBER, R. T., AND WYTHER, J. H. 1969. Organic chelators: Factors affecting primary production in the Cromwell Current upwelling. journal of Experimental Marine Biology and Ecology 3, 191-199. BERNTZEN, A. K., AND MUELLER, J. R. 1964. In vitro cultivation of Spirometra mansanoides (Cestoda) from the procercoid to the early adult. Journal of Parasitology 50, 705-711. CHOWDHURY, A. B., DASGUFTA, B., AND RAY, H. N. 1962. On the nature and structure of the calcareous corpuscles in Tuenia saginata. Parasitology 52, 153-157. EVANS, V. J. 1956. Chemically defined media for
195
the cultivation of long-term strains from four mammalian species. Cancer Research 16, 77. FREY-WYSSLING, A. 1935. Die unentbehrlichen Ebmente der Pflanzennahrung. Naturwissenschaften 23, 767-769. of Chemistry, GLASSER, F. 1972. Department University of Aberdeen, Aberdeen, Scotland, U.K., personal communication. GRAY, S. J., AND STERLING, K. 1950. The tagging of red cells and plasma proteins with radioactive chromium. Journal of Clinical Investigation 29, 1064. International Centre for Diffraction Data 1971. “Index (Inorganic) to the Powder Diffraction File 1971,” ASTM Publication PD lS-21i. International Centre for Diffraction Data, Swathmore, Pennsylvania. JEEVARATNAM, J., AND GLASSER, F. P. 1961. The system CaO-GazOa. Journal of the American Ceramic Society 44, 563-566. JEEVARATNAM, J., GLASSER, F. P., AND GLASSER, L. S. 1963. Crystallography of the CaGanOa polymorphs. Zeitschrift fiir Kristallogruphie 118, 257-262. KEGLEY, L. M., BALDWIN, J., BROWN, B. W., AND BERNTZEN, A. K. 1970. Mesocestoides corti: Environmental cation concentration in calcareous corpuscles. Experimental Parasitology 27, 88-94. KEGLEY, L. M., BROWN, B. W., AND BERNTZEN, A. K. 1969. Mesocestoides co&: Inorganic components in calcareous corpuscles. Experimental Parasitology 25, 85-92. MOELLER, T., AND SCHNIZLEIN, J. G. 1947. Contributions to the chemistry of indium. VIII. Journal of Physical Chemistry 51, 771-775. ROTHMAN, A. . 1963. Electron microscopic studies of tapeworms: The surface structures of Hymenolepio diminuta (Rudolphi, 1819) Blanchard, 1891. Transactions of the American Microscopic Society 82, 2230. STEINBERG, R. A. 1928. Correlations between biological essentiality and atomic structure of the chemical elements. Jozrrnal of Agricultural Research 57, 851-858. UNDERWOOD, E. J. 1971. “Trace Elements in Human and Animal Nutrition.” Academic Press, New York. VOGE, M., AND COULOMBE, L. S. 1966. Growth and asexual multiplication in vitro of Mesocestoides tetruthyridia. American Journal of Tropical Medicine and Hygiene 15, 902-907. VON BRAND, T., MERCADO, T. I., NYLEN, M. U., AND SCOTT, D. B. 1960. Observation on function, composition, and structure of cestode calcareous corpuscles. Experimental Parasitology 9, 205-214.
196
BALDWIN,
BERNTZEN,
VON BRAND, T., NYLEN, M. U., SCOTT, D. B., AND MARTIN, G. N. 1965a. Observation on calcareous corpuscles of larval Echlnoccus granukxus of various geographic origins. PTOC. Sot. Exp. Biol. Med. 120, 383-385. VON BRAND, T., NYLEN, M. U., MARTIN, G. N., AND CHURCHWELL, F. K. 1967. Composition and crystallization patterns of calcareous corpuscles
AND
BROWN
of cestodes grown in different classes of hosts. Journal of Parasitology 53, 638-687. VON BRAND, T., WEINBACH, E. C., AND CLAGGETT, C. E. 1965b. Incorporation of phosphate into soft tissues and calcareous Taenia taeniaeformis. and Physiology
corpuscles
Comparative
14, 11-20.
of larval
Biochemistry
PARASITOLOGY
4,
(1978)
190-196
Mesocestoides co&: Cation Concentration in Calcareous Corpuscles of Tetrathyridia Grown in Vitro JANNET L. BALDWIN,* * Oregon
ALLEN K. BERNTZEN,"
AND BRUCE W.
BROWN
Zoological Research Center, Portland, Oregon 97221, and t Department Chemistry, Portland State University, P.O. Box 751, Portland, Oregon 97207, U.S.A. (Accepted
for publication
5 December
+,I
of
1977)
BALDWIN, J. L., BERNTZEN, A. K., AND BROWN, B. W. 1978. Mesocestoides corti: Cation concentration in calcareous corpuscles of tetrathyridia grown in vitro. Experimental Parasitology 44, 190-196. The tetrathyridia larval stage of the cestode Mesocestoides codi has been maintained for 5 days in vitro in the presence of Cr( III), Cu( II), Ga( III), In( III) Tl( I), VO( II), Zn( II), and Zr (IV) cations. With the exception of the VO( II) culture, all tetrathyridia concentrated these cations in the calcareous corpuscles. Vanadium, as the vanadyl ion, appeared to have a toxic effect on the organisms and no vanadium was detected in these corpuscles by emission spectrographic analysis. Incorporation of the other ions has been shown by emission spectrographic analysis and, in the case of Ga, In, and Tl, compounds containing these ions were identified by X-ray powder diffraction. The identification by emission spectrography of cations whose compounds were not identifiable by X-ray diffraction has led to the speculation that these ions may have been incorporated into the organic portion of the calcareous corpuscles rather than the inorganic portion. INDEX DESCRIPTORS: Mesocestoides co&i; Tetrathyridia; Cestode; Tapeworm; Calcareous Corpuscles, organic, inorganic portions; X-ray diffraction; Emission spectrography; fn vz’tro cultivation; Cation concentration; Vanadyl ion toxicity; Peromyscus maniculatrcs; Mice.
Calcareous corpuscles comprise a major portion of the larval cestode tissue. Inorganic substances are localized in these corpuscles along with an organic base containing deoxyribonucleic acid, ribonucleic acid, glycogen, proteins, mucopolysaccharides, and alkaline phosphatase (Chowdhury et al. 1962; von Brand et al. 1960). Variations in the inorganic compositions of calcareous corpuscle has been noted between species of cestodes, the same species from different classes of hosts, and the same 1 Author to whom be addressed.
requests
for reprints
should
species from different geographic locations (von Brand et al. 1965a; von Brand et al. 1967 ) . Total phosphate concentration in the corpuscles may be altered by a change in the nutritional state of the host (von Brand et al. 196513). Experiments with Tuenia taeniaformis indicate the possibility that one of the functions of the calcareous corpuscle is to provide a phosphate reserve for the metabolic needs of the cestode (von Brand et al. 1965b). Chowdhury et al. (1962) have suggested that the calcareous corpuscles may serve as elements of a very rudimentary skeletal system for the cestode, with the alkaline phosphatase taking part in energy transport 190
OOI4-4894/78/0442-0I90$02.60/0 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Mesocestoides
Corti:
CATIONS
during cellular metabolism. Another passible role of the corpuscle may be in storage, transformation, and distribution of carbohydrates, proteins, and other necessary materials, The buffering capacities of the corpuscles may be important in the host-parasite relationship. Kegley et al. (1970) have shown that Mesocestoides corti tetrathyridia concentrate divalent cations in their calcareous corpuscles, both in viva and when exposed to these ions in their in vitro environment. In the present study we have exposed to additional cations, the tetrathyridia maintained the culture for several days, isolated the corpuscles, and analyzed their content. Analysis was carried out by emission spectrography for elemental determination and X-ray diffraction for compound identification. MATERIALS
AND
METHODS
The strain of Mesocestoides corti tetrathyridia used in these studies were originally isolated from the liver of Peromyscus municulatus collected in Coos County, Oregon, U.S.A. Tetrathyridia were propagated in the laboratory by means of interperitoneal injection into Swiss albino mice. Infected mice were maintained on Purina Lab Chow ad lib. Tetrathyridia were recovered at autopsy from mice infected for 5-8 months. Preparation of the tetrathyridia for in vitro culture was accomplished by repeated washings with a sterile saline solution (0.85% NaCl) containing the antibiotics streptomycin sulfate and penicillin G (Sigma Chemical Co., St. Louis, Missouri). Medium 115 developed by Berntzen and Mueller (1964) was used in place of the NCTC 109 (Grand Island Biological Co., Grand Island, New York) developed by Evans (1956) because of the ease of preparation and modification. M. co& tetrathyridia have been maintained equally well for short periods of
IN
CALCAREOUS
191
CORPUSCLES TABLE
I
Compounds Used to Prepare in Vitro &fesocestoides co& Tetrathyridia Ion
added
Compound
used
Cr(II1) cu (II) Ga(II1) In(II1) Tl (1) vo (II) Zn (II) Zr (IV)
Media for
Cation concentration (mM/liter)
Crz03
10
CuSOh. 5HzO Gas (Sod 3
10 20 20 20 20 10 20
Inz(SO.i)3 T&304 VOSOa.7HzO ZnSOc.7Hz0 Zr(S0412
time in both NCTC 109 and M 115 (Voge and Coulombe 1966). To the basic M 115 was added a solution of Ficoll ( Sigma Chemical Co. ) at the 20% level to replace the horse serum used in the previous study (Kegley et al. 1970). Ficoll, a nonionic synthetic polymer of sucrose, was added in order to minimize the addition of other cations to the system. No noticeable changes in the growth or development of the tetrathyridia were noted due to the addition of the Ficoll. The Ficoll solution was prepared by dissolving 8 g of the powdered Ficoll in 100 ml of distilled water. The Ficoll solution was then autoclaved in order to sterilize it before adding to the rest of the media. The cations were added to the system by the incorporation into stock solution A of M I.15 before combining with the rest of the media; see Table I for compounds used to prepare the media. Due to the relative insolubility of copper, chromium, and zinc compounds in the media it was necessary to add these ions at the 10 mM concentration, while all the other ions were incorporated at the 20 mM concentration. The removal of CaClz and NaH2P04 from stock solution A facilitated the addition of the copper, gallium, indium, thallium, and zinc compounds. It was necessary to initially dissolve the Cr203 in a minimum
192
BALDWIN,
BERNTZEN,
quantity of 0.01 M Sigma 7-9 buffer (Sigma Chemical Co.) before the addition to stock solution A in order to prevent precipitation. Once all the cations were dissolved in stock solution A, it was combined according to the method of Berntzen and Mueller (1964). The Ficoll solution was then added and streptomycin sulfate and penicillin G were incorporated. Cultures were grown for 5 days at 37 C under a gas phase of 10% COz, 10% 02, and SOY, Nz. Culture methods were similar to those used by Voge and Coulombe (1966). Upon termination of the culture the tetrathyridia were washed free of the media with distilled water and frozen. Residual worm tissue was removed from the corpuscles by digestion with 1% trypsin, pH 7, as described by Kegley et al. ( 1969). Isolated corpuscles were freed of any residual tissue by alternating washing and centrifugation with saline, distilled water, ethanol, and diethyl ether. Corpuscles were then air dried. Samples of the dried corpuscles were ashed at 800 C for a period of 0.5 hr prior to emission spectrographic analysis and a period of 18 hr prior to X-ray diffraction analysis. Methods of analysis were similar to those used by Kegley et al. ( 1969). RESULTS
Results of emission spectrographic analysis of the calcareous corpuscles from the Mesocestoides corti tetrathyridia grown in vitro in media containing various cations are given in Table II. With the exception of the tetrathyridia grown in media containing vanadyl ions, all corpuscles show elements that were incorporated to at least moderate levels. In some cases extraneous cations which were contained in compounds used to prepare the media were also concentrated in trace amounts. X-ray diffraction analysis, given in Table III, confirms the presence of incorporated ions by the identification of compounds such as CaGa201, InPOa, and TlpOa.
AND
BROWN
Since X-ray diffraction analysis is successful only for crystalline components of the ignited corpuscles it is not surprising that all incorporated ions were not detected. Trace components may have been incorporated into the hydroxyapatite, dolomite, or oxide structures by an isomorphic replacement process such as that proposed for nickel, lead, and tin cations in a previous study (Kegley et al. 1970). The possibility also has arisen that some of the cations, such as copper and chromium, may be incorporated into the organic portion of the corpuscle. Upon ignititon of the corpuscles at high temperatures these organic compounds would be destroyed leaving these cations in the ash as an amorphous powder. No powder pattern would be identifiable from these remains. All the X-ray diffraction patterns contain additional lines which remain as yet unidentified. These lines may be evidence of additional compounds but an acceptable comparison has not been found with published patterns in the JCPDS fiile (International Centre for Diffraction Data 1971). DISCUSSION
It has been shown by Kegley et al. (1970) that divalent cations are incorporated into the structure of the calcareous corpuscles of Mesocestoides corti tetrathyridia. In the present study, the following cations were incorporated into the in vitro culture media; Cu( II), Ga( III), In( III), Tl(I), VO(II), Zn(II), and Zr( IV). With the exception of the VO( II) culture, all organisms in culture remained active and appeared normal. Moderate concentrations of chromium, as shown by emission spectrographic analysis, without the identification of a chromium compound by X-ray diffraction, may be the result of the chromium being incorporated into the organic portion of the corpuscle rather than the inorganic portion. Cestode calcareous corpuscles are known to contain organic and inorganic fractions.
Mesocestoides
TABLE Environmental Emission
incorporated
into medium used to grow
Normalb
Cu(I1)
Cr(II1)
Ga(II1)
Al Ca Cr CU Fe Ga In
+ +++ + + + ___ ___
+ +++ _--
+ +++ ++
+ +++ ---
nfg Mn Na Ni P Si Sn ‘I’1
+++ + + ___
+++ + + _--
V zn &.
++ + _____ --____-
II
Cation Concentration in Mesocestoides corti Tetrathyridia: Spectrographic Data of Isolated Calcareous Corpuscles”
Element
Elements found
+++ + _- _- -
++ ++ --___ __-----
+” + ----+++ + + +” ++ ++ --_- _-$” ---
In(II1)
Tl(U
+ ++ ---
+ +-t+ ---
+ + +++ ---
+ + --+++
-t + -----
+++ + + ---
+++ + + ---
++ ++ --_- -------
++ ++ --___ ----_ ---
The organic portion consists of a protein base with such substances as glycogen, mucopolysaccharides, lipides, and alkaline phosphatase incorporated into it. (Chowdbury et al. 1962; von Brand et al. 1965a).
Identified
by X-Ray
Cation added to medium Normala Chromium (III) Copper (11) Gallium (III) Indium (III) Thallium (I) Vanadium VO (II) Zinc (II) Zirconium (IV)
Diffraction
M. corti larvae VO (II)
Zn (II)
Zr (IV)
+ +++ _--
+ +++ ---
+ +++ __-
$”
+”
+ +” _+ ---
-+ ---
+++ + + ---
+++ + + ---
+++ + + ___
++ ++ -_+++ -------
-t+ ++ ----_-- __ ---
a - - -, Not found; +, trace; + f, moderate; f + +, major component. b From Kegley et al. (1970). Listed here for comparison with tetrathyridia without added cations. c Present in compounds used to build media.
Compounds
193
corti: CATIONS IN CALCAREOUS CORPUXLES
-r_ --+++ + +
++ ++ -__ -----
++; ++ ___ ___ ---
++ ___
--++
grown in NCTC
199 medium
Chromium in man and animals is not easily absorbed an anionic hexavalent chromium. Absorbed hexavalent chromium passes through the red blood cell membrane and becomes bound to the globulin
TABLE
III
Analysis
of Calcareous
Corpuscles
from Mesocestoides corti
Compounds identified (heated to 800 C) Ca,(P04)30H, CaO, Cas(P04)30H, CaO, Cab(POa)30H, MgO, Cas(POd)sOH, CaO, CaO, MgO, InlO Ca,(POS30H, CaO, Ca,(POb)sOH, CaO, Cab(P04)80H,b CaO, Ca,(POa)aOH, CaO,
MgO, CaSOa, FenOa MgO, Fe203 MgSO+ Fe203, CaO,b CaSO2 MgO, CaSOd, Fez03, CaGazOdc MgO, MgO, MgO, MgO,
CaS04, Fez03, T1203 CaSOa, FezOl CaS04, MgS04 CaS04
a See footnote b in Table II. b Due to a sl-ift in d-spacings only a questionable match was achieved. c Identified from d-spacings reported in a phase study of the system CaO-Ga203.
194
BALDWIN,
BERNTZEN,
fraction of hemoglobin. Trivalent chromium, on the other hand, is unable to pass through the membrane and combines with the 8-globulin fraction of plasma proteins (Gray and Sterling 1950). Identification of only a moderate concentration of chromium in the corpuscles may be due to the use of Cr( III) to prepare the media. Copper was found in high concentration in the corpuscIes by emission spectrograp hit analysis. No copper-containing compounds were identified by X-ray diffraction. A change in crystalline components in the ignited corpuscles is noted by the questionable match of CaO and CaSOd and the addition of MgS04 to the mixture. The problems associated with unambiguously identifying trace amounts of CaS04 have been discussed previously (Kegley et aZ. 1969). Copper, like chromium, is also known to be closely associated with proteins and enzymes (Underwood 1971) and may have been incorporated into the organic portion of the corpuscles. Addition of gallium (III) to the growth media resulted in relatively high concentrations of gallium in the corpuscles. X-ray powder diffraction analysis confirms the presence of CaGazOd. CaGaxOJ has been synthesized by the combination of Gaz03 and CaC03 quenched at temperatures over 1200 C (Jeevaratnam and Glasser 1961; Jeevaratnam et al. 1963). Ashing corpuscles at 800 C for 18 hr appears to have produced the same compound. It is thought that this may be possible due to the finegrained crystalline structure of the corpuscles ( Glasser 1972 ) . Trivalent indium produced the most marked changes in the crystalline structure of the calcareous corpuscles. Hydroxyapatite can no longer be identified in the powder pattern In203, CaO, and MgO are the only identified compounds. Although some reaction is known to take place between InzOa and MgO forming Mg(InOz)z, the necessary temperatures (I.300 C) are considerably in excess of the 800 C used
AND
BROWN
in our study (Moeller and Schnizlein 1947). Tetrathyridia grown in media containing thalIium( I ) concentrated the foreign cation in the calcareous corpuscle to a considerable degree as shown by emission spectrographic analysis and by the identification of T&O, in the powder pattern. The tetrathyridia grown in the media containing VO( II) started to balloon similar to the situation described by Voge and Coulombe ( 1966 ) and by the end of the 5 days all were inactive. An additional culture was grown at the 2 mM VO ( II) concentration with the same effect being noted after only a few days in culture, Vanadium was not detected in either sample of corpuscles by emission spectrographic means and no change in the powder pattern was apparent. Zinc and zirconium also produced effects similar to chromium; only a moderate concentration of each ion was found and no zinc or zirconium compounds were identified in the powder pattern of the isolated corpuscles. Zinc is known to be a component of many metaloenzymes such as aIkaline cestode calcareous corpuscles (Chowdhury et al. 1962; von Brand et al. 1960). It is possible that the zinc has been incorporated into the organic portion of the corpuscles, as may also be the situation with both copper and chromium. As observed previously (Kegley et al, 1970) trace elements in compounds used to build the media appear in the calcareous corpuscles and are occasionally identified in moderate concentrations by emission spectrography ( see Table II ) . Research has attempted to find a relation between the position of elements in the periodic table and the essentiality of those elements in plant and animal cells (FreyWyssling 1935; Steinberg 1928). In the tetrathyridia of M. co&, the calcareous corpuscles have been shown to serve as a reservoir for calcium, magnesium, and phosphate ions (von Brand et al. 1956b).
Mesocestoides
corti: CATIONS IN CALCAREOUS CORPUSCLES
Attempts to develop media for phytoplankton culturing (Barber and Wyther 1969) suggest a mechanism for environmental cation accumulation by the tetrathyridia. Regardless of the cation introduced into the medium, the phytoplankton failed to grow well without the addition of a chelating agent. In the case of the tetrathyridia, the constituents of the NCTC 109 medium (Kegley et al. 1970) or the Ml15 medium used here may supply the agents that convert the cations into a chemical form that facilitates the absorption into the interior through the acellular cuticulate membrane ( Rothman 1963). The incorporation of divalent environmental cations in the inorganic layers of the calcareous corpuscles by an isomorphic replacement process (Kegley eZ al. 1970) also explains the efficient concentration of Cu(I1) and Zn(I1) in this study of Mesocestoides corti. The incorporation of other cations, with the exception of vanadyl which was not detected, must proceed by a more complex route. ACKNOWLEDGMENTS The authors wish to acknowledge the Oregon Zoological Research Center and Portland State University for providing the research space and equipment necessary for the completion of this project. This work was supported in part by the U.S. National Institutes of Health through Grant No. ROl-AI-09860-03 TMP. A. K. Berntzen died prior to completion of this work. REFERENCES BARBER, R. T., AND WYTHER, J. H. 1969. Organic chelators: Factors affecting primary production in the Cromwell Current upwelling. journal of Experimental Marine Biology and Ecology 3, 191-199. BERNTZEN, A. K., AND MUELLER, J. R. 1964. In vitro cultivation of Spirometra mansanoides (Cestoda) from the procercoid to the early adult. Journal of Parasitology 50, 705-711. CHOWDHURY, A. B., DASGUFTA, B., AND RAY, H. N. 1962. On the nature and structure of the calcareous corpuscles in Tuenia saginata. Parasitology 52, 153-157. EVANS, V. J. 1956. Chemically defined media for
195
the cultivation of long-term strains from four mammalian species. Cancer Research 16, 77. FREY-WYSSLING, A. 1935. Die unentbehrlichen Ebmente der Pflanzennahrung. Naturwissenschaften 23, 767-769. of Chemistry, GLASSER, F. 1972. Department University of Aberdeen, Aberdeen, Scotland, U.K., personal communication. GRAY, S. J., AND STERLING, K. 1950. The tagging of red cells and plasma proteins with radioactive chromium. Journal of Clinical Investigation 29, 1064. International Centre for Diffraction Data 1971. “Index (Inorganic) to the Powder Diffraction File 1971,” ASTM Publication PD lS-21i. International Centre for Diffraction Data, Swathmore, Pennsylvania. JEEVARATNAM, J., AND GLASSER, F. P. 1961. The system CaO-GazOa. Journal of the American Ceramic Society 44, 563-566. JEEVARATNAM, J., GLASSER, F. P., AND GLASSER, L. S. 1963. Crystallography of the CaGanOa polymorphs. Zeitschrift fiir Kristallogruphie 118, 257-262. KEGLEY, L. M., BALDWIN, J., BROWN, B. W., AND BERNTZEN, A. K. 1970. Mesocestoides corti: Environmental cation concentration in calcareous corpuscles. Experimental Parasitology 27, 88-94. KEGLEY, L. M., BROWN, B. W., AND BERNTZEN, A. K. 1969. Mesocestoides co&: Inorganic components in calcareous corpuscles. Experimental Parasitology 25, 85-92. MOELLER, T., AND SCHNIZLEIN, J. G. 1947. Contributions to the chemistry of indium. VIII. Journal of Physical Chemistry 51, 771-775. ROTHMAN, A. . 1963. Electron microscopic studies of tapeworms: The surface structures of Hymenolepio diminuta (Rudolphi, 1819) Blanchard, 1891. Transactions of the American Microscopic Society 82, 2230. STEINBERG, R. A. 1928. Correlations between biological essentiality and atomic structure of the chemical elements. Jozrrnal of Agricultural Research 57, 851-858. UNDERWOOD, E. J. 1971. “Trace Elements in Human and Animal Nutrition.” Academic Press, New York. VOGE, M., AND COULOMBE, L. S. 1966. Growth and asexual multiplication in vitro of Mesocestoides tetruthyridia. American Journal of Tropical Medicine and Hygiene 15, 902-907. VON BRAND, T., MERCADO, T. I., NYLEN, M. U., AND SCOTT, D. B. 1960. Observation on function, composition, and structure of cestode calcareous corpuscles. Experimental Parasitology 9, 205-214.
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VON BRAND, T., NYLEN, M. U., SCOTT, D. B., AND MARTIN, G. N. 1965a. Observation on calcareous corpuscles of larval Echlnoccus granukxus of various geographic origins. PTOC. Sot. Exp. Biol. Med. 120, 383-385. VON BRAND, T., NYLEN, M. U., MARTIN, G. N., AND CHURCHWELL, F. K. 1967. Composition and crystallization patterns of calcareous corpuscles
AND
BROWN
of cestodes grown in different classes of hosts. Journal of Parasitology 53, 638-687. VON BRAND, T., WEINBACH, E. C., AND CLAGGETT, C. E. 1965b. Incorporation of phosphate into soft tissues and calcareous Taenia taeniaeformis. and Physiology
corpuscles
Comparative
14, 11-20.
of larval
Biochemistry