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Differences in esterase frequencies in five strains of Biomphalaria glabrata (Say)
International Journal for Parasitology, 1973, Vol. 3, pp. 43-46. Pergamon Press. Printed in Great Britain
DIFFERENCES IN ESTERASE FREQUENCIES FIVE STRAINS OF BIOMPHALARIA GLABRATA
IN (SAY)
R. D. BAIR and F. J. ETGES Department
of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221, U.S.A. (Receiued 19 May 1972)
Abstract BAIRR. D. and ETGESF. J. 1973. Differences in esterase frequencies in five strains of Biomphalariaglabrata (Say). International Journalfor Parasitology, 3: 4346. Vertical polyacrylamidegel electrophoresis of extracts from hepatopancreas tissue of individual snails of five laboratory strains of Biomphalariaglabrata has shown considerable variation in the frequency of occurrence of ten esterases. Differences in esterase frequencies seemed correlated with strain differences in certain morphological, physiological, and biochemical aspects of the snail strains; however, no causal relationships are apparent in these systems. INDEX KEY WORDS: Biomphalariaglabrata; esterase; Schistosoma mansoni; polymorphism; Mollusca; snail.
INTRODUCTION WRIGHT et al. (1966)
described considerable electrophoretic variation in hepatopancreas esterases of the snail Biomphalaria sudanica collected in Khartoum, Sudan and subsequently maintained in the laboratory for 9 yr. Wright & File (1968) pointed out the importance of population differences in the study of relationships between parasitic organisms and their hosts. Wright & File (1968), examining digestive gland esterases of five species of laboratoryreared Bulinus, concluded that electrophoretic separation of these enzymes provides a sensitive indication of genetic diversity between and within populations. Coles (1970) studied population and species variation in digestive gland esterases of two species of Bulinus and three species of Biomphalaria. In addition to finding obvious differences in esterase patterns between the species of snails, Bulinus africanus geographic strains from South and East Africa also had distinctly different esterase patterns, as did two strains of Biomphalaria pfe$eri.
The present study examines qualitative differences in hepatopancreas esterases in five laboratory strains of the neotropical snail Biomphalaria glabrata. Previous work and unpublished data from this laboratory have shown these strains to differ in several biological aspects, aside from enzyme polymorphism. Gilbertson & Etges (1967), working with three of the B. glabrata strains used in the present study, demonstrated considerable variation in the specificity of vertebrate red cell agglutinins of the hemolymph from these strains of snails. Gilbertson (1966) reported significant differences in hemolymph total protein, and demonstrated variation in susceptibility to infection with a single strain of Puerto Rican Schistosoma mansoni. Glaudel (1972), using the same snails employed in the present study, found some consistent differences in mortality rates of snails during both the prepatent and patent periods of infection as well as differences in their susceptibility to S. mansoni infection. MATERIALS
AND
METHODS
All B. gZabrata used in this study were laboratory-bred, fed with leaf lettuce and measured approximately 15 mm dia. The Puerto Rican strain of B. glabrata (PR) was obtained in 1958 from the National Institutes 43
I.I.P. VOL. 3. 1973
R. D. BAIR and F. J. ETCHES
44
of Health. These snails are moderately susceptible to infection with S. munsoni and have normal pigmentation. The Venezuelan albino strain of B. gfnbraru (VA) was acquired from the Tropical Research Medical Laboratory, San Juan, Puerto Rico, in 1962. This strain lacks normal pigmentation and is moderately susceptible to infection. The Bahian Brazilian strain of B. glubratu (BAH) was supplied by Dr. W. L. Paraense in 1964. These snails are highly refractory to infection (Paraense & Correa, 1963). A strain of B. glabratu (UCLA) was obtained from the University of California at Los Angeles in 1970; this strain is highly susceptible to infection and appears to die sooner than the other strains after the onset of patency. Puerto Rican B. glubrutu (PR-l), acquired from Harvard University’s School of Public Health in 1971, also are very susceptible to infection, but die more frequently than snails of other strains during the prepatent period. The hepatopancreas was dissected from 25 individual snails of each strain, and ground with a glass hand homogenizer in 100-150 ~1 of 0.05 M Tris Borate EDTA (pH 8.9)-5 % sucrose. Suspensions were centrifuged at 4-8°C at N 15,000 g for 5 min and the supematants subjected to vertical polyacrylamide-gel electrophoresis in 5 % gels at N 24 V/linear cm of gel. The buffer used during electrophoresis was 0.1 M Tris Borate EDTA (pH 8.9). Following 90 min of electrophoresis, gels were either stained in 100 ml of 0.1 M Tris-HCl (pH 7.1) containing 20 mg each of Q- and g-napthylacetate and 30 mg of Fast Blue RR, or with 20 ~1 of fl-napthylbutyrate and 30 mg of Fast Blue RR. Gels were incubated at room temperature for 20-30 min with Q- and p-napthylacetate. This staining procedure modified from that of Brewer (1970). permits tentative identification of carbonic anhydrase on the basis of a pink-purple color reaction in addition to revealing other esterases. Esterase activity determined with &napthylbutyrate was done at room temperature for 12 h. Infected snails were obtained by en masse exposure to large numbers of miracidia from the National Institutes of Health strain of Puerto Rican S. munsoni. Miracidia were obtained from eggs isolated from livers of Cox (Swiss) random bred white female mice. Densitometric tracings of electrophoretograms were done with a Densicord electrophoresis densitometer (Photovolt, Corp). Brewer’s (1970) recommendation that the most anodal band be numbered ‘1’ and the numbering continued in the cathodal direction was adopted in the present study. Variation in experimental conditions during electrophoresis resulted in some variation in the mobility of similar bands from one gel run to another. In order to compensate for such errors, the fourth most anodal band was used as a reference and the distances that all other esterases migrated from the origin were compared to its migration. This procedure provided an accurate and easily compared relative mobility value for each enzyme. Only the presence or absence of major enzymes was recorded. Minor variations in the migration of esterases, the presence of lightly staining, easily obscured bands and variation in the density of staining were not analyzed.
RESULTS
The representative densitometer tracing presented in Fig. 1 shows excellent separation of esterases, the relative position of each band, and the relative degree of esterase activity with a- and Bnapthylacetate substrates. The third and fourth bands of activity in all strains had a very pink-purple color, indicating carbonic anhydrase activity. All other sites were gray-brown in color. Table 1 gives the frequency of each enzyme in each strain. Comparison of gels incubated with a- and p-napthylacetate with those incubated in P-napthylbutyrate showed the same enzyme bands, with some variation in density of staining at most sites of activity. In gels stained with /I-napthylbutyrate, bands of activity were less 9
ci 4
6
-
0.5
I.0 Relative
FIG. 1. Densitometric
scan of esterases
+
mobility
of the hepatopancreas
of Biomphufuriu glubrutu.
I.J.P. VOL.
3. 1973
ESTERASES OF
Biomphalariaghbrata
45
defined and therefore more difficult to analyze. Accordingly, data presented in Table 1 are derived from gels stained with a- and /?-napthylacetate. The PR-1 strain showed the least variation of all strains examined (Table 1). With the exception of the sixth esterase, all enzymes were present in each PR-1 snail. Over-all, the VA snails were the most polymorphic, PR and UCLA snails had similar polymorphism, and the BAH snails exhibited moderate heterogeneity. Occasionally, four additional very lightly staining esterases could be detected in BAH extracts in particular. TABLE~--F~EQUENCYOFOCCURRENCEOFHEPATOPANCREASESTERASESIN 25 IND~IDUA~~~MEACHOFFIVE LABORATORY STRAINS OF 3iomp~Iar~g~abra~a.(No. OF SNAILS WITH ~TER~E~OTAL E~MINED.)
Frequency of occurrenceof enzymenumber
B. glabrata
strain
1
2
3
PR-1 UCLA PR BAH VA
l-00 O-88 0.92 0.96 1.00
I-00 0.84 0.92
l-00 o-92 O-80 076 0.72
1.00
0.72
4
5
6
la0 la0
140 o-92
O-80
0.96 044 o-92
@SO 0.72 0.96
0.76 0.72 I.00 0.80
7
8
9
10
I-00
140
la0
1.00
The seventh through the tenth esterases were always present in PR-1. UCLA, PR and BAH extracts. Only a few individuals from the VA strain lacked the seventh and ninth esterases, while each snail had esterases eight and ten. Most variation in esterases of all strains was present in the first six bands. The two most frequent deficiencies of esterases were the fourth one in the BAH strain and the ninth in the VA strain.
DISCUSSION The present study attempts to quantify variability in hepatopancreas esterases of five neotropical strains of B. glubrcztu. While several workers (Wright et al., 1966; Wright & File, 1968; Coles, 1970) have described hepatopancreas esterase polymorphism in schistosome vector snails, none have attempted to quantify variation between snail strains. Hepatopancreas esterases of the PR-1 strain show a strong degree of monomo~hism indicating that these snails are rather homogeneous genetically. Glaudel(l972) found that PR-1 B. glabrata die more frequently during the prepatent period of infection with Puerto Rican S. mansoni than the other three susceptible strains. It is suggested that such inbred snails may be less hardy than other snail strains which have a greater degree of heterosis, indicated by their enzyme variability. PR-1 snails with mature S. ma~soni infections have two bands of peroxidase activity in the hepatopancreas, while ?JC and UCLA snails have one peroxidase (Bair & Etges, unpublished). The additional band of peroxidase activity arises from hemprotein of the hemolymph. This condition may be related to changes in hepatopancreas arteries and veins in PR-2 snails described by Pan (1965) and to their prolonged survival of patency observed by Glaudel(l972). The eighth and tenth esterases were always present in each snail of each strain. All snails, except some individuals of the VA strain, showed esterases seven and nine. VA snails are pure albinos, but any relationship between the occasional absence of esterases and albinism is unknown. UCLA B. glabrata were shown to be far more susceptible to infection with our S. mansoni strain than PR snails, but their survival rates following infection were
46
R. D. BAIR and F. J. ETOES
I.I.P. VOL. 3. 1973
essentially equivalent. While differing in susceptibility to infection, PR and UCLA snails have similar esterase polymorphism, indicating that susceptibility to infection is not related to hepatopancreas esterases. The third and fourth enzymes from all snail strains are likely to be carbonic anhydrases. Definitive identification of carbonic anhydrase awaits specific inhibition studies. The lack of any apparent changes in esterases from the hepatopancreas of infected snails suggests that these enzymes are not affected by pathology resulting from the presence of S. mansoni. The present study and those of Wright et al. (1966), Wright & File (1968) and Coles (1970) demonstrate important strain differences in schistosome vector snails. These studies indicate that enzyme electrophoresis could be used in work on the population dynamics of snails, the genetics of snails, and could provide valuable genetic markers in laboratorycultured snail strains. Acknowledgements-We thank Drs. Eli Chernin and E. H. Michelson of the Harvard School of Public Health, and Dr. Austin J. MacInnis, University of California at Los Angeles for supplying breeding stocks of snails used in this study. We are also indebted to Dr. Bruce L. Umminger for reviewing the manuscript.
REFERENCES BREWERG. F. 1970. An Introduction to Isozyme Techniques. Academic Press, New York, 186 pp. COLESG. C. 1970. Enzyme electrophoresis and speciation of Schistosoma intermediate hosts. Parasitology 61: 19-25. GILBERTXIND. E. 1966. A study of the proteins and amino acid of the hemolymph of infected and uninfected schistosome vector snails. Ph.D. Thesis, University of Cincinnati. GILBERTSON D. E. & ETGESF. J. 1967. Haemagglutinins in the haemolymph of planorbid snails. Annals of Tropical Medicine & Parasitology 61: 144-147. GLAUDELR. J. 1972. Studies on behavioral physiology of free-living stages of schistosomes (Trematoda: Schistosomatidae). M.S. Thesis, University of Cincinnati. PAN C. 1965. Studies on the host-parasite relationship between Schistosoma mansoni and the snail Australorbis glabratus. American Journal of Tropical Medicine & Hygiene. 14: 931-976. PARAENSEW. L. & CORREAL. R. 1963. Variation in susceptibility of Australorbis glabratus to a strain of Schistosoma mansoni. Revista do Institute de Medicina Tropical de SZo Paulo. 5: 15-22. WRIGHTC. A., FILE S. K. & ROSSG. C. 1966. Studies on the enzyme systems of planorbid snails. Annals of Tropical Medicine &Parasitology, 66: 522-525. WRIGHT C. A. & FILE S. K. 1968. Digestive gland esterases in the genus Bulinus (Mollusca, Planorbidae). Comparative Biochemistry & Physiology 27: 871-874.
DIFFERENCES IN ESTERASE FREQUENCIES FIVE STRAINS OF BIOMPHALARIA GLABRATA
IN (SAY)
R. D. BAIR and F. J. ETGES Department
of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221, U.S.A. (Receiued 19 May 1972)
Abstract BAIRR. D. and ETGESF. J. 1973. Differences in esterase frequencies in five strains of Biomphalariaglabrata (Say). International Journalfor Parasitology, 3: 4346. Vertical polyacrylamidegel electrophoresis of extracts from hepatopancreas tissue of individual snails of five laboratory strains of Biomphalariaglabrata has shown considerable variation in the frequency of occurrence of ten esterases. Differences in esterase frequencies seemed correlated with strain differences in certain morphological, physiological, and biochemical aspects of the snail strains; however, no causal relationships are apparent in these systems. INDEX KEY WORDS: Biomphalariaglabrata; esterase; Schistosoma mansoni; polymorphism; Mollusca; snail.
INTRODUCTION WRIGHT et al. (1966)
described considerable electrophoretic variation in hepatopancreas esterases of the snail Biomphalaria sudanica collected in Khartoum, Sudan and subsequently maintained in the laboratory for 9 yr. Wright & File (1968) pointed out the importance of population differences in the study of relationships between parasitic organisms and their hosts. Wright & File (1968), examining digestive gland esterases of five species of laboratoryreared Bulinus, concluded that electrophoretic separation of these enzymes provides a sensitive indication of genetic diversity between and within populations. Coles (1970) studied population and species variation in digestive gland esterases of two species of Bulinus and three species of Biomphalaria. In addition to finding obvious differences in esterase patterns between the species of snails, Bulinus africanus geographic strains from South and East Africa also had distinctly different esterase patterns, as did two strains of Biomphalaria pfe$eri.
The present study examines qualitative differences in hepatopancreas esterases in five laboratory strains of the neotropical snail Biomphalaria glabrata. Previous work and unpublished data from this laboratory have shown these strains to differ in several biological aspects, aside from enzyme polymorphism. Gilbertson & Etges (1967), working with three of the B. glabrata strains used in the present study, demonstrated considerable variation in the specificity of vertebrate red cell agglutinins of the hemolymph from these strains of snails. Gilbertson (1966) reported significant differences in hemolymph total protein, and demonstrated variation in susceptibility to infection with a single strain of Puerto Rican Schistosoma mansoni. Glaudel (1972), using the same snails employed in the present study, found some consistent differences in mortality rates of snails during both the prepatent and patent periods of infection as well as differences in their susceptibility to S. mansoni infection. MATERIALS
AND
METHODS
All B. gZabrata used in this study were laboratory-bred, fed with leaf lettuce and measured approximately 15 mm dia. The Puerto Rican strain of B. glabrata (PR) was obtained in 1958 from the National Institutes 43
I.I.P. VOL. 3. 1973
R. D. BAIR and F. J. ETCHES
44
of Health. These snails are moderately susceptible to infection with S. munsoni and have normal pigmentation. The Venezuelan albino strain of B. gfnbraru (VA) was acquired from the Tropical Research Medical Laboratory, San Juan, Puerto Rico, in 1962. This strain lacks normal pigmentation and is moderately susceptible to infection. The Bahian Brazilian strain of B. glubratu (BAH) was supplied by Dr. W. L. Paraense in 1964. These snails are highly refractory to infection (Paraense & Correa, 1963). A strain of B. glabratu (UCLA) was obtained from the University of California at Los Angeles in 1970; this strain is highly susceptible to infection and appears to die sooner than the other strains after the onset of patency. Puerto Rican B. glubrutu (PR-l), acquired from Harvard University’s School of Public Health in 1971, also are very susceptible to infection, but die more frequently than snails of other strains during the prepatent period. The hepatopancreas was dissected from 25 individual snails of each strain, and ground with a glass hand homogenizer in 100-150 ~1 of 0.05 M Tris Borate EDTA (pH 8.9)-5 % sucrose. Suspensions were centrifuged at 4-8°C at N 15,000 g for 5 min and the supematants subjected to vertical polyacrylamide-gel electrophoresis in 5 % gels at N 24 V/linear cm of gel. The buffer used during electrophoresis was 0.1 M Tris Borate EDTA (pH 8.9). Following 90 min of electrophoresis, gels were either stained in 100 ml of 0.1 M Tris-HCl (pH 7.1) containing 20 mg each of Q- and g-napthylacetate and 30 mg of Fast Blue RR, or with 20 ~1 of fl-napthylbutyrate and 30 mg of Fast Blue RR. Gels were incubated at room temperature for 20-30 min with Q- and p-napthylacetate. This staining procedure modified from that of Brewer (1970). permits tentative identification of carbonic anhydrase on the basis of a pink-purple color reaction in addition to revealing other esterases. Esterase activity determined with &napthylbutyrate was done at room temperature for 12 h. Infected snails were obtained by en masse exposure to large numbers of miracidia from the National Institutes of Health strain of Puerto Rican S. munsoni. Miracidia were obtained from eggs isolated from livers of Cox (Swiss) random bred white female mice. Densitometric tracings of electrophoretograms were done with a Densicord electrophoresis densitometer (Photovolt, Corp). Brewer’s (1970) recommendation that the most anodal band be numbered ‘1’ and the numbering continued in the cathodal direction was adopted in the present study. Variation in experimental conditions during electrophoresis resulted in some variation in the mobility of similar bands from one gel run to another. In order to compensate for such errors, the fourth most anodal band was used as a reference and the distances that all other esterases migrated from the origin were compared to its migration. This procedure provided an accurate and easily compared relative mobility value for each enzyme. Only the presence or absence of major enzymes was recorded. Minor variations in the migration of esterases, the presence of lightly staining, easily obscured bands and variation in the density of staining were not analyzed.
RESULTS
The representative densitometer tracing presented in Fig. 1 shows excellent separation of esterases, the relative position of each band, and the relative degree of esterase activity with a- and Bnapthylacetate substrates. The third and fourth bands of activity in all strains had a very pink-purple color, indicating carbonic anhydrase activity. All other sites were gray-brown in color. Table 1 gives the frequency of each enzyme in each strain. Comparison of gels incubated with a- and p-napthylacetate with those incubated in P-napthylbutyrate showed the same enzyme bands, with some variation in density of staining at most sites of activity. In gels stained with /I-napthylbutyrate, bands of activity were less 9
ci 4
6
-
0.5
I.0 Relative
FIG. 1. Densitometric
scan of esterases
+
mobility
of the hepatopancreas
of Biomphufuriu glubrutu.
I.J.P. VOL.
3. 1973
ESTERASES OF
Biomphalariaghbrata
45
defined and therefore more difficult to analyze. Accordingly, data presented in Table 1 are derived from gels stained with a- and /?-napthylacetate. The PR-1 strain showed the least variation of all strains examined (Table 1). With the exception of the sixth esterase, all enzymes were present in each PR-1 snail. Over-all, the VA snails were the most polymorphic, PR and UCLA snails had similar polymorphism, and the BAH snails exhibited moderate heterogeneity. Occasionally, four additional very lightly staining esterases could be detected in BAH extracts in particular. TABLE~--F~EQUENCYOFOCCURRENCEOFHEPATOPANCREASESTERASESIN 25 IND~IDUA~~~MEACHOFFIVE LABORATORY STRAINS OF 3iomp~Iar~g~abra~a.(No. OF SNAILS WITH ~TER~E~OTAL E~MINED.)
Frequency of occurrenceof enzymenumber
B. glabrata
strain
1
2
3
PR-1 UCLA PR BAH VA
l-00 O-88 0.92 0.96 1.00
I-00 0.84 0.92
l-00 o-92 O-80 076 0.72
1.00
0.72
4
5
6
la0 la0
140 o-92
O-80
0.96 044 o-92
@SO 0.72 0.96
0.76 0.72 I.00 0.80
7
8
9
10
I-00
140
la0
1.00
The seventh through the tenth esterases were always present in PR-1. UCLA, PR and BAH extracts. Only a few individuals from the VA strain lacked the seventh and ninth esterases, while each snail had esterases eight and ten. Most variation in esterases of all strains was present in the first six bands. The two most frequent deficiencies of esterases were the fourth one in the BAH strain and the ninth in the VA strain.
DISCUSSION The present study attempts to quantify variability in hepatopancreas esterases of five neotropical strains of B. glubrcztu. While several workers (Wright et al., 1966; Wright & File, 1968; Coles, 1970) have described hepatopancreas esterase polymorphism in schistosome vector snails, none have attempted to quantify variation between snail strains. Hepatopancreas esterases of the PR-1 strain show a strong degree of monomo~hism indicating that these snails are rather homogeneous genetically. Glaudel(l972) found that PR-1 B. glabrata die more frequently during the prepatent period of infection with Puerto Rican S. mansoni than the other three susceptible strains. It is suggested that such inbred snails may be less hardy than other snail strains which have a greater degree of heterosis, indicated by their enzyme variability. PR-1 snails with mature S. ma~soni infections have two bands of peroxidase activity in the hepatopancreas, while ?JC and UCLA snails have one peroxidase (Bair & Etges, unpublished). The additional band of peroxidase activity arises from hemprotein of the hemolymph. This condition may be related to changes in hepatopancreas arteries and veins in PR-2 snails described by Pan (1965) and to their prolonged survival of patency observed by Glaudel(l972). The eighth and tenth esterases were always present in each snail of each strain. All snails, except some individuals of the VA strain, showed esterases seven and nine. VA snails are pure albinos, but any relationship between the occasional absence of esterases and albinism is unknown. UCLA B. glabrata were shown to be far more susceptible to infection with our S. mansoni strain than PR snails, but their survival rates following infection were
46
R. D. BAIR and F. J. ETOES
I.I.P. VOL. 3. 1973
essentially equivalent. While differing in susceptibility to infection, PR and UCLA snails have similar esterase polymorphism, indicating that susceptibility to infection is not related to hepatopancreas esterases. The third and fourth enzymes from all snail strains are likely to be carbonic anhydrases. Definitive identification of carbonic anhydrase awaits specific inhibition studies. The lack of any apparent changes in esterases from the hepatopancreas of infected snails suggests that these enzymes are not affected by pathology resulting from the presence of S. mansoni. The present study and those of Wright et al. (1966), Wright & File (1968) and Coles (1970) demonstrate important strain differences in schistosome vector snails. These studies indicate that enzyme electrophoresis could be used in work on the population dynamics of snails, the genetics of snails, and could provide valuable genetic markers in laboratorycultured snail strains. Acknowledgements-We thank Drs. Eli Chernin and E. H. Michelson of the Harvard School of Public Health, and Dr. Austin J. MacInnis, University of California at Los Angeles for supplying breeding stocks of snails used in this study. We are also indebted to Dr. Bruce L. Umminger for reviewing the manuscript.
REFERENCES BREWERG. F. 1970. An Introduction to Isozyme Techniques. Academic Press, New York, 186 pp. COLESG. C. 1970. Enzyme electrophoresis and speciation of Schistosoma intermediate hosts. Parasitology 61: 19-25. GILBERTXIND. E. 1966. A study of the proteins and amino acid of the hemolymph of infected and uninfected schistosome vector snails. Ph.D. Thesis, University of Cincinnati. GILBERTSON D. E. & ETGESF. J. 1967. Haemagglutinins in the haemolymph of planorbid snails. Annals of Tropical Medicine & Parasitology 61: 144-147. GLAUDELR. J. 1972. Studies on behavioral physiology of free-living stages of schistosomes (Trematoda: Schistosomatidae). M.S. Thesis, University of Cincinnati. PAN C. 1965. Studies on the host-parasite relationship between Schistosoma mansoni and the snail Australorbis glabratus. American Journal of Tropical Medicine & Hygiene. 14: 931-976. PARAENSEW. L. & CORREAL. R. 1963. Variation in susceptibility of Australorbis glabratus to a strain of Schistosoma mansoni. Revista do Institute de Medicina Tropical de SZo Paulo. 5: 15-22. WRIGHTC. A., FILE S. K. & ROSSG. C. 1966. Studies on the enzyme systems of planorbid snails. Annals of Tropical Medicine &Parasitology, 66: 522-525. WRIGHT C. A. & FILE S. K. 1968. Digestive gland esterases in the genus Bulinus (Mollusca, Planorbidae). Comparative Biochemistry & Physiology 27: 871-874.