Electrophoretic studies on the digestive gland esterases of some biomphalarid and lymnaeid snails

Bull. Org. mond. Sante )1971, 45, 819-825Bull. Wld Hlth Org. Electrophoretic Studies on the Digestive Gland Esterases of some Biomphalarid and Lymnaeid Snails* EMILE A. MALEK1 & SHARON K. FILE1 Because ofthe problems encountered in the classification ofsnails ofmedical importance, biochemical methods have been sought to help clarify the situation. Of these, the separa- tion ofthe enzymes of adult snails by electrophoresis seems the most promising but very few attempts have been made so far to use the results for taxonomic studies. The purpose of the present study was to evaluate the use of the enzyme systems of neotropical planorbid and of lymnaeid snails to elucidate their taxonomy and also snail-schistosome relationships at the species and population levels. The findings show the characteristic electrophoretic patterns ofdigestive gland esterases of the planorbid and lymnaeid snails used, as well as their variation and the level of such variation among certain populations and the consistency of the patterns among others. The results also show that, in general, the extent of variation between some populations of the same species is greater than the differences between species of the same group. However, at the specific level, there are similarities suggesting close relationships between some populations of Biomphalaria glabrata and B. tenagophila on the one hand, and of certain populations of B. peregrina and of B. obstructa on the other hand. The present study has thrown some light on the question of electrophoretic variation in enzymes, and the ways in which this can be applied to studies of the genetics of snails. A correlation is suggested between certain patterns that indicate biochemical similarities or differences among the planorbid snail populations and the susceptibility of the species or the population to infection with the schistosomes. The systematics of freshwater snails of medical importance is in a chaotic state. This is especially true with the lower taxa, and clarification has been attempted by several different methods. The criteria used are morphological, serological, and biochemical. Wright & Ross (1965, 1966) found that the planorbid egg proteins, when subjected to electrophoresis, are sensitive indicators of differences at the population level. Recently Wright et al. (1966) attempted a study of the enzyme systems of adult planorbid snails to elucidate aspects of their systematics, and to examine some of the biochemical factors that may influence the capacity of these snails to act as hosts for certain trematodes, in particular the schistosomes. * This investigation was supported by Public Health Service Research Career Award K6-AI-18,424 and by Research Grant AI-02898, from the National Institutes of Allergy and Infectious Diseases. 1 Department of Parasitology, School of Public Health and Tropical Medicine, Tulane Medical School, New Orleans, La., USA. It was established in the above report that the esterases of the digestive gland from mature snails, maintained on a standard diet, were very useful for comparative purposes. It was the purpose of the present study to evaluate the use of the enzyme systems of planorbid snails in a study of the taxonomy and host-parasite relation- ships. In this evaluation, special emphasis was placed on determining whether differences could be recognized in the electrophoretic patterns of enzymes among different neotropical species of Biomphalaria, among different populations of the same species, and among individuals of the same field-collected or laboratory-reared populations. The biomphalarids used are actual and potential hosts of Schistosoma mansoni in South America, in some Caribbean islands, and in countries bordering the Gulf of Mexico. The study was also extended to cover certain species and populations of lymnaeid snails, some of which are intermediate hosts of the mammalian 2773 - 819- 820 E. A. MALIK & S. K. FILE schistosome Heterobilharzia americana, which is prevalent in the southern USA from Texas to North Carolina and South Carolina. MATERIALS AND METHODS The planorbid material used in this study was obtained from various parts of the neotropics. Nine populations of Biomphalaria glabrata were used; one was collected at Paulista Pemambuco, Brazil, and the snails were reared in the laboratory for 3 years; the second was originally from Puerto Rico and has been reared for about 17 years; the third was collected in St Lucia, West Indies, and reared for 2 years; the fourth is an albino Puerto Rican- Venezuelan cross from the Parasitic Diseases Labor- atory of the National Institutes of Health, Bethesda, Md.; the fifth was brought from Dique, Bahia, Brazil, by Dr Air Barretto; the sixth was sent by Dr Lobato Paraense from Belo Horizonte, Minas Gerais, Brazil; the seventh was collected at Agua Branca, Minas Gerais, Brazil; the eighth was sent from La Victoria, Venezuela, by Dr J. Herrer Faria and Mr P. Chrochiechowski; and the ninth, from Olinda, Pernambuco, Brazil, was sent to us by Dr Frederico Barbosa of Recife, Pernambuco, Brazil. Two populations of Biomphalaria tenagophila were studied: one was collected from a watercress planta- tion at Jacarapagua, Guanabara, Brazil, and reared in the laboratory for 3 years, and the other, from Dom Bosco, Minas Gerais, Brazil, was sent to us by Dr Lobato Paraense. One population of Biomphala- ria straminea from Olinda, Pernambuco, Brazil, was sent by Dr F. Barbosa, and one of Biomphalaria peregrina from Gaspar Lopes, Minas Gerais, was sent by Dr L. Paraense. Biomphalaria obstructa was collected in the field from Spillway near Norco and New Orleans, Louisiana, USA, and from drainage ditches and swamps on Carmen Island, Campeche, Mexico. All the lymnaeid snails used in this study were collected in the USA, some in Louisiana (New Orleans and Baton Rouge), some in Michigan (Clinton, Bass Lake, and near Ann Arbor), and some in Texas (Houston). The laboratory-bred snails were maintained on a lettuce diet, and field- collected specimens were fed on lettuce until studied or were dissected at once. The animals were kept at 4°C for 30 min before dissection. The digestive gland was removed free of any other tissues; in some of the immature lymnaeids, however, the ovotestis was not removed. The tissue from each individual snail was homogenized manu- ally in distilled water in a glass homogenizer. This material was stored in the freezer at -20°C. Material that had been frozen for up to 6 months was found to be similar to fresh unfrozen prepara- tions. Before use, each extract was centrifuged and the supernatant fluid was inserted into a template slot in a starch gel. The starch was prepared accord- ing to the method of Smithies (1955), the method that was also used by Wright et al. (1966) (10.5% Connaught hydrolysed starch in 0.03 M tris-boric acid buffer, pH 8.0). The cooked starch was poured into a Shandon agar-electrophoresis tray containing eight 2.5 x 7.5-cm microscope slides to a depth of 1 mm. Whatman No. 3 filter-paper was used as a bridge between the gel and the electrode buffer (0.3 M tris-boric acid buffer, pH 8.0). The gels were stored at 4°C for 12-18 hours before they were used. Electrophoresis was carried out in a Shandon Kohn Mk II tank. During electrophoresis, a current of 5 mA was applied for I hour giving a potential of 10 volts per cm at room temperature. The gels were then immersed in a solution consisting of 100 ml of 0.1 M tris-maleate buffer, pH 7.5, 1 ml of a 1% solution of substrate in acetone solution, and 80 mg of Fast Blue RR salt. The incubation time varied between 15 and 60 min depending upon the substrate. Inhibitors were added to the super- natant material and preincubated or they were added to the incubation media. The gels were photographed fresh, then were allowed to dry, or they were embedded in glycerol-gelatin and dried. RESULTS Biomphalarid snails Photographs of a selection of slides are shown in the figures. Five electrophoretic patterns (Fig. 1) were observed with the biomphalarids examined. In these patterns certain anodal bands representing fractions are included. The numbering of the bands follows the speed of separation, band no. I being the fastest running fraction and band no. 5 the slowest running. pattern 1 shows bands 1, 2, 3, 4, 5 pattern 2 shows bands Is, 2,1 3, 4 pattern 3 shows bands 1, 3, 4W,2 5 pattern 4 shows bands 1, 2, 3, 4 pattern 5 shows bands Is, 3, 4, 5 1 Often covered by Is and not resolved. 2 Probably different from the intense No. 4; the two are never present together. 12 3 4 5 Fig. 1. The five electrophoresis patterns found after separating the digestive gland esterases of Biomphalaria species. Puerto Rico Puerto Rico Puerto Rico Puerto Rico Puerto Rico Paulista, Brazil Paulista. Brazil Fig. 2. Electrophoresis patterns found after separating the digestive gland esterases of some laboratory- reared populations of Biomphalaria glabrata. B.glabrata, Dique, Brazil B. glabrata, Agua Branca, Brazil B. glabrata, Belo Horizonte, Brazil B. glabrata, La Victoria, Venezuela B. tenagophila, Guanabara, Brazil B. peregrina, Minas Gerais, Brazil B. straminea, Pernambuco, Brazil B. obstructa, New Orleans, USA I, ;....... F1.i I:!..' 1. i .I Fig. 3. Electrophoresis patterns found after separating the digestive gland esterases of Biomphalaria species collected from different areas. F. cubensis, New Orleans, USA F. cubensis, New Orleans, USA F. cubensis, Houston, USA F. cubensis, Houston, USA F. cubensis, Baton Rouge, USA F. cubensis, Baton Rouge,.USA - F. humils, Clinton, USA F. humilis, Bass Lake, US Fig. 4. Electrophoresis patterns found after separating the digestive gland esterases of Fossaria species collected from different areas. A B C ZJ Ef ...... ...... 821DIGESTIVE GLAND ESTERASES OF SNAILS: ELECTROPHORETIC STUDIES Table 1. Proportions of electrophoretic patterns of digestive gland esterases (see Fig. 1 ) obtained for certain neotropical planorbid snail populations Population B. glabrata (Puerto Rico) B. glabrata (Paulista, Brazil) B. glabrata (St. Lucia) B. glabrata (albino, Puerto Rican x Venezuelan) B. glabrata (Dique, Brazil) B. glabrata (Belo Horizonte, Brazil) B. glabrata (Agua Branca, Brazil) B. glabrata La Victoria, Venezuela B. glabrata Olinda, Brazil B. tenagophila (Dom Bosco, Brazil) B. tenagophila (Jacarapagua, Brazil) B. straminea Olinda, Brazil B. peregrina (Gaspar Lopes, Brazil) B. obstructa (New Orleans, USA) B. obstructa (Carmen Island, Mexico) No. of snails examined - 74 95 17 11 58 44 I 16 85 15 Patterns obtained, and percentage of each a 1 2 3 4 5 37 16 38 5 3 27 67 1 3 2 + + + + + _ 100 100 100 - - + + 10 26 14 100 - + 15 122 100 12 a Whenever the number of snails was small, the pattern is indicated either as observed (+), or not observed (-). Differentiation of the 5 patterns, therefore, depends on the presence or absence of bands 4 or 5, the Is variant of band 1, and band 4W. Band 3 is present in all patterns, while band 2 is absent in pattern 5 and seems to be absent or very weak in pattern 3. The percentage ofeach pattern, in the biomphalarid species and populations examined, is shown in Table 1: when the number of snails examined was small, percentages were not calculated. It is evident that variations were encountered among some populations of Biomphalaria glabrata, but not with B. tenagophila. Laboratory-reared B. glabrata, stocks from Paulista, Pernambuco, Brazil (Fig. 2), those from Puerto Rico, and the albino B. glabrata, Puerto Rican-Venezuelan cross showed all 5 patterns. However, B. glabrata from Dique, Bahia, Brazil, and from La Victoria, Vene- zuela (Fig. 3), consistently showed pattern 3, while B. glabrata from Belo Horizonte and Agua Branca, Minas Gerais, Brazil, consistently showed pattern 4. B. glabrata from St Lucia showed all the patterns except pattern 4 and B. glabrata from Olinda, Pernambuco, Brazil, exhibited patterns 1, 2, and 3. It is to be noted that no significant anatomical differences were observed between any of the above populations of B. glabrata. Four other species of biomphalarids were also studied. B. tetiagophila both from Dom Bosco, E. A. MALIK & S. K. FILE Minas Gerais, and from Jacarapagua, Guanabara, Brazil, showed pattern 3. No variation was observed among individuals of the same population of B. obstructa. Thus all the B. obstructa collected in the field from Spillway, New Orleans, La., USA, were of pattern 4 (Fig. 3), while all of the laboratory- reared B. obstructa (stock from Carmen Island, Mexico) were of pattern 3. B. straminea from Olinda, Pernambuco, Brazil, consistently exhibited pattern 1, while B. peregrina from Gaspar Lopes, Minas Gerais, Brazil, showed pattern 3 only. With the biomphalarid populations examined, a varying number of cathodal bands appeared. Besides being very faint their variability seems to be without any definite pattern, and thus they cannot be used for comparison. Lyilnaeid snails Of the lymnaeid snails studied, there were 4 popu- lations of Fossaria cubensis (=Lymnaea (Fossaria) ciubensis) from New Orleans, La. (Audubon Park, 10 snails; and from near Huey Long Bridge, 100 snails), from Baton Rouge, La., (42 snails), and from Houston, Tex. (6 snails); two populations of Fossaria humilis ( =Lymnaea (Fossaria) humilis) from Michigan (38 snails from Clinton, and 12 from Bass Lake); two populations of Pseudosuccinea columella (=Lymnaea (Pseudosuccinea) columella) from New Orleans, La. (25 snails), and from Baton Rouge, La. (27 snails); and one population of laboratory-reared Stagnicola palustris (=Lymnaea (Stagnicola) palustris) from near Ann Arbor, Mich. (30 snails). Variation was observed among the population of Fossaria cubensis from New Orleans. Three distinct patterns of anodal bands could be recognized, and are designated A, B, and C in Fig. 4. Pattern A, the predominant one, was found in 65 % of the specimens studied, pattern B in 10%, and pattern C in 10%. No distinct pattern was observed in the remaining 15%0 of the specimens. Pattern A has a massive band close to the starting line, and two minor bands of moderate mobility. The pattern exhibited by the population from Houston, Tex., is probably the same as pattern A for the New Orleans popula- tion except that the main band is slightly closer to the starting line. The population from Baton Rouge was not homogeneous and showed two patterns that differed in the number of bands and their mobility. The two populations of Fossaria humilis from Clinton and Bass Lake, Mich., showed essentially similar patterns. Homogenates containing specimens from both produced no extra bands. It is also of interest to note that the two popula- tions of Pseudosuccinea columella from New Orleans and Baton Rouge showed similar and consistent patterns. No variation was observed among the speci- mens of Stagnicola palustris that were investigated. Pronounced cathodal bands were obtained with the lymnaeid species and populations studied, especially with Pseudosuccinea columella and Stagni- cola palustris. Five of these cathodal bands were in the patterns for both these species: with Stagnicola palustris they are bands no. 12-16. Anodal band no. 11 is a more pronounced fraction and is closer to the starting line than was shown by Norris & Morrill (1964) for the same species. In our results for this species, anodal band no. 3 is very faint while cathodal bands 14, 15, and 16 are very pronounced. Table 2 shows the results of inhibition tests in the case of esterases of the biomphalarid digestive gland, carried out with various inhibitors. The esterases were inhibited by 10-3M physostigmine sulfate, by 1O-2M and 1O-4M PCMB ((carboxyphenyl) chloro- mercury sodium salt); by 1O-3M sodium fluoride, and by 1O-3M DFP (diisopropyl phosphorofluoridate), and were not inhibited by 1O-6M physostigmine sulfate, 10-5M PCMB, or 1O-3M EDTA (ethylene- Table 2. Results of inhibition tests of esterases of biomphalarid digestive gland, preincubated for 15 min at 37°C; pH adjusted to 7.5 Inhibitor Concentration Results a physostigmine sulfate 10-3M ++ 10 6M NaF 10-3M + PCMB 10-2M + + 10-4M ++ 10-5M EDTA 10-3M DFP 10-3M + a + = 50 % of the bands inhibited ++ = all bands inhibited - = no inhibition. 822 DIGESTIVE GLAND ESTERASES OF SNAILS: ELECTROPHORETIC STUDIES diaminetetraacetic acid). These results were to be expected; to determine the nature of the various enzymes more refined and detailed tests should be used. DISCUSSION The separation of esterases of the digestive gland of the biomphalarid species studied did achieve some positive results. That one or more of 5 electro- phoretic patterns were exhibited by Biomphalaria glabrata, and one or more of the same patterns were shown by the other populations of the biomphalarid species, was expected on account of the close relation- ships between species of this neotropical planorbid group. However, the results also show that there were pronounced intra- and interpopulation varia- tions among the snails investigated. Norris & Morrill (1964) indicated that differences in band patterns may be due to differences or errors in the isolation procedure or to dilution of non-enzymatic substances and may even be caused by autolysis products arising from proteolytic activities in the homogenate. In our studies, however, the patterns reported have been selected from the results of a number of runs in order to rule out some of the above possibilities. Moreover, the extracts were mixed and subjected to electrophoresis to ensure the validity of the results. Variations were particularly noticeable among the 9 populations of B. glabrata. Wright et al. (1966) also encountered variations in the digestive gland esterases of B. sudanica, even between individuals of comparable age and dietary background. On the other hand consistent patterns were exhibited by Biomphalaria tenagophila, B. peregrina, B. straminea, and four of the B. glabrata populations. B obstructa from New Orleans showed a consistent pattern (pattern 4), and the population of the same species from Carmen Island, Campeche, Mexico, showed pattern 3. Since only 12 specimens of the latter population were used, it is felt that more material should be examined from this and other populations. It should be noted that no morpho- logical differences were found between individuals of the two populations of B. obstructa. Electrophoretic pattern 3 was exhibited by B. tenagophila from two localities, by B. peregrina, by one population of B. obstructa, and by two popula- tions of B. glabrata from Dique, Brazil, and from La Victoria, Venezuela. Some possible explanations can be given for the observed similarities. B. peregrina and B. tenagophila are both South American, and within that area their geographical distribution over- laps. B. glabrata is distinguished from other species of neotropical Biontphalaria by the presence ofa renal ridge on the ventral surface of the kidney. Such a ridge or a remnant of it is occasionally present in B. tenagophila also (Barbosa, 1964). This might explain some relationships between B. tenagophila and certain populations of B. glabrata. It is not surprising that similarities were observed between B. peregrina and one population of B. obstructa. Certain biomphalarid species (among which is B. obstructa) in northern South America, in some of the Caribbean islands, and in countries bordering the Gulf of Mexico show relationships with the southern form, B. peregrina (Malek, 1969). The present study has thrown some light on electrophoretic variation in enzymes and the ways in which this can be applied to a study of the genetics of snails: variant enzymes, being the direct products of genes, can be used as useful genetic markers. It may be concluded from the results of the present study that the populations that are homogeneous for patterns 1, 3, or 4 are homozygous forms. The fact that we did not find a population showing only pattern 2 indicates that pattern 2 is heterozygous, and is the result of " hybrid " molecules demonstrated as characteristic bands. It is logical to assume that biochemical similarities or differences between snail populations might be related to susceptibility of the snails to infection with the schistosomes and might also be related to the magnitude of the infection the schistosomes produce in the snail and later in the mammal. Thus with regard to the biomphalarids studied in the present work, it is postulated on the basis of the results obtained that there might be a correlation between pattern 3 and poor susceptibility, or nonsuscepti- bility, of the snail to infection with Schistosoma mansoni. Pattern 3 alone was demonstrated by B. glabrata from Dique, by B. tenagophila from two localities, by B. obstructa from Mexico, and by B. glabrata from La Victoria. It is known that some populations of B. tenagophila are nonsusceptible to infection with S. mansoni, and others are only very slightly susceptible. Several populations of B. obstructa were found to be nonsusceptible to infec- tion with 3 different strains of S. mansoni, one from Puerto Rico, one from St Lucia, and one from Minas Gerais, Brazil (Malek, 1967a). The popula- tion of B. obstructa from Carmen Island, Mexico, was tested and found to be nonsusceptible to a 823 E. A. MALIK & S. K. FILE Puerto Rican and a St Lucian strain (Malek, un- published data). As regards B. glabrata, it has been demonstrated that populations of this species show differences in their susceptibility to various strains of S. n7ansoni. Bahian strains of B. glabrata, includ- ing the one from Dique used in the present study, showed a very low susceptibility, or none at all, to infection with S. mansoni from Paulista, Pernambuco (Barbosa & Barreto, 1960), and to a Belo Horizonte strain of S. mansoni (Paraense & Correa, 1963). Thus the above postulate linking poor susceptibility to pattern 3 seems to fit the facts. One cannot include the population of B. glabrata from La Victoria, Venezuela, which also showed pattern 3 only, since its susceptibility was not tested. No correlation, however, can be drawn between susceptibility and the occurrence of pattern 4. The populations that showed only this pattern were those of B. glabr-ata from Belo Horizonte and Agua Branca in Brazil, and of B. obstructa from Spillway near New Orleans, La. The first two populations are known to be highly susceptible to S. mansoni from Belo Horizonte (Paraense & Correa, 1963). The natural infection rates in material collected by the writer on several occasions from Agua Branca were up to 50%. B. obstructa from Spillway and from several other localities in Louisiana proved to be nonsusceptible to infection with 3 strains of S. n1ian/sonii (Malek, 1967a). With the lymnaeids tested in the present study, no attempt should be made to link susceptibility of the snail and the electrophoretic pattern obtained for the digestive gland esterases until more work has been done. All Fossaria cubensis and Pseudosuccinea col/amella from Louisiana are highly susceptible to infection with Heterobilharzia americana; Fossaria hitmilis from Michigan is poorly susceptible and Stagnicola palustris is nonsusceptible to infection with the same schistosome (Malek, 1967b). On the other hand Stagnicola palustris is the host for another mammalian schistosome, Schistosomatium douthitti. The findings with the lymnaeids were, otherwise, very interesting. Pseudosuccinea columella from New Orleans and from Baton Rouge, Fossaria humilis from either Clinton or Bass Lake, Mich., and Stagnicola palustris showed consistent and distinctive patterns, which can be very useful in their identifica- tion. Variation occurred with Fossaria cubensis from New Orleans, but 65% of the snails studied showed one pattern. A condition that might have accounted for the variation was that the ovotestis was not removed completely, portions of the latter organ being left with the digestive gland. Another possible explanation is the age of the snails used: the majority of the snails were not mature. Wright et al. (1966) considered that age of the snails was very important, and that most of the consistent patterns were given by fully mature specimens. It is evident, therefore, that the work with Fossaria cubensis should be extended to include fully mature snails and pure homogenates of the digestive gland. RURES ME ETUDE ELECTROPHORETIQUE DES ESTERASES DES GLANDES DIGESTIVES DE MOLLUSQUES BIOMPHALARIA ET LYMNAEA Jusqu'ici, on n'a consacre que peu de travaux a l'etude par electrophorese des systemes enzymatiques des mol- lusques d'eau douce importants en sante publique. Le present article decrit les recherches entreprises dans ce domaine afin de determiner si les caracteristiques elec- trophoretiques des enzymes peuvent servir a mieux connaitre la taxonomie des mollusques et les relations mollusque-parasite. On a choisi comme materiel les esterases des glandes digestives de Biomphalaria et Lymnaea adultes. L'origine de ce materiel et les methodes utilisees sont decrites en detail. L'etude de diverses especes de Biomphalaria permet d'identifier cinq types d'image electrophoretique. On note des variations suivant les populations chez B. glabrata. On obtient en revanche des traces electrophoretiques constants avec B. tenagophila, B. peregrina, B. straminea et quatre populations de B. glabrata. 11 en va de meme pour B. obstructa (New Orleans) et B. obstructa (Carmen Island). Aucune difference morphologique n'a ete observee entre individus des deux populations de B. obstructa. En ce qui concerne les mollusques Lymnaea, on dcele 3 aspects electrophoretiques distincts chez une population de Fossaria cubensis (New Orleans) et 2 chez la meme espece originaire de Baton Rouge. Des images constantes et caracteristiques sont observees chez deux populations de Fossaria humilis (Michigan), deux populations de Pseudosuccinea columella (Louisiane) et une population de Stagnicola palustris elevee en laboratoire. On enregistre des bandes cathodiques particulierement nettes avec 824 DIGESTIVE GLAND ESTERASES OF SNAILS: ELECTROPHORETIC STUDIES 825 Pseuldosu ccinea coluniella et Stagnicola palustris. La regularite et l'aspect caracteristique de ces images peuvent beaucoup aider a identifier ces especes de mollusques. On donne d'autre part les resultats d'epreuves d'inhi- bition des esterases de glandes digestives de mollusques Bioiuphalaria realisees avec differents inhibiteurs. Les auteurs attirent I'attention sur les correlations qui semblent se faire jour entre certaines images electro- phoretiques (indices d'analogies ou de differences biochi- miques entre populations de mollusques) et la receptivite d'espece ou de population a l'infection schistosomienne. On releve par ailleurs des similitudes electrophoretiques donnant a penser qu'il existe des affinites etroites entre certaines populations de B. glabrata et B. tenagophila d'une part et certaines populations de B. peregrina et B. obstructa d'autre part. REFERENCES Barbosa, F. S. (1964) Rev. Inst. Med. trop. S. Paulo, 6, 64-70 Barbosa & Barretto, A. C. (1960) Exp. Parasit., 9, 137- 140 Malek, Emile A. (1967a) Amer. J. trop. Med. Hyg., 16, 715-717 Malek, Emile A. (1967b) J. Parasit., 53, 700-702 Malek, Emile A. (1969) Malacologia, 7, 183-209 Norris, E. & Morrill, J. B. (1964) Acta Embryol. Morph. exp. (Palermo), 7, 29-41 Paraense, W. L. & Correa, L. R. (1963) Rev. Inst. Med. trop. S. Paulo, 5, 15-22 Shaw, C. R. (1965) Science, 149, 936-942 Smithies, 0. (1955) Biochem. J., 61, 629 Wright, C. A., File, S. K. & Ross, G. C. (1966) Ann. trop. Med. Parasit., 60, 522-525 Wright, C. A. & Ross, G. C. (1965) Bull. Wld Hlth. Org., 32, 709-712 Wright, C. A. & Ross, G. C. (1966) Bull. Wld Hlth Org., 35, 727-731 10