667

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 73, No. 6, 1979

Enzyme analyses of Buhnus africanus group snails ( : ) from Tanzania

D. ROLLINSON AND V. R. SOUTHGATE Experimental Unit, British Muesum (Natural History), London SW7 5BD Downloaded from https://academic.oup.com/trstmh/article/73/6/667/1885199 by guest on 29 September 2021

Summary and suggested that such methods could be of 38 population samples of snails of the particular use for local taxonomic problems. africanus group, collected from three separate areas The present study was undertaken to establish of Tanzania, have been examined. Enzymes in whether B. africanus group snails, collected from crude digestive gland extracts of individual snails coastal and inland areas of Tanzania, could be have been analysed by isoelectric focusing in distinguished by their enzyme types and whether polyacrylamide gels. The enzymes studied were: the application of biochemical methods would help malate dehydrogenase (MDH); phosphogluco- to elucidate the relationships within and between mutase (PGM); glucosephosphate isomerase (GPI); populations. acid phosphatase (A#) and hydroxybutyrate dehydrogenase (HBDH). Materials and Methods Samples of B. nasutus were clearly differentiated Snails were collected in October, 1977, from a from other species and enzyme differences were variety of aquatic habitats located in three separate apparent between samples from the lake and coastal areas of Tanzania, as indicated on the accompanying areas. Similarly, although clear distinctions could sketch map (Fig. 1). 11 population samples of not always be made, samples of B. africanus, B. B. africanus grouu snails collected from the Tanaa globosus and B. ugandae were characterized by their region, five fx%m habitats close to Dar es Salaam a:d enzyme types. Individual variation was detected a further 22 from the lake region around Mwanza within populations and the significance of enzyme survived and formed the basis of this studv. The polymorphisms in relation to identification has been identification of the snails, determined by eximina- considered. No correlation was found between snail tion of the shell and copilatory organ, ;he locality enzyme type and susceptibility to Schistosoma and the tvue of habitat are detailed in Table I. haematobium or S. bovis. Before analysis, snails were screened for shedding of cercariae. Hamsters were exposed to schistosome Introduction Snails belonging to the Bulinus africanus species group are of particular interest because they com- monly act as intermediate hosts for some Schisto- so& spp. Discrimination of species within the Fig. 1 groun is difficult due to the variabilitv of both kternal and external .morphological Characters. Although many populations appear to exhibit clear-cut species characters, others possess characters which are either intermediate or which combine the ‘.\. morphological peculiarities of one species with Mwanza \. those of another. In Tanzania, four species of the B. africanus group, B. africanus, B. nasutus, B. globosus and B. ugandae, are known to occur: their distribution has been described by STURROCK (1965). WEBBE (1958), in a survey of the Handeni and Korogwe districts of Tanzania, recorded both globosus- nasutus intermediates and nasutiform africanus. The difficulties that exist in making reliable species identifications were emphasized by MANDAHL- BARTH (1965). The medley of forms encountered in East Africa led WRIGHT (1961) to suggest that B. africanus group snails probably originated in this area, greater distinctions becoming apparent as the snails radiate outwards from their centre of evolution. Recently, WRIGHT & ROLLINSON (1979) reported the use of isoelectric focusing of enzymes as an aid to the characterization of members of the B. Fig. 1. Sketch-map of Tanzania showing the three areas from which africanus group from diverse geographical areas, snails were collected. 668 ENZYME ANALYSES 0~ Bulinus FROM TANZANIA

Table I-Locality, type of habitat and enzyme type of population samples of B. africanus group snails

Sample Lab. No. Species Ref. Locality Habitat MDH PGM GPI AcP HBDH Tanga B. nasutus 497 Talawani S 1 cl 1 .: >> 510 Pangani 33 1 cl 3 ,> 519 Talawani N 3 1 cl : 4 ,, 520 Mn’garoni Rd 1 cl 1 5 ,, 521 Takindu Lake z 1 cl 1 Downloaded from https://academic.oup.com/trstmh/article/73/6/667/1885199 by guest on 29 September 2021 6 Mn’garoni Pond n >, 523 1 cl 1 524 Takindu River ; 1 cl ti B. gi;bosus 514 Lake Kilimele c2 ii 9 3, 518 Kulumuzi River : $2 10 ,, 525 Lake Masaika 1 $3 2,10,11~12,13 11 498 Nairobi Dam 1 $2 c2 10 Dar es Salaam ” 12 B. nasutus 527 Lukware l/4 cl 13 ,, 528 Morogoro F ; 1 cl/c2 3,4f5 14 J, 529 ,, 3 1 cl/c2 31415 15 J, 532 >J E 3 cl 16 J, 533 ,, D 3 : cl : Mwanza 17 B. nasutus 535 Misungwi A 3 c3 2 18 ,, 536 Misungwi B : c3 19 ,, 538 Mwuamba A s c3 ; J, 539 Mwuamba B 3 : c3 E >> 542 Misungwi Dam 3 1 c3 ; J, 545 Misungwi F 3 1 c3 2 2 >> 547 G 3 1 c3 24 ,> 555 Fula” 3 1 c3 i 25 >> 557 Nyanguge A 1 c3 >> 558 B z 1 c3 ; ;; >> 560 Fela” 3 1 c3 2 28 >9 564 Sese A 1 c3 >> 466 B ; c3 ; zz ,J 569 Se)s)e A 3 : c3 2 31 550 Nyakoto c3 B. o&anus 535 Misungwi A 3 a c3 2 3: 9, 537 J, C 3 c3 34 ,J 544 E 3 273 c4 : 35 568 Sese’i3 3 2 c4 6 36 B. gi;bosus 551 Nyakbungo 3 c3 - 37 B. ugandae 552 Kigoto 3 ; c3 7 38 >> 553 Butinba 3 2 c3 7 P - pool L - lake R- river/stream BP -borrow pit P/R - pool at time of sampling, occasionally river

cercariae and the resulting adult worms were isomerase (GPI), acid phosphatase (Acl?) and identified. Snails were killed after a short period in hydroxybutyrate dehydrogenase (HBDH). MDH, laboratory culture and usually after egg masses had AcP and HBDH were separated on pH 4 to 9 gels, been laid. whereas pH 4 to 7 gels were used for resolving Five enzymes in the digestive gland and gonad GPI and PGM. Egg proteins from 29 of the of individual snails were analvsed bv isoelectric populations were also analysed by cellulose acetate focusing in polyacrylamide gel following-the methods electrophoresis (WRIGHT & Ross, 1965). of WRIGHT & ROLLINSON (1979). The number of snails available for analysis from each population Results sample varied from between one and 30. The 1. Enzyme analyses enzymes were: malate dehydrogenase (MDH), The enzyme types were distinguished by the phosphoglucomutase (PGM), glucosephosphate differing p1 values of the bands of enzyme activity D. ROLLINSON AND V. R. SOUTHGATE 669

PH PH Downloaded from https://academic.oup.com/trstmh/article/73/6/667/1885199 by guest on 29 September 2021

7 7 7 7 10101010181818183535 7 7 7 7 10101010181818183535

Plate 1. MDH of B. africnrrfrs group snails separated by isoelectric Plate 2. PGM of B. africa~tur group snails separated by isoelectric focusing. Samples 7, 18 and 35, MDH-3; sample 10, MDH-1. focusing. Samples 7, 18 and 35, PGM-a-note variation in the intensity of the most alkaline fraction, sample 10, PGM-3.

PH PH 5.0- a I

5.5- b I

6.0- c I 6.5- 6 6 1212 121212 12 242424333333 C2 C3 C2 - C2lC3 - C2 C3C2K3 C2 10

Plate 3. GE’1 of B. n/Gurus snails separated by isoclectrlc focusing. Plate 4. AcP of sample 10 scparatcd by isoelectric focusing. The Samples 6, 12, 24, GPI-1, in addition sample 12 includes GPI-4 and three zones, a, b and c are indicated. AcP-~2, AcP-c3 and possibly the heterozygote GPI-l/GPI-4. Sample 33-GPI-1. The extra the heterozygote are seen. bands between pH 6.0 and 6.5 of the first two snails of sample 6 are attributable to parasites.

-0 (v yi 1 fi a 10 Plate 5. HBDH of sample 10 separated by isoelectric focusing, includes HBDH-2, -10, -11, -12 and -13. 670 ENZYME ANALYSES OF Bulinus FROM TANZANIA which made up the pattern in the stained gel. As AcP-Acid phosphatase separations produce a certain enzyme types of B. africanus group snails series of bands within the pH range 4.5 snd 7.0. have been oreviouslv described (WRIGHT & ROLLIN- The over-all enzyme pattern of all the samples was SON, 1979), the sake notation‘has been followed. typical of B. africanus group snails and can be Where new enzyme types have been mentioned, the divided into three zones of activity a, b and c pI values of the bands of enzyme activity have also (Plate 4). The variation in the banding pattern been given. encountered both between and within population The enzyme types of MDH, PGM, GPI, AcP samples made interpretation difficult. To facilitate and HBDH encountered in each snail population analysis, particular attention was paid to the most are detailed in Table I. alkaline bands of activity in zone c, the enzyme types, previously undescribed, being grouped Downloaded from https://academic.oup.com/trstmh/article/73/6/667/1885199 by guest on 29 September 2021 MDH-Two enzyme types, MDH-1 and MDH-3, according to the p1 value of these fractions. illustrated in Plate 1 were observed. MDH-1 was B. nasutus samples 1 to 7 and 12 to 16 were confined to the four B. globosus populations (8, 9, designated AcP-cl with a p1 value of 6.3. Addition- 10, 11) from the Tanga region. No MDH hetero- ally, samples 13 and 14 included snails which geneity within the populations was observed possessed- a more alkaline fraction, AcP-c2, p1 value 6.5. B. nasutus oooulations 17 to 31 were PGM-Two PGM types were encountered, PGM-2 characterized by AcP-d separating out at pH 6.7. and PGM-3 (Plate 2). PGM-3 occurred in the four Samples 8, 9 and 11 of B. globosus possessed populations of B. globosus (8, 9, 10, 11) possessing AcP-c2 whereas 10 also possessed AcP-c3 and MDH-1. Although no polymorphism was found what appeared to be an AcP-c2, AcP-c3 heterozvgote

Table II-HBDH-types of samples of B. africanus group snails

HBDH-type PH Sample No. 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 1 I I 1-7, 12, 15-16 2 I I 17-31 3 I I 13, 14 4 I I 13, 14 5 I I I 13, 14 6 1 32-35 7 I I 37-38 I I I 98 10 I I I I1 :: I I I I 10 13 I I 10 D. ROLLINSON AND V. R. SOUTHGATE 671

divisions, particularly when snails from the same area have been compared. Hence, samples of B. nasutus from the Tanga area were clearly differ- entiated from B. globosus from that region, none of the five enzyme loci studied being held in common. This sunnorts the results of PRINGLE et al. (1971) who were unable to induce hybridization between these two species in the laboratory. Of particular interest is the apparent geographical distribution of the enzyme types, B. nasutus from the lake region possessed MDH-3, PGM-2, GPI-1, Downloaded from https://academic.oup.com/trstmh/article/73/6/667/1885199 by guest on 29 September 2021 as did the other samples of this species, but the AcP and HBDH-types clearly distinguished snails from the lake region from those collected elsewhere. MANDAHL-BARTH (1960) recognized two subspecies, B. nasutus nasutus and B. nasutus productus, the latter differing from the nominate form by the longer, more slender shell, the higher spire and its inland distribution. Similar morphological distinct- ions were noted between the populatyons sampled here and the enzyme data complement these observations. B. nasutus samples from the Dar es A A Salaam area possessed the same enzyme types as those occurring in the Tanga region. In addition, Plate 6. Egg protein profiles separated by cellulose acetate electro- however, GPI-4 was identified in sample 12 and phoresis. A-E. na~ufu (sample 291, B-B. a/rica!tur (sample 11). in the two particularly polymorphic samples, 13 and 14, novel types of AcP and HBDH were observed; these enzyme types were not found in samples of patterns as depicted in Plate 5. HBDH-11 was B. nasutus from other areas. identified in 15 snails, HBDH-10 in eight, HBDH-2 Enzyme differences between samples of B. in three, HBDH-13 in two and HBDH-12 in one. africanus and B. nasutus from the lake region were not great. Samples of B. nasutus, which were 2. Egg Proteins particularly homogeneous, differed only in their Among the 29 samples which were examined, GPX and HBDH loci from the other snail samples in addition to small nooulation differences, two from this area. However, the validity of these distinct egg protein profiles were recognizable distinctions was strengthened by the analysis of (Plate 6). One was typical of samples of B. nasutus samples 17 (B. nasutus) and 32 (B. africanus) which and the other was common to the samples of B. had been collected from the same habitat; no africanus, B. globosus and B. ugandae. evidence of hybridization was found. The close relationship of ‘B. africanus’ group snails in this area is perhaps reflected in the similarity of their 3. Infection with Schistosoma species enzvme complement. B. uxandue (37, 38) was only Cercariae isolated from samples 32 and 33 of distinguished from samples of B. ~f&an& (32, ST) B. africanus developed into adult worms in hamsters bv the oossession of HBDH-7. Four enzvmes were -,- I and were subsequently identified as S. bovis. examined in B. globosus (36) and, apart from varia- Similarly, B. nasutus (29) was found to have been tion in the GPI-2 pattern in two snails, no differences harbouring S. haematobium. In the laboratory, were detected between this sample and B. africanus B. globosus (11) has been successfully infected with (32.,-~,- 35’1., Furthermore, GPI-2 was the onlv enzvme Tanzanian strains of both S. bovis and S. haema- held in common between samples of B: glodosus tobium. from the Tanga region and B. globosus (36). Until Parasites within snail digestive glands used for more material has been analysed the validity of this enzyme analyses were found to contribute to the identification must be held in question. Samples final enzyme pattern, The differing pI values of the 34 and 35 of B. africanus differed from 32 and 33 of enzymes of the snaiIs and parasites allowed each this species by the possession of AcP-c4; sample 34 to be distinguished (Plate 3) (see WRIGHT et al., was further distinguished by the occurrence of 1979). GPI-3. The value of electrophoretic data grows as both Discussion the number of enzyme loci examined and the To understand relationships between populations sample size increases. Difficulties encountered in it is necessary to assess how different they are the transportation of wild caught bulinid snails genetically and how much variation exists from reduce the sample size available for analysis in the individual to individual within a population. laboratory. Undoubtedly, this decreases the amount Enzyme electrophoresis is a suitable method of of variation detected within populations. However, auoroachinn this nroblem. in seven of the 38 samples, individual variation was - The snails exammed in this survey were originally observed and in three of these, variation was found identified using morphological criteria. For the for two enzyme loci. This does not take into account most part the enzyme data has supported these the differences occurring in the ‘a’ and ‘b’ zones of 672 ENZYME ANALYSES OF Bulinus FROM TANZANIA

the AcP pattern. Even though a meaningful ap- 2750046 from the Edna McConnell Clark Founda- praisal of allelic frequencies cannot be made. it is tion. necessary to be aware of the enzyme polymorphisms occurring within populations. The finding of References GPI-1, GPI-2 heterozygotes in samples 9 and 11 of Mandahl-Barth, G. (1960). Intermediate hosts of B. globosus preclude the use of GPI-1 as a ‘species Schistosomu in Africa, some recent information. marker’ for ‘B. nasutus’. Indeed nothing can be Bulletin of the World Health Organization, 22, gained by choosing one particular enzyme locus for 565-573. snail identification; although PGM-2 seems to Mandahl-Barth, G. (1965). The species of the differentiate samples of B. nasutu~ from other genus Bulinus, intermediate hosts of Schistosomu. B. africanus group snails in the Tanga region, it Bulletin of the World Health Organization, 33, Downloaded from https://academic.oup.com/trstmh/article/73/6/667/1885199 by guest on 29 September 2021 does not in the lake area. 33-44. No correlation was found by WRIGHT & ROLLIN- Pringle, G., Otieno, L. H. & Chimtawi, M. B. SON (1979) between snail enzyme type and suscepti- (1971). Notes on the morphology, susceptibility bility to species of schistosomes, and no association to Schistosomu huemutobium and genetic relation- is apparent from the present data. S. bovis isolated ships of Bulinus (Physopsis) globosus globosus and from B. africanus (32), characterized by MDH-3, B. (P.) nasutus nusufus from north-eastern PGM-2. GPI-1. AcP-c3. and HBDH-6. also Tanzania. Annuls of Tropicai Medicine and developed in B: globosus _ (1 l), characterized by Parasitology, 65, 211-219. MDH-1, PGM-3, GPI-l-2, AcP-c2, HBDH-10. Sturrock, R. F. (1965). The development of irriga- Similarly, both B. globosus (11) and B. nusutus 29 tion and its influence on the transmission of (MDH-3, PGM-2, GPI-1, AcP-c3 and HBDH-2) bilharziasis in Tanganyika. Bulletin of the World were susceptible to a Tanzanian strain of S. huemu- Health Organization, 32, 225-236. tobium. Webbe, G. (1958). A bilharzia and molluscan survey B. ufricunus group snails are hermaphrodite and in the Handeni and Korogwe districts of Tangan- potentially self-fertilizing, they only have limited yika. Journal of Tropical Medicine and Hygiene, possibilities for active dispersal and discrete gene 61, 37-42. pools are quite likely to occur. Isoelectric focusing Wright, C. A (1961). Taxonomic problems in the of enzymes provides a suitable means of character- molluscan genus Bulinus. Trunsuctions of the izing and differentiating populations occurring Royal Society of Tropical Medicine and Hygiene, within small geographical areas. 55. 225-231. Wright, C. A. & Rollinson, D. (1979). Analysis of enzymes in the Bulinus africanus group (Mollusca : Acknowledgements Planorbidae) by isoelectric focusing. Journal of We wish to thank Drs. J. E. and R. McMahon and Natural History, 13, 263-273. Mr. N. Kolstrup of the Helminthiasis Research Wright, C. A., Rollinson, D. & Goll, P. H. (1979). Unit, Tanga, Mr. A. Gray of the University of Dar Parasites in Bulinus senegulensis (Mollusca : es Salaam, and Dr. D. Matovu and staff of the East Planorbidae) and their detection. Parusitolopy,-< _ African Institute of Medical Research, Mwanza, 79, 95-105. ’ for their invaluable help and assistance. We are Wright, C. A. & Ross, G. C. (1965). Electrophoretic indebted to Miss J. R. Lines, Mr. M. Anderson and studies of some planorbid egg proteins. Bulletin Mr. R. J. Knowles for technical assistance. Finally, of the World Health Organization, 32, 709-712. we would like to thank Dr. C. A. Wright for helping with the identification of material and for his comments on the manuscript. This study was supported in part by Grant Accepted for publication 22nd March, 1979.