Preliminary morphological data on the Simulium damnosum complex

WORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTE VBC/sc/76.11 ORIGINAL: FRENCH PRELIMINARY MORPHOLOGICAL DATA ON THE SIMULIUM DAMNOSUM COMPLEX •0 ~, << · '/ L J'..J,.'\,, by 'I .. D. Quillevere, Y. Sechan, and B. Pendriez OCCGE/ORSTOM 1. The Simulium damnosum complex in West Africa Simulium damnosum is represented in West Africa by seven cytotypes. To begin with, Vajime (1972) regrouped them into four "species" on the basis of their chromosomal affinities. These four species, "Bille-Yah", "Bandama-Soubre", "Nile-Sirba", and "Dieguera", are distinguished by the presence of characteristic fixed chromosome inversions. Within each of these "species" the cytotypes were differentiated on the basis of one or two floating inversions. In 1975 Vajime & Dunbar, on the basis of statistical calculations, divided the Sirba cytotype into two parts, known as "Sirba" and "Sudan", and raised the eight cytotypes to species rank, giving them a binomial designation. For our part, in view of the great complexity and variability of the chromosomal combinations that are present (Quillevere, 1974 and 1975) and in view also of our morphological (Quillevere et al., 1976a and b), ecological (Quillevere et al., 1976c and d), and epidemiological (Quillevere et al., 1976d; Philippon et al., 1975) studies we considered it preferable to abide by the hypothesis formulated in 1972 by Vajime. For so long as a precise and full investigation has not been carried out into the genetics, morphology, ecology and vector potential of the different cytotypes it will be impossible to assign them species, sub-species or population rank. Six cytotypes are thus present in the Ivory Coast: Nile, Sirba, Bandama, Soubre, Bille and Yah. 2. Value of morphological studies While the chromosomal identifications of the various cytotypes are still the most precise and the most objective, they suffer from the major disadvantage that only sufficiently old larvae can be determined. The eggs, young larvae, pupae and adults cannot be identified by this method, which furthermore requires considerable skill. Finally, chromosome investi- gations require costly apparatus and experienced specialists. It has proved absolutely essential as part of the Onchocerciasis Control Progrannne to study the possibilities of identifying the various cytotypes morphologically at their various stages of development. It is, in particular, of first-class importance to identify female vectors of Cnchocerca volvulus that can travel several score or even several hundreds of kilometres. 3. Techniques of morphological study 3.1 Light microscopy The techniques we use are the conventional o~es. The material is usually harvested in ethyl alcohol at 70° and then cleared with the Marc Andre reagent in the cold for 24 hours before being mounted in polyvinyl alcohol between slide and cover slip. The issue of this document does not constitute formal publication. It should not be reviewed, abstracted or quoted without the agreement of the World Health Organization. Authors alone are responsible for views expressed in signed articles. Ce document ne constitue pas une publication. II ne doit faire l'objet d'aucun compte rendu ou resume ni d'aucune citation sans l'autorisation de !'Organisation Mondiale de la Sante. Les opinions exprimees dans les articles signes n'engagent que leurs auteurs. VBC/sc/76.11 page 2 The larvae are fixed alive in modified Carnoy fluid at low temperature. They are then Feulgen-stained and dissected for the removal of the salivary glands which are used for chromosome determinations. The rest of the larval body is placed in 70° ethyl alcohol for morphological study. After drying, preparations are observed with a Leitz Orthoplan microscope fitted with a camera and a drawing tube, 3.2 Electron microscopy For study under the scanning electron microscope material was harvested in 70°ethyl alcohol or in Duboscq Brazil fixative which gives greater rigidity. The material was then dehydrated in a succession of alcohol baths and finally in acetone baths. Adults with a well chitinized integument are simply dried in the open air, whereas more fragile material (eggs, larvae, pupae) is dried in a critical point apparatus with liquid carbon dioxide under high pressure. After drying the preparations were stuck with shellac to a metal capsule in an SEE 4B evaporator. Observations are made on the cathode screen of a Jeol J.S.M. Sl microscope with a camera attachment. 4. Results obtained 4.1 The eggs In view of their small size the eggs were examined solely by electron microscopy. No differences were found in the appearance of the cuticle in the eggs taken from the various cytotaxonomic zones. Eggs laid by engorged females were studied under a magnification of x 10 OOO. At higher magnifications the images obtained were too fuzzy. Williams (personal communication) seems to have obtained more satisfactory results with magnifications of the order of x 25 OOO. 4.2 Larvae Larvae have been the subject of numerous studies under the light microscope and by electron microscopy. We have always kept the bodies of larvae after study of their chromosomes, This has enabled us to investigate the larval morphology of the six cytotypes present in the Ivory Coast throughout their habitat. The results of this study are being published (Quillevere et al., 1976a) and so here the results obtained will be only briefly described: the dorsal tubercles and tegumentary scales are very different in different cytotaxonomic pairs and sometimes have been dependent on the cytotype. Bille and Yah have large dorsal tubercles and posterior scales of two types, one piliform and the other spatuliform, Bandama and Soubre have no dorsal tubercles and their scales are small, particularly in the case of Bandama whose posterior scales are pyriform and anterior scales rounded. Nile and Sirba have dorsal tubercles of varying sizes - usually larger in Sirba than in Nile; all the posterior scales are lanceolate and elongated while the thoracic scales are spatuliform. the position of the premandibular setae also differs in different cytotaxonomic pairs. the other morphological characters studied (anal sclerite, mouthparts and head capsule) showed no constant distinguishing features. 4.3 Pupae VBC/sc/76.11 page 3 So far pupae have only been studied under the electron microscope. However, the few examples of each cytotaxonomic pair that have been studied show certain interesting differences in the abdominal cuticular hooks. Numerous specimens have been prepared for light microscopy and are now being studied. 4.4 Adults In the case of the adults our efforts have been concentrated particularly on the females, which are the only vectors of O. volvulus. The numerous mounted morphological specimens prepared from the females of various cytotypes have already shown up two particularly interesting characters, the antennae and the maxillae. A provisional report has been made on this work (Quillevere et al., 1976b). In Nile and Sirba antennal segments 4-8 are very flattened compared with segments 1-3 and 9-11. The antenna averages 500 µ in length; it is tapering and the ratio between the diameters of the 3rd and lOth segments is 1.5. The ratio between the lengths of the antenna as a whole and of the five flattened segments is 2.7. There are always two apical setae, coloration varies considerably and depending on the area of the survey ranges from pale yellow to dark brown, particularly in the apical part. As for the number of maxillary teeth, in all the Nile-Sirba zones where we have worked we have found two very distinct populations: one in which the average number of maxillary teeth is 47 (extreme values 44-49), corresponding to the Nile cytotype, and the other in which the average number of maxillary teeth is 52 (extreme values 49-57), corresponding to the Sirba cytotype. These results were recently confirmed by the study of "pure" Nile and Sirba breeding sites where examination of the maxillae showed that only one of the two populations was present. In Bandama and Soubre the antennal segments are not flattened. The antenna measures over 700 µ in Bandamaand over 600 µ in Soubre. It is more tapering in Soubre (ratio between the diameters of the 3rd and lOth segments 1.5) than in Bandama where the ratio is 1.25. The ratio between the length of the antenna as a whole and the length of the segments 4-8 is about 2.3. The number of apical setae varies from 2 to 4. In Bandama it is usually 3 and in Soubre 2. Antennal coloration is also quite variable. In Bandama the antennae are often very dark in their apical portion, where the hair insertions form small white spots on a black ground. In Soubre the antennae range from brown to pale yellow in colour according to region. In the Bandama breeding sites the average number of maxillary teeth is 44, with extreme values of 39 and 46, whereas in Soubre sites it is 48, with extreme values of 45 and 52. We also noted that in areas where Bandama-Soubre hybrids are very numerous, as on the Sassandra, a single peak is obtained, corresponding to 46 maxillary teeth. In Bille and Yah the average length of the antennae is 550).l. The ratio between the diameters of the 3rd and lOth segments is 1.35 and segments 4-5 are someeimes flattened. The ratio between total antennal length and the length of segments 4-8 may be as high as 2.5. The number of apical setae varies considerably, particularly in Yah where it ranges from Oto 4. Coloration of the antennae is quite uniform; they are usually light brown and abundantly covered with hairs. It was impossible to study the maxillae in detail in Bille and Yah, but it seems that in Yah the average number of maxillary teeth is 49 or so as against 46 in Bille. However, these results still require confirmation. Other morphological characters are now being studied: the genitalia and hind tarsi of the females also seem to have interesting features. Finally numerous mounted specimens of males of the various cytotypes are now undergoing morphological and biometric study. VBC/sc/76.11 page 4 5. Discussion The value of these results is indisputable and well demonstrates the complementary nature of chromosomal and morphological taxonomic techniques. Of course, in morphology care must be taken not to draw general conclusions from results, since it is well known that morphological characters may vary greatly from one geographical region to another. It is for that reason that each cytotype has been studied at various points in its habitat. It would therefore be advisable to consider the results as being valid for the Ivory Coast alone until investigations have been extended to other regions of Africa, However, even now these results enable us to study more precisely the ecology and vector potential of the various cytotypes, something which has not been possible on the basis of chromosomal identification alone. 6. Conclusion We now have available simple morphological characters enabling us in the majority of cases to identify larval and adult "species" and sometimes even the various cytotypes. We have seen how important these identifications are because h~nceforward all work on the ecology, behaviour and epidemiology can be carried out on each of the cytotypes separately. It goes without saying that we shall continue the investigation of new characters with a view to improving to the utmost the morphological identification of the various members of the S. damnosum complex. Of course, morphological investigations will have to be extended to other countries in West Africa and also to countries in Central and East Africa so that we can obtain an overall view of the epidemiology of onchocerciasis. 7. Acknowledgements We wish to thank in particular our colleagues at the Bouake Institute and in particular the Director of the Institute, B. Phillipon, ORSTOM medical entomologist, who is constantly giving us guidance and advice on our work. We also wish to thank the consultants and entomologists who are our colleagues in the Onchocerciasis Control Programme and with whom we are working in close contact. REFERENCES Philippon, B., Quillevere, D., Sechan, Y., & Pendriez, B. (1975) Biologie, ecologie et caracteristiques vectrices des vecteurs d'onchocercose des savanes ouest-africaines, Oncho/WP/75.20, 33 pp Quillevere, D. (1974) Etude cytotaxonomique du complexe Simulium damnosum en Afrique Occidentale. III. Cartes chromosomiques et repartition geographique des cytotypes, Roneoed OCCGE report, No. 79/0ncho/Tech/74, 41 pp Quillevere, D. (1975) Etude du I. Techniques d 1 etude. Parasitol., 13(2), 87-100 complexe Simulium damnosum en Afrique de l 10uest. Identification des cytotypes. Cah. ORSTOM, ser. Ent. med. Quillevere, D., Razet, P. & Le Piver, M. M. (1976a) Etude du complexe Simulium damnosum en Afrique de l'Ouest. III. Etude de la morphologie larvaire des cytotypes presents en Cote d'Ivoire. Cah. ORSTOM, ser, F.nt. med, Parasitol. (In press) Quillevere, D., Sechan, Y. & Pendriez, B. (1976b) Determination morphologique des femelles du complexe Simulium damnosum en Afrique de l'Ouest. Roneoed OCCGE report, No. 18/0nch/ Rapf76, 5 pp. 7 pl. vBc/sc/16.11 page 5 Quillevere, D., Gouzy, M., Sechan, Y. & Pendriez, B. (1976c) Etude du complexe Simulium damnosum en Afrique de l'Ouest. IV. Analyse de l'eau des gites larvaires en saison seche. Cah. ORSTOM, ser. Ent. med. Parasitol. (In preparation) Quillevere, D., Pendriez, B. & Sechan, Y. (1976d) Rapport annuel 1975 sur l'etude du complexe S. damnosum. Roneoed OCCGE Report, No. 1/0ncho/Rap/76, 9 pp Vajime, C. G. (1972) Le complexe Simulium (Edwardsellum) damnosum: rapport sur les etudes cytotaxonomiques effectuees jusqu'en avril 1972. WHO/Oncho/72.100, 13 pp Vajime, C. G. & Dunbar, R. W. (1975) Chromosomal identification of eight species of the sub- genus Edwardsellum near and including Simulium (Edwardsellum) damnosum Theobald (Diptera Simuliidae) Tropenmed. Parasit., 26(1), 111-138 * * *