Artificial feeding of East African female Simuliidae (Diptera), including vectors of human onchocerciasis.

HAEMOGLOBINOPATHIES AND MALARIA IN THAILAND 463 Our results conflict with studies of frequency of HbE gene distribution in relation to the incidence of P. falciparum infections in Thailand.r We are convinced, however, that the interpretation of the gene distribution depends not only on the prevailing malaria incidence, but also on the interaction of the abnormal genes among the populations concerned. The apparent correlation of malaria incidence and a given gene may be purely coincidental or a third unknown factor may be involved which is not P. falciparum. If correlation between these abnormal gene frequencies and malarial incidence is real, it should be possible to demonstrate this clearly in many malarious areas in Thailand. r Flatz, G., Pik, K. & Sumdharagiati, B. (1964) Lancet, 2, 385. The results of our studies of fatal cases of P. falci- parum infection with parasitaemia higher than 50 000 mm3 in infants and children do not indicate any clear difference between the series of normal and abnormal haemoglobins. Details of these studies will be provided at a later stage. * * We thank Dr Bunloo Siripanit, Director of Saraburi Hospital, and Dr Narong Sadudee, Director of Paputha- bath Hospital, for allowing us to study their patients, Professor B. G. Maegraith and Dr D. J. Weatherall, University of Liverpool, for their valuable suggestions, Dr Udom Vutheepoom, Director of Nakom Nayok Hospital, and the staffof the School of Tropical Medicine, Bangkok, whose help made this survey possible. Artificial Feeding of East African Female Simuliidae (Diptera), including Vectors of Human Onchocerciasis* by JOHN N. RAYBOULD, Research Officer, and ALPHONCE S. K. YAGUNGA, Technician, East African Institute of Malaria and Vector-borne Diseases, Amani, Tanzania There is little doubt that many aspects of Simulium biology will remain obscure until successful culturing or colonization can be achieved.a One of the main obstacles to laboratory colonization has been to persuade Simulium females to feed on blood under artificial conditions. This difficulty has applied both to wild-caught females and to those reared in the laboratory. However, during the past few years several different investigators have devised new tech- niques for the artificial feeding of simuliids that have met with considerable success. b, c, d, e Wenk b succeeded in feeding Boophthora erythro- * This investigation was supported, in part, by a grant from the World Health Organization. a Muirhead-Thomson, R. C. (1966) Blackflies. In: Smith, C. N., ed., Insect colonization and mass production, New York, Academic Press, pp. 127-144. b Wenk, P. (1965) Z. Tropenmed. Parasit., 16, 207-226. c McMahon, J. P. & Nelson, G. S. (1967) Trans. roy. Soc. trop. Med. Hyg., 61, 21-22. d Field, G., Duplessis, R. J. & Breton, A. P. (1967) J. med. Ent., 4, 304-305. 6 McMahon, J. P. (1968) Bull. WldHith Org., 38,957-966. cephala and a number of other simuliids on citrated or defibrinated blood, on rabbits' ears and on man. The present paper describes the results obtained using the latter 2 methods for feeding East African Simuliidae. Species of Simuliidae used in these investigations The species used in these investigations were vectors, or potential vectors, ofhuman onchocerciasis. They were collected either at Amani in the eastern Usambara Mountains in north-east Tanzania, or at Njombe in the south-west of the country. Members ofthe Simulium neavei Roubaud complex. Feeding trials were carried out on 2 members of the S. neavei complex from Amani: Simulium woodi de Meillon and Simulium nyasalandicum' de Meillon (Amani form).f S. woodi is probably the only im- portant vector of human onchocerciasis in the area, while S. nyasalandicum (Amani form) feeds on cows f Lewis, D. J. (1961) Proc. roy. ent. Soc. Lond. B. 30, 107-111. 2314B 464 NOTES and occasionally on man,9 and it is a possible secondary vector of human onchocerciasis.h Simulium damnosum Theobald. An anthropophilic form of S. damnosum from Njombe was used in these investigations. Whether or not human oncho- cerciasis occurs at Njombe is not yet known, but this species is capable of supporting the development of Onchocerca volvulus larvae to the infective stage.~ Simulium vorax Pomeroy. This species was col- lected at Amani where it normally feeds on cows g but occasionally bites man and it has been shown to be a potential vector of 0. volvulus.j Methods Collection offemale Simuliidae. Wild-caught flies were used in the feeding trials. Females of S. woodi and S. damnosum were caught, as they came to feed, by human bait subjects. They were usually collected individually in 5 cm by 1 cm glass tubes with plastic caps, but if they were particularly numerous a suction-bottle was used. Great care was taken to collect each fly before it started to bite. The biting sites of S. nyasalandicum and S. vorax on cows are specific: k the former species was col- lected with a suction-bottle as it landed on the ventral surface of an animal, while the latter was taken from the ears. Maintenance offemale Simuliidae in the laboratory. As may be seen from the accompanying table, most feeding trials were carried out on the same day as the flies were collected, but a few flies were kept alive in the laboratory for a number of days before being used in a feeding experiment. These simuliids were maintained in the laboratory by the method described by McMahon.e They were fed on 25% sugar solution and then starved for 24 hours prior to the experiment. Feeding female Simuliidae on a human arm. The technique employed was essentially the same as that g Raybould, J. N. (1967) Ann. trop. Med. Parasit., 61, 76-88. h Wegesa, P. (1968) Artificial infection of Simulium nyasalandicum (Amani form) and Simulium adersi with Onchocerca volvulus. In: Annual Report of the East African Institute of Malaria and Vector-borne Diseases, January- December 1967, East African Community, p. 67. Wegesa, P., personal communication. IWegesa, P. (1967) Ann. trop. med. Parasit., 61, 89-92. k Raybould, J. N. (1966) Preliminary observations on Simuliidae feeding on cattle at Amani. In: Annual report of the East African Institute of Malaria and vector-borne dis- eases, January-December 1965, East African Common Ser- vices Organization, p. 41. originally devised by Wenk.b A nylon or fine cotton- netting sleeve was fitted around the forearm and tied just above the elbow and around the wrist. Flies were released into the sleeve from a sucking-tube. Blood-fed flies could normally be recognized by their distended abdomens. However, to obtain a more accurate assessment of the number of flies which had taken blood, each fly was squashed with a flat instrument on to a piece of filter-paper. These experiments were carried out under natural lighting. Feeding female Simuliidae on the ears of a rabbit. Before the experiment was commenced, the rabbit was confined in a specially designed box (Fig. I) recommended for this purpose by Mr J. P. McMahon. The author has been unable to find out who was originally responsible for the design of this box, but Mr McMahon first saw it in use in Nairobi in 1929. This method of restraining the animal was found to be much more convenient and less time-consuming than anaesthetization with sodium pentobarbital (Nembutal). The feeding technique employed was similar to that devised by Wenk.b A suitably prepared sleeve of nylon or fine cotton-netting was placed over the rabbit's ear and closed at each end with a rubber band (Fig. 2). The required number of flies were released into the sleeve using a sucking-tube. As the flies fed only on the outsides of the ears (and not on the insides, which are practically hairless), the ears were carefully shaved before the sleeve was placed in po- sition. All feeding trials were carried out under natural lighting. Results It may be seen from the table that all 4 species were successfully fed by both methods. These results showed that it is possible to feed both freshly caught flies and (at least in the case of S. woodi, S. nyasa- landicum and S. vorax) also flies that have been kept in the laboratory for a number of days. A much larger proportion of freshly caught flies took a blood- meal, however, than did flies that had been kept in captivity. The results given in the table represent the means of a number of tests. Results of individual feeding trials, however, were very variable and unpredictable for all 4 species: on certain occasions all flies refused to feed, while on other occasions most did so. Some of the flies which initially refused to feed 465ARTIFICIAL FEEDING OF EAST AFRICAN FEMALE SIMULIIDAE FIG. I BOX FOR HOLDING RABBIT DURtNG FEEDING TRIALS a CROSS - (e) a Partition A Is divided Into an upper and a lower section. The rabbit's head Is placed In position before the upper section of the partition Is slid into place. Partition B Is used to alter the size of the box according to the size of the rabbit. would do so if kept alive in the laboratory and the species tested by Wenk b are reported to have offered a blood-meal on a subsequent occasion. done. When fed on the ear of a Tabbit, all 4 species bit S. woodi and S. nyasalandicum (Amani form). only the outside of the ear, and not the inside as About equal numbers of S. woodi fed on a human FIG. 2 A FEEDING TRIAL IN PROGRESS 466 NOTES BLOOD-FEEDING OF WILD-CAUGHT SIMULIUM FEMALES IN THE LABORATORY Source No. inNo. Total No. No. of flies No. of flies Total No. PercentageSpecies of fleus of days In Method of tests of flies partially fully d of flies of flieses captivity__a_ __j fed Iengorged fed fed l|. Arm 8 95 3 41 44 46 S. woodl Amani - Ear 9 122 1 70 71 58 1, 2,4 Arm 3 63 1 5 6 10 - Arm 8 107 2 12 14 13 S. nyasalandicum Amani - Ear 8 110 0 62 62 56 1, 3,4 Ear 3 52 1 9 10 19 - Arm 4 89 NRC NRC 45 51 S. damnosum Njombe - Ear 4 110 NRc NRc 40 36 - Arm 3 88 2 34 36 41 - Ear 3 89 1 36 37 42 S. vorax Amani 1 Arm 2 60 0 7 7 12 1 Ear 2 61 2 9 11 18 a This column shows the number of days the flies were kept in the laboratory prior to being offered a blood-meal. Where several figures are given these refer to different tests. b Arm refers to human arm; ear refers to rabbit's ear. c NR = not recorded. Although no accurate record was taken, many flies of this species took only a small blood-meal. arm and on the ears of a rabbit. S. nyasalandicum, however, showed a definite preference for a rabbit's ear as compared with a human arm. Almost all the flies of both species that fed became fully engorged and were readily recognized by their distended abdo- mens. S. damnosum. A few S. damnosum females became fully engorged but many took only a partial blood- meal. When feeding on a rabbit's ear, this species tended to bite in the vicinity of any small cuts made when the ear was shaved. S. vorax. Results with S. vorax were particularly variable. Nearly all the flies that fed became fully engorged. Discussion The results of these preliminary investigations show that both feeding techniques are satisfactory for all 4 species of Simulium tested. The human-arm method can be used for feeding the flies on a person infected with onchocerciasis and is, therefore, of particular value for transmission experiments. An important merit of both methods is their simplicity. However, they suffer from the disadvantage that the results are very variable and unpredictable. Further investigations on the effect of light intensity and other factors (as were carried out by McMahon e in connexion with his membrane-feeding technique) are required in the hope that more consistent results can be obtained. Tests on newly emerged flies are also required to determine whether they feed as readily as wild-caught specimens. The fact that S. damnosum and members of the S. neavei complex can be fed in the laboratory on the human arm and the ear of a rabbit, and also by the membrane feeding techniques of McMahon,e sug- gests that blood-feeding under artificial conditions is no longer likely to prove a major block to the laboratory colonization of these species. A form of S. damnosum has already been reared from egg to adult under artificial conditions.' The major obsta- cle to the laboratory culturing of the vectors of human onchocerciasis, therefore, now appears to be that of inducing the flies to mate in captivity. * * We wish to express our thanks to Dr J. L. M. Lelijveld, Director of the East African Institute of Malaria and Vector-bome Diseases, for his support and encourage- ment; and to Mr R. A. Kupe, Mr S. Fedha and Mr K. Madeni for their invaluable help. I Raybould, J. N. (1967) Bull. Wid Hlth Org., 37, 447-453.