Hybrid compatibilities and susceptibility of Culex pipiens fatigans Wied. to Wuchereria bancrofti (Cobbold) in East Africa*

Bull. Org. mond. Sante 1972, 4f, 801-805 Bull. Wld Hlth Org.f Hybrid compatibilities and susceptibility of Culex pipiens fatigans Wied. to Wuchereria bancrofti (Cobbold) in East Africa* S. A. MAGAYUKA1 & G. B. WHITE 2 Tests with colonies ofCulex pipiens fatigans from six localities in Kenya and Tanzania revealed high levels (60-90 %) of susceptibility to infection with Wuchereria bancrofti. Twenty-six of 30 possible crosses between these colonies were performed and revealed no cases of incompatibility. All but 2 crosses produced near-normal sex ratios of F1 adults, with a slight excess offemales in 16 of 20 detailed assessments. The implications of these results for control work and filariasis transmission are discussed. From other data it is concluded that the strain of C. p. fatigans present in Kenya and Tanzania is homologous with populations found elsewhere in Africa and in southern Asia. The taxonomic division of the polytypic species Culexpipiens Linnaeus into a number ofmorphologi- cally or biologically recognizable subspecies (Mat- tingly, 1967; Laven, 1967c), and at the same time into more than 20 reproductively incompatible strains spread through 5 continents (Laven, 1967c, 1969a, 1969b), is confusing to applied entomologists. Most perplexing is the discordance of these two disparate systems of classification, whereby distinct subspecies may regularly hybridize successfully in nature (Barr, 1967), while allopatric strains of a single subspecies may not be interfertile (Dobrotworsky, 1955; Vinogradova, 1961; Laven, 1967c). The C. pipiens complex is rich in evolutionary interest, especially because the incompatibility of strains has been shown to depend upon cytoplasmic and not genic factors (Ghelelovitch, 1952; Laven, 1953,1957,1967a; Smith- White & Woodhill, 1954). Urgent reasons for understanding these pheno- mena stem from the sanitary and medical importance of certain mosquitos of the C. pipiens complex that are noxious biters and vectors of viruses and filariae. Where separate subspecies or incompatible strains exist they may exhibit contrasting susceptibilities to insecticides or to parasite infections, possibly together * From the East African Institute of Malaria & Vector- Borne Diseases, Amani, Tanga, Tanzania. Scientific Assistant. Research Officer/Entomologist. Present address: Well- come Parasitology Unit No. 2, Haile Sellassie I University, P.O. Box 1176, Addis Ababa, Ethiopia. with medically important differences in ecology and behaviour. Such characteristics are likely to be of particular significance in populations of the sub- species C. p. fatigans Wiedemann (= quinquefasciatus Say), which constitutes a major public health prob- lem throughout much of the tropics. Accordingly we have investigated a number of East African C. p. fatigans populations to ascertain their genetical com- patibilities and levels of susceptibility to Wuchereria bancrofti (Cobbold) infection. MATERIALS AND METHODS C. p. fatigans mosquitos from a number of East African localities were colonized indoors under am- bient conditions at the Muheza Ubwari field station of the East African Institute of Malaria and Vector- Borne Diseases (for details of climate see Gillies, 1954). Insectary temperatures varied from 26°C to 32°C and the relative humidity was usually 85-90 Y. Colonies from the following places were established successfully Malindi (3°1O'S, 40°05'E), Taveta (3°30'S, 37°45'E), and Thika (1°05'S, 37°05'E) in Kenya, Ifakara (8O10'S, 36035'E), Umbugwe (4°00'S, 35'50'E), and Tanga (5°05'S, 39°05'E) in Tanzania. Unfortunately several attempts to esta- blish colonies from Uganda failed. Colonies originated from one or only a few egg rafts, obtained from females captured resting indoors, and were maintained in separate rooms to prevent confusion. Larvae were reared in stream 'water in 30-cm diameter bowls covered with mosquito 2859 - 801- 802 S. A. MAGAYUKA & G. B. WHITE netting, using a diet of desiccated meat meal.1 Pupae were transferred to 30 x 30 x 30-cm cages with wooden frames covered with plastic gauze. After emergence adult mosquitos had continuous access to a food source of 10% sugar solution on cotton-wool pads which were removed 12-24 hours before offer- ing a blood meal in the form of a guineapig sedated with pentobarbital. Eggs were collected subsequently from water in a 250-ml beaker or similar receptacle. For crossing experiments, pupae of a particular colony were first placed in a small empty cage and the resulting adult males and females were sepa- rated within 12 hours of emergence, before the onset of reproductive activity. The desired combinations of these males and females were then mixed in a colony cage and allowed to consort for at least 72 hours, during which time mating could occur, before the females were offered a blood meal. Egg rafts resulting from a test cross were placed in indi- vidual bowls and the resulting larvae were reared carefully to adulthood. The yield of F1 adult males and females was counted. The testes of hybrid males were inspected for the presence ofmotile spermatozoa 24 hours or more following emergence of the males. The criteria used in examining the male reproductive tract were drawn from experience of conditions found in fertile and sterile hybrid males of the Anopheles gambiae complex (Davidson, 1964). On several occasions during 1970-71, groups of female mosquitos from each C. p. fatigans colony were allowed to engorge blood at 21.00 hours from a volunteer W. bancrofti carrier (a 19-year-old male) whose infection was thought to have been contracted via C. p. fatigans in urban Tanga. Microfilaria counts in finger-prick blood taken at the times when mosqui- tos were feeding on this volunteer showed that his microfilaraemia was in the range 50-90 microfilariae/ 100 ,ul. Batches of potentially infected mosquitos were held in small cages on a sugar maintenance diet for 13 days to permit filarial development. Sur- viving C. p. fatigans females were completely dis- sected and thoroughly examined on the 14th day and the presence of filarial larvae was recorded. RESULTS Cross-nmting tests All but 4 of the 30 possible crosses between these 6 populations were performed successfully without 1 Wegesa, P. (1964) A comparison of the efficacy of meat and yeast asfood materialfor Anopheles gambiae larvae in the laboratory (unpublished document WHO/Mal/471.64). revealing any cases of incompatibility (Table 1). Eggs from all these crosses gave a high hatch rate. Among 20 crosses assessed in detail the yield of F1 adults varied from 21 per raft (Taveta male x Malindi female) to 62 per raft (Tanga x Ifakara) and averaged 46 per raft. The ratio of male to female progeny reaching adulthood ranged from 0.33 (Malindi x Ifakara) to 1.94 (Tanga x Taveta) with a mean sex ratio of 0.85. The 26 batches of hybrid males, examined when 24 hours old or more, all had testes and reproductive tracts of normal appearance in which motile sperma- tozoa were observed. Table 1. Results of crosses between six East African populations of C. p. fatigans Ratio of Cross No of rafts No. of Fi males! (male x female) tested a adults per femalesraft among Fi adults a Ifakara x Malindi 0 - - Tanga 8 61 0.81 Taveta 5 58 0.86 Thika nc many nc Umbugwe 5 58 0.71 Malindi x Ifakara 5 23 0.33 Tanga 9 49 0.74 Taveta nc many nc Thika 5 30 0.70 Umbugwe nc many nc Tanga x Ifakara 8 62 0.93 Malindi 0 - - Taveta 7 46 1.94 Thika 0 - - Umbugwe nc many nc Taveta x Ifakara 9 51 0.73 Malindi 7 21 0.83 Tanga 2 48 0.77 Thika nc many nc Umbugwe 0 - - Thika x Ifakara 4 39 1.05 Malindi 5 23 0.49 Tanga 2 22 1.00 Taveta 5 32 0.62 Umbugwe 2 59 0.93 Umbugwe x Ifakara 7 56 0.67 Malindi nc many nc Tanga 3 58 0.94 Taveta 6 54 0.55 Thika 2 61 1.09 a nc = not counted. INFECTION OF C. P. FATIGANS WITH WUCHERERIA BANCROFTI IN EAST AFRICA Susceptibility to W. bancrofti The females of all six C. p. fatigans populations proved capable of supporting the larval development of W. bancrofti up to the infective stage. The approxi- mate proportions of females harbouring stage-3 lar- vae 13-14 days after feeding on the carrier ranged from 60% to 90% (Table 2). Thus, although only 3 Table 2. Proportions of C. p. fatigans (six populations) in which infective filarial larvae were present 13-14 days after feeding at night (21.00 hours) on a W. ban- crofti microfilaria-carrier No. of females ProportionColony examined infective Ifakara 16 75 Malindi 17 88 Tanga 8 63 Taveta 56 80 Thika 24 83 Umbugwe 23 70 of these colonies (Ifakara, Malindi, and Tanga) came from actual filariasis foci, all populations showed a similarly high level of vectorial susceptibility to an urban strain of W. bancrofti. DISCUSSION C. p. fatigans mosquitos abound among most urban, suburban, and agricultural communities in Kenya, Tanzania, and Uganda. General appearances suggest the presence of uniform highly domestic C. p. fatigans populations everywhere, although Teesdale (1959) suspected that " out-door " adults might form a different population around Mombasa. Recently Highton & van Someren (1970) reported the introduction of numerous alien C. p. fatigans and some C. p. pipiens on aircraft arriving in Nairobi. The present study of 6 sample populations was inspired chiefly by the revelation that a number of mating barriers exist between African strains of C. p. fatigans (Laven, 1969a, 1969b) similar to the range of incompatibilities previously recorded among strains of C. p. pipiens and C. p. molestus in other continents (Vinogradova, 1965; Laven, 1967c). In- compatibilities between C. p. fatigans populations had not been revealed by some previous studies in- volving two West African populations from Nigeria and British Guiana (Service, 1956) and two Pacifico- American populations (Rozeboom, 1958), although Krishnamurthy (1961) observed one-way incom- patibility between two Indian strains and Roubaud (1956) was the first to demonstrate a case of incom- patibility in Africa, between C. p. fatigans males from Brazzaville and females from Dakar, but not with the reciprocal cross. Using populations of widely spaced origins in Africa, Laven (1969a) demonstrated 5 cases of in- compatibility out of 30 possible crosses between 6 strains from East, West, and Central Africa. In a further study restricted to the Nile valley in Egypt, Laven (1969b) tested 13 strains by performing 113 of the 156 possible crosses and observed 7 cases of uni- directional incompatibility. Some of these noninter- breeding strains occurred naturally less than 100 km apart. Laven's progress indicates that public health wor- kers in Africa should be generally alert to the exis- tence of distinct C. p. fatigans strains in different areas, with the prospect that such strains may exhibit different ecological preferences or levels of suscepti- bility to parasites or insecticides. The present results, from investigations of only two biological charac- teristics, show no such heterogeneity in C. p. fatigans populations over a large part of Kenya and Tanzania. This encourages one to expect that well chosen con- trol measures should be similarly effective throughout this part of East Africa. Insecticide resistance is not a current problem in this geographical region (White, 1971a) although it must be appreciated that, if they were to appear, insecticide resistance genes would probably spread rapidly through these interfertile C. p. fatigans populations under any insecticidal challenge. On the other hand, the release of a suitable cytoplasmically incompatible strain for genetical sup- pression of indigenous C. p. fatigans (Laven, 1967b) would be equally feasible in all parts of this region. None of the crosses in this study gave an F1 sex ratio distorted to the degree reported by Umino (1965), who obtained only females from a male C. p. pallens x female C. p. molestus cross in Japan. The majority of crosses gave sex ratios close to the normal value of 1:1 (Qutubuddin, 1953), although there was a moderate preponderance of females in most cases. The highest and lowest sex ratios (1.94 and 0.33) obtained in the present series of crosses might have resulted from differential accidental mortality of male and female developmental stages or from an intrinsic genetical cause. 803 804 S. A. MAGAYUKA & G. B. WHITE C. p. fatigans is likely to account for an increasing proportion of the transmission of filariasis as East African towns continue expanding at the expense of their rural surroundings, where anopheline filariasis vectors have hitherto played the principal vectorial role (Hamon et al., 1967; White, 1971b). It is un- welcome to find that C. p. fatigans strains in filariasis- free areas (Taveta, Thika, Umbugwe) supported levels of filaria development of similar high order to that occurring in populations from places where C. p. fatigans is known to be an active vector (Malindi and Tanga: see Nelson et al., 1962; White, 1971b). Since mosquito susceptibility to filariae is governed by inheritable factors (MacDonald, 1967) these data indicate a widespread high frequency of the hypo- thetical gene(s) in C. p. fatigans for supporting W. bancrofti development. Furthermore, the urban Tanga W. bancrofti strain was found to be well adapted to development in C. p. fatigans populations from rural nonfilarious areas (Taveta, Umbugwe). A C. p. fatigans population from Ifakara was in- cluded in the present experiments because Laven (1967a, 1967c) had previously employed an Ifakara strain and found it fully compatible with strains from Douala (Cameroon), Freetown (Sierra Leone), Bobo Dioulasso (Upper Volta), Thies (Senegal), Brazza- ville (Congo), Penang (Malaysia), and Rangoon (Bur- ma), but not fertile when crossed Ifakara female x New Halfa (Sudan) male. It can be concluded, therefore, that the single strain of C. p. fatigans that is distributed throughout much, if not the whole, of Kenya and Tanzania is homologous with C. p. fati- gans populations occurring in several other parts of Africa and in southern Asia. ACKNOWLEDGEMENTS We are indebted to Mr A. Mtango and Mr M. Fumba for their assistance in this work, and to Mr V. K. Prashar of the Klenya Medical Research Laboratory (Division of Insect-Borne Diseases) for sending mosquito eggs from Kenya. Thanks are due to the Secretary-General of the East African Community for granting permission to publish this paper. RESUME APTITUDE A L'HYBRIDATION ET RECEPTIVITE DE CULEX PIPIENS FATIGANS WIED. A WUCHERERIA BANCROFTI (COBBOLD) EN AFRIQUE ORIENTALE Afin de preciser la place et le role epid6miologique qu'il convient d'attribuer a Culex pipiens fatigans en Afrique orientale, on a 6tudi6 les possibilit6s d'hybrida- tion et la receptivite a Wuchereria bancrofti de colonies de cet insecte originaires de six endroits du Kenya et de Tanzanie. Sur 30 croisements possibles entre les 6 populations, 26 ont ete r6alises avec succes sans que se manifestent des cas d'incompatibilit6. Dans leur quasi-totalite, ces croisements ont donne' une descendance F1 comportant un rapport normal des sexes. Des insectes femelles de chacune des populations, infect6s sur un porteur de microfilaires de W. bancrofti, ont temoign6 d'un degr6 61ev6 (60 a 90 °/) de r6ceptivite a l'infection. On conclut que les populations de C. p. fatigans repar- ties sur la majeure partie des territoires du Kenya et de la Tanzanie constituent, sous le rapport de leur aptitude a l'hybridation et de leur r6ceptivit6 a W. bancrofti, un groupe relativement homogene. Les observations faites par d'autres auteurs suggerent en outre qu'elles sont etroitement apparentees aux populations de C. p. fatigans rencontr6es dans un certain nombre d'autres regions d'Afrique et en Asie meridionale. REFERENCES Barr, A. R. (1967) Bull. Wld Hlth Org., 37, 293-296 Davidson, G. (1964) Riv. Malar., 43, 167-183 Dobrotworsky, N. V. (1955) Proc. Linn. Soc. (N.S. W), 80, 33-43 Ghelelovitch, S. (1952) C.R. Acad. Sci. (Paris), 234, 2386-2388 Gillies, M. T. (1954) Bull. ent. Res., 45, 361-374 Hamon, J. et al. (1967) Bull. Wid Hlth Org., 37, 217-237 INFECTION OF C. P. FATIGANS WITH WUCHERERIA BANCROFTI IN EAST AFRICA 805 Highton, R. B. & Someren, E. C. C., van (1970) Bull. Wld Hlth Org., 42, 334-335 Laven, H. (1953) Z. Vererbungsl., 85, 118-136 Laven, H. (1957) Z. Vererbungsl., 88, 478-516 Laven, H. (1967a) Bull. Wld Hlth Org., 37, 263-266 Laven, H. (1967b) Nature (Lond.), 216, 383-384 Laven, H. (1967) Speciation and evolution in Culex pipiens. In: Wright, J. W. & Pal, R., ed., Genetics of insect vectors of disease, Amsterdam, Elsevier Laven, H. (1969a) Mosquito News, 29, 70-74 Laven, H. (1969b) Mosquito News, 29, 74-83 MacDonald, W. W. (1967) The influence ofgenetic and other factors on vector susceptibility to parasites. In: Wright, J. W. & Pal, R., Genetics of insect vectors of disease, Amsterdam, Elsevier Mattingly, P. F. (1967) Bull. Wld Hlth Org., 37, 257- 261 Nelson, G. S. et al. (1962) Trans. roy. Soc. trop. Med. Hyg., 53, 202-217 Qutbuddin, M. (1953) Bull. ent. Res., 43, 549-565 Roubaud, E. (1956) C. R. Acad. Sci. (Paris), 242, 1557-1559 Rozeboom, L. E. (1958) Amer. J. trop. Med. Hyg., 7, 526-530 Service, M. W. (1956) Nature (Lond.), 178, 1065 Smith-White, S. & Woodhill, A. R. (1954) Proc. Linn. Soc. (N.S. W.), 79, 163-176 Teesdale, C. (1959) Bull. ent. Res., 50, 191-208 Umino, T. (1965) Jap. J. sanit. Zool., 16, 282-290 Vinogradova, E. B. (1961) Rev. Ent. URSS, 40, 63-75 Vinogradova, E. B. (1966) Tr. zool. Inst. (Leningr.), 36, 31-57 White, G. B. (1971a) E. Afr. med. J., 48, 266-274 White, G. B. (1971b) Trans. roy. Soc. trop. Med. Hyg., 6, 819-829 9