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The Anopheles gambiae complex: a new species from Ethiopia
TRANSACTIONS OFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(1998)92,231-235
The Anopheles
gambiae
complex:
231
a new species from Ethiopia
Richard H. Huntl, Maureen Coetzeel and Messay Fetten& ‘Medical Entomology, Department of Tropical Diseases,School of Pathology of the South African Institute for Medical Research and the University of the Witwatersrand,Johannesburg, South Africa; 2Entomology and Vector Control Unit, Environmental Health School, Jimma Institute of Health Sciences,Jimma, Ethiopia Abstract Historically, members of theAnopheZes gambiae complex from Ethiopia have been identified chromosomally as either A. arubiensis or A. quadriunnulutus. Recent collections from the Jimma area in Ethiopia, southwest ofAddis Ababa, revealed 29 specimens ofA. quadriannulutus based on the standard polymerase chain reaction (PCR) identification method. ‘Wild’ females were induced to lay eggs and the progeny reared as individual families. Resulting adults were cross-mated to a laboratory colony strain of A. quadriunnulutus originating from the Kruger National Park, South Africa. Hybrid progeny were obtained only from the colony femalexEthiopian male cross. This cross produced a female/male sex ratio of 0.48. Male offspring were sterile and ovarian polytene chromosomes from hybrid females showed typical asynapsis as expected in interspecific crosses within the A. gumbiae complex. The X chromosomes, although apparently having homosequential banding patterns, were usually totally asynapsed. All autosomes were homosequential.The lack of inversion heterozygotes, in both the wild and hybrid samples, may simply be a reflection of the small sample size. Until such time as the Ethiopian species can be formally described and assigned a scientific name, it is provisionally designated Anopheles quadriannulatus species B because of its close similarity to this species. Keywords:
malaria, Anophelesgambiae complex, Anophelesquadriannulatus, new species, Ethiopia
Introduction Populations of the Anopheles gumbiue Giles complex from Ethiopia were first identified by cross-mating studies as A. urubiensis Patton (=sp. B of the A. gumbiue complex) and A. quudriunnulatus (Theobald) (=sp. C of the A. gumbiue complex) (Ross Institute Reports, unpublished, 19651969, 1973; TURNER, 1972) and chromosomal studies (M. Coluzzi, in Ross Institute Report, unpublished, 1968; WHITE et al., 1980; WHITE, 198 1). The distribution of A. quudriunnulutus is considered to be relict (WHITE, 1974; COLUZZI et al., 1979), havine been recorded in Ethionia. Pemba and Zanzibar Islands, Zimbabwe, Mozambique; Swaziland and South Africa (see GILLIES & COETZEE, 1987). The southern African populations are widespread, can readily be found in association with cattle, and breed in open sunlit stagnant pools in sandy river beds or next to roads. Ethiopian populations, on the other hand, are found in both animal shelters and mixed human/animal dwellings but nothing appears to be known about the larval habitats (ZAHAR, 1985). Little is known about the Pemha/Zanzibar Islands populations other than that they are zoonhilic and exouhilic UAHAR. 1985) and thev have not’been recorded there since ‘the original redort of ODETOYINBO&DAVIDSON(~~~~).MNZAVA&KLAMA (1986) found no specimen of A. quadriunnulatus on Zanzibar Island, despite using outdoor calf-baited traps. Chromosomal studies on Ethiopian A. quudriannulatus are briefly mentioned by WHITE et al. (1980) and WHITE (198 1). No difference in the banding arrangements between it and the published description ofk. auadriunnulutus from Zimbabwe (COLUZZI & SABATINI, 1968) has been reported. WHITE ( 198 1) published a photograph of chromosomes from what was-apparently a wild F. hvbrid female of A. auadriunnulutus from Ethiopia shcwmg 2 autosomal arms asynapsed with inversion loops but the X chromosome was homosequential and completely synapsed. The X chromosome, however, had been montaged on to the photograph of the autosomes and was at a different magnification. No other information is available on the cytogenetics of the Ethiopian population. The polymerase chain reaction (PCR) is now widely used for identification of members of the A. gumbiue Address for correspondence: Dr R. H. Hunt, Department of Medical Entomology, SAIMR, l? 0. Box 1038, Johannesburg 2000, South Africa.
complex (SCOTT et al., 1993; TOWNSON & ONAPA, 1994). PCR identification of Ethiopian populations has been carried out in recent years, with only typical A. arabiensis and A. quadriannulatus bands appearing on the gels (FETTENE, 1996). This paper presents the results of a study carried out in the Jimma area of Ethiopia with the objective of defining the relationship between a southern African population of A. quadriannulutus and that in Ethiopia. Materials and Methods Mosquitoes were collected from 5 villages outside Jimma (7”39’N, 36”47’E), 335km south-west of Addis Ababa, Ethiopia. They came from habitations where cattle were kept in close proximity to humans and were collected by hand aspirator and placed in polvstvrene cups for transport to the insect&y in Johannesburg, South Africa. There. individual A. gambiae comnlex females were isolated in tubes for egg-laying. Those that laid eggs and survived were refed and kept until half gravid, whereupon their ovaries were dissected and the bodies stored for PCR identification and enzyme-linked immunosorbent assay (ELBA) for sporozoite detection. Dead specimens were stored desiccated for the latter 2 tests. Each egg batch was kept separate and the larvae reared to adults. Ten adults per egg batch, with larval and pupal pelts, were preserved as museum specimens for morphological study (HUNT & COETZEE, 1986). The remaining adults from each family were placed in individual cages until PCR identification had been carried out. Those identified adults not used in the crossmating experiments were pooled in an attempt to establish a laboratory colony. The cross-mating studies were carried out using a laboratory colony ofA. quadriunnukztus (SKUQUA) originating from Skukuza camp, Kruger National Park, South Africa. This colony was established in 1995 and has been checked regularly for purity using chromosome morphology, isoenzyme electrophoresis and PCR. On emergence from the pupae, Ethiopian males and females were separated into individual cages where males or virgin females from the laboratory colony were placed appropriately. Eggs resulting from the crosses were treated as above for individual families. The internal genitalia of sexually mature adult males were dissected and examined for form and sperm content (DAVIDSON et al., 1967). Females were fed to obtain half gravid ovaries for chromosomal studies. Sex ratios were noted. Chromosomal preparations were made according to
RICHARDH. HUNT ETAL.
232
HUNT (1987) and PCR identifications used the protocol of SCOTT et al. (1993). ELISASwere carried out according to WIRTZ et al. (1987). Results A. quadriunnulatus, identified by PCR (Fig. l), was
collected from only 2 of the 5 villages sampled, i.e. Minko and Babo, close to Serbo town about 18 km east of Jimma. Twenty-nine individuals were collected: 3 in human only? 3 in mixed human/cattle, and 23 in cattle only habitations. All the females laid eggsand the larvae were successfully reared to adults. All ELISASon the A. quadriannulatus specimens were negative for Plasmodium fuhparum circumsporozoite protein compared with the A. arabiensissample from the same area, among which 4 of 148 females were positive (2.7%).
Fig. 2. Atrophied testes of hybrid A. quadrimnulatus from the cross SKUQUA female x Ethiopian male.
Fig. 1. Polymerase chain reaction gel showing the identification of Ethiopian Anopheles quadriannulatus. Lane 1: negative control; lane 2: A. arubiensiscontrol; lane 3: A. gambiue control; lane 4: A. mews control; lane 5: A. quadriannulatus SKUQUA; lane 6: Ethiopian A. quadriunnulatus extracted DNA; lane 7: hybrid SKUQUA x Ethiopia non-extracted DNA, lane 8: 1 kb molecular marker. The additional band in lane 7 was randomly expressed in some Ethiopian specimens but not in others.
Of the 29 females that laid eggsand were identified by PCR, 10 survived and ovaries were obtained for chromosomal study. The banding arrangements of all the chromosomes were homosequential with those found in A. quadriannulatus (see COLUZZI & SABATINI, 1968). No inversion polymorphism was seen in the Ethiopian sample. The X chromosome was standard for inversion f, as it was in the SKUQUAcolony. Crosses carried out between the Ethiopian sample and the laboratory colony resulted in hybrid progeny from only the SKUQUAfemales x Ethiopian males. The sex ratio of this cross was 0.48 (117 females/56 males). The Fl hybrid females backcrossed to SKUQUAmales produced a 0.97 sex ratio (29 females/28 males). Fl hy-
males
brid males were sterile with atrophied testes (Fig. 2) corresponding with testis type II of-DAVIDSON er al: (1967). F2 hvbrid males anneared fertile with normal testes and accessory glands. &-ther crosses with males were not attempted. Chromosomal studies on the Fl females revealed extensive asynapsis of the X chromosomes, which were usually totally asynapsed (Fig. 3a), although one specimen was seen where the X chromosomes were partially synapsed (Fig. 3b). The autosomes showed partial asynapsis but no heterozygous loop, as seen in hybrids of speciesthat differ by fixed paracentric inversions (Fig. 4). Attempts to establish a laboratory colony of the Ethiopian population were unsuccessful. Discussion
Recognition that the taxon A. gumbiue was a group of cryptic specieswas initially based on cross-mating characteristics (MIJIKHEAD-THOMSON, 1948; DAVIDSON. 1962; PATERSON,1962). The fact-that males from in: terspecific crosses are sterile was used extensively for routine
identification
of populations
of ‘A. gambiae’
from all over Africa by the Ross Institute at the London School of Hygiene and Tropical Medicine (University of London; seethe unpublished Ross Institute Reports). The cross-mating method requires the maintenance of laboratory stocks of all species in the group and is timeconsuming and laborious. It was, nevertheless, used as the ‘gold standard’ until replaced by polytene chromosome morphology which was shown to be a reliable tool for identifying the recognized species in the complex (COLUZZI & SABATINI, 1967,1968, 1969). PATERSON (1964) used the lack of sterile males in progeny broods from wild-caught females to show lack of mating be-
Fig. 3. The X chromosomes of hybrid A. quadriannulam females from the cross SKUQUA female x Ethiopian male, showing (a) almost total asynapsis which occurred in the majority of the hybrids, and (b) semi-asynapsis seen in one individual.
A NEW MEMBER
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GAMBIAE
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233
Fig. 4. The autosomes of hybrid A. quadriunnuZutusfemales from the cross SKUQUA female x Ethiopian male, showing homologous arms with homosequential banding sequences.Asynapsis was consistently present at the centromeric regions of both chromosomes (indicated by open arrows). Intermittent asynapsis is indicated by closed arrows. tween A. gambiae, A. arabiensis and A. quadriannulatus in sympatric populations in Zambia. While A. quadriannulatus from Ethiopia was identified as such on crossmating evidence (Ross Institute Report, unpublished,
1973), at that time no A. quadriannulatus from southern Africa was available as a colony in London. Crosses of Ethiopian samples were made only with A. gambiae, A. arabiensis, A. rrzerusDiinitz and A. melas Theobald and,
234
by inference, identifications were recorded as ‘probably sp. C’. In the present study, using the criteria on which the whole group was differentiated, we are faced with evidence of male sterility and chromosomal asynapsis from cross-mating studies between the 2 allopatric populations of ‘A. quadriannzdutus’.This allopatric situation is comparable to that found between A. melas and A. merus, the West and East African saltwater species (DAVIDSONet al., 1967), as well as between A. bwambae White from Uganda and A. quadriannulatus with which it shares the most chromosomal homology (DAVIDSON & WHITE, 1972; DAVIDSON& HUNT, 1973). Homosequential polytene chromosomes in more than one species of Anopheles are well known, e.g. A. atroparZeus Van Thiel and A. Zabranchiue Falleroni (see COLUZZI, 1970), A. funestus Giles and A. vaneedeni Gillies & Coetzee (seeGREEN& HUNT, 1980). Similar situations are found in Drosophila, where many homosequential species are known (CARSONet al., 1967; CRADDOCK, 1974). Chromosomal studies on the members of the A. gambiae complex, however, have so far shown either fixed paracentric inversion differences between species (COLUZZI & SABATINI. 1967.1968.1969) or nolvmornhic inversions with di&erentfrequencies between subpopulations of A. gambiae in West Africa (the ‘incipient soecies’ of COLUZZI et al.. 1979. 1985). This is the first record of 2 members of ihe complex having homosequential chromosome banding patterns and apparently no polymorphic inversion difference. It is not surprising, therefore, that the original chromosomal studies resulted in identifications ofA. quadriannulatus from Ethiopia (M. Coluzzi, in Ross Institute Report, unpublished 1968; WHITE et al., 1980; WHITE, 1981). Only 2 polymorphic inversions have been reported in A. quadtiunnulatus from southern Africa, xf and 2Ri (COLUZZI & SABATINI, 1968), with Xfbeing fairly common and 2Ri extremely rare (unpublished data). More extensive sampling of the Ethiopian populations may reveal polymorphic inversions not seen in the present study because of the small sample size. The use of PCR for identification of the named species of the A. gumbiae group has become standard practice in many laboratories. It is accurate (>99%) and has many advantages over both the chromosomal and isoenzyme electrophoretic methods (PASKEWITZet al., 1993; VAN RENSBURGet al.. 1996). It does. however. have the disadvantage of not being able to provide any additional information on the population genetics of the samples tested. The amplification of additional bands, such as the A. quadriannulatus band found in many A. merus specimens, does not pose a problem as long as the A. merusband amplifies, but will result in misidentification if the deoxyribonucleic acid (DNA) is degraded and only the smaller, 153 base-pair fragment ofA. quadriannulatus is present (VAN RENSBURGet al., 1996). In the present study, the Ethiopian ‘A. quadriannulatus’ produced a fragment that appears to be identical in size to that found in the South African populations. The result is that neither PCR nor polytene chromosome morphology can be used as a method for separating the 2 taxa and we do not know whether both occur in Ethiopia or, for that matter, in southern African regions where A. quadriannulatus is known to occur. An apparently identical PCR product size does not necessarily indicate an identical base pair sequence in the fragment. The production of similar fragment sizes in 2 different svecies of the A. pambiae comvlex has already been recorded, i.e. A. kerus and A. melas. It is necessary, therefore, that an alternative identification system be found, preferably a primer sequence, that enables the new taxon to be identified using established molecular procedures. Once this is done, it will be possible to establish the extent of the distribution of the new species. These results bring into sharp focus the conceptual issues underlying genetically based species identifica-
RICHARD H. HUNT ETAL.
tion methods and their use for establishing the evolutionary status of a taxon. They clearly indicate that the genetical characters we use to recognize the species in the A. gumbiae complex are, in fact, simply fortuitous markers of genetic discontinuity and are not in any way involved in the speciation process. It is possible that species exist within the complex that exhibit none of the currently used criteria of sterile male hybrids, asynapsed chromosomes, inversion arrangements and polymorphisms, isoenzyme polymorphisms or DNA sequences. These considerations become particularly important when genetical studies are undertaken on populations that may in fact consist of more than one species. In summary, the key genetic crossing characteristics between recognized members of theA. gambiae complex have been found in 2 apparently allopatric populations sharing the A. quadria%ulatus -chromosomal arrangements and indistinguishable in terms of PCR nroduct size. They differ in”3 important ways: in the c;oss between A. quadriannulatus and the Ethiopian population (i) resultant males are sterile, (ii) there is marked sex ratio distortion, and (iii) there is extensive asynapsis in the ovarian polytene chromosomes. We conclude, therefore, that the Ethiopian population is a different species.Until such time as a formal description can be produced and a scientific name assigned, we designate it asAnopheles quadricmnulatus species B because of its close similarity to this species. The southern African population is designated-as A. quadriannulatus species A, described by THEOBALD (1911) from the tvne localitv of Onderstepoort, South‘ Africa (MATTINGLY, 1977): Acknowledgements We thank the field collecting teams and the Research and Publications Office of the Jimma Institute of Health Sciences (JIHS) in Ethiopia for their help with the fieldwork and Dr F. Ayele, Head, Jimma Zone Health Department, JIHS, for facilitating the visit by R. H. H. to Jimma; L. Koekemoer and D. Home, Department of Medical Entomology, SAIMR, South Africa, for the PCR identifications of the ‘wild’ material; Dr C. Curtis, London School of Hygiene and Tropical Medicine, UK, for providing copies of the unpublished Ross Institute Reports; and Professor H. Paterson, University of Queensland, Australia, for constructive comments on the manuscript. The project was funded by the Research Foundation of the SAIMR. Carson, H. L., Clayton, F. E. & Stalker, H. D. (1967). Karyotypic stability and speciation in Hawaiian DrosophiZu. Proceedings of the National Academy of Sciences of the USA, 57, 1280-l 285. Coluzzi, M. (1970). Sibling species in Anopheles and their importance in malariology. Miscellaneous Publications of the Entomological Socieey ofAmerica, 7,63-77. Coluzzi, M. & Sabatini, A. (1967). Cytogenetic observations on species A and B of the Anopheles gumbiae complex. Parassitologia, 9, 73-88. Coluzzi, M. & Sabatini, A. (1968). Cytogenetic observations on species C of the Anopheles gambiae complex. Parassitolcgia, 10, 155-166. Coluzzi, M. & Sabatini, A. (1969). Cytogenetic observations on the salt water species, Anopheles menus and Anopheles melas, of the nambiae complex. Parassitoloaia, 11, 177-187. Coluzzi, M., Sabatini, A.,‘Petrarca, V. & Di Deco, M. A. (1979). Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complex. Transactions of the Royal Society of Tropical Medicine and Hygiene, 13,483497. Coluzzi, M., Petrarca, V. & Di Deco, M. A. (1985). Chromosomal inversion, intergradation and incipient speciation in Anopheles gambiae. Bolletino di Zoologia, 52,45-63. Craddock, E. M. (1974). Degrees of reproductive isolation between closely related species of Hawaiian Drosophila. In: Genetic Mechanisms of Speciation in Insects, White, M. J. D. (editor). Sydney: Australia & New Zealand Book Company, pp. 111-139. Davidson, G. (1962). Anopheles gumbiae complex. Nature, 196, 907. Davidson, G. & Hunt, R. H. (1973). The crossing and chromosome characteristics of a new, sixth species in the Anoph-
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elesgambiae complex. Parassitologia, 15, 12 l-l 28. Davidson, G. &White, G. B. (1972f.The crossing characteristics of a new, sixth species in the Anopheles gambiae complex. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66,531-53X Davidson, G., Paterson, H. E., Coluzzi, M., Mason, G. F. & Micks, D. W. (1967). The Anopheles gambiae complex. In: Genetics of Insect Vectors of Disease, Wright, J. W. & Pal, R. (editors). Amsterdam: Elsevier, pp. 21 l-250. Fettene, M. (1996). Insecticide susceptibility tests of Anopheles gambiae s. 1. populations in Jimma zone, South Western Ethiopia. MSc Dissertation, London School of Hygiene and Tropical Medicine, University of London. Gillies, M. T. & Coetzee, M. (1987). A Supplement to the Anophelinae of Africa south of the Sahara. Publications of the South African Institute for Medical Research, no. 55. Green, C. A. & Hunt, R. H. (1980). Interpretation of variation in ovarian polytene chromosomes ofAnopheZesfunestus Giles, A. narensis Gilles, andA. aruni? Genetica, 51, 187-195. Hum, R. H. (1987). Location of genes on chromosome arms in the Anopheles gambiae group of species and their correlation to linkage data for other anopheline mosquitoes. Medical and Veterinary Entomology, 1,81-88. Hunt, R. H. & Coetzee, M. (1986). Field sampling ofAnopheles mosquitoes for correlated cytogenetic, electrophoretic and morphological studies. Bulletin of theW%rld Health Organization, 64,897-900. Mattingly, - _. I? F. (1977). Names for the Anopheles pambiae complex. Mosquito‘Systematics, 9, 323-328. Mnzava, A. E. I? & Kilama, W. L. (1986). Observations on the distribution of the Anopheles gambiae complex in Tanzania. Acta Tropica, 43,277-282. Muirhead-Thomson, R. C. (1948). Studies on Anopheles gambiae and A. melas in and around Lagos. Bulletin ofEntomological Research, 38, 527-558. Odetoyinbo, J. A. & Davidson, G. (1968). The Anopheles gambiae complex and its role in malaria transmission in the islands of Zanzibar and Pemba, United Republic of Tanzania. Geneva: World Health Organization, mimeographed documents WHOiMAu68.660 andWHONBC168.89. Paskewitz, S. M., Ng, K., Coetzee, M. & Hunt, R. H. (1993). Evaluation of the polymerase chain reaction method for identifying members of the Anopheles gambiae (Diptera: Culicidae) complex in southern At%ca.Journal of Medical Entomology, 30, 953-957. Paterson, H. E. (1962). Status of the East African salt-waterbreeding variant of Anopheles gambiae Giles. Nature, 195, 469-470. Paterson, H. E. (1964). Direct evidence for the specific distinctness of forms A, B and C of the Anopheles gambiae com-
plex. Rivista di Malariologia, 43, 191-196. Scott, J. A., Brogdon, W. G. & Collins, E H. (1993). Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. American Journal of Tropical Medicine and Hygiene, 49, 520-529. Theobald, F. V. (1911). The Culicidae or mosquitoes of the Transvaal. South Afiica&erinary Divison Report, 1,232-272. Townson, H. & Onapa, A. W. (1994). Identification by rDNAI’CR of Anopheles bwambae, a geothermal spring species of the An. gambiae complex. Insect Molecular Biology, 3, 279-282. Turner, R. L. (1972). Malaria epidemiological observations from unsprayed study districts in Ethiopia. Mosquito News, 32,608613. Van Rensburg,A. J., Hunt, R. H., Koekemoer, L. L., Coetzee, M., Shiff, C. J. & Minjas, J. (1996). The polymerase chain reaction method as a tool for identifying members of the Anopheles gambiae complex (Diptera: Culicidae) in northeastern Tanzania. Journal of the American Mosquito Control Association, 12,27 l-274. White, G. B. (1974). Anopheles gambiae complex and disease transmission in Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68,278-298. White, G. B. (198 1). Academic and applied aspects of mosquito cytogenetics. In: Insect Cytogenetics (Symposia of the Royal Entomological Society of London, no. lo), Blackman, R. L., Hewitt, G. M. & Ashburner, M. (editors). London: Blackwell Scientific Publications. DD. 245-274. White, G. B., Tessfaye, F., Boreham, I? F. L. & Lemma, G. (1980). Malaria vector canacitv of Anopheles arabiensi and An. quadriannulatus in Ethiopia: chromosomal interpretation after 6 years storage of field preparations. Transacttons of the Royal Society of Tropical Medicine and Hygiene, 14, 683-684. Wirtz, R. A., Zavala, F., Charoentit, Y., Cambell, G. H., Burkot, T. R., Schneider, I., Esser, K. M, Beaudoin, R. L. & Andre, G. R. (1987). Comparative testing of Plasmodium falciparum sporozoite monoclonal antibodies for ELISA development. Bulletin of the World Health Oranization, 65, 39-45.
Zahar, A. R. (1985). Vktor bionomics in the epidemiology and control of malaria. Part 1. The WHO African Region and the southern WHO Eastern Mediterranean Region. Geneva: World Health Organization, mimeographed documents VBC185.3 and MAP185.3.
Received 2 December 1997; revised SJanuary ed for publication 7January 1998
1998; accept-
The Anopheles
gambiae
complex:
231
a new species from Ethiopia
Richard H. Huntl, Maureen Coetzeel and Messay Fetten& ‘Medical Entomology, Department of Tropical Diseases,School of Pathology of the South African Institute for Medical Research and the University of the Witwatersrand,Johannesburg, South Africa; 2Entomology and Vector Control Unit, Environmental Health School, Jimma Institute of Health Sciences,Jimma, Ethiopia Abstract Historically, members of theAnopheZes gambiae complex from Ethiopia have been identified chromosomally as either A. arubiensis or A. quadriunnulutus. Recent collections from the Jimma area in Ethiopia, southwest ofAddis Ababa, revealed 29 specimens ofA. quadriannulutus based on the standard polymerase chain reaction (PCR) identification method. ‘Wild’ females were induced to lay eggs and the progeny reared as individual families. Resulting adults were cross-mated to a laboratory colony strain of A. quadriunnulutus originating from the Kruger National Park, South Africa. Hybrid progeny were obtained only from the colony femalexEthiopian male cross. This cross produced a female/male sex ratio of 0.48. Male offspring were sterile and ovarian polytene chromosomes from hybrid females showed typical asynapsis as expected in interspecific crosses within the A. gumbiae complex. The X chromosomes, although apparently having homosequential banding patterns, were usually totally asynapsed. All autosomes were homosequential.The lack of inversion heterozygotes, in both the wild and hybrid samples, may simply be a reflection of the small sample size. Until such time as the Ethiopian species can be formally described and assigned a scientific name, it is provisionally designated Anopheles quadriannulatus species B because of its close similarity to this species. Keywords:
malaria, Anophelesgambiae complex, Anophelesquadriannulatus, new species, Ethiopia
Introduction Populations of the Anopheles gumbiue Giles complex from Ethiopia were first identified by cross-mating studies as A. urubiensis Patton (=sp. B of the A. gumbiue complex) and A. quudriunnulatus (Theobald) (=sp. C of the A. gumbiue complex) (Ross Institute Reports, unpublished, 19651969, 1973; TURNER, 1972) and chromosomal studies (M. Coluzzi, in Ross Institute Report, unpublished, 1968; WHITE et al., 1980; WHITE, 198 1). The distribution of A. quudriunnulutus is considered to be relict (WHITE, 1974; COLUZZI et al., 1979), havine been recorded in Ethionia. Pemba and Zanzibar Islands, Zimbabwe, Mozambique; Swaziland and South Africa (see GILLIES & COETZEE, 1987). The southern African populations are widespread, can readily be found in association with cattle, and breed in open sunlit stagnant pools in sandy river beds or next to roads. Ethiopian populations, on the other hand, are found in both animal shelters and mixed human/animal dwellings but nothing appears to be known about the larval habitats (ZAHAR, 1985). Little is known about the Pemha/Zanzibar Islands populations other than that they are zoonhilic and exouhilic UAHAR. 1985) and thev have not’been recorded there since ‘the original redort of ODETOYINBO&DAVIDSON(~~~~).MNZAVA&KLAMA (1986) found no specimen of A. quadriunnulatus on Zanzibar Island, despite using outdoor calf-baited traps. Chromosomal studies on Ethiopian A. quudriannulatus are briefly mentioned by WHITE et al. (1980) and WHITE (198 1). No difference in the banding arrangements between it and the published description ofk. auadriunnulutus from Zimbabwe (COLUZZI & SABATINI, 1968) has been reported. WHITE ( 198 1) published a photograph of chromosomes from what was-apparently a wild F. hvbrid female of A. auadriunnulutus from Ethiopia shcwmg 2 autosomal arms asynapsed with inversion loops but the X chromosome was homosequential and completely synapsed. The X chromosome, however, had been montaged on to the photograph of the autosomes and was at a different magnification. No other information is available on the cytogenetics of the Ethiopian population. The polymerase chain reaction (PCR) is now widely used for identification of members of the A. gumbiue Address for correspondence: Dr R. H. Hunt, Department of Medical Entomology, SAIMR, l? 0. Box 1038, Johannesburg 2000, South Africa.
complex (SCOTT et al., 1993; TOWNSON & ONAPA, 1994). PCR identification of Ethiopian populations has been carried out in recent years, with only typical A. arabiensis and A. quadriannulatus bands appearing on the gels (FETTENE, 1996). This paper presents the results of a study carried out in the Jimma area of Ethiopia with the objective of defining the relationship between a southern African population of A. quadriannulutus and that in Ethiopia. Materials and Methods Mosquitoes were collected from 5 villages outside Jimma (7”39’N, 36”47’E), 335km south-west of Addis Ababa, Ethiopia. They came from habitations where cattle were kept in close proximity to humans and were collected by hand aspirator and placed in polvstvrene cups for transport to the insect&y in Johannesburg, South Africa. There. individual A. gambiae comnlex females were isolated in tubes for egg-laying. Those that laid eggs and survived were refed and kept until half gravid, whereupon their ovaries were dissected and the bodies stored for PCR identification and enzyme-linked immunosorbent assay (ELBA) for sporozoite detection. Dead specimens were stored desiccated for the latter 2 tests. Each egg batch was kept separate and the larvae reared to adults. Ten adults per egg batch, with larval and pupal pelts, were preserved as museum specimens for morphological study (HUNT & COETZEE, 1986). The remaining adults from each family were placed in individual cages until PCR identification had been carried out. Those identified adults not used in the crossmating experiments were pooled in an attempt to establish a laboratory colony. The cross-mating studies were carried out using a laboratory colony ofA. quadriunnukztus (SKUQUA) originating from Skukuza camp, Kruger National Park, South Africa. This colony was established in 1995 and has been checked regularly for purity using chromosome morphology, isoenzyme electrophoresis and PCR. On emergence from the pupae, Ethiopian males and females were separated into individual cages where males or virgin females from the laboratory colony were placed appropriately. Eggs resulting from the crosses were treated as above for individual families. The internal genitalia of sexually mature adult males were dissected and examined for form and sperm content (DAVIDSON et al., 1967). Females were fed to obtain half gravid ovaries for chromosomal studies. Sex ratios were noted. Chromosomal preparations were made according to
RICHARDH. HUNT ETAL.
232
HUNT (1987) and PCR identifications used the protocol of SCOTT et al. (1993). ELISASwere carried out according to WIRTZ et al. (1987). Results A. quadriunnulatus, identified by PCR (Fig. l), was
collected from only 2 of the 5 villages sampled, i.e. Minko and Babo, close to Serbo town about 18 km east of Jimma. Twenty-nine individuals were collected: 3 in human only? 3 in mixed human/cattle, and 23 in cattle only habitations. All the females laid eggsand the larvae were successfully reared to adults. All ELISASon the A. quadriannulatus specimens were negative for Plasmodium fuhparum circumsporozoite protein compared with the A. arabiensissample from the same area, among which 4 of 148 females were positive (2.7%).
Fig. 2. Atrophied testes of hybrid A. quadrimnulatus from the cross SKUQUA female x Ethiopian male.
Fig. 1. Polymerase chain reaction gel showing the identification of Ethiopian Anopheles quadriannulatus. Lane 1: negative control; lane 2: A. arubiensiscontrol; lane 3: A. gambiue control; lane 4: A. mews control; lane 5: A. quadriannulatus SKUQUA; lane 6: Ethiopian A. quadriunnulatus extracted DNA; lane 7: hybrid SKUQUA x Ethiopia non-extracted DNA, lane 8: 1 kb molecular marker. The additional band in lane 7 was randomly expressed in some Ethiopian specimens but not in others.
Of the 29 females that laid eggsand were identified by PCR, 10 survived and ovaries were obtained for chromosomal study. The banding arrangements of all the chromosomes were homosequential with those found in A. quadriannulatus (see COLUZZI & SABATINI, 1968). No inversion polymorphism was seen in the Ethiopian sample. The X chromosome was standard for inversion f, as it was in the SKUQUAcolony. Crosses carried out between the Ethiopian sample and the laboratory colony resulted in hybrid progeny from only the SKUQUAfemales x Ethiopian males. The sex ratio of this cross was 0.48 (117 females/56 males). The Fl hybrid females backcrossed to SKUQUAmales produced a 0.97 sex ratio (29 females/28 males). Fl hy-
males
brid males were sterile with atrophied testes (Fig. 2) corresponding with testis type II of-DAVIDSON er al: (1967). F2 hvbrid males anneared fertile with normal testes and accessory glands. &-ther crosses with males were not attempted. Chromosomal studies on the Fl females revealed extensive asynapsis of the X chromosomes, which were usually totally asynapsed (Fig. 3a), although one specimen was seen where the X chromosomes were partially synapsed (Fig. 3b). The autosomes showed partial asynapsis but no heterozygous loop, as seen in hybrids of speciesthat differ by fixed paracentric inversions (Fig. 4). Attempts to establish a laboratory colony of the Ethiopian population were unsuccessful. Discussion
Recognition that the taxon A. gumbiue was a group of cryptic specieswas initially based on cross-mating characteristics (MIJIKHEAD-THOMSON, 1948; DAVIDSON. 1962; PATERSON,1962). The fact-that males from in: terspecific crosses are sterile was used extensively for routine
identification
of populations
of ‘A. gambiae’
from all over Africa by the Ross Institute at the London School of Hygiene and Tropical Medicine (University of London; seethe unpublished Ross Institute Reports). The cross-mating method requires the maintenance of laboratory stocks of all species in the group and is timeconsuming and laborious. It was, nevertheless, used as the ‘gold standard’ until replaced by polytene chromosome morphology which was shown to be a reliable tool for identifying the recognized species in the complex (COLUZZI & SABATINI, 1967,1968, 1969). PATERSON (1964) used the lack of sterile males in progeny broods from wild-caught females to show lack of mating be-
Fig. 3. The X chromosomes of hybrid A. quadriannulam females from the cross SKUQUA female x Ethiopian male, showing (a) almost total asynapsis which occurred in the majority of the hybrids, and (b) semi-asynapsis seen in one individual.
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Fig. 4. The autosomes of hybrid A. quadriunnuZutusfemales from the cross SKUQUA female x Ethiopian male, showing homologous arms with homosequential banding sequences.Asynapsis was consistently present at the centromeric regions of both chromosomes (indicated by open arrows). Intermittent asynapsis is indicated by closed arrows. tween A. gambiae, A. arabiensis and A. quadriannulatus in sympatric populations in Zambia. While A. quadriannulatus from Ethiopia was identified as such on crossmating evidence (Ross Institute Report, unpublished,
1973), at that time no A. quadriannulatus from southern Africa was available as a colony in London. Crosses of Ethiopian samples were made only with A. gambiae, A. arabiensis, A. rrzerusDiinitz and A. melas Theobald and,
234
by inference, identifications were recorded as ‘probably sp. C’. In the present study, using the criteria on which the whole group was differentiated, we are faced with evidence of male sterility and chromosomal asynapsis from cross-mating studies between the 2 allopatric populations of ‘A. quadriannzdutus’.This allopatric situation is comparable to that found between A. melas and A. merus, the West and East African saltwater species (DAVIDSONet al., 1967), as well as between A. bwambae White from Uganda and A. quadriannulatus with which it shares the most chromosomal homology (DAVIDSON & WHITE, 1972; DAVIDSON& HUNT, 1973). Homosequential polytene chromosomes in more than one species of Anopheles are well known, e.g. A. atroparZeus Van Thiel and A. Zabranchiue Falleroni (see COLUZZI, 1970), A. funestus Giles and A. vaneedeni Gillies & Coetzee (seeGREEN& HUNT, 1980). Similar situations are found in Drosophila, where many homosequential species are known (CARSONet al., 1967; CRADDOCK, 1974). Chromosomal studies on the members of the A. gambiae complex, however, have so far shown either fixed paracentric inversion differences between species (COLUZZI & SABATINI. 1967.1968.1969) or nolvmornhic inversions with di&erentfrequencies between subpopulations of A. gambiae in West Africa (the ‘incipient soecies’ of COLUZZI et al.. 1979. 1985). This is the first record of 2 members of ihe complex having homosequential chromosome banding patterns and apparently no polymorphic inversion difference. It is not surprising, therefore, that the original chromosomal studies resulted in identifications ofA. quadriannulatus from Ethiopia (M. Coluzzi, in Ross Institute Report, unpublished 1968; WHITE et al., 1980; WHITE, 1981). Only 2 polymorphic inversions have been reported in A. quadtiunnulatus from southern Africa, xf and 2Ri (COLUZZI & SABATINI, 1968), with Xfbeing fairly common and 2Ri extremely rare (unpublished data). More extensive sampling of the Ethiopian populations may reveal polymorphic inversions not seen in the present study because of the small sample size. The use of PCR for identification of the named species of the A. gumbiae group has become standard practice in many laboratories. It is accurate (>99%) and has many advantages over both the chromosomal and isoenzyme electrophoretic methods (PASKEWITZet al., 1993; VAN RENSBURGet al.. 1996). It does. however. have the disadvantage of not being able to provide any additional information on the population genetics of the samples tested. The amplification of additional bands, such as the A. quadriannulatus band found in many A. merus specimens, does not pose a problem as long as the A. merusband amplifies, but will result in misidentification if the deoxyribonucleic acid (DNA) is degraded and only the smaller, 153 base-pair fragment ofA. quadriannulatus is present (VAN RENSBURGet al., 1996). In the present study, the Ethiopian ‘A. quadriannulatus’ produced a fragment that appears to be identical in size to that found in the South African populations. The result is that neither PCR nor polytene chromosome morphology can be used as a method for separating the 2 taxa and we do not know whether both occur in Ethiopia or, for that matter, in southern African regions where A. quadriannulatus is known to occur. An apparently identical PCR product size does not necessarily indicate an identical base pair sequence in the fragment. The production of similar fragment sizes in 2 different svecies of the A. pambiae comvlex has already been recorded, i.e. A. kerus and A. melas. It is necessary, therefore, that an alternative identification system be found, preferably a primer sequence, that enables the new taxon to be identified using established molecular procedures. Once this is done, it will be possible to establish the extent of the distribution of the new species. These results bring into sharp focus the conceptual issues underlying genetically based species identifica-
RICHARD H. HUNT ETAL.
tion methods and their use for establishing the evolutionary status of a taxon. They clearly indicate that the genetical characters we use to recognize the species in the A. gumbiae complex are, in fact, simply fortuitous markers of genetic discontinuity and are not in any way involved in the speciation process. It is possible that species exist within the complex that exhibit none of the currently used criteria of sterile male hybrids, asynapsed chromosomes, inversion arrangements and polymorphisms, isoenzyme polymorphisms or DNA sequences. These considerations become particularly important when genetical studies are undertaken on populations that may in fact consist of more than one species. In summary, the key genetic crossing characteristics between recognized members of theA. gambiae complex have been found in 2 apparently allopatric populations sharing the A. quadria%ulatus -chromosomal arrangements and indistinguishable in terms of PCR nroduct size. They differ in”3 important ways: in the c;oss between A. quadriannulatus and the Ethiopian population (i) resultant males are sterile, (ii) there is marked sex ratio distortion, and (iii) there is extensive asynapsis in the ovarian polytene chromosomes. We conclude, therefore, that the Ethiopian population is a different species.Until such time as a formal description can be produced and a scientific name assigned, we designate it asAnopheles quadricmnulatus species B because of its close similarity to this species. The southern African population is designated-as A. quadriannulatus species A, described by THEOBALD (1911) from the tvne localitv of Onderstepoort, South‘ Africa (MATTINGLY, 1977): Acknowledgements We thank the field collecting teams and the Research and Publications Office of the Jimma Institute of Health Sciences (JIHS) in Ethiopia for their help with the fieldwork and Dr F. Ayele, Head, Jimma Zone Health Department, JIHS, for facilitating the visit by R. H. H. to Jimma; L. Koekemoer and D. Home, Department of Medical Entomology, SAIMR, South Africa, for the PCR identifications of the ‘wild’ material; Dr C. Curtis, London School of Hygiene and Tropical Medicine, UK, for providing copies of the unpublished Ross Institute Reports; and Professor H. Paterson, University of Queensland, Australia, for constructive comments on the manuscript. The project was funded by the Research Foundation of the SAIMR. Carson, H. L., Clayton, F. E. & Stalker, H. D. (1967). Karyotypic stability and speciation in Hawaiian DrosophiZu. Proceedings of the National Academy of Sciences of the USA, 57, 1280-l 285. Coluzzi, M. (1970). Sibling species in Anopheles and their importance in malariology. Miscellaneous Publications of the Entomological Socieey ofAmerica, 7,63-77. Coluzzi, M. & Sabatini, A. (1967). Cytogenetic observations on species A and B of the Anopheles gumbiae complex. Parassitologia, 9, 73-88. Coluzzi, M. & Sabatini, A. (1968). Cytogenetic observations on species C of the Anopheles gambiae complex. Parassitolcgia, 10, 155-166. Coluzzi, M. & Sabatini, A. (1969). Cytogenetic observations on the salt water species, Anopheles menus and Anopheles melas, of the nambiae complex. Parassitoloaia, 11, 177-187. Coluzzi, M., Sabatini, A.,‘Petrarca, V. & Di Deco, M. A. (1979). Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complex. Transactions of the Royal Society of Tropical Medicine and Hygiene, 13,483497. Coluzzi, M., Petrarca, V. & Di Deco, M. A. (1985). Chromosomal inversion, intergradation and incipient speciation in Anopheles gambiae. Bolletino di Zoologia, 52,45-63. Craddock, E. M. (1974). Degrees of reproductive isolation between closely related species of Hawaiian Drosophila. In: Genetic Mechanisms of Speciation in Insects, White, M. J. D. (editor). Sydney: Australia & New Zealand Book Company, pp. 111-139. Davidson, G. (1962). Anopheles gumbiae complex. Nature, 196, 907. Davidson, G. & Hunt, R. H. (1973). The crossing and chromosome characteristics of a new, sixth species in the Anoph-
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Received 2 December 1997; revised SJanuary ed for publication 7January 1998
1998; accept-