362
SAMT
VOL 76
7 OKT 1989
3. Schoub BD, Smith AN, Lyons SF er al. Epidemiological considerations of
the present Starus and future growth of the acquired immunodeficiency
syndrome epidemic in South Mrica. S Afr Med] 1988; 74: 153-157.
4. Sherr L. Evaluation of the UK Health Educarion Campaign. Psychol Healch
1987; 1: 61-72.
5. Sherr L. Evaluation of the National AIDS Counselling Training Workshops. Paper presented at the Global Impact of AIDS Conference, Barbican,
London, 8-10 March 1988.
6. Miller D. The worried well. In: Miller D, Weber J, Green J, eds. The
Managemenc of AIDS Paciencs. London: Macmillan, 1986: 169-173.
.
7. Fineberg HV. Education to prevent AIDS: prospects and obstacles. Saence
1988; 239: 592-596.
8. Morlet A, Guinan J, Diefenthalec I, Gold J. The impacr of the 'Grim
Reaper' national AIDS educational campaign on the A1bion Street (AIDS)
Centre and the AIDS horline. Med] Ausc 1988; 148: 282-285.
9. Lehmann P, Hausser D, Somaini B, GutzWiller J. Campaign against AIDS
in Switzerland: evaluation of a nationwide educational programme. Br Med]
1987; 295: 1118-1120.
.
10. Ginzberg HM, Fleming PL, Miller KD. Selected public health observations
derived from the Multicenter AIDS Cohort Study. ] AcqulTed Immune
Deficiency Syndrome 1988; 1: 2-7.
.
.
11. Miller D. Clinic anendance as the result of pubhc health education campaigns on AIDS. Paper presented at the HIrd International Conference on
AIDS, Washington, DC, 1-5 June 1987.
12. Miller D. Worried well. Paper presented at the IVth International Conference on AIDS, Stockholm, Sweden, 12-16 June 1988.
13. Metz J. Guest Editorial. The Leech 1987; 256: 2, 1-2.
A parasitological, cytogenetic and biochemical
study of Anopheles gambiae (Diptera: Culicidae)
from the People's Republic of Congo
RICHARD H. HUNT,
MAUREEN COETZEE
Summary
A sample of 41 live adult members of the Anopheles gambiae
complex were obtained from Yaka Yaka, near Brazzaville,
People's Republic of Congo. They were collected resting in
human habitations. Thirty-seven specimens were identified
as A. gambiae s.s. using cytogenetic and electrophoretic
criteria. Of the identified sample, one specimen was heterozygous for a previously undescribed inversion on chromosome
arm 3. One female and the progeny of a second were found
to be polymorphic for a superoxide dismutase electromorph
previously only found in A. bwambae and A. arabiensis.
Thirty-four specimens were examined for plasmodia. Five
had oocysts on their stomachs and 2 had sporozoites in the
salivary glands.
S AIr Med J 1989: 76: 362-364.
The Anopheles gambiae Giles group of mosquitoes include
major vectors of malaria and filariasis in most of Africa. The
six species comprising the group are difficult to identify
because of their morphological similarity.I-J The unreliability
of morphological characteristics as markers of species in
members of this group was demonstrated by Cambournac e£
al.,4 who showed that the morphologically defined subspecies
A. quadriannulacus davidsoni Ribeiro et al. 5 from the Cape
Verde islands is a geographically isolated population of
A. arabiensis Patron. They based their argument for this
synonomy on the fact that the Cape Verde population has
Medical Entomology, Department of Tropical Pathology,
School of Pathology, South African Institute for Medical
Research and University of the Witwatersrand, Johannesburg
RICHARD H. HUNT, M.Se., PH.D., F.R.E.S.
MAUREEN COETZEE, M.Se., PH.D., F.RE.S
polytene chromosomes that are homosequential with those of
A. arabiensis. The species-specific inversion arrangements
found in the polytene chromosomes are currently used as the
basis for species identification of the groUp.6-9 These chromosomal arrangements have been shown to be consistently reliable
indicators of species identity by all workers on the group. 10
Possible exceptions are studies in West Africa, which showed
assortative mating with respect to polymorphic inversions in
populations of A. gambiae. I1,12
An alternative method of identification is based on enzyme
electromorph differences in the various species.J,IJ-Js Presence
or absence of particular alleles and their relative frequencies
can vary geographically in a single species. For this reason,
diagnostic electromorphs should be checked in chromosomally
identified samples. This must be done before electrophoresis is
used for specific identification in any locality for which electromorph frequency data are unavailable.
Material and methods
A sample of 41 wild female mosquitoes were received in May
1985 from Yaka Yaka, a village about 30 km west of Brazzaville,
People's Republic of Congo (04°22'S, 15°09'E). The females
were captured while resting inside houses.
On arrival in the laboratory, they were placed in tubes lined
with moist filter paper for egg-laying. Females that laid eggs
were offered a blood meal and ovaries obtained 33 hours later
for chromosomal studies. Stomachs and salivary glands of all
the specimens were dissected and examined for oocysts and
sporozoites. The dissected bodies were stored in liquid nitrogen
for electrophoretic examination. Progeny obtained from individual females were used for identification purposes where
necessarl and morphological specimens preserved for the collection. I -I8
The cytogenetic methods follow those described by Hune 9,20
and Green and Hunt 21 and the electrophoretic methods of
SAMJ
VOL 76
7 aCT 1989
363
Mahon et al. 13 and Miles. I '. 15 At least 2 controls, A. merus
Donitz and A. gambiae, were used on each electrophoretic gel;
in some cases 4 controls were used. Controls came from colony
material of A. gambiae from The Gambia, West Africa,
A. arabiensis from Zimbabwe, A. merus from Natal, South
Africa, and A. quadn'annulacus Theobald from Transvaal, South
Africa.
Hind-leg banding patterns of identified females were examined using the method of Coetzee et al. 22
Results
Thiny-seven of the specimens were identified as A. gambiae
using the X chromosome arrangement6 and inversion 20p on
chromosome arm 2 16 (Fig. 1). Twenty-two of them were
identified both chromosomally and electrophoretically, while
15 were only identified electrophoretically. Four specimens
died in the laboratory before producing eggs or being identified.
Fig. 2. Acrylamide gel showing superoxide dismutase electromorphs in a sample of A. gambiae from Yaka Yaka, Republic of
Congo. Specimen Y40 exhibits the three bands at the 100/105
locus to be expected in an animal heterozygous for a dimeric
enzyme. '6 (Hb = human blood marker; KGB = A. arabiensis
control; GAG = A. gambiae control; QUAD = A. quadriannulatus
control; Y = Yaka Yaka).
TABLE I. COMPARISON OF THE FREQUENCIES OF SIX
GENE LOCI IN A SAMPLE OF A. GAMBIAE GROUP
MOSQUITOES FROM THE REPUBLIC OF CONGO WITH
THREE MEMBERS OF THE COMPLEX FROM WEST AFRICA
Enzyme.
Yaka Yaka
locus
A. gambiae*
sample
A. arabiensis* A. me/as*
Fig. 1. Full polytene chromosome karyotype of A. gambiae
heterozygous for inversion 3i from Yaka Yaka, Republic of Congo.
The electromorphs of AAT (aspartate aminotransferase EC
2.6.1.1), ODH (octanol dehydrogenase EC 1.1.1.73) and EST
(nonspecific esterase EC 3.1.1.1) 1,2 and 3 were as expected
for A. gambiae in West Africa in all specimens examined. The
gene frequencies are shown in Table I. There was, however,
an unusual heterozygous SOD (superoxide dismutase EC
1.15.1.1) electromorph in I of the wild-caught females and in
I of the families from a second female. These 2 specimens
were heterozygous for the 100/105 alleles, resulting in a.
typical dimeric electromorph (Fig. 2). One of the specimens
with the polymorphic SOD was identified chromosomally and
had the normal A. gambiae karyotype. Both these specimens
had the other diagnostic electromorphs expected in A. gambiae.
Chromosomally, all specimens had the typical A. gambiae X
chromosome and arm 2 inversion arrangements. The population
was polymorphic for the 3a inversion. One specimen was
heterozygous for a previously undescribed inversion on arm 3
(Fig. 1), which is designated as 3i.. Because only the one
hete'rozygote was seen, the precise breakpoints of the inversion
could not be determined.
All the identified specimens had the leg banding patterns
expected in A. gambiae. Details of these data have been
published elsewhere. 23
Thirty-four of the wild-caught specimens were dissected
and examined for plasmodia and filaria. No filariallarvae were
seen. Five stomachs were found to have oocysts on their
surface and 2 specimens had sporozoites in the salivary glands.
AAT
105
100
95
ODH
105
100
95
90
SOD
105
100
95
EST 1
110
105
100
95
90
85
80
75
70
EST 2
110
105
100
95
90
EST 3
110
105
100
95
90
85
80
75
N=96
0
1,00
0
N=332
0,03
0,94
0,03
0
N=332
0
1,00
0
N=338
0
0,004
0,11
0,66
0,22
0,005
0
0
0
N=338
0,02
0,44
0,51
0,03
0
N=338
0,02
0,17
0,26
0,31
0,13
0,08
0,03
0
*Data from Miles.'"
N=92
0
0,94
0,06
N=85
0
0,99
0,01
0
N= 89
0,01
0,99
0
N=60
0,017
0,025
0,208
0,55
0,192
0,008
0
0
0
N=57
0
0,35
0,491
0,132
0,026
N=85
0
0,018
0,076
0,10
0,423
0,329
0,053
0
N= 54
0
1,00
0
N= 192
0
0,20
0,76
0,04
N= 192
0
1,00
0
N= 194
0
0,008
0,55
0,43
0,01
0
0
0
0
N= 194
N=84
0
1,00
0
N= 114
0,01
0,04
0,94
0,01
N=98
0
0,90
0,10
N= 110
0
0
0
0
0
0
0,06
0,88
0,06
N= 110
Overlaps
with
EST 3
Overlaps
with
EST 3
N= 194
0
0,16
0,66
0,16
0,02
0
0
0
N= 110
0
0,06
0,80
0,08
0,06
0
0
0
I
I
......
364
SAMT
VOL 76
7 OKT 1989
One sporozoite-poslOve specimen had died before dissection
so no firm conclusion could be made as to the presence or
absence of oocysts on the abdomen. The other was positive for
both sporozoites and oocysts.
Discussion
Although only A. gambiae has been identified from Yaka Yaka,
it is possible that A. arabiensis and A. melas Theobald may also
occur there. However, A. melas is a saltwater coastal species
and has never been recorded so far inland.
The sporozoite rate of 5,9% found in this study is very
similar to that found by Carnevale er al. 24 in their study on
A. gambiae in a village south-west of Brazzaville. In May of
1977 and 1978 they recorded sporozoite rates of 6,1 % and 3, I %
respectively.
The 100/105 SOD heterozygote is particularly interesting
since, until recently, the 105 SOD electromorph l5 was considered monomorphic and diagnostic for A. bwambae White.
The distribution of this member of the A. gambiae group is
limited to the Semliki forest in Uganda. The electromorph has
since been reported as a polymorphism in A. arabiensis in East
Africa25 and now in the Yaka Yaka population of A. gambiae.
These findings highlight the need for correlated chromosomal and electrophoretic studies on any population of the
A. gambiae group before electrophoresis is considered as a
routine method of identification. The inclusion of electromorphs as characters in identification keys, as used by White,26
is brought into question.
We thank Mr I. Davidson for collecting the material and Mr A.
von Maltitz for help in transporting the specimens. Professor H.
E. Paterson and Mr A. Comel are thanked for comments on the
manuscript. This study formed part of a Ph.D. thesis (R.H.H.)
submitted through the Department of Zoology, University of the
Wirwatersrand. One of us (M.C.) was suppotted in part by a
short-term research grant from the South African Medical
Research Council.
REFERENCES
I. Coluzzi M. Sibling species in Anopheles and their imponance in malariology.
Mise Publ Entomol Soc Am 1970; 7: 63-77.
2. Green CA. Malaria epidemiology and anopheline cytogenetics. In: Pal R,
Kitzmiller JB, Kanda T, eds. Cycogene,;,;s and Gene,;,;s of VeuOTS. Amsterdam: E1sevier, 1981: 21-29.
3. Miles SJ. Inversions, e1ectromorphs and the identification of individual
mosquitoes. In: Pal R, Kitzmiller .lB, Kanda T, eds. Cycogene,ics and
Gene';,;s of VeccOTS. Amsterdam: E1seVler, 1981: 61-64.
4. Carnbournac FJC, Petrarca V, Coluzzi M. Anopheles arabiensis in the Cape
Verde Archipelago. Parassicologia 1982; 24: 265-267.
5. Ribeiro H, Rarnos H da C, Pires CA, Capela RA. Description and biometric
study of Anopheles (Cellia) quadnannulaeus davidsoni ssp. n., a seventh
member of the Anopheles gambiae Giles complex (Diptera: Culicidae) endemic
to the Cape Verde Archipelago. Garcia de Orea Ser Zoo11979; 8: 75-88.
6. Coluzzi M, Sabatini A. Cytological observations on species A and B of the
Anopheles gambiae complex. Parassicologia 1967; 9: 73-88.
7. Coluzzi M, Sabatini A. Cytological observations on species C of the Anopheles
gammae complex. Parassicologia 1968; 10: 155-165.
8. Coluzzi M, Sabatini A. Cytological observations on the saltwater species
Anopheles merus and Anopheles melas of the gambiae complex. Parassicologia
1969; 11: 177-187.
9. Hunt RH. Cytological srudies on a new member of the Anopheles gambiae
complex. Tram R Soc Trop Med Hyg 1972; 66: 532.
10. GiIlies MT, Coetzee M. A Supplement CO ,he Anophelinae of Africa Sourh of
,he Sahara. Jobannesburg: SAIMR, 1985 (No. 55.).
I!. Bryan JH, di Deco MA, Petrarca V, Coluzzi M. Inversion polymorphism
and incipient speciation in Anopheles gambiae Ulr. in The Gambia, West
Mrica. Genetica 1982; 59: 167-176.
12. Coluzzi M, Petrarca V, di Deco MA. Chromosomal inversion intergradation
and incipient speciation in Anopheles gambiae. Boll Zoo11985; 52: 45-63.
13. Mahon RJ, Green CA, Hunt RH. Diagnostic alIozymes for routine identification of adults of the Anopheles gambiae complex (Diptera: Culicidae). Bull
Entomol Res 1976; 66: 25-31.
14. Miles SJ. Enzyme variation in the Anopheles gambiae Giles group of species
(Diptera: Culicidae). Bull Emomol Res 1978; 68: 85-96.
15. Miles SJ. A biochemical key to adult members of the Anopheles gambiae
group of species (Diptera: Culicidae). J Med Emomol 1979; IS: 297-299.
16. Hunt RH. Location of genes on chromosome arms in the Anopheles gambiae
. group of species and their correlation to linkage data for other anopheline
mosquitoes. Med Vel Emomo11987; 1: 81-88.
17. Hunt RH, Coetzee M. Field sampling of Anopheles mosquitoes for correlated
cytogenetic, eleCtrophoretic and morphological srudies. Bull WHO 1986; 64:
897-900.
18. Hunt RH, Coetzee M. Chromosomal and electrophoretic identification of a
sample of Anopheles gambiae group mosquitoes from the island of Grand
Comoros, Indian Ocean. J Med Emomol 1986; 23: 655-660.
19. Hunt RH. A cytological technique for the srudy of the Anopheles gambiae
complex. Parassicologia 1973; 15: 137-139.
20. Hunt RH. Narural and induced chromosomal rearrangements in three
members of the Anopheles gambiae complex. M.Sc. dissertation, University
of the Witwatersrand, Jobannesburg, 1984.
21. Green CA, Hunt RH. Interpretation of variation in ovarian polytene chromosomes of Anophelesfunescus Giles,A. parensis GiIlies andA. aruni? Gene'ica
1980; 51: 187-195.
22. Coetzee M, Newberry K, Durand D. A preliminary reporr on morphological character distinguishing imporrant malaria vectors in the Anopheles
gambiae Giles complex in southern Mrica. Mosq Sysc 1982; 14: 88-93.
23. Coetzee M. Comparative morphology of four members of the Anopheles
gambiae group in southern Mrica. Mosq Sysc 1989 (in press).
24. Carnevale P, Bosseno M-F, Zoulani A, Michel R, Molez J-F. La dynamique
de la transmission du paludisme humain en zone de savane herbeuse et de
forc~t degradee des environs nord et sud de Brazzaville, R.P. du Congo. Cah
ORSTOM Ser Em Med el Parasicol1985; 23: 95-115.
25. Marchand RP, Mnzava AEP. A field test of a biochemical key to identify
members of the Anopheles gambiae group of species in north-east Tanzania. J
Trop Med Hyg 1985; 88: 205-210.
26. White GB. Anopheles bwambae sp.n., a malaria vector in the Semliki Valley,
Uganda, and its relationships with other sibling species of the A. gambiae
complex (Diptera: Culicidae). Sysc Emomo11985; 10: 501-522.
a