Distribution and Chromosomal Characterization of the Anopheles

Am. J. Trop. Med. Hyg., 78(1), 2008, pp. 169–175
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene
Distribution and Chromosomal Characterization of the Anopheles gambiae
Complex in Angola
Maria Calzetta, Federica Santolamazza, Gian Carlo Carrara, Pedro J. Cani, Filomeno Fortes, Maria Angela Di Deco,
Alessandra della Torre,* and Vincenzo Petrarca
Dipartimento di Scienze di Sanità Pubblica, Università degli Studi di Roma “La Sapienza”; Ministry of Health, National Program of
Malaria Control, Luanda, Angola; Istituto Pasteur-Fondazione Cenci-Bolognetti; Dipartimento di Genetica e Biologia Molecolare,
Università degli Studi di Roma “La Sapienza,” Rome, Italy
Abstract. Mosquitoes of the Anopheles gambiae complex (N ⳱ 1,336) were sampled (2001–2005) across Angola to
identify taxa, study inversion polymorphisms, and detect the circumsporozoite protein of Plasmodium falciparum.
Anopheles gambiae s.s. was found in all sites; it was characterized as M-form in localities of the tropical dry and
semi-desertic belts, whereas the S-form was predominant in comparatively more humid and less anthropized sites. Both
forms were characterized by low degrees of chromosomal polymorphism based solely on the 2La inversion, a pattern
usually associated with An. gambiae populations from forested, humid, and derived savanna areas. Unexpectedly, this
pattern was also observed in M-form populations collected in dry/pre-desertic areas, where this form largely predominates over An. arabiensis, which was also detected in central/inland sites. Anopheles melas was found in northern coastal
sites. Three of 534 An. gambiae s.s. were positive for P. falciparum CS-protein, whereas none of the 105 An. melas were
positive.
sites, covering predominantly the western and central areas of
the Country. Anopheles gambiae s.l. was sampled at many of
those sites, but no attempt was made to methodically differentiate the member species of the complex, except to discriminate between generic “fresh-water species” and An.
melas, and to report in the Cacuaco area a few records as
“Anopheles gambiae species A” (⳱An. gambiae s.s.), through
the crossing experiment technique. However, based on ecoclimatic speculations, Ribeiro and Ramos stated that “species
A will prove to be more common in the West Africa biomes
in Angola, while species B (⳱An. arabiensis) is expected to
be more associated with the Rhodesian Highland and SW
Arid Zone.” Moreover, in the same article Ribeiro and Ramos (citing Ribeiro and others, unpublished reports) reported
Plasmodium sporozoite indexes of fresh-water An. gambiae
(presumably An. gambiae s.s.) ranging from 0–8%.
Very recently, Cuamba and others reported the results of
the PCR–RFLP-based species identification of about 400 An.
gambiae s.l., collected in 2001 in Angola at three central and
western localities, and at Luanda, the capital city.6 Cuamba
and others identified An. gambiae s.s. M molecular form
(94%), S-form (6%), a single An. arabiensis larval specimen
in the site of Samba (Luanda), where An. melas was also
found. No data on the chromosomal inversion patterns of the
collected samples were reported. Cuamba and others also
assessed, through an ELISA-based method, the Plasmodium
falciparum infection rates of 580 An. gambiae s.s., reporting
an overall infection rate of less than 2%.
The end of wartime in 2002 opened up the perspective of
updating entomological data in the region. Here we present
the results of sampling activities (2001–2005) aimed at collecting entomological baseline information, especially in and
around urban coastal sites, mainly to study the distribution
and chromosomal characterization of the taxonomic units of
the An. gambiae complex, with particular reference to the M
and S molecular forms of An. gambiae s.s.
INTRODUCTION
Information on malaria vectors in Angola and in the whole
central part of the African continent is limited, especially
concerning the member species of the Anopheles gambiae
complex. In particular, the distribution and the chromosomal
inversion patterns of An. gambiae molecular forms in West
Africa has only been extensively analyzed in northern and
southern Guinea savanna areas, where the M-form and Sform show high frequencies of paracentric inversion polymorphisms, and in forested and humid areas north of the Equator,
where they are characterized by a low degree of polymorphism, mainly based on the 2La inversion, or even monomorphism.1,2
Based on chromosomal data, Coluzzi (1982) proposed that
co-adapted inversions may act as selective forces leading to
the formation of new species within An. gambiae s.s.3,4 If
inversions have had a crucial role as selective forces, the presence of uninverted homokaryotypic M and S in forested and
humid areas of west-central Africa could be interpreted as a
convergence due to the role of the standard arrangement on
chromosome-2 in the ecotypic adaptation to these environments. In this case, the core of the speciation process would
have been in west African savanna areas, and the southern
humid areas would have been re-colonized afterward.1 However, the scarceness of data on the distribution and the absence of data on chromosomal inversion polymorphisms of
molecular forms in west-African dry areas south of the Equator, do not allow further speculations on the speciation process within An. gambiae s.s. and on the role of the inversions
in the ecotypic adaptation to these areas.
The few existing papers on the Angolan mosquito fauna
were published mainly by Portuguese scientists before the
beginning of the war events (November 1975), which came to
an end in 2002. The major pre-war article was by Ribeiro and
Ramos,5 who sampled species of the genus Anopheles at 147
MATERIALS AND METHODS
* Address correspondence to Alessandra della Torre, Piazzale Aldo
Moro 5, Rome, Italy 00185. E-mail: alessandra.dellatorre@
uniromal.it
Study area and climate. The sampling activity was carried
out in peri-urban and rural sites belonging to 11 Provinces of
169
170
CALZETTA AND OTHERS
Angola (Table 1, Figure 1), which are situated in four main
different eco-climatic domains (sampling sites between parentheses):
1. The Provinces of Cabinda (Cabinda), Zaire (Soyo District:
Kikudo, Matajor), Cuanza Norte (N’dalatando), Malanje
(Malanje), and Lunda Sul (Saurimo) are located in the
northern tropical humid zone, characterized by areas ranging from broadleaf evergreen forest to savanna. The northern Cabinda coastal enclave and the Soyo District are located in an oil well area; the other localities are in rural
zones.
2. The Provinces of Luanda (Samba, Nazarè, Bairro Pinto,
Mateba, Cazenga, Viana, Mussulo) and Bengo (Cabungo)
are located in the northern-central part of the coast,
characterized by tropical dry climate and mangrove
swamps along the sea-shore, and grassland and woodland further inland. The collection sites were mainly located in peri-urban areas, except for Cabungo, a rural
zone, and for the peninsular strip of Mussulo, a tourist area
of Luanda.
3. The Provinces of Benguela (Cavaco) and Namibe
(Namibe) are located on the southern coastal area, characterized by an even drier climate, which degrades into
desert in the extreme south; the collecting sites were in
peri-urban rural areas.
4. The Provinces of Huambo (Cossango, Camussamba) and
Huila (Lubango) are located on an inland plateau (Bié
plateau) that extends as far as 2⁄3 of the country (average
altitude of 1200–1600 m above sea-level), characterized by
evergreen tropical forest in the north, humid savanna in
the center and southeast, and desert steppe in the southwest.
Annual rainfall ranges from 1750–1250 mm in northern and
central areas, to 600–400 mm in the extreme southeast, up to
250–100 mm in the southwest and less than 100 mm in the
extreme southwest. The rainy season is from November to
March in the extreme southwest, from September to May in
the northeast, and from November to April on the inland
plateau, where the dry and cool season is from May to October. In Luanda the rainy season is from October to April/
May, and the average rainfall (about 600 mm per year) is
lower than surrounding areas.
Mosquito sampling and identification. Collections of indoor-resting adult An. gambiae s.l. were carried out from June
2001 to March 2005: date details are given for each collecting
site in Table 1. Sampling was performed by hand-operated
aspirators on domestic walls (IR-HC) or inside bed-nets (IRNET), or by pyrethrum spray collections (IR-PSC) (Table 1).
The living mosquitoes were kept in moistened cool-boxes until they reached the half-gravid gonotrophic stage, suitable for
polytene chromosome analysis. Either whole female mosquitoes or their dissected ovaries were dropped in Carnoy’s fixative (one part of glacial acetic acid in three parts of absolute
ethanol). Specimens were stored at −20°C until processing.
The non-half–gravid mosquitoes were kept in vials with desiccant. Larval collections were carried out at Samba
(Luanda): larvae were bred to adults and then processed as
previously outlined. Anophelines were identified using the
morphologic identification keys of Gillies and de Meillon7
and Gillies and Coetzee.8 Anopheles gambiae s.l. specimens
were identified by species and molecular forms following the
PCR-RFLP protocol by Fanello and others.9
Cytogenetic analysis. The half-gravid Carnoy’s fixed An.
gambiae s.l. females and ovaries were processed for ovarian
polytene chromosome analysis following della Torre.10 Para-
TABLE 1
Relative frequencies of Anopheles gambiae species and molecular forms sampled in sites in Angola listed north to south
Province
Cabinda
Zaire
Luanda
Bengo
Cuanza
Norte
Malanje
Lunda Sul
Huambo
Benguela
Huila
Namibe
Locality
Lat.
An. gambiae s.s.
Long.
Ecoclimatic
belt*
Date
Sampling
methods†
An. gambiae s.l.
total
An.
melas
An.
arabiensis
N°
M form
S form
Cabinda
Kikudo, Matajor
Samba
Nazarè,
Bairro Pinto
Nazarè
Mateba
Mateba,
Cazenga,
Viana
Mateba
Mussulo
Mussulo
Cabungo
N’dalatando
5°33⬘S
6°07⬘S
8°49⬘S
8°45⬘S
12°11⬘E
12°22⬘E
13°13⬘E
13°23⬘E
1
1
2
2
Feb–Mar 2003
Apr 2002
Jun–Jul 2001
Apr 2002
IR-HC
IR-PSC
LC
IR-NET
124
75
70
24
0.0
30.7
0.0
0.0
0.0
0.0
0.0
0.0
124
52
70
24
0.0
0.0
100.0
100.0
100.0
69.3
0.0
0.0
8°45⬘S
8°45⬘S
13°23⬘E
13°23⬘E
2
2
Apr 2003
Apr 2002
IR-NET
IR-NET
12
457
75.0
20.6
0.0
0.0
3
363
25.0
79.4
0.0
0.0
8°45⬘S
13°23⬘E
2
May 2002
IR-NET
IR-PSC
21
61.9
0.0
8
38.1
0.0
8°45⬘S
8°53⬘S
8°53⬘S
8°34⬘S
9°17⬘S
13°23⬘E
13°07⬘E
13°07⬘E
13°30⬘E
14°59⬘E
2
2
2
1–2
1
Apr 2003
May 2002
Sep 2003
Apr 2002
Apr 2003
IR-NET
IR-HC
IR-NET
IR-NET
IR-HC
71
43
24
1
14
94.4
18.6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
4
35
24
1
14
5.6
81.4
100.0
0.0
7.1
0.0
0.0
0.0
100.0
92.9
Malanje
Saurimo
Cossango
Camussamba
Cavaco
Lubango
Namibe:
Bairro Aida,
Bairro St. Rita
9°32⬘S
9°39⬘S
11°13⬘S
12°04⬘S
12°33⬘S
14°55⬘S
16°20⬘E
20°26⬘E
15°10⬘E
15°48⬘E
13°26⬘E
13°30⬘E
1
1
4
4
3
4
Jun 2003
Apr 2002
Dec 2003
Jan 2004
Mar 2005
Apr 2002
6
18
1
1
106
1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
100.0
0.0
100.0
6
18
1
0
106
0
0.0
0.0
0.0
0.0
100.0
0.0
100.0
100.0
100.0
0.0
0.0
0.0
15°11⬘S
12°09⬘E
3
Jun–Jul 2001
and 2002
IR-NET
IR-PSC
IR-NET
IR-NET
IR-NET
–
IR-PSC
IR-NET
IR-HC
267
0.0
0.4
266
99.6
0.0
* Eco-climatic belt (tropical climate): 1 ⳱ humid; 2 ⳱ dry; 3 ⳱ pre-desert; 4 ⳱ highlands; see the text for details.
† Sampling methods: IR-HC ⳱ hand-operated aspirators, indoor collections; IR-NET ⳱ indoor resting mosquitoes in bed-nets; IR-PSC ⳱ pyrethrum spray collection; LC ⳱ larval collection.
ANOPHELES GAMBIAE COMPLEX IN ANGOLA
171
FIGURE 1. Relative frequencies of indoor-resting Anopheles gambiae M-form, An. gambiae S-form, An. Melas, and An. arabiensis at sites
across Angola. Smaller cyclograms refer to collections with a sample size < 10. * The Luanda cyclogram refers to the sum of the Nazaré and Bairro
Pinto samples.
centric inversion karyotypes were scored according to the nomenclature of Coluzzi and others11 and Petrarca and others.12
Observed karyotype frequencies were tested against HardyWeinberg expectations by ␹2 test.
Plasmodium infection rates. Plasmodium falciparum circumsporozoite protein (Pf-CSP) detection was carried out
following Beier and others,13 on a subsample of An. gambiae
s.l. collected in 2001–2005 in sites of the Provinces Zaire,
Luanda, Bengo, Lunda Sul, Benguela, Huila, and Namibe.
Blood-meal identification. A sub-sample of blood-fed An.
gambiae s.l. females collected in the Luanda Province in 2002
was analyzed for host preference: blood from the mosquito
stomach was absorbed and dried on filter paper and the
blood-meal source was determined following Beier and others,14 using human, bovine, swine, and canid antisera. Forty
An. gambiae s.s. from Cavaco (Benguela) were tested with
human and rat antisera.
RESULTS
Distribution of Anopheles gambiae species and molecular
forms. A total of 1,336 An. gambiae s.l. specimens from 22
sites of Angola were identified by species/form: 3 were An.
arabiensis, 214 An. melas, 904 An. gambiae M-form, and 215
An. gambiae S-form. No hybrids between these species/forms
were found. The relative frequencies of the species and molecular forms at the collection sites are reported in Table 1
and summarized in Figure 1.
Two of the three An. arabiensis specimens were sampled in
highland sites (Provinces of Huambo and Huila); the third
specimen was found in the arid area of Namibe, where An.
gambiae M-form largely prevailed (99.5%).
Anopheles melas was found in most coastal sites where
mangrove brackish swamps were present. More in detail, this
species was found in sympatry with the S-form of An. gambiae
s.s. in the Zaire Province sites (30.7%) and with the M-form
in most of the sampling sites in the Luanda area with frequencies ranging from 19–94%.
Anopheles gambiae s.s. was found in all the localities
sampled; the two molecular forms have been found in sympatry in a single sampling site (N’dalatando, Cuanza Norte
Province). The M-form was mainly recorded in coastal localities of Luanda, Benguela, and Namibe Provinces, whereas the
S-form was found in northern coastal sites (i.e., Cabinda and
Zaire Provinces) and inland localities.
172
CALZETTA AND OTHERS
Chromosomal characterization. The single karyotyped An.
arabiensis was inverted homozygote for the inversion 2Rb
(Figure 2A); all the rest of the complement were standard
(i.e., uninverted for any polymorphic inversions).
The 50 karyotyped specimens of Anopheles melas were
polymorphic for the inversion 2Rm1 (Figure 2B), with a mean
frequency of the inverted arrangement of 33%; the three
2Rm1 karyotypes were in Hardy-Weinberg equilibrium
[␹2 ⳱ 0.85, degrees-of-freedom (df) ⳱ 1, P ⳱ 0.36], even
when lumping together all the samples. A single specimen from Kikudo (Soyo District, Province of Zaire) was
heterozygous for an inversion provisionally named 2Rm1#9,
a short inversion based on the inverted arrangement
2Rm1.
We karyotyped 259 An. gambiae s.s., 236 of which were
subsequently discriminated as M-form (191 from Luanda
area, 39 from Cavaco, and 6 from Namibe) and 12 as S-form
(9 from Kikudo and 3 from Cabinda); 11 have not been molecularly identified, mostly due to unavailability of the carcasses. All of them were standard homozygotes for chromosomal arms 3R, 3L, and 2R (i.e., uninverted for any of the
known polymorphic inversions), whereas all samples showed
the common 2La/+ polymorphism (Figure 2C), with a mean
frequency of the inverted arrangement of 13.9%. No significant differences were recorded among the 2La frequencies of
the M-populations from Luanda area, Cavaco and Namibe
(2La mean frequency ⳱ 13.6%). The M-form populations
were in Hardy-Weinberg equilibrium. The small S-form
sample had a 2La frequency of 20.8%, not significantly
greater than that of the M-form (Fisher exact probability test
P ⳱ 0.23).
Plasmodium infection rates. We analyzed for the presence
of Pf-CSP a total of 712 An. gambiae s.l. specimens. All 105
An. melas and the single An. arabiensis tested were Pf-CSP
negative. Three of 606 An. gambiae s.s. specimens were positive: 1 of 355 M-form from Luanda area (0.28%; standard error [SE] ⳱ 0.28), 1 of 130 M-form from Namibe
(0.77%; SE ⳱ 0.77), and 1 of 27 S-form from Zaire Province
(3.70%; SE ⳱ 3.63). The Pf-CSP index was not significantly different between the M- and S-form samples (0.41%,
SE ⳱ 0.29 and 3.70%, SE ⳱ 3.63, respectively; Fisher exact
probability test, P ⳱ 0.15).
Host preferences. Blood-meal identification of 141 An.
gambiae M-form from the Luanda area and 40 from Cavaco
(Benguela Province), showed that 61% and 70%, respectively, of the indoor-resting females had fed on humans,
whereas the rest had bitten animals other than bovines, swine,
canids (at Luanda) and rats (at Cavaco); the difference was
not significant. Fifteen of 25 (60%) indoor-resting An. melas
of the coastal area of Luanda had fed on humans.
DISCUSSION
Anopheles arabiensis. Our data show that An. arabiensis is
largely outnumbered by the M-form of An. gambiae s.s. in the
drier coastal sites, in spite of the eco-climatological conditions
apparently very suitable for arabiensis, which could suggest
better fitness and possibly a process of competitive exclusion
of the M-form over An. arabiensis, as suggested by Touré and
others.15 The single An. arabiensis from Namibe was among
the southernmost records of this species in west Africa (see
Coetzee and others, for references)16: the Namibe area is
FIGURE 2. Schematic representation of the paracentric inversions recorded in samples of Anopheles arabiensis (A), An. melas (B), and An.
gambiae s.s. (C) in Angola. The homozygote 2Rb/b is shown for An. arabiensis. For An. melas, the polymorphic inversions 2Rm1 and 2Rm1#9
are drawn on the species-specific 2Rm/m arrangement. For An. gambiae s.s., the polymorphic inversion 2La is drawn on the standard (i.e.,
uninverted) 2L arrangement.
ANOPHELES GAMBIAE COMPLEX IN ANGOLA
characterized by a nearly desert climate (annual rainfall:
about 100 mm), which is known to be tolerable for this species, particularly in East Africa (see Petrarca and others, for
references).12 The other two specimens were found at highland sites (> 1.000 m asl; annual rainfall > 1000 mm), in agreement with other findings at mid/high altitude areas of Ethiopia,17 Madagascar,18 and Mozambique.19 Cuamba and others
did not report the presence of An. arabiensis in the highland
zone (i.e., Huambo), while reporting a single specimen of this
species from the Luanda area.6 Apart from obvious samplesize biases, these differences could possibly be due to the
different sampling periods (e.g., late versus early rainy season), which could reflect differentiated seasonal prevalence
for this species. An additional, reasonable hypothesis is that
the minimal presence of An. arabiensis in indoor samples
could also reflect a marked exophilic behavior (as it was observed on the Madagascar Plateau),18 which seems to be supported by the fact that the single An. arabiensis found by
Cuamba and others was a larva.6
Anopheles melas. As expected, An. melas was found in the
coastal sites characterized by the presence of brackish
swampy zones with mangroves.7 In more detail, this species
was found in sympatry with An. gambiae M-form in most of
the coastal sampling sites of the Luanda area, as observed also
by Cuamba and others,6 and by Wondji and others on the
Atlantic shores of southern Cameroon.2 The indoor relative
frequencies of Luandan An. melas varied from zero to more
than 90%, even on limited temporal and/or spatial scales (i.e.,
Mateba, Mussulo and peri-urban sites of Luanda city; see
Table 1 and Figure 1), not an uncommon finding when dealing with a species that is known to have non-uniform population dynamics (see references in Bryan and others.20) In the
sites of Zaire Province, An. melas was sympatric with the
S-form of An. gambiae; to our knowledge, this is the first time
such an event is reported south of Ghana coasts: in fact, Yawson and others21 reported the presence of a single An. melas
specimen together with 133 M- and S-forms at a site of coastal
Ghana.
The paracentric inversion set-up of Angolan An. melas has
shown remarkable similarities with coastal populations sampled at the Congo River mouth in 19903 (Petrarca & Coene,
unpublished data). In fact, although separated by more than
10 years in time and about 100 km in space, the Congolese
and Angolan populations share the 2Rm1 and 2Rm1#9 floating inversions, which have thus proven to make stable local
polymorphisms. They have not been recorded elsewhere in
west Africa20,22–25 (Petrarca and others, unpublished data).
Although no Pf-CSP positive An. melas individuals have
been found in the sub-sample analyzed, the high indoor relative frequency reached by this species in some sites, associated with a human blood index around 60%, suggests a potential role of this species in local malaria transmission.7,8,20
Anopheles gambiae s.s. Anopheles gambiae s.s. was present
at all sampled sites; both molecular forms were found, with
M-form being the most abundant taxon. The two molecular
forms apparently showed a complementary distribution. In
fact, we found the M-form only in the sites in the Luanda area
(with the exception of a single S-specimen collected in
Cabungo, Bengo Province), Benguela, and Namibe Provinces. Although Cuamba and others reported the presence of
both forms in the Benguela Province,6 the prevalence of Mform in coastal sites in the Luanda area and southward is a
173
consistent finding, which could be attributed to a better adaptation of this form to the anthropized environment where
the collections have been carried out. Conversely, the S-form
was the only one observed in our samples from northern
coastal sites (i.e., Cabinda and Zaire) and in the small samples
from humid inland sites (i.e., at Malanjie, Lunda Sul, and
Huambo Provinces). This may possibly reflect a better adaptation of the S-form to more humid and rural environments,
although the co-occurrence of both forms in the inland areas
is supported by our finding of one M-specimen in Cuanza
Norte and by the report of several M-specimens in Huambo
by Cuamba and others.6 Although these results are probably
affected by collection and/or season biases, the observed distribution of the two molecular forms is likely to reflect a
definite prevalence of one form over the other in different
eco-climatic conditions, which is consistent with previous observations in other west-African areas, where M-form predominates in urban or peri-urban settings, whereas the Sform is usually more abundant in rural environments.1,2,26
Both forms had Pf–CSP-positives, with non-significant differences between M- and S-form. The overall Pf–CSPpositivity (0.59%, SE ⳱ 0.34) was non-significantly different
from that reported by Cuamba and others (1.90%, SE ⳱
0.57),6 and within the range of the sporozoite rates reported
by Ribeiro & Ramos for “fresh-water An. gambiae” (presumably An. gambiae s.s.).5
The cytogenetic analysis of the Angolan An. gambiae s.s.
populations showed that both molecular forms are characterized by the same low degree of chromosomal polymorphism
based on the floating inversion 2La only, and also share similar frequencies of its inverted arrangement. Populations of
An. gambiae s.s. very similar to the Angolan ones for the same
inversion set-up have been recorded in sites generally characterized by a humid tropical climate, like in some West African
sites (southern Ghana,27 southern Cameroon,2 and Kinshasa
area, Democratic Republic of Congo [Petrarca & Coene, unpublished data]), and especially in extensive areas of East
Africa, namely eastern Mozambique,19 coastal Madagascar,28
and Comoro archipelago.29 Thus, the inversion set-up of the
Angolan samples is strictly comparable to that of the An.
gambiae s.s. populations from rain forest areas and humid
and derived savannas.1,30 However, it must be noted that,
although the central and northern areas of western Angola
actually lie in the humid tropical belt (where the S-form predominates), the coastal sites (Luanda and Cavaco) and particularly the southernmost collection locality (Namibe) lie in
comparatively more arid or even pre-desertic areas, where
the potential larval sites are almost exclusively associated
with, or even provided by, human activities, where the Mform thrives. In fact, the ability of the M-form to exploit
man-made larval breeding sites in dry areas has already been
shown in Mali and Burkina Faso,15,31,32 where Sudan-savanna
or Sahelian rice fields are almost exclusively colonized by this
form. In these regions the ability and the highly successful adaptation of the M-form to arid habitats have been associated with 2R inversion polymorphisms typical of this
form, such as 2Rbc/+ and 2Ru/+. On the contrary, the very
low degree of inversion polymorphism characterizing the
M-populations from the comparatively drier zones of Angola
suggests that the M-form local adaptation to man-made larval sites and arid conditions could be completely independent
from the 2R inversions. Moreover, these M-populations
174
CALZETTA AND OTHERS
also show unexpected relatively low frequencies of the inverted arrangement of the catholic 2La/+a polymorphism,
which is known to be associated with an adaptation to aridity.11,33,34
If these preliminary observations were confirmed on a
larger scale, it would open a new debate on the role of chromosomal inversions in the ecotypic adaptation to the ecological conditions in south dry areas and on the speciation process
within An. gambiae s.s.
CONCLUSION
In conclusion, our results provide new information on the
Anopheles gambiae complex in Angola, highlighting regional
peculiarities, such as the unexpected predominance of an almost chromosomally monomorphic M-form over An. arabiensis in dry/pre-desertic areas. However, knowledge of the
bionomics of the Angolan member species of the An. gambiae
complex and their genetic make-up still requires a significant strengthening. In particular, it would be of great interest:
i) to clarify how the M-form of An. gambiae is capable of
exploiting dry and pre-desertic areas without making use of
paracentric inversions as tools for ecotypic adaptation; ii) to
extend the temporal/spatial sampling activity, particularly
toward the inland and highland zones, to study the temporal/
spatial distribution boundaries of the species/forms1; iii) to
carry out a genetic characterization and comparison of the
S-populations of An. gambiae and of An. arabiensis from Angola with those from East Africa, with which they share
similarities (e.g., a generalized low degree of chromosomal polymorphism; presence of An. arabiensis on highlands);
and iv) to study with much higher detail the trophic behavior
and the abilities of the species/forms in transmitting Plasmodium.
Received March 12, 2007. Accepted for publication July 2, 2007.
Acknowledgments: Collections have been done in a framework of
collaboration among University of Rome “La Sapienza,” Italy, and
representatives of the Italian Ministry of Foreign Affairs and Angolan Ministry of Health. We thank André Francisco Sebastião, Mpova
Zambote, Alberto Bunga, and Manuel Alfredo Paulo (Instituto Nacional da Saúde Publica-Luanda) for technical assistance during the
field collections. We are especially grateful to Stefano Ferroni,
Project Manager of the “Programma di Cooperazione Socio-Sanitaria
AID 5810.” We thank Joao Pinto for commenting on the manuscript.
We thank Mr. Gianni Petrangeli and Mrs. Graziella Croce for laboratory support and Mario Coluzzi for advice and support.
Work was funded by Istituto Pasteur-Fondazione Cenci-Bolognetti,
Italian Ministry for University and Research (funds MIUR/PRIN),
University of Rome “La Sapienza” (Faculty funds), and by the
UNDP/World Bank/WHO Special Programme for Research and
Training in Tropical Diseases (TDR).
Authors’ addresses: Maria Calzetta, Federica Santolamazza, Gian
Carlo Carrara, Maria Angela Di Deco, Alessandra della Torre, and
Vincenzo Petrarca, Dipartimento di Scienze di Sanità Pubblica, Sezione
di Parassitologia, Università “La Sapienza,” Piazzale Aldo Moro, 5,
00185 Roma, Italy, Telephone: +39-06-4455780, Fax: +39-06-49914653,
E-mails: Maria.Calzetta@uniroma1.it, Federica.Santolamazza@
uniroma1.it, carbriga@alice.it, Mariaangela.Dideco@uniroma1.it,
Alessandra.dellatorre@uniroma1.it, and Vincenzo.Petrarca@
uniroma1.it. Filomeno Fortes and Pedro J. Cani, Ministério da Saúde–
Programa Nacional de Controle da Malária, Luanda, Angola.
Reprint requests: Alessandra della Torre, Dipartimento di Scienze di
Sanità Pubblica, Sezione di Parassitologia, Università “La Sapienza”,
Rome, Italy. E-mail: alessandra.dellatorre@uniroma1.it.
REFERENCES
1. della Torre A, Tu Z, Petrarca V, 2005. On the distribution and
genetic differentiation of Anopheles gambiae s.s. molecular
forms. Insect Biochem Mol Biol 35: 754–769.
2. Wondji C, Frederic S, Petrarca V, Etang J, Santolamazza F, della
Torre A, Fontenille D, 2005. Species and populations of the
Anopheles gambiae complex in Cameroon with special emphasis on chromosomal and molecular forms of Anopheles gambiae s.s. J Med Entomol 42: 998–1005.
3. Coluzzi M, Sabatini A, della Torre A, Di Deco MA, Petrarca V,
2002. A polytene chromosome analysis of the Anopheles gambiae species complex. Science 298: 1415–1418.
4. della Torre A, Costantini C, Besansky N, Caccone A, Petrarca V,
Powell JR, Coluzzi M, 2002. Speciation within Anopheles gambiae—the glass is half full. Science 298: 115–117.
5. Ribeiro H, Ramos Hd C, 1975. Research on the mosquitoes of
Angola. Garcia de Orta, Sér Zool 4: 1–40.
6. Cuamba N, Kwang SC, Towson H, 2006. Malaria vectors in Angola: distribution of species and molecular forms of the Anopheles gambiae complex, their pyrethroid insecticide knockdown resistance (kdr) status and Plasmodium falciparum
sporozoite rates. Malar J 5: 2.
7. Gillies MT, de Meillon B, 1968. The Anophelinae of Africa South
of the Sahara. Johannesburg: South African Institute for Medical Research.
8. Gillies MT, Coetzee M, 1987. A Supplement to the Anophelinae of
Africa South of the Sahara. Johannesburg: South African Institute for Medical Research.
9. Fanello C, Santolamazza F, della Torre A, 2002. Simultaneous
identification of species and forms of the Anopheles gambiae
complex by PCR-RFLP. Med Vet Entomol 16: 461–464.
10. della Torre A, 1997. Polytene chromosome preparation from
Anopheline mosquitoes. Crampton JM, Beard CB, Louis C,
eds. Molecular Biology of Insect Disease Vectors: A Methods
Manual. London: Chapman & Hall, 329–336.
11. Coluzzi M, Sabatini A, Petrarca V, Di Deco MA, 1979. Chromosomal differentiation and adaptation to human environments
in the Anopheles gambiae complex. Trans R Soc Trop Med
Hyg 73: 483–497.
12. Petrarca V, Nugud AD, Elkarim Ahmed MA, Haridi AM, Di
Deco MA, Coluzzi M, 2000. Cytogenetics of the Anopheles
gambiae complex in Sudan, with special reference to Anopheles arabiensis: relationships with East and West African populations. Med Vet Entomol 14: 149–164.
13. Beier JC, Perkins PV, Wirtz RA, Whitmire RA, Mugambi M,
Hockmeyer WT, 1987. Field evaluation of an enzyme-linked
immunoadsorbent assay (ELISA) for Plasmodium falciparum
sporozoite detection in Anopheline mosquitoes from Kenya.
Am J Trop Med Hyg 36: 459–468.
14. Beier JC, Perkins PV, Wirtz RA, Koros J, Diggs D, Gargan TP II,
Koech DK, 1988. Bloodmeal identification by direct enzymelinked immunoadsorbent assay (ELISA), tested on Anopheles
(Diptera: Culicidae) in Kenya. J Med Entomol 25: 9–16.
15. Touré YT, Petrarca V, Traoré SF, Coulibaly A, Maiga HM, Sankaré O, Sow M, Di Deco MA, Coluzzi M, 1998. Distribution
and inversion polymorphism of chromosomally recognized
taxa of the Anopheles gambiae complex in Mali, West Africa.
Parassitologia 40: 477–511.
16. Coetzee M, Craig MH, Le Sueur D, 2000. Mapping the distribution of members of the Anopheles gambiae complex in Africa
and adjacent islands. Parasitol Today 16: 74–77.
17. Mekuria Y, Petrarca V, Tasfamariam T, 1982. Cytogenetic
studies on the malaria vector mosquito Anopheles arabiensis
Patton in the AwashValley, Ethiopia. Parassitologia 24: 237–
243.
18. Ralisoa Randrianasolo BO, Coluzzi M, 1987. Genetical investigations of zoophilic and exophilic Anopheles arabiensis from
Antananarivo area (Madagascar). Parassitologia 29: 93–97.
19. Petrarca V, Carrara GC, Di Deco MA, Petrangeli G, 1984. Osservazioni citogenetiche e biometriche sui membri del complesso Anopheles gambiae in Mozambico. Parassitologia 26:
247–259.
20. Bryan JH, Petrarca V, Di Deco MA, Coluzzi M, 1987. Adult
behaviour of members of the Anopheles gambiae complex in
ANOPHELES GAMBIAE COMPLEX IN ANGOLA
21.
22.
23.
24.
25.
26.
27.
the Gambia with special reference to An. melas and its chromosomal variants. Parassitologia 29: 221–249.
Yawson AE, McCall PJ, Wilson MD, Donnelly MJ, 2004. Species
abundance and insecticide resistance of Anopheles gambiae in
selected areas of Ghana and Burkina Faso. Med Vet Entomol
18: 372–377.
Bafort JM, Petrarca V, 1983. Contribution to the knowledge of
Anopheles melas and An. gambiae in West Africa. Ann Soc
Belg Med Trop 63: 167–170.
Petrarca V, Carrara GC, Di Deco MA, Petrangeli G, 1983. Il
complesso Anopheles gambiae in Guinea Bissau. Parassitologia 25: 29–39.
Fonseca LF, Di Deco MA, Carrara GC, Dabo I, Do Rosario V,
Petrarca V, 1996. Anopheles gambiae complex near Bissau
City, Guinea Bissau, West Africa. J Med Entomol 33: 939–945.
Akogbeto M, Di Deco MA, 1995. Répartition des membres du
complexe Anopheles gambiae et de leurs variants chromosomiques au Bénin et au Togo, Afrique occidentale. J Afr Zool
109: 443–454.
Kristan M, Fleischmann H, della Torre A, Stich A, Curtis CF,
2003. Pyrethroid resistance/susceptibility and differential urban/rural distribution of Anopheles arabiensis and An. gambiae
s.s. malaria vectors in Nigeria and Ghana. Med Vet Entomol
17: 326–332.
Appawu MA, Baffoe Wilmont A, Afari EA, Nkrumah FK, Petrarca V, 1994. Species composition and inversion polymor-
28.
29.
30.
31.
32.
33.
34.
175
phism of the Anopheles gambiae complex in some sites of
Ghana, west Africa. Acta Trop 56: 15–23.
Petrarca V, Sabatinelli G, Touré YT, Di Deco MA, 1998. Morphometric multivariate analysis of field samples of adult
Anopheles arabiensis and Anopheles gambiae (Diptera: Culicidae). J Med Entomol 35: 16–25.
Petrarca V, Sabatinelli G, Di Deco MA, Papakay M, 1990. The
Anopheles gambiae complex in the Federal Islamic Republic of
Comoros (Indian Ocean): some cytogenetic and biometric
data. Parassitologia 32: 371–380.
Coluzzi M, Petrarca V, Di Deco MA, 1985. Chromosomal inversion intergradation and incipient speciation in Anopheles gambiae. Boll Zool 52: 45–63.
Petrarca V, Petrangeli G, Rossi P, Sabatinelli G, 1986. Etude
chromosomique d’Anopheles gambiae et Anopheles arabiensis
à Ouagadougou (Burkina Faso) et dans quelques villages voisins. Parassitologia 28: 41–61.
Robert V, Petrarca V, Carnevale P, Ovazza L, Coluzzi M, 1989.
Analyse cytogénétique du complexe Anopheles gambiae dans
la région de Bobo-Dioulasso (Burkina Faso). Ann Parasitol
Hum Comp 64: 290–311.
Rishikesh N, Di Deco MA, Petrarca V, Coluzzi M, 1985. Seasonal
variations in indoor resting Anopheles gambiae and Anopheles
arabiensis in Kaduna, Nigeria. Acta Trop 42: 165–170.
Coluzzi M, 1992. Malaria vector analysis and control. Parasitol
Today 8: 113–118.

Download Report