Advances in the study of Anopheles funestus, a major vector of malaria in Africa

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1 Insect Biochemistry and Molecular Biology 34 (2004) Advances in the study of Anopheles funestus, a major vector of malaria in Africa M. Coetzee a,b,, D. Fontenille c a Vector Control Reference Unit, National Institute for Communicable Diseases, P.O. Box 1038, Johannesburg 2000, South Africa b Department of Clinical Microbiology and Infectious Diseases, School of Pathology of the National Health Laboratory Service, University of Witwatersrand, Johannesburg, South Africa c Laboratoire de Lutte Contre les Insectes Nuisibles, Institut de Recherche pour le Developpement (LIN-IRD), 911 Avenue Agropolis, BP 64501, Montpellier Cedex 5, France Received 8 March 2004; accepted 18 March 2004 Abstract The recent literature on cytogenetic and molecular studies of Anopheles funestus, a major vector of malaria in Africa, is reviewed. Molecular data from West and Central Africa suggest a new species in the group closely allied to Anopheles rivulorum. Cytogenetic and molecular studies of populations from West, Central, East and southern Africa indicate considerable genetic structuring within An. funestus itself, which may well restrict the spread of pyrethroid resistance that has been demonstrated in southern Africa. # 2004 Elsevier Ltd. All rights reserved. Keywords: Anopheles funestus; Anopheles rivulorum; Cytogenetics; Chromosomes; Microsatellites; Molecular; PCR; Insecticide resistance 1. Introduction Anopheles funestus is one of the two major vectors of malaria parasites in Africa, in some cases far outstripping An. gambiae in its ability to transmit Plasmodium falciparum (Gillies and De Meillon, 1968). In many places, An. funestus is the major vector responsible for malaria transmission and sometimes for malaria epidemics (Fontenille et al., 1990; Hargreaves et al., 2003). Parasite rates of 22% (De Meillon, 1933) and 27% (Swellengrebel et al., 1931) have been recorded in South Africa and more recently 11% in Tanzania (Shiff et al., 1995). In West Africa, rates of 2.6% and 3.3% have been observed in Senegal (Dia et al., 2003; Fontenille et al., 1997), between 2.8% and 14.6% in Burkina Faso (Costantini et al., 1999) and around 5% in Cameroon (Antonio-Nkondjio et al., 2002). In Burkina Faso, Costantini et al. (1999) Corresponding author. Vector Control Reference Unit, National Institute for Communicable Diseases, P.O. Box 1038, Johannesburg 2000, South Africa. Fax: address: maureen.coetzee@nhls.ac.za (M. Coetzee). recorded up to 50% of An. funestus in one village in November 1991 (n ¼ 56) positive for P. falciparum circumsporozoite protein. Despite its obvious importance as a vector, An. funestus has been sadly neglected for almost half a century, with most of the research focusing on members of the An. gambiae complex. Undoubtedly, this has been due to the adaptability of the An. gambiae complex to laboratory conditions and the ease with which species in the group can be colonized. In the last five years, however, molecular and cytogenetic studies on An. funestus and its allied species have become more prevalent in the literature. This paper reviews the progress to date and discusses the implications of the research findings. 2. The Anopheles funestus group An. funestus belongs to a group of nine species that are morphologically very similar in the adult stage. Four species, An. funestus, An. vaneedeni, An. parensis and An. aruni, have identical morphology at all life /$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi: /j.ibmb

2 600 M. Coetzee, D. Fontenille / Insect Biochemistry and Molecular Biology 34 (2004) stages and are known as the Funestus sub-group (Gillies and De Meillon, 1968; Gillies and Coetzee, 1987). Of the other species in the group, An. leesoni is the most distinct at both egg and larval stage, while An. confusus is easily identified on larval characteristics. Anopheles rivulorum and An. brucei also have distinctive larvae although these two species are virtually indistinguishable from each other. The ninth species, An. fuscivenosus, is known only from the adult stage and by chromosomal banding arrangements that distinguish it from the other members of the group (Gillies and De Meillon, 1968; Green, 1982). Distribution maps, reproduced from Gillies and De Meillon (1968), are given in Fig. 1. The morphological identification of members of the An. funestus group entails obtaining egg batches from wild females and rearing the progeny through to adults (apart from An. leesoni, whose eggs are distinct), so that fourth instar larvae and adults can be used in the identification process (Gillies and Coetzee, 1987). Laboratory rearing of larvae is both difficult and time-consuming, taking four weeks or more to obtain the necessary specimens, another reason perhaps for the Fig. 1. Distribution of Anopheles funestus group in Africa.

3 M. Coetzee, D. Fontenille / Insect Biochemistry and Molecular Biology 34 (2004) lack of attention paid to this group by researchers in Africa. As with other species complexes, the biology and vectorial capacity of the members of the An. funestus group are very different. Apart from An. funestus, which is highly anthropophilic, the rest of the group are mainly zoophilic. An. funestus is a very efficient vector of human Plasmodium throughout its distribution, while An. rivulorum has been implicated in transmission at one locality in Tanzania (Wilkes et al., 1996). Experimental transmission has been demonstrated in An. vaneedeni (De Meillon et al., 1977), but this species is not known to transmit malaria parasites in nature. Historical examples show that in order to conduct an efficient vector control program, precise identification of species is necessary to avoid misidentification of non-vector species as the vector An. funestus (Hargreaves et al., 2000, 2003). In South Africa and Tanzania, indoor spraying was implemented to eliminate An. funestus. However, some specimens remained, suggesting failure of the control program. Subsequent careful identification revealed that these mosquitoes were in fact An. parensis, An. rivulorum or An. vaneedeni, none of which are involved to any great extent in the transmission of human Plasmodium (Gillies and De Meillon, 1968; De Meillon et al., 1977). More recently, An. parensis was the most common member of the An. funestus group found resting inside human dwellings in a village in Kenya, but was not implicated in malaria transmission (Kamau et al., 2003a) Cytogenetics In 1980, the first paper appeared on the cytogenetics of the An. funestus group (Green and Hunt, 1980), and two years later, Green (1982) published chromosome maps for seven members of the group detailing fixed and polymorphic inversions occurring within and between the species. Cytogenetics too has its drawbacks for species identification, requiring considerable skill in interpreting the banding arrangements on the polytene chromosomes, and this technique was not applied widely to the An. funestus group until very recently. Investigations of an outbreak of malaria in a rural area of northern South Africa led to the discovery of an outdoor resting and feeding population of An. funestus that apparently was not involved in transmission (De Meillon et al., 1977). This population was designated Anopheles aruni? because of morphological similarities to this species, although An. aruni had only ever been recorded from Zanzibar and possibly one specimen from mainland Tanzania (Gillies and De Meillon, 1968). Cross-mating studies showed that An. funestus and An. aruni? were separate species based on sterile hybrid males and asynapsis of the polytene chromosomes (De Meillon et al., 1977; Green and Hunt, 1980). Chromosomal analysis revealed that these two species had homosequential banding arrangements and differed only by a single polymorphic inversion found in An. aruni? (Green and Hunt, 1980). An. aruni? was later named An. vaneedeni based on morphological differences found in the adult stage (Gillies and Coetzee, 1987). The chromosomes of An. parensis were also described in Green and Hunt (1980), differing from An. funestus/aruni? by one fixed inversion on arm 3. Green (1982) went on to publish chromosomal maps for other members of the group, An. rivulorum, An. confusus, An. fuscivenosus and An. leesoni, demonstrating that An. leesoni was genetically distinct and should not be included in the An. funestus group. He hypothesized that An. leesoni was more closely related to the oriental Anopheles minimus group and this has subsequently been supported by molecular studies (see below) Molecular Identification of the An. funestus group The first molecular studies of the An. funestus group were published by Koekemoer et al. (1998), who showed that restriction enzymes could be used to distinguish between the morphologically similar and chromosomally homosequential species An. funestus and An. vaneedeni. Using PCR primers for the D3 region in the 28S ribosomal gene, amplified products digested with HpaII restriction endonuclease produced distinct fragments on an agarose gel, giving easy differentiation of the two species (Koekemoer et al., 1998). This study was followed by single-strand conformation polymorphism analysis for identification of four members of the An. funestus group (Koekemoer et al., 1999) where An. funestus, An. rivulorum, An. leesoni and An. vaneedeni/parensis could be easily separated using polyacrylamide gel electrophoresis. This technique has the disadvantage of overlap of banding patterns for An. vaneedeni and An. parensis, is somewhat more difficult to perform than agarose gel methods and is more expensive using polyacrylamide gels. While rdna internal transcribed spacer (ITS2) sequences have provided valuable techniques for identification of individual species (Koekemoer et al., 1999; 2002a), what is really needed in a control program is a multiplex system that can identify all potential members of the group, such as that developed by Scott et al. (1993) for the An. gambiae complex. A cocktail polymerase chain reaction assay to identify five members of the An. funestus group was developed by Koekemoer et al. (2002b) and has been used to good effect in many subsequent studies. Evaluation of the multiplex PCR method for identification of five members of the An. funestus group (Koekemoer et al., 2002b) did not produce species distribution records that were different from those

4 602 M. Coetzee, D. Fontenille / Insect Biochemistry and Molecular Biology 34 (2004) presented in Gillies and De Meillon (1968), except for the presence of An. parensis in Ethiopia (Weeto et al., 2004). Interestingly, South Africa is the only country where more than two members of the group were found. All five species were found in sympatry at one locality in northern Kwazulu/Natal (Weeto et al., 2004), four of these being collected inside pyrethroid sprayed houses near the Mozambique border (Hargreaves et al., 2000). Studies in West Africa on the An. funestus group using ITS2 sequence differences revealed that An. rivulorum from West Africa is not the same as An. rivulorum from East or southern Africa (Hackett et al., 2000). Recent studies in Cameroon have confirmed these observations (Cohuet et al., 2003), suggesting that the taxon An. rivulorum comprises more than one species. The role of the new species, provisionally called An. rivulorum-like, in malaria transmission remains to be determined. 3. Anopheles funestus sensu stricto 3.1. Cytogenetic studies Following Green and Hunt (1980), there were no further publications on the cytogenetics of An. funestus for 12 years, until Boccolini et al. (1994) and then Costantini et al. (1999) documented chromosomal and bionomic heterogeneities within An. funestus populations in Burkina Faso. Cytogenetic studies conducted in several African countries and in Madagascar have shown the presence of at least 11 paracentric chromosomal inversions on chromosomes II and III (Green and Hunt, 1980; Boccolini et al., 1994, 2002; Lochouarn et al., 1998; Dia et al., 2000; Kamau et al., 2002, 2003b). Chromosomal maps are presented by Sharakhov et al. (2001b, 2002) using arm nomenclature different from the original map produced by Green and Hunt (1980). The first study (Sharakhovet al., 2001b) gives inversion breakpoints and compares the linear and spatial organization of banding arrangements of An. funestus with An. gambiae. In situ hybridization of a sub-telomeric satellite DNA fragment of An. gambiae hybridized to the chromosomes of An. funestus showed, not surprisingly, marked species-specific differences. The second study (Sharakhovet al., 2002) mapped An. funestus cdnas to An. gambiae chromosomes and found substantial shuffling of gene order along corresponding chromosome arms. At least 70 chromosomal inversions were found to be fixed between the two species, the highest rate of rearrangement of any eukaryotes studied to date. In Senegal, An. funestus populations with different chromosomal arrangements showed different anthropophilic rates (Lochouarn et al., 1998), and in Burkina Faso, specimens with inverted karyotypes correlated with biological characteristics such as vectorial capacity, and were found in higher frequencies in indoor, human-fed samples (Costantini et al., 1999). Inversions, therefore, at least in West Africa, could be valuable markers of vector ability, because carriers of different chromosomal arrangements could be more or less prone to becoming infective. Hardy Weinberg disequilibrium and linkage disequilibrium between inversions observed in populations from Burkina Faso led Costantini et al. (1999) to describe two chromosomal forms that they called Kiribina and Folonzo, based on the presence and association of paracentric inversions. The strong lack of hybrid heterokaryotypes in areas where both forms are present led these authors to hypothesize incipient speciation within An. funestus. In Senegal, three chromosomal populations were recognized. In the village of Kouvar, two of these forms were sympatric, and the observed strong deficit of heterokaryotypes suggests, as in Burkina Faso, the presence of two genetically distinct populations (Dia et al., 2000). In Cameroon, northern populations are related to the Kiribina form, and in the south to the Folonzo form. A cline of inversion frequencies is observed among six locations from the humid forest in the south to the dry savannas in the north, with strong heterozygote deficiency in the Tibati village situated in between north and south, in an area where both forms occur. All these data suggest restricted gene flow between chromosomal forms of An. funestus (Cohuet et al., in press). Analysis of the population structure of Kenyan An. funestus suggested limited gene flow between coastal and western samples, 700 km apart, with chromosomal inversion polymorphisms differing significantly between the two populations (Kamau et al., 2002). Interpretation of allopatric data is more difficult because genetic drift, founder effect and selection pressure may all play a role in contributing to the observed discontinuities. Inferences of specific status are therefore difficult to draw in these circumstances. Subsequent analysis of samples from 10 sites in Kenya (Kamau et al., 2003b) suggests that the Rift Valley acts as a barrier to gene flow between subpopulations of An. funestus. Observations of sympatric populations from Kenya (Kamau et al., 2002), Angola (Boccolini et al., 2002) and Madagascar (Le Goff et al., unpublished data) revealed no evidence for reproductive isolation based on chromosomal polymorphisms, with heterokaryotypes observed at their expected frequencies in these populations. These data may suggest that population genetic structuring in West Africa could be different to that of East Africa.

5 M. Coetzee, D. Fontenille / Insect Biochemistry and Molecular Biology 34 (2004) Microsatellite and other DNA data The recent development and use of microsatellite markers (Sinkins et al., 2000; Sharakhovet al., 2001a; Cohuet et al., 2002), which are supposed to be neutral, allowed a re-analysis of the cytogenetic data and the incipient speciation hypothesis. Studies were conducted in Senegal and in Cameroon using a set of nine microsatellite markers spread over the entire genome of An. funestus. Four and 10 different geographical populations have been sampled in these two countries, respectively. Results showed that gene flow existed between populations within each country as well as gene exchanges between chromosomal forms. No evidence for population subdivision was observed in populations where strong deficits in heterokaryotypes were obtained. Isolation by distance between geographical populations was nonetheless detected, confirming the ability of microsatellite markers to detect population subdivision (Cohuet et al., unpublished data). Similar results were obtained in Kenya when comparing two populations from the coast and two population from western Kenya. Most of the differentiation observed seemed to be due to geographic distance (Braginets et al., 2003). Temu et al. (2004) used microsatellite DNA markers to study the population structure of An. funestus from Kenya, Uganda, Malawi, Mozambique and South Africa, showing discontinuities between southern African and East African populations. Samples from Malawi were different to both southern and East African samples. The apparent restricted gene flow shown in the above studies may simply indicate that dispersal of An. funestus adults is not widespread as reported in many early studies (Gillies and De Meillon, 1968). Or it may indicate the existence of discrete, non-mating populations that could be regarded as separate species. The latter is difficult to prove in allopatric populations, but the work of Costantini et al. (1999) and others have shown a lack of gene flow between sympatric populations of An. funestus based on chromosome inversion polymorphisms. Microsatellite data do not show the same genetic discontinuities that chromosomal inversion data do. These results suggest that heterozygote deficits at chromosomal loci are possibly locus-specific and partly dependent on environmental selection of the inversions themselves (or the genes they contain) rather than population subdivision or incipient speciation. On the other hand, it may be due to the limited number of microsatellite markers used in such studies. Molecular analysis of five members of the An. funestus group using restriction fragment length polymorphisms (RFLP) has demonstrated not only unique differences between the species, but population differences within An. funestus from East, West and southern Africa (Garros et al., 2004). Comparison of the ITS2 and D3 fragments of the rdna sequence revealed that An. leesoni and An. rivulorum exhibited a higher similarity to the Asian An. minimus group, and especially species A and C of the Minimus complex (85% and 91%), than to the An. funestus group. Intraspecific variation within An. funestus populations was found using PCR-RFLP of the D3 fragment, differentiating three clusters from West/Central, East and southern Africa, respectively Insecticide resistance The possibility of resistance to insecticides used in malaria control was first suggested in An. funestus from Nigeria, where a population in the north was not eliminated by three years of continuous spraying with DDT, BHC and dieldrin (Bruce-Chwatt et al. in Service, 1960). In Mali, Toure (1982) showed resistance to DDT, dieldrin and malathion. In the review by Brown (1986), An. funestus is recorded as being resistant to dieldrin in an additional four countries (Ghana, Benin, Cameroon and Kenya), but no references are provided. No further records exist until Hargreaves et al. (2000) reported resistance to pyrethroids in South Africa. The DDT house-spraying for vector control, initiated in the early 1950s, had effectively eradicated An. funestus from South Africa. In 1996, a policy decision was taken to move away from DDT and use pyrethroid insecticides instead for house spraying. The malaria control program authorities were not to know that pyrethroid resistance had developed in the An. funestus populations across the border in southern Mozambique, and that the return of this species to South Africa was to cause the biggest malaria epidemic in South Africa in over 50 years (Hargreaves et al., 2000, 2003). At the peak of the epidemic in 2000, the SSCP molecular identification method for members of the An. funestus group had just been published (Koekemoer et al., 1999) and established as a routine method in the laboratory. This enabled the South Africans to identify the vector species and to demonstrate that while four members of the group could be collected in window exit traps, only An. funestus was involved in transmitting P. falciparum (Hargreaves et al., 2000). Subsequently, spraying of traditional houses with DDT and Western-style houses with pyrethroids, producing a mosaic effect, eliminated An. funestus and reduced the number of malaria cases by 75% over two years (Hargreaves et al., 2003). The above underlines the critical need for species identification of the vector involved in transmission. It also highlights the need for gene flow studies to determine the likelihood of the genes for insecticide resistance spreading beyond the study areas.

6 604 M. Coetzee, D. Fontenille / Insect Biochemistry and Molecular Biology 34 (2004) Molecular and biochemical studies have shown that the resistance mechanism in An. funestus is metabolic and mediated by the mono-oxygenases (Brooke et al., 2001). There is also cross-resistance to the carbamate insecticide propoxur, which further limits the number of insecticides available for malaria vector control. 4. Conclusions The information being generated by the renewed interest in the An. funestus group indicates that this group may be as complex and problematic as the An. gambiae group. The existence of a species complex within An. funestus s.s. will not be surprising considering that almost every anopheline taxon studied so far has exhibited this phenomenon (e.g. An. maculipennis, Swellengrebel and De Buck, 1938; An. punctulatus, Bryan, 1970; An. pharoensis, Miles et al.,1983; An. pseudopunctipennis, Coetzee et al., 1999; An. marshallii, Lambert and Coetzee, 1982; An. nili, Kengne et al., 2003). Direct observations of vector mosquito mating behaviour in nature is hampered by their preference for operating in the dark, and their small physical size makes it difficult to see them at night. Molecular technology can provide alternative, indirect methods for studying aspects such as gene flow between subpopulations of the same species, and lack of exchange of genetic material between sympatric, non-panmictic populations. 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