Urban malaria in sub-Saharan Africa: dynamic of the vectorial system and the entomological inoculation rate.

Sub-Saharan Africa is registering one of the highest urban population growth across the world. It is estimated that over 75% of the population in this region will be living in urban settings by 2050. However, it is not known how this rapid urbanization will affect vector populations and disease transmission. The present study summarizes findings from studies conducted in urban settings between the 1970s and 2020 to assess the effects of urbanization on the entomological inoculation rate pattern and anopheline species distribution. Different online databases such as PubMed, ResearchGate, Google Scholar, Google were screened. A total of 90 publications were selected out of 1527. Besides, over 200 additional publications were consulted to collate information on anopheline breeding habitats and species distribution in urban settings. The study confirms high malaria transmission in rural compared to urban settings. The study also suggests that there had been an increase in malaria transmission in most cities after 2003, which could also be associated with an increase in sampling, resources and reporting. Species of the Anopheles gambiae complex were the predominant vectors in most urban settings. Anopheline larvae were reported to have adapted to different aquatic habitats. The study provides updated information on the distribution of the vector population and the dynamic of malaria transmission in urban settings. The study also highlights the need for implementing integrated control strategies in urban settings.

Polymorphic chromosomal inversions in Anopheles moucheti, a major malaria vector in Central Africa.

Anopheles moucheti Evans (Diptera: Culicidae) is a major vector of malaria in forested areas of Central Africa. However, few genetic tools are available for this species. The present study represents the first attempt to characterize chromosomes in An. moucheti females collected in Cameroon. Ovarian nurse cells contained polytene chromosomes, which were suitable for standard cytogenetic applications. The presence of three polymorphic chromosomal inversions in An. moucheti was revealed. Two of these inversions were located on the 2R chromosome arm. The homology between the 2R chromosome arms of An. moucheti and Anopheles gambiae Giles was established by fluorescent in situ hybridization of six An. gambiae genic sequences. Mapping of the probes on chromosomes of An. moucheti detected substantial gene order reshuffling between the two species. The presence of polytene chromosomes and polymorphic inversions in An. moucheti provides a new basis for further population genetic, taxonomic and ecological studies of this neglected malaria vector.

Optimization of breeding output for larval stage of Anopheles gambiae (Diptera: Culicidae): prospects for the creation and maintenance of laboratory colony from wild isolates.

Domesticating anopheline species from wild isolates provides an important laboratory tool but requires detailed knowledge of their natural biology and ecology, especially the natural breeding habitats of immature stages. The aim of this study was to determine the optimal values of some parameters of Anopheles gambiae larval development, so as to design a standard rearing protocol of highland isolates, which would ensure: the biggest fourth instars, the highest pupae productivity, the shortest duration of the larval stage and the best synchronization of pupation. The density of larvae, the size of breeding water and the quantity of food supplied were tested for their effect on larval growth. Moreover, three cheap foodstuffs were selected and tested for their capability to improve the breeding yield versus TetraMin® as the standard control. The larval density was a very sensitive parameter. Its optimal value, which was found to be ≈1 cm-2 surface area, yielded a daily pupation peak of 38.7% on day 8 post-oviposition, and a global pupae productivity of 78.7% over a duration range of three days. Anopheles gambiae's larval growth, survival and developmental synchronization were density-dependent, and this species responded to overcrowding by producing smaller fourth instars and fewer pupae, over elongated immature lifetime and duration range of pupae occurrence, as a consequence of intraspecific competition. While shallow breeding waters (<3 cm) produced a higher number of pupae than deeper ones, no effect of the breeding habitat's absolute surface area on larval development was observed. Increasing the daily food supply improved the pupae productivity but also boosted the water pollution level (which was assessed by the biological oxygen demand (BOD) and the chemical oxygen demand (COD)) up to a limit depending on the food quality, above which a rapid increase in larval mortality was recorded. The food quality that could substitute the manufactured baby fish food was obtained with weighed mixture of 1 wheat+1 shrimp+2 fish. On establishing an anopheline mosquito colony in the laboratory, special care should be taken to design and maintain the appropriate optimal values of larval density, water depth, daily diet quantity and nutritional quality.

La presence des bovins comme hotes alternatifs peut-elle modifier le comportement trophique des vecteurs du paludisme en zone de foret?

Des enquetes entomologiques longitudinales ont ete effectuees de novembre 1998 a novembre 2000 en vue de determiner le comportement trophique des anopheles en presence de bovins comme hotes alternatifs a l`homme dans deux localites de la zone de foret du sud Cameroun; Obala et Nkolbisson. Les collectes des moustiques ont eu lieu a l`interieur des habitations par pulverisation intra domiciliaire et a l`exterieur a l`aide de captures sous moustiquaire piege avec comme appats l`homme et le veau. L`origine du repas de sang et l`infectivite des moustiques ont ete identifiees par la technique ELISA (Enzyme Linked Immunosorbent Asay). Au total; 992 anopheles appartenant a 8 especes ont ete collectees au cours de cette etude. An. funestus etait l`espece la plus abondante a Obala tandis qu`An. nili predominait a Nkolbisson. Le taux d`anthropophilie etait plus eleve chez les moustiques collectes a l`interieur des habitations que chez ceux collectes a l`exterieur sous moustiquaire piege. An. gambiae et An. funestus ont montre une nette preference pour l`homme tandis qu`An. nili a pique indifferemment l`homme et le boeuf. An. gambiae etait de loin le vecteur principal dans les deux sites d`etude avec 24 cas d`infections sur les 42 trouves. Ces resultats montrent que les vecteurs en zone de foret peuvent diversifier la source de leur repas de sang en presence d`hotes alternatifs tels que les bovins. Toutefois; il serait important de comprendre si l`introduction de l`elevage de bovins dans cette region pourrait influencer le cycle de transmission du paludisme

Epidemiological Importance of the Anopheles Nili Group of Malaria Vectors in Equatorial Villages of Cameroon; Central Africa

From March 1999 to March 2007; entomological surveys were conducted in eight riverside locations of the equatorial domain in Cameroon; to assess the contribution of Anopheles nili group to malaria transmission. Results showed that the anthropophilic Anopheles nili; Anopheles carnevalei and Anopheles ovengensis were involved in 5-68of the Plasmodium falciparum entomological inoculation rates (EIRs) in all the study sites. Alongside; vectors such as Anopheles Gambiae; Anopheles moucheti; Anopheles funestus; Anopheles marshallii and Anopheles hancocki also took a substantial part to these transmission rates in sympatric conditions. Anopheles nili s.s. was the major vector in villages situated near the Sanaga river; with more than 100 infective bites/person/year. Its true Plasmodium falciparum infections rates ranged from 1.4to 6.1. Anopheles carnevalei and Anopheles ovengensis were mainly distributed in the equatorial cross-bordering areas; where they contributed for less than 30of the Plasmodium falciparum EIRs in villages located along the Nten river and Congo river networks. These findings confirm Anopheles nili s.s. as a major malaria vector mosquito; and also provided more data regarding the epidemiological role of Anopheles carnevalei and Anopheles ovengensis througout the central African region

Field Evaluation of Three Commercial Repellents Formulations against Afrotropical Vectors Mosquitoes in Camerooun

Three synthetic commercial insert repellents; Buzz-Off TM (30DEET); Cinq-sur-CinqTM (25IR3535 + essential oils) and prebutix TM(25IR3535) were tested in field conditions for their efficacy and persistence against mosquitoes in the forest area of Cameroon. During 48-days; 7; 569 mosquitoes belonging to four genera were collected: Mansonia spp (67.3); Anopheles spp (27.4); Aedes spp(3.8); and Culex spp (1.5). At the end of the 8-hours exposure period; the DEET-based repellent; Buzz Off TM produced the highest protection against Aedes spp; Culex spp and Mansonia spp. For Anophelines; results were more variable : PrebutixTM showed better protection against An. mouchetti; Cinq-sur-CinqTM was more efficacious against An. Funestus and An.ziemani; whereas Buzz-OffTM appeared more effective against An. Gambiae s.s. and An. nili. Efficacy and persistence parameters were estimated only for An. moucheti and Mansonia spp. The effective dosages (ED50 and ED95) as well as the effective half-life obtained with the DEET-based repellent were highly variable among replicates in the case of An. moucheti. For mansonia spp; the estimated EDO value for the DEET -based repellent was = 0.06mg/cm2. For the two IR3535-based repellents; the ED50 values varied from 0.06 to 0.10 mg/cm2; and 0.15 to 0.20 mg/cm2 for An. moucheti and Mansonia spp; respectively. Globally; the ED95 values values of the three repellents were around 1mg/cm2 except that of Cinq-sur-CinqTM which was ? 0.3mg/cm2 in the case of An. moucheti. The estimated effective half-lifes of the three repellents were approximately between 3 and 5h. Our results highlight the heterogeneity in the response of different mosquito species when exposed to the tested insect repellents; showing the relevance of evaluating efficacy and persistence profiles of different formulations in specific environmental contexts

Molecular differentiation of three closely related members of the mosquito species complex, Anopheles moucheti, by mitochondrial and ribosomal DNA polymorphism.

Distinction between members of the equatorial Africa malaria vector Anopheles moucheti (Evans) s.l. (Diptera: Culicidae) has been based mainly on doubtful morphological features. To determine the level of genetic differentiation between the three morphological forms of this complex, we investigated molecular polymorphism in the gene encoding for mitochondrial cytochrome oxidase b (CytB) and in the ribosomal internal transcribed spacers (ITS1 and ITS2). The three genomic regions revealed sequence differences between the three morphological forms similar in degree to the differences shown previously for members of other anopheline species groups or complexes (genetic distance d = 0.047-0.05 for CytB, 0.084-0.166 for ITS1 and 0.03-0.05 for ITS2). Using sequence variation in the ITS1 region, we set up a diagnostic polymerase chain reaction (PCR) for rapid and reliable identification of each subspecies within the An. moucheti complex. Specimens of An. moucheti s.l. collected in Cameroon, the Democratic Republic of Congo (DRC), Uganda and Nigeria were successfully identified, demonstrating the general applicability of this technique.

Insertion polymorphism of transposable elements and population structure of Anopheles gambiae M and S molecular forms in Cameroon.

The insertion polymorphism of five transposable element (TE) families was studied by Southern blots in several populations of the M and S molecular forms of the mosquito Anopheles gambiae sensu stricto from southern Cameroon. We showed that the mean TE insertion site number and the within-population insertion site polymorphism globally differed between the M and S molecular forms. The comparison of the TE insertion profiles of the populations revealed a significant differentiation between these two molecular forms (0.163 < Phi(ST) < 0.371). We cloned several insertions of a non-LTR retrotransposon (Aara8) that were fixed in one form and absent in the other one. The only insertion that could be clearly located on a chromosome arm mapped to cytological division 6 of chromosome X, confirming the importance of this region in the ongoing speciation between the M and S molecular forms.

[Malaria vectors: from the field to genetics. Research in Africa]. / Vecteurs de paludisme: du terrain à la génétique moléculaire. Recherches en Afrique.

Only about 60 Anopheline species transmit malaria among more than 3,000 mosquito species recorded in the world. In Africa, the major vectors are Anopheles gambiae,An. arabiensis, An. funestus, An. nili and An. moucheti. They all belong to species complexes or groups of closely related species that are very difficult to set apart on morphological grounds, but which may have highly variable behaviours and vectorial capacities. Understanding this complexity is of major importance in vector control programs or for implementing any public health intervention program such as drugs or vaccine trials. Among the seven species of the complex,Anopheles gambiaes.s. shows a huge chromosomal polymorphism related to adaptation to specific natural or anthropic environments, from equatorial forested Africa to dry sahelian areas. Recent studies conducted in West and Central Africa suggest an incipient speciation into 2 molecular forms provisionally called M and S. A similar evolutionary phenomenon is observed in An. funestus, in which sympatric populations carrying specific chromosomal paracentric inversions showed restricted gene flow. Distribution of species from An. nili group and An. moucheti complex is restricted to more humid regions of Africa. However in some areas these species play the major role in malaria transmission. Comprehensive knowledge of transmission cycles and of behavioural and underlying genetic heterogeneities that exist within and among natural vector populations will thus benefit the whole area of malaria control and epidemiology. Molecular and genetic studies, as well as in depth monitoring of vector biology, have been recently facilitated by advances in functional and comparative genomics, including recent publication of the nearly complete genome sequence of An. gambiae. Challenge for the next years is to answer to the very simple question: why is an insect a vector?

Description and bionomics of Anopheles (Cellia) ovengensis (Diptera: Culicidae), a new malaria vector species of the Anopheles nili group from south Cameroon.

Mosquito species of the Anopheles nili group (Diptera: Culicidae) transmit malaria to humans along rivers in Africa. To date, the An. nili group includes the species Anopheles nili s.s. and its pale-winged variant known as the "Congo form," Anopheles somalicus and Anopheles carnevalei. Larval and adult mosquito collections in the forest region of Campo, in southern Cameroon, uncovered an additional morphological variant provisionally called "Oveng form" that was subsequently found to be genetically distinct from the other members of the An. nili group. In this study, we provide further biological data that characterizes this new taxon and justifies elevation to specific rank. We propose calling this new species Anopheles ovengensis, after its geographical origin. We present a morphological description of the adult female and fourth instars and original data on the biology, ecology, and role as a human malaria vector of this new species in its type location. We provide dichotomous keys for identification of adult females and fourth instars that can be used at least in tropical areas of west and central Africa.

[Development of a PCR (polymerase chain reaction) specific for complex Anopheles nili (Theobald) 1904 species in Cameroon ]. / Développement d'une PCR (polymerase chain reaction) spécifique d'espèces du complexe Anopheles nili (Théobald) 1904 au Cameroun.

The objective of this study was to develop new molecular tools for the identification of members of An. nili group, a malaria vector in Africa. Our strategy was based on the sequence analysis of portions of the rDNA. The ITS2 fragment of An. nili collected in Cameroon was sequenced and compared. The analysis of these sequences has revealed a great variability of ITS2 sequence. Three molecular forms: An. nili typical form, An. nili Oveng form and An. carnevalei were observed within the six morphological types. Specific primers were selected on ITS2 sequence to develop an allele-specific PCR giving 3 size bands. 169 specimens of An. nili collected in Cameroon were successfully tested. This method has been validated on specimens collected in others localities of tropical Africa. The multiplex PCR developed was very sensitive practical and applicable on large scale.

[Systematics and biology of Anopheles vectors of Plasmodium in Africa, recent data]. / Systématique et biologie des anophèles vecteurs de Plasmodium en Afrique, données récentes.

Renewed interest in research on Plasmodium vectors in Africa and development of genetic and molecular biology techniques has been spearheaded by the WHO and the PAL+ program of the French research ministry. New findings have led to a better understanding of the systematics and biology of the main vector groups. The purpose of this article is to describe the newest data on the Anopheles gambiae complex and the M and S forms of An. gambiae s.s., on species in the An. funestus group and genetic polymorphism of An. funestus, on the two probable species in the An. moucheti complex, and on An. mascarenesis.

Molecular identification of the Anopheles nili group of African malaria vectors.

Distinction between members of the Anopheles nili group of mosquitoes (Diptera: Culicidae), including major malaria vectors in riverside villages of tropical Africa, has been based mainly on doubtful morphological characters. Sequence variations of the ribosomal DNA second internal transcribed spacer (ITS2) and D3 28S region between morphological forms revealed four genetic patterns corresponding to typical An. nili (Theobald), An. carnevalei Brunhes et al., An. somalicus Rivola & Holstein and the newly identified variant provisionally named Oveng form. Primers were designed based on ITS2 fixed nucleotide differences between haplotypes to develop a multiplex PCR for rapid and specific identification of each species or molecular form. Specimens of the An. nili group from Cameroon, Burkina Faso, Ivory Coast and Senegal were successfully identified to species, demonstrating the general applicability of this technique based on criteria described in this paper.

Chromosomal inversion polymorphism of Anopheles funestus from forest villages of South Cameroon.

The polymorphism of paracentric inversions of Anopheles funestus polytene chromosomes was studied in three villages (Nkoteng, Obala, and Simbock) located in a forest area of South Cameroon in order to analyse the genetic structure of these populations. A total of 146-210 chromatids could be scored from specimens collected over about two years. A low degree of chromosomal polymorphism was observed with two floating inversions on chromosomal arm 2 (2h and 2d), and three fixed arrangements on arms 3 (3a and 3b), and 5 (5a). Such arrangement of inversions has never been recorded elsewhere so far. The chromosome analysis indicated that the population from Obala was in Hardy-Weinberg equilibrium, whereas the samples from Nkoteng and Simbock showed a significant excess and deficit of heterokaryotypes, respectively. Significant differences in inversion frequencies on chromosomal arm 2 among villages lying in contrasting eco-climatic settings suggested an adaptive role of these inversions.