Colonization of Anopheles coustani, a neglected malaria vector in Madagascar.

Anopheles coustani has long been recognized as a secondary malaria vector in Africa. It has recently been involved in the transmission of both Plasmodium falciparum and P. vivax in Madagascar. As most secondary malaria vectors, An. coustani mainly bites outdoors, which renders the control of this mosquito species difficult using classical malaria control measures, such as the use of bed nets or indoor residual spraying of insecticides. For a better understanding of the biology and vector competence of a vector species, it is useful to rear the species in the laboratory. The absence of a colony hinders the assessment of the bionomics of a species and the development of adapted control strategies. Here, we report the first successful establishment of an An. coustani colony from mosquitoes collected in Madagascar. We used a forced copulation procedure as this mosquito species will not mate in cages. We describe our mosquito colonization procedure with detailed biological features concerning larval to adult development and survival, recorded over the first six critical generations. The procedure should be easily applicable to An. coustani from different African countries, facilitating local investigation of An. coustani vector competence and insecticide resistance using the colony as a reference.

Title: Colonisation d'Anopheles coustani, vecteur négligé du paludisme à Madagascar. Abstract: Anopheles coustani est reconnu depuis longtemps comme un vecteur secondaire du paludisme en Afrique. Il a récemment été impliqué dans la transmission de Plasmodium falciparum et de P. vivax à Madagascar. Comme la plupart des vecteurs secondaires du paludisme, An. coustani pique principalement à l'extérieur, ce qui rend difficile le contrôle de cette espèce de moustique par les mesures classiques de lutte contre le paludisme telles que l'utilisation de moustiquaires ou la pulvérisation intradomiciliaire d'insecticides à effet rémanent. Pour une meilleure compréhension de la biologie et de la compétence vectorielle d'une espèce vectrice, il est utile d'élever l'espèce en laboratoire. L'absence de colonie gêne l'évaluation de la bionomie d'une espèce et le développement de stratégies de contrôle adaptées. Nous rapportons ici le premier établissement réussi d'une colonie d' An. coustani issue de moustiques collectés à Madagascar. Nous avons utilisé une procédure de copulation forcée car cette espèce de moustique ne s'accouple pas en cage. Nous décrivons notre procédure de colonisation des moustiques avec des caractéristiques biologiques détaillées concernant le développement et la survie des stades larvaires aux adultes, enregistrées au cours des six premières générations critiques. La procédure devrait être facilement applicable aux An. coustani de différents pays africains, facilitant les enquêtes locales sur la compétence vectorielle d'An. coustani et sa résistance aux insecticides, en utilisant une colonie comme référence.

Differential contribution of Anopheles coustani and Anopheles arabiensis to the transmission of Plasmodium falciparum and Plasmodium vivax in two neighbouring villages of Madagascar.

BACKGROUND: Malaria is still a heavy public health concern in Madagascar. Few studies combining parasitology and entomology have been conducted despite the need for accurate information to design effective vector control measures. In a Malagasy region of moderate to intense transmission of both Plasmodium falciparum and P. vivax, parasitology and entomology have been combined to survey malaria transmission in two nearby villages. METHODS: Community-based surveys were conducted in the villages of Ambohitromby and Miarinarivo at three time points (T1, T2 and T3) during a single malaria transmission season. Human malaria prevalence was determined by rapid diagnostic tests (RDTs), microscopy and real-time PCR. Mosquitoes were collected by human landing catches and pyrethrum spray catches and the presence of Plasmodium sporozoites was assessed by TaqMan assay. RESULTS: Malaria prevalence was not significantly different between villages, with an average of 8.0% by RDT, 4.8% by microscopy and 11.9% by PCR. This was mainly due to P. falciparum and to a lesser extent to P. vivax. However, there was a significantly higher prevalence rate as determined by PCR at T2 ([Formula: see text] = 7.46, P = 0.025). Likewise, mosquitoes were significantly more abundant at T2 ([Formula: see text] = 64.8, P < 0.001), especially in Ambohitromby. At T1 and T3 mosquito abundance was higher in Miarinarivo than in Ambohitromby ([Formula: see text] = 14.92, P < 0.001). Of 1550 Anopheles mosquitoes tested, 28 (1.8%) were found carrying Plasmodium sporozoites. The entomological inoculation rate revealed that Anopheles coustani played a major contribution in malaria transmission in Miarinarivo, being responsible of 61.2 infective bites per human (ib/h) during the whole six months of the survey, whereas, it was An. arabiensis, with 36 ib/h, that played that role in Ambohitromby. CONCLUSIONS: Despite a similar malaria prevalence in two nearby villages, the entomological survey showed a different contribution of An. coustani and An. arabiensis to malaria transmission in each village. Importantly, the suspected secondary malaria vector An. coustani, was found playing the major role in malaria transmission in one village. This highlights the importance of combining parasitology and entomology surveys for better targeting local malaria vectors. Such study should contribute to the malaria pre-elimination goal established under the 2018-2022 National Malaria Strategic Plan.

Hemocyte-targeted gene expression in the female malaria mosquito using the hemolectin promoter from Drosophila.

Hemocytes, the immune cells in mosquitoes, participate in immune defenses against pathogens including malaria parasites. Mosquito hemocytes can also be infected by arthropod-borne viruses but the pro- or anti-viral nature of this interaction is unknown. Although there has been progress on hemocyte characterization during pathogen infection in mosquitoes, the specific contribution of hemocytes to immune responses and the hemocyte-specific functions of immune genes and pathways remain unresolved due to the lack of genetic tools to manipulate gene expression in these cells specifically. Here, we used the Gal4-UAS system to characterize the activity of the Drosophila hemocyte-specific hemolectin promoter in the adults of Anopheles gambiae, the malaria mosquito. We established an hml-Gal4 driver line that we further crossed to a fluorescent UAS responder line, and examined the expression pattern in the adult progeny driven by the hml promoter. We show that the hml regulatory region drives hemocyte-specific transgene expression in a subset of hemocytes, and that transgene expression is triggered after a blood meal. The hml promoter drives transgene expression in differentiating prohemocytes as well as in differentiated granulocytes. Analysis of different immune markers in hemocytes in which the hml promoter drives transgene expression revealed that this regulatory region could be used to study phagocytosis as well as melanization. Finally, the hml promoter drives transgene expression in hemocytes in which o'nyong-nyong virus replicates. Altogether, the Drosophila hml promoter constitutes a good tool to drive transgene expression in hemocyte only and to analyze the function of these cells and the genes they express during pathogen infection in Anopheles gambiae.

Evolution of sexually-transferred steroids and mating-induced phenotypes in Anopheles mosquitoes.

Human malaria, which remains a major public health problem, is transmitted by a subset of Anopheles mosquitoes belonging to only three out of eight subgenera: Anopheles, Cellia and Nyssorhynchus. Unlike almost every other insect species, males of some Anopheles species produce steroid hormones which are transferred to females during copulation to influence their reproduction. Steroids are consequently a potential target for malaria vector control. Here, we analysed the evolution of sexually-transferred steroids and their effects on female reproductive traits across Anopheles by using a set of 16 mosquito species (five Anopheles, eight Cellia, and three Nyssorhynchus), including malaria vector and non-vector species. We show that male steroid production and transfer are specific to the Cellia and therefore represent a synapomorphy of this subgenus. Furthermore, we show that mating-induced effects in females are variable across species and differences are not correlated with sexually-transferred steroids or with Anopheles ability to transmit human malaria. Overall, our findings highlight that Anopheles mosquitoes have evolved different reproductive strategies, independently of being a malaria vector or not.

Efficient ΦC31 integrase-mediated site-specific germline transformation of Anopheles gambiae.

Current transgenic methodology developed for mosquitoes has not been applied widely to the major malaria vector Anopheles gambiae, which has proved more difficult to genetically manipulate than other mosquito species and dipteran insects. In this protocol, we describe ΦC31-mediated site-specific integration of transgenes into the genome of A. gambiae. The ΦC31 system has many advantages over 'classical' transposon-mediated germline transformation systems, because it allows integration of large transgenes at specific, characterized genomic locations. Starting from a general protocol, we have optimized steps from embryo collection to co-injection of transgene-containing plasmid and in vitro-produced ΦC31 integrase mRNA. We also provide tips for screening transgenic larvae. The outlined procedure provides robust transformation in A. gambiae, resulting in homozygous transgenic lines in ∼2-3 months.