Coustani Group mosquito (Diptera: Culicidae) diversity and malaria transmission in the Democratic Republic of the Congo

(2025) 24:364 Hendershot et al. Malaria Journal https://doi.org/10.1186/s12936-025-05608-y Malaria Journal Open Access RESEARCH Coustani Group mosquito (Diptera: Culicidae) diversity and malaria transmission in the Democratic Republic of the Congo A. L. Hendershot1,9, F. T. Wat’senga2,10, T. Mampangulu2, O. Lodi3, I. T. Nsungu4, E. Z. Manzambi2, A. Okito3, P. Nzila2, M. A. Aubourg1, X. Yu1, J. Likwela5, C. Fornadel6,11, R. M. Oxborough7, N. F. Lobo1 and S. R. Irish8,12* Abstract Background The Democratic Republic of Congo is one of the countries with the heaviest burdens of malaria. To ensure that vector control tools, particularly insecticide-treated nets, remain effective as a prevention tool, entomological surveillance has been conducted throughout the country. In one location, early biting was noted by a majority of mosquitoes, morphologically identified as Anopheles paludis, a species in the Coustani Group, which had previously been found to transmit malaria in this country. The aim of this study was to better understand the Coustani Group species diversity, bionomics, and role in local malaria transmission in the DRC using both field and molecular approaches. Methods Human landing collections and pyrethrum spray catches were conducted in Lodja, Kapolowe, and on one occasion, in Kenge. The mosquitoes were morphologically identified and processed in the laboratory. Enzyme linked immunosorbent assays and PCRs were conducted to determine whether mosquitoes carried Plasmodium sporozoites, and ITS2 and CO1 sequencing was conducted to identify species. Results Anopheles paludis mosquitoes (as morphologically identified) were not found with sporozoites in Lodja, and only one specimen was found with sporozoites in Kapolowe. When the ITS2 and CO1 sequencing was conducted, there appeared to be two species groups in Lodja (where only An. paludis was identified morphologically) and four in Kapolowe (where An. paludis and Anopheles caliginosus were identified morphologically). There appeared to be differences between the species groups in terms of their biting times. Conclusions The Coustani Group mosquitoes tested in this study did not appear to be major vectors of malaria, due to the low levels of detection of sporozoites in mosquitoes collected in and around houses in DRC, which differs from some studies previously conducted in DRC. However, this conclusion is complicated by the detection of four species groups, which may have different biting behaviour. Further work is necessary to ensure correct identification of species in order to understand the contribution of these species to malaria in DRC. Keywords Anopheles paludis, Coustani Group, Democratic Republic of Congo, Lodja, Kapolowe *Correspondence: S. R. Irish Full list of author information is available at the end of the article © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. Hendershot et al. Malaria Journal (2025) 24:364 Background Malaria is a major cause of morbidity and mortality in the Democratic Republic of Congo (DRC), which has an estimated 13% of the world’s malaria cases and 11% of the malaria deaths [1]. Towards reducing malaria associated morbidity and mortality, the national programme has developed a strategy which emphasizes vector control in the form of continued distribution of long-lasting insecticidal nets (LLINs), the introduction of indoor residual spraying (IRS), and the promotion of larval source management. In order to ensure that these vector control tools are being implemented appropriately, alongside monitoring entomological impacts, entomological monitoring is conducted at several sites throughout DRC [2]. Anopheles gambiae sensu lato (s.l.) and Anopheles funestus are the primary vectors of malaria in the DRC, though several potential secondary vectors have been identified, including those within the Coustani Group [3–5]. The Coustani Group is composed of nine known species, of which Anopheles coustani, Anopheles ziemanni, Anopheles tenebrosus, Anopheles symesi, Anopheles paludis, and Anopheles caliginosus have been identified within the DRC [6]. Historically, members of the Coustani Group were considered of negligible importance to malaria transmission, given their propensity for exophilic, opportunistic biting and long gonotrophic cycle, but more recent evidence has shown some species to be anthropophilic, endophilic, and abundant relative to local primary vector species [7–9]. One such site is Lodja (Sankuru Province, DRC). In 2013 and 2014, limited human landing collections conducted in Lodja found high numbers of mosquitoes identified as An. paludis, often in greater numbers than An. gambiae s.l. [10, 11]. Additionally, in some cases the biting times for An. paludis showed a distinct early biting peak, with the majority of biting occurring outdoors in the early evenings between 1800 and 2200 h. A second site, Kapolowe (Haut Katanga Province, DRC) also had high numbers of An. paludis collected in 2014 [11]. Anopheles paludis has often been found with low sporozoite rates and has generally been considered a secondary vector [12–14]. However, the most recent work on An. paludis in DRC is that of Karch et al. [15], who found it to be an important vector in western DRC, with sporozoite rates of 6.2%. While this was based on only 97 specimens dissected, other reports of high sporozoite rates had also been reported from Gemena (Sud Ubangi Province, DRC) [15, 16]. As the reported peak of biting was prior to the time when LLINs might be used, and was primarily outdoors, further investigation was considered necessary to determine whether LLINs alone were a sufficient vector control intervention for malaria control in this part of the country. Page 2 of 14 Therefore, the objective of this work was to understand the Coustani Group species diversity, bionomics, and role in local malaria transmission in the DRC using both field and molecular approaches. Methods Study site Anopheles specimens were collected (...truncated)