Ogola et al. Parasites & Vectors (2018) 11:577 https://doi.org/10.1186/s13071-018-3171-3 RESEARCH Open Access Insights into malaria transmission among Anopheles funestus mosquitoes, Kenya Edwin O. Ogola1, Ulrike Fillinger1, Isabella M. Ondiba2, Jandouwe Villinger1, Daniel K. Masiga1, Baldwyn Torto1 and David P. Tchouassi1* Abstract Background: Most malaria vectors belong to species complexes. Sibling species often exhibit divergent behaviors dictating the measures that can be deployed effectively in their control. Despite the importance of the Anopheles funestus complex in malaria transmission in sub-Saharan Africa, sibling species have rarely been identified in the past and their vectoring potential remains understudied. Methods: We analyzed 1149 wild-caught An. funestus (senso lato) specimens from 21 sites in Kenya, covering the major malaria endemic areas including western, central and coastal areas. Indoor and outdoor collection tools were used targeting host-seeking and resting mosquitoes. The identity of sibling species, infection with malaria Plasmodium parasites, and the host blood meal sources of engorged specimens were analyzed using PCR-based and sequencing methods. Results: The most abundant sibling species collected in all study sites were Anopheles funestus (59.8%) and Anopheles rivulorum (32.4%) among the 1062 successfully amplified specimens of the An. funestus complex. Proportionally, An. funestus dominated in indoor collections whilst An. rivulorum dominated in outdoor collections. Other species identified were Anopheles leesoni (4.6%), Anopheles parensis (2.4%), Anopheles vaneedeni (0.1%) and for the first time in Kenya, Anopheles longipalpis C (0.7%). Anopheles funestus had an overall Plasmodium infection rate of 9.7% (62/636), predominantly Plasmodium falciparum (59), with two infected with Plasmodium ovale and one with Plasmodium malariae. There was no difference in the infection rate between indoor and outdoor collections. Out of 344 An. rivulorum, only one carried P. falciparum. We also detected P. falciparum infection in two non-blood- fed An. longipalpis C (2/7) which is the first record for this species in Kenya. The mean human blood indices for An. funestus and An. rivulorum were 68% (93/136) and 64% (45/70), respectively, with feeding tendencies on a broad host range including humans and domestic animals such as cow, goat, sheep, chicken and pig. Conclusions: Our findings underscore the importance of active surveillance through application of molecular approaches to unravel novel parasite-vector associations possibly contributed by cryptic species with important implications for effective malaria control and elimination. Keywords: Anopheles funestus group, Anopheles longipalpis C, Malaria parasite transmission, Molecular approaches, Entomological surveillance, Kenya * Correspondence: [email protected] 1International Centre of Insect Physiology and Ecology (icipe), Nairobi, Kenya Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ogola et al. Parasites & Vectors (2018) 11:577 Page 2 of 10 Background species, Plasmodium infection rates and blood-feeding Malaria remains the most important vector-borne dis- patterns from specimens sampled from varied ease in tropical countries of the world, especially in malaria-endemic locations in coastal, western and Rift sub-Saharan Africa (SSA). The scale-up of long lasting Valley areas of Kenya. insecticidal net (LLIN) distribution has contributed to a decline in malaria transmission, number of cases and Methods mortality within the last decade [1], prompting efforts Anopheles samples toward a target of 2030 for elimination. Sustained mal- We analysed female specimens of the An. funestus com- aria reduction has proved challenging in many plex collected from randomly selected consenting house- disease-endemic countries of Africa. Whilst in many set- holds in diverse sites in the different epidemiological tings LLIN coverage is high, malaria transmission per- risk zones for malaria in Kenya [15]. These included sists [2] and there is need for a better understanding of Kwale County (Kidomaya, Marigiza, Fihoni), Kilifi the vector species composition. Surveillance of vector County (Jaribuni, Malindi), Taita-Taveta County populations becomes increasingly important to inform (Kiwalwa, Njoro), Tana River County (Tana Delta) in the fine-tuning of control strategies in the event of changing coastal region and Busia County (Bunyala, East Bukusu, local conditions [3] or to discern cryptic species contrib- Samia, Lwanya-Bumala), Kisumu County (Ahero) and uting to stealth transmission. Siaya County (Usenge, West Alego, Mageta) in the Lake The major African malaria vectors belong to two spe- Victoria basin in western Kenya and more seasonal mal- cies complexes: Anopheles gambiae and Anopheles funes- aria transmission areas including the arid and semi-arid tus [4, 5]. Few studies in Kenya have examined the areas of northern and central Kenya such as Baringo contribution of Anopheles funestus sibling species to County (Kapkuikui, Kamnarok) (Fig. 1). Samples from malaria transmission, despite its well-established role in the study sites were mainly collected in 2017, except for malaria transmission [6]. It is well known that An. funes- those from Ahero, Usenge and Kamnarok (2015), and tus (sensu stricto) (hereafter referred to as An. funestus) Mageta (2014). is the primary vector species in the An. funestus group Mosquito collections were undertaken both indoors [5]. Recent reports of novel Plasmodium falciparum and outdoors with CDC light traps targeting sporozoite associations in An. vaneedeni [7], as well as host-seeking mosquitoes for most sites (Bunyala, East yet-to-be-identified species [8, 9], suggest the possibility Bukusu, Samia, Lwanya-Bumala, West Alego, Kidomaya, of adaptive changes in vector systems among the group. Marigiza, Fihoni, Jaribuni). This was achieved by deploy- This underscores the importance of active surveillance ing 10 traps per site (5 indoors and 5 outdoors) in separ- of vector populations for potential emerging threats as ate randomly selected households. At the sites in Ahero, malaria is being controlled. Tana Delta, Kapkuikui, collections were limited to out- Bionomic traits and susceptibility to Plasmodium in- doors only using CDC light traps (10) while in Kam- fection vary among the 13 sibling species known to narok, Usenge, Mageta, Njoro and Kiwalwa, collections occur throughout the Afrotropical region in the An. were limited to indoors using pyrethrum spray catches funestus group: An. funestus, An. funestus-like, An. (PSC) or backpack aspiration (ASP) targeting resting aruni, An. confusus, An. parensis, An. vaneedeni, An. mosquitoes. CDC light traps were set at 18:00 h and re- longipalpis types A and C, An. leesoni, An. rivulorum, trieved at 06:00 h the following day with PSC and ASP An. rivulorum-like, An. brucei and An. fuscivenosus [5]. collections conducted between 06:00 and 08:00 h. Indoor Five species have been documented to occur in Kenya, and outdoor catches were not undertaken systematically namely An. funestus, An. rivulorum, An. leesoni, An. to compare trapping efficiency indoors and outdoors at parensis and An. vaneedeni [6, 10]. Of these, P. falcip- a particular site, but different tools and locations were arum infection has been found in An. funestus as the used in an attempt to identify the mosquito species main vector [11, 12] and to a minor extent in An. rivu- richness. lorum [13, 14] and An. leesoni [9]. The often small sam- ple sizes of An. funestus (s.l.) mosquitoes analyzed by molecular means in most of these studies preclude a de- DNA extraction and identification of Anopheles funestus tailed understanding of the occurrence and ecological sibling species distribution of member species in this complex and cor- We extracted genomic DNA from individual whole ollary, their vectoring potential of malaria parasites. specimens using the Qiagen DNeasy Blood and Tissue This study focused on the An. funestus group in rela- Kit (Qiagen, Hilden, Germany) as per the manufacturer’s tion to possible ongoing variations in vector-parasite as- instructions. The DNA was used for identification of the sociations in Kenya. Specifically, we examined the sibling species as well as probing for Plasmodium infec- identity and occurrence in the An. funestus group sibling tion and host blood meal sources. Ogola et al. Parasites & Vectors (2018) 11:577 Page 3 of 10 Fig. 1 Map of Kenya showing the sampling locations Molecular identification of member species of the An. of denaturation at 95 °C for 30 s, annealing at 46 °C for funestus complex involved polymerase chain reaction 30 s and extension at 72°C for 40 s, and a final extension (PCR) amplification of the polymorphic ITS2 region of at 72 °C for 10 min. Thermal reactions were conducted ribosomal DNA using established primers [16, 17]. The on SimpliAmp Thermal Cycler (Applied