Vol. 11(1), pp. 1-9, January 2019 DOI: 10.5897/JPVB2018.0341 Article Number: 69934FA59973 ISSN: 2141-2510 Copyright ©2019 Journal of Parasitology and Author(s) retain the copyright of this article http://www.academicjournals.org/JPVB Vector Biology Full Length Research Paper A pilot study on isolation of Asaia and detecting its co- presence with Plasmodium falciparum in two major malaria vectors in Senegal Hubert Bassene1,2#, El Hadji Amadou Niang1,3,4*#, Florence Fenollar2, Souleymane Doucoure1,2, Badara Samb4, Ousmane Faye4, Didier Raoult3, Cheikh Sokhna1,2 and Oleg Mediannikov3 1Vecteurs-Infections Tropicales et Méditerranéennes (VITROME), Campus International UCAD-IRD, Dakar, Sénégal. 2Aix Marseille Univ, IRD, AP-HM, SSA, VITROME, IHU-Méditerranée Infection, Marseille, France. 3Aix-Marseille Univ, IRD, AP-HM, MEPHI, IHU-Méditerranée Infection, Marseille, France. 4Laboratoire d‟Ecologie Vectorielle et Parasitaire, Faculté des Sciences et Techniques, Université Cheikh Anta Diop (UCAD) de Dakar, Sénégal. Received 18 August, 2018; Accepted 19 December, 2018 The declining of malaria creates the opportunity to accelerate its elimination. However, the elimination program is threatened by the spread of insecticide resistance among Anopheles (An.) mosquitoes, stressing the urgent need for new vector control strategies. Recent evidence of stable infection of anopheles’ symbionts that could affect the development of Plasmodium parasites within mosquitoes, has paved the way for the use of such organisms to target and control malaria vector populations. We have isolated a new Asaia strain from a wild population of Anopheles gambiae s.l. and, for the first time, from natural population of Anopheles funestus, and insectary colony of Anopheles coluzzii. The new anopheline strain has been named Asaia (A.) aff. bogorensis GD01 because of its close relationship with A. aff. bogorensis isolated from plant flowers. We highlighted an antagonistic effect of A. aff. bogorensis GD01 on the sporogonic development of Plasmodium falciparum within the two major malaria vectors (An. gambiae and An. funestus) in Senegal. The putative impact of A. aff. bogorensis GD01 infection on the P. falciparum sporogonic development offers an opportunity for new malaria vector control approaches that can be added to the limited arsenal required to fulfil the World Health Organization (WHO) recommendations for searching new tools to achieve malaria elimination goal. Although this is an innovative and promising malaria control tool, more investigations are required to better characterize Asaia vs. Plasmodium interactions, before any Asaia-based intervention. This is required to ensure their safe use as an alternative or complementary vector control strategy to achieve malaria elimination goal. Key words: Asaia, Plasmodium falciparum, malaria, Anopheles gambiae, Anopheles funestus, Senegal. INTRODUCTION Among the arthropod-borne diseases affecting human, than 600,000 annual deaths (World Health Organization malaria is the primary global health problem with over [WHO], 2016). Developing countries of the WHO African *Corresponding author. E-mail: [email protected] #Contributed equally Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 2 J. Parasitol. Vector Biol. region are the most affected, accounting for almost 90% antagonistic effect of Asaia aff. bogorensis GD01 on the of the global deaths (WHO, 2016). They also suffer a sporogonic cycle of P. falciparum within wild populations huge economic burden due to the disease (Sachs and of two major malaria vectors (Anopheles gambiae and Malaney, 2002). Nowadays, efforts to control malaria Anopheles funestus) in central and south-eastern integrate preventive strategies, such as insecticide-based Senegal and its high and stable presence in an vector control interventions with rapid diagnostic testing Anopheles coluzzii insectary strain. and effective drug treatments. These efforts are enhanced by strengthening surveillance and healthcare systems and research infrastructure (Breman et al., 2004; MATERIALS AND METHODS WHO, 2017). Despite the global progress in the fight against malaria Study sites recorded so far, recurrent problems, including the Wild populations of An. funestus and An. gambiae s.l. were increase and the spread of resistance phenomena collected using the pyrethroid spray method from four different among vectors and parasites as well as the lack of Senegalese sites located in the two administrative regions of effective vaccines, highlight the need for a new Kedougou and Fatick, about 500 km apart from each other. Dielmo generation of malaria control tools. Genetically modifying and Ndiop, located in the Fatick region, which host one of the longest epidemiological cohort studies on malaria, are two mosquitoes that transmit malaria to become resistant to Sudanese savannah villages in central Senegal, near the Gambian parasites has been successfully attempted (Grossman et border (Trape et al., 2011). Kedougou, Bandafassi and al., 2001). However, genetic manipulation tends to Tomboronkoto were chosen close to the Malian and Guinean reduce mosquito fitness and thus the chance to borders in the South-Eastern Senegal. The region belongs to the successfully spread genes of interest among natural Sudano-Guinean savannah ecological zone and has the highest populations (Favia et al., 2007). malaria incidence (PNLP, 2015). Recent evidences of the stable natural infection by endosymbionts in human malaria vectors, which appears Samples collection to affect the development of the Plasmodium parasites, has shown the potential for the use of such organisms to A total of 1,184 females of wild anopheline populations were tested. control population of malaria vectors (Baldini et al., 2014; Of these, 375 An. funestus and 362 An. gambiae s.l. were from Fatick. While Kedougou‟s sample included only 447 females of An. Shaw et al., 2016). gambiae s.l. The same specimens were tested to detect both P. Asaia species are facultative intracellular, Gram- falciparum and/or A. aff. bogorensis GD01 infections. In addition to negative, aerobic, rod-shaped and peritrichously the wild anopheline populations, 23 males and 127 females of an flagellated Alphaproteobacterium from the An. coluzzii insectary colony, maintained in our laboratory in Dakar Acetobacteraceae family, primarily isolated from the (Boudin et al., 2005) were included in the study to assess the tropical flowers of Bauhinia purpurea and Plumbago frequency of the A. aff. bogorensis GD01 in the mass rearing conditions. auriculata (Katsura et al., 2001; Moore et al., 2002; Yamada et al., 2000; Yukphan et al., 2004). Later, several strains of the genus were described as Isolation and cultivation of the new Asaia strain extracellular endosymbiotic bacteria in the salivary glands, gut and the reproductive organs of both males An. gambiae mosquitoes collected during routine entomological surveillance in Dielmo during the summer of 2011 were used as and females of anopheline mosquitoes. Indeed, Favia et isolation sources. Prior to isolation, 11 alive individual mosquitoes al. (2007) reported a laboratory reared Anopheles were washed in a 10% solution of a commercial disinfectant- stephensis strain stably harbouring an acetic acid detergent (Amphomousse, Hydenet S.A., Sainghin-en-Melantois, bacterium of the Asaia genus (Favia et al., 2007). France), then rinsed with sterile water and placed in a 1% solution Furthermore, the location of these strains in the of sodium hypochlorite for 1 min. After the final rinse in sterile phosphate-buffered saline, mosquitoes were placed in a sterile 1.5 reproductive organs suggests their potential vertical and µl plastic tube and triturated with a sterile micro-pestle in 600 µl of sexual transmissions to females‟ offspring and mating MEM tissue culture medium. The shell-vial culture approach (Kelly partners, respectively. Since some Asaia strains have et al., 1991) for intracellular bacteria was employed to isolate the been suspected to affect the development of Plasmodium new Asaia strain. Isolation material, consisting of 200 µl of the falciparum within its invertebrate host (Favia et al., 2008; supernatant of crushed mosquitoes, was inoculated into three Pumpuni et al., 1996), they may be good candidates for different cell line monolayers respectively made of Rhipicephalus microplus tick (BME/CTVM2), Aedes albopictus (C6/36) and use as novel malaria vector control tool in combination Xenopus laevis (XTC2) cells, then incubated at 28°C and 5% CO2 with, or as an alternative to the existing core interventions atmosphere and checked every day to assess microbial growth. to increase the efficiency of malaria control and The catalase and oxidase activities were determinate as previously elimination programs. However, to successfully use such (Holt et al., 1994). The supernatant of the C6/36 infected cells, the an alternative tool, better understanding the nature of only cell line producing intracellular gram-negative bacteria growth interactions between Anopheles - Asaia - P. falciparum is at day 7 post-inoculation, was collected and streaked on a 5% sheep's blood-enriched Columbia agar (BioMérieux) at 28°C and critical. 5% CO2 atmosphere. Microbial growth of the isolate occurred the In this study, we present the first data on a possible 3rd day post-cultivation. Regarding molecular characterization, the Bassene et al. 3 grown bacteria DNA was extracted,