Background Infections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour. Methodology/Principal findings Prior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2–3 days. The cell-free culture system (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK2 and DMEM supplemented with 10% NCS and seeded on LLC-MK2 systems. Conclusions/Significance The present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening.

39 40 Keywords: 41 O. volvulus 42 in vitro culture 3 43 growth 44 development 45 motility 46 moulting 47 mating 48 competition Abstract 53 Background: Infections with Onchocerca volvulus nematodes remain a threat in Sub-54 Saharan Africa after two decades of ivermectin mass drug administration. Despite this 55 effort, there is still an urgent need for understanding the parasite biology, especially 56 mating behaviour and nodule formation, as well as development of more potent drugs 57 that can clear the developmental (L3, L4, L5) and adult stages of the parasite and 58 inhibit parasite's reproductive and behavioural pattern. 59 Methodology/Principal Findings: Prior to culture, freshly harvested O. volvulus L3 60 larvae from dissected Simulium were purified by centrifugation using a 30% Percoll 61 solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both 62 cell-free and cell-based co-culture systems, and monitored daily by microscopic visual 63 inspection. Exhausted culture medium was replenished every 2-3 days. The cell-free 64 culture system supported the viability and motility of O. volvulus larvae for up to 84 65 days (DMEM-10%NCS), while the co-culture system (DMEM-10%FBS-LLC-MK 2 ) 66 extended the worm survival period to 315 days. Co-culture systems alone promoted 67 the two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates 68 observed in DMEM-10%FBS-LLC-MK 2 (69.2±30 %), while no moult was observed in 69 DMEM-10%NCS-LEC condition. O. volvulus adult worms mating and even mating 70 competitions were observed in DMEM-10% FBS -LLC-MK 2 co-culture system. Early 71 nodulogenesis was observed in both DMEM-10% FBS-LLC-MK 2 and DMEM-72 10%NCS-LLC-MK 2 systems. 73 Conclusions/Significance: The present study describes an in vitro system in which 74 O. volvulus L3 larvae can be maintained in culture leading to the development of 75 reproductive adult stages. Thus, this platform gives potential for the investigation of 5 76 mating, mating competition and early stage of nodulogenesis of O. volvulus adult 77 worms that can be used as additional targets for onchocercacidal drug screening. 78 Author summary 79 River blindness affects people living in mostly remote and underserved rural 80 communities in some of the poorest areas of the world. Although significant efforts 81 have been achieved towards the reduction of disease morbidity, onchocerciasis still 82 affect million of people in Sub-Saharan Africa. The current control strategy is the 83 annual mass administration of ivermectin which have accumulated several drawbacks 84 overtime: as the sole microfilaricidal action of the drug, very long treatment period (15-85 17 years) and reports of ivermectin losing its efficacy; Therefore, raising the urgent 86 need for new onchocercacidal molecules. Our study has established an in vitro 87 platform capable of supporting the growth and development of all developmental 88 stages of O. volvulus (L3 infective stage, L4, L5 and adult worms), moreover the 89 platform pro...