bioRxiv preprint doi: https://doi.org/10.1101/694687; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 2 3 4 5 Lek-associated movement of a putative Ebolavirus reservoir, the Hammer-headed fruit bat 6 (Hypsignathus monstrosus), in northern Republic of Congo 7 8 9 10 Sarah H. Olson1*, Gerard Bounga2, Alain Ondzie2, Trent Bushmaker3, Stephanie N. Seifert3, 11 Eeva Kuisma2, Dylan W. Taylor1, Vincent J. Munster3, and Chris Walzer1 12 13 Affiliations: 14 1Wildlife Conservation Society, 2300 Southern Boulevard, Bronx, New York, NY, 10460 15 2Wildlife Conservation Society, 151 Avenue du General de Gaulle, BP14537, Brazzaville, 16 Republic of Congo 17 3Virus Ecology Section, Laboratory of Virology, Division of Intramural Research, National 18 Institutes of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain 19 Laboratories, 903 S. 4th Street, Hamilton, MT, USA 20 * Corresponding author bioRxiv preprint doi: https://doi.org/10.1101/694687; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 21 Funding statement: This study was made possible by the joint support of the Laboratory of 22 Virology, Virus Ecology Unit (NIH) at Rocky Mountain Laboratories and the Wildlife 23 Conservation Society as well as donations from Julie Maher’s WildView fund. Funding also 24 came from the United States Fish and Wildlife Service (Great Ape Conservation Fund). The 25 study also benefitted from intellectual contributions from the PREDICT project of the United 26 States Agency for International Development (USAID) Emerging Pandemic Threats Program. 27 The Virus Ecology Section is supported by the Division of Intramural Research of the NIAID. bioRxiv preprint doi: https://doi.org/10.1101/694687; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 28 Abstract 29 The biology and ecology of Africa’s largest fruit bat remains largely understudied and 30 enigmatic despite at least two highly unusual attributes. The acoustic lek mating behavior of the 31 hammer-headed bat (Hypsignathus monstrosus) in the Congo basin was first described in the 32 1970s. Then in the 2000s, molecular testing implicated this species and other fruit bats as 33 potential reservoir hosts for Ebola virus and it was one of only two fruit bat species 34 epidemiologically linked to the 2008 Luebo, Democratic Republic of Congo, Ebola outbreak. 35 Here we share findings from the first pilot study of hammer-headed bat movement using GPS 36 tracking and accelerometry units and a small preceding radio-tracking trial at an apparent lekking 37 site. The radio-tracking revealed adult males had high rates of nightly visitation to the site 38 compared to females (only one visit) and that two of six females day-roosted ~100 m west of 39 Libonga, the nearest village that is ~1.6 km southwest. Four months later, in mid-April 2018, 40 five individual bats, comprised of four males and one female, were tracked from two to 306 41 days, collecting from 67 to 1022 GPS locations. As measured by mean distance to the site and 42 proportion of nightly GPS locations within 1 km of the site (percent visitation), the males were 43 much more closely associated with the site (mean distance 1.4 km; 51% visitation), than the 44 female (mean 5.5 km; 2.2% visitation). Despite the small sample size, our tracking evidence 45 supports our original characterization of the site as a lek, and the lek itself is much more central 46 to male than female movement. Moreover, our pilot demonstrates the technical feasibility of 47 executing future studies on hammer-headed bats that will help fill problematic knowledge gaps 48 about zoonotic spillover risks and the conservation needs of fruit bats across the continent. 49 bioRxiv preprint doi: https://doi.org/10.1101/694687; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 50 Introduction 51 Lek mating behavior is a very rare mating strategy among the more than 1200 known 52 species of bats [1]. In the early 1970s it was the strong suspicion of lek behavior that led to Dr. 53 Jack Bradbury’s interest in Hypsignathus monstrosus, or the hammer-headed bat, and he 54 conducted the first detailed observational, acoustic, growth-rate, and radio-tracking movement 55 study of H. monstrosus at a research station near Makoko, Gabon [2]. He concluded the 56 population there used a classical lek mating system, as defined by a narrow set of distinguishing 57 characteristics, as well as other traits commonly found among lekking species. In brief the 58 ‘classical’ criteria of a lek species included, “(1) no male parental care, (2) an arena to which 59 females come and on which most of the mating occurs, (3) the display sites of males contain no 60 significant resources required by females except the males themselves, and (4) the female has an 61 opportunity to select a mate once she visits the arena” [3]. Curiously, in West Africa this same 62 species is reported to use a harem-based reproductive strategy suggesting the potential for intra- 63 species behavioral plasticity (Dr. Dina Dechmann personal communication). 64 In Central Africa, the visitation of demographically mixed H. monstrosus at cacophonous 65 lekking sites is an opportunity to reliably sample large numbers of individuals. Importantly, this 66 species was epidemiologically linked to a human outbreak of Ebola virus (Luebo, Democratic 67 Republic of Congo) [4] and has had repeated positive detections for Zaire ebolavirus antibodies 68 and RNA [4–7]. An inhabitant of Central and West Africa, its geographical distribution overlaps 69 with previous Ebola virus outbreaks within Africa [8]. Such aggregations of bats, at breeding or 70 feeding sites, and their movements in between are important targets for epidemiological study as 71 they may be key in determining spillover risk by increasing contact rates among individuals. For bioRxiv preprint doi: https://doi.org/10.1101/694687; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 72 example, a biannual birth pulse with concurrent changes in immune response would introduce 73 many susceptible individuals into a population and create a spillover pathway [9,10]. 74 There is one study that reported visual observations of a month-long seasonal migratory 75 movement of H. monstrosus from the Congo river upstream to the Lulua river in the Democratic 76 Republic of Congo (DRC) [4], but little else is known about where these bats go and when. 77 Beyond aggregations, movement data is critical to monitor animal responses to environmental 78 change and to understand bat-human interfaces. In Australia, movement data helped reveal 79 foraging shifts of flying-foxes from native (in decline) to non-native plants that may be bringing 80 bats into closer contact with horses and human populations [11]. Furthermore, a few studies have 81 suggested correlative associations between logging of fruit bat habitat and Ebola virus disease 82 outbreaks, however, the findings to date remain inconclusive due to data limitations [12–15]. 83 Here we present an analysis of hammer-headed bat movement in northern Republic of 84 Congo from two pilot studies. The purpose of the pilots was to begin elucidating the movement 85 ecology of a population of hammer-headed bats already the focus of a longitudinal virological 86 study that began in 2011. That study includes monthly tree phenology observations that began in 87 2016 and local daily rainfall and temperature data collections since 2015. The distinctive 88 honking sounds made by the congregations of males and the amount of bat activity during the 89 initial and subsequent missions led us to believe the site is a lek [2]. And upon listening to calls 90 recorded and digitized by the Macaulay Library, we recognized that we often hear the ‘staccato 91 buzz’ performances, which Dr. Bradbury observed to occur when a female passes within meters 92 of a male, for example, Hypsignathus monstrosus (ML170632). We began with an affordable 93 VHF radio-tracking study that helped inform the selection of a base station methodology that we 94 then used for the GPS-based second pilot. With data collections completed, here we describe our bioRxiv preprint doi: https://doi.org/10.1101/694687; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 95 findings and revisit Dr. Bradbury’s definition of a lekking species to support or reject our initial 96 hypothesis of the site as lek.