Fruit bats in flight: a look into the movements of the ecologically important Eidolon helvum in Tanzania

Background Many ecologically important plants are pollinated or have their seeds dispersed by fruit bats, including the widely distributed African straw-colored fruit bats (Eidolon helvum). Their ability to fly long distances makes them essential for connecting plant populations across fragmented landscapes. While bats have been implicated as a reservoir of infectious diseases, their role in disease transmission to humans is not well understood. In this pilot study, we tracked E. helvum to shed light on their movement patterns in Tanzania and possible contact with other species. Methods Tracking devices were deployed on 25 bats captured in the Morogoro Municipal and Kilombero District area near the Udzungwa Mountains of Tanzania. Nightly flight patterns, areas corresponding to foraging bouts and feeding roosts, and new day roosts were determined from bat movement data and characterized according to their proximity to urban built-up and protected areas. Sites for additional environmental surveillance using camera traps were identified via tracking data to determine species coming in contact with fruits discarded by bats. Results Tracking data revealed variability between individual bat movements and a fidelity to foraging areas. Bats were tracked from one to six nights, with a mean cumulative nightly flight distance of 26.14 km (min: 0.33, max: 97.57) based on data from high-resolution GPS tags. While the majority of their foraging locations were in or near urban areas, bats also foraged in protected areas, of which the Udzungwa Mountains National Park was the most frequented. Camera traps in fruit orchards frequented by tracked bats showed the presence of multiple species of wildlife, with vervet monkeys (Chlorocebus pygerythrus) observed as directly handling and eating fruit discarded by bats. Conclusions Because we observed multiple interactions of animals with fruits discarded by bats, specifically with vervet monkeys, the possibility of disease spillover risk exists via this indirect pathway. With flight distances of up to 97 km, however, the role of E. helvum in the seed dispersal of plants across both protected and urban built-up areas in Tanzania may be even more important, especially by helping connect increasingly fragmented landscapes during this Anthropocene epoch.


Background
The chiropteran order is diverse with over a thousand species of bats occupying different ecological niches and providing multiple ecosystem services. Fruit bats in particular are important pollinators and seed dispersers for many plants of ecological and economic importance [1][2][3][4][5]. The ability of fruit bats to traverse great distances for foraging or during migration helps connect plant populations across fragmented landscapes to maintain gene flow [6][7][8][9][10]. Maintaining this connectivity is crucial due to threats of on-going fragmentation and degradation of forest ecosystems, leading to impeded movement of plant materials and animals among habitat patches [11][12][13]. In addition, hunting and habitat loss are leading to the extinction of many seed dispersers throughout the tropics, including birds, mammals, and reptiles, affecting not only their numbers, but also the plants they disperse [14,15]. The importance of fruit bats and the ecosystem services they provide is therefore magnified when landscape fragmentation and declines in bird and mammal species facilitating seed dispersal are taken into consideration [15].
Eidolon helvum, the straw-colored fruit bat, is an important long distance seed disperser in tropical Africa by virtue of the diversity of plants upon which it forages and pollinates and its ability to retain seeds in its gastrointestinal tract for long periods of time and disperse them over distances greater than 70 km [16][17][18]. Strawcolored fruit bats are gregarious, spending daytime rest periods in large social groups often exceeding 1000 to 5000 animals at centralized roosting locations. The strong flight capabilities of E. helvum allow the species to take advantage of distant resources during nighttime feeding forays and return to these day roosts daily over extended periods of time, rather than frequently moving colony roost locations nomadically in search of nutritional resources [17]. With colony numbers that can reach a few thousand to several million bats [17,[19][20][21][22], their sheer numbers also enhance the extent of the ecosystem services they provide. Numbers of E. helvum bats in colonies vary across the year, likely in response to food availability [9,21]. Movement patterns have also been found to change seasonally [17]. Eidolon helvum bats have been observed to migrate over 2000 km across many different habitat types including Zambezian woodland, forest-savanna mosaic, and lowland and riverine forests [23].
Bats are also transmission hosts of zoonotic viruses that can cause severe diseases in humans, such as Marburg virus, ebolaviruses, Nipah virus, Hendra virus, other paramyxoviruses, and coronaviruses, including Severe acute respiratory syndrome-like coronaviruses (SARSlike CoVs) and Middle East respiratory syndrome coronavirus (MERS-CoV) [24][25][26][27][28][29][30][31][32][33][34][35][36]. The suitability of bats as hosts for diverse viruses is likely associated with their evolutionary age, genetic diversity, broad geographical distribution, and social, biological, and immunological features [37][38][39]. With over 1000 recognized species, bats are the second largest order of mammals, after rodents, whose origins can be traced back to 50 million years ago [40,41]. As social animals often living together in large numbers in close physical proximity, their colonies can facilitate the circulation and transmission of viruses and even promote virus amplification during bats' breeding seasons [42]. Bats are unique in that they are the only mammals capable of powered flight, and it is hypothesized that the immune and metabolic changes associated with flight facilitate the large diversity of zoonotic viruses in bats as a result of altered host-virus interactions [43,44]. Despite the implication of bats as a source of disease outbreaks in humans, the role of bats in disease emergence is not well understood; with not enough known about bat biology and insufficient measures having being put in place with respect to their conservation [45].
Previous studies have looked at genetic and epidemiological connectivities among E. helvum populations in Africa, as well as their role as seed dispersers in Tanzania [4,46,47]. While Tanzania has at least two documented E. helvum colonies in Dar es Salaam and Morogoro [22,48], little is known about the movements of bats in these colonies. We aimed to understand the movement and foraging behavior of E. helvum in Tanzania, using Global Positioning System (GPS) loggers, and to investigate how this behavior relates to the connectivity of landscapes and the bats' interactions with other species, including humans. Our objectives were to explore the fine-scale movement ecology of E. helvum in Tanzania and to also use this information to inform environmental surveillance of indirect contact of bats with other species. We hope that the insights gained from this work will contribute to the existing body of knowledge on E. helvum and be helpful for both the conservation of fruit bats and for informing on the reduction of bat-human interaction and pathogen transmission risk.

Study sites and animals
We tracked the movements of straw-colored fruit bats (E. helvum) living in two colonies in Morogoro Region, Tanzania in November 2016. The first site (−6.8233, 37 . Both colony locations were amid human dwellings and a school. While a formal estimate of the population of bats at these colony sites was not carried out, they were roughly estimated to be at least several thousand in number, and the Morogoro colony's roost size was previously estimated at 10,000 [22]. The bats were captured with synthetic mist nets suspended between two metal or wooden poles placed near tree roosts, carefully removed from the nets, and temporarily restrained in a closed cotton bag. Handling, sedation and reversal (dexmedetomidine/atipamezole), and release were performed in accordance with the University of California, Davis, Institutional Animal Care and Use Committee (IACUC) (protocol number: 19300). Only adult male and female bats not observed to be pregnant were considered for GPSlogger attachment, in order to ensure that the weight of the GPS-loggers did not exceed 5-10% of bat body mass [50,51]. All work in this study was conducted with approvals and permissions from the Tanzania Wildlife Research Institute (2016-290-NA-2011-29).

GPS logger attachment
We used two types of GPS loggers: 15 g solar (e-obs, Munich, Germany) and 3.5 g PinPoint Argos satellite tags (Lotek Wireless Inc.), and aimed to attach GPS loggers to at least eight bats at each study site to collect sufficient movement data for analyses. This minimum number was determined based on the percentages of successfully tracked E. helvum bats in previous studies (for tags attached to bats via adhesives) [16,17,23]. Tags were attached to bats by clipping the hair between their shoulder blades, applying Skin-Tac-H Adhesive wipe (Torbot Group Inc.) on the clipped area, and then affixing tags with cyanoacrylate glue [51]. To compare longevity of tag application, we attached one Argos satellite tag using a collar tied with a degradable suture, Vicryl® (polyglactin 910), so that while the collar might stay on longer than loggers attached with adhesive glue, it would eventually drop-off with no long-term adverse effects on bat health [16].

GPS logger setup
The solar e-obs tags collected acceleration-informed GPS fixes between 17:00-07:00, with acceleration data recorded upon 3-axes: X = left-right, Y = backward-forward, and Z = up-down. We programmed tags to collect acceleration data all day at intervals of 60 s at a byte count of 1135 (16.67 Hz). GPS fixes were collected at intervals of 45 min during GPS on-times, unless the bat was moving at a speed of 50 cm/s or more, in which case collection intervals changed to every 30 s. Loggers started collecting data immediately after bats were released with affixed loggers. To download the GPS and associated acceleration data, we walked at least once each day through the bat colonies with the e-obs base station connected to a directional high-gain antenna. The base station automatically downloaded the data when it connected with a logger. We programmed the Argos satellite tags at different intervals, starting with every 1.5 h in the beginning of the study, to every 30 min, as our study progressed, in order to balance tag battery life with the number of discrete GPS fixes recorded and with the days the tag remained attached to the bat. Data from the Argos satellite tags were downloaded remotely from the Argos website (http://www.argossystem.org/).

Analyses of movement data Estimation of foraging areas/feeding roost locations and new day roosts
The GPS points collected from e-obs tags were categorized as 'flying' or 'not-flying' based on the variances of their temporally corresponding acceleration bursts [16]. Previous studies have differentiated acceleration bursts with higher variances in their Y-(backward-forward) and Z-(up-down) axes, corresponding to 'flying' behavior, from those with lower variances ('not-flying' behavior) by k-means clustering [16]. We compared the classification of GPS points into 'flying' and 'not-flying' behavior using XZ, YZ, and XYZ axes, and observed no significant difference between them (Additional file 1: Figure S1 and Table S1). Using the GPS classification based on the YZ axes (Additional file 2: Figure S2), we further subsetted sequential 'not-flying' GPS fixes within a radius corresponding to the distance a bat could travel at its average speed (determined by averaging the ground speeds recorded for each GPS fix) and with a time interval greater than or equal to 30 s. These subsetted GPS fixes were considered to comprise both foraging bouts (i.e. moving within a food tree) as well as night feeding roosts (Fig. 1). Points falling in the vicinity of the original colony where bats were tagged were excluded from classification as foraging areas/ feeding roosts or new day roosts. A day roost location was one where a bat was recorded as present between sunrise and sunset.
The mean and range of cumulative distance flown each night as well as each hour was determined. In addition, the maximum straight-line distance between day roosts and the most distant foraging sites from the colony location was determined. The utilization distribution of each bat was calculated and mapped using classic kernel density estimation (KDE) with vertices set to 50 and 95% location probabilities respectively, using the adehabitatHR R package [52].

Distance of foraging areas/feeding roost locations from urban built-up and protected areas
We measured the distance of each foraging area/feeding roost location to its nearest urban built-up and protected area (national park or reserve) using GDAL v2.4.1 (https:// gdal.org) and GRASS GIS v7.0.5 (https://grass.osgeo.org) [53]. For determining urban built-up areas, we used the Global Urban Footprint (GUF), which is high resolution human settlement/urban footprint data derived from TerraSar-X satellite imagery [54,55]. Originally at a resolution of 12 X 12 m, we aggregated urban built-up areas into grids of 100 X 100 m such that any larger grid containing at least one smaller grid classified as built-up was assigned as urban [17]. In order to determine the distances of foraging locations from protected areas in Tanzania, we used data on game reserves, nature reserves, forest reserves, and national parks from the World Database of Protected Areas (WDPA) [56]. The GUF and WDPA data were accessed in February 2016 and 2019, respectively.
Site characterization of foraging areas/feeding roost locations and environmental surveillance A convenience sample of locations identified as foraging areas/feeding roosts (or in proximity with them) or new day roosts by the tracking data were visited to determine types of trees at the site, presence of human dwellings, and to confirm whether there were any wild or domestic animals or signs of them at the sites. Where possible, we talked with the local community members about their observations on bat activity at these sites. Two remotetriggered field camera traps (Reconyx™ Hyperfire HC600) were set up at the horticultural garden at the Sokoine University of Agriculture (SUA), Morogoro, Tanzania (Fig. 6), in order to understand the potential for other species to contact fruits discarded by bats at this foraging site. The SUA horticultural gardens were chosen because they were identified as being visited by the tracked bats, who fed upon mangoes at this site. Fruit bat activity at this site was confirmed by the presence of mangoes eaten and subsequently discarded by the bats under the mango trees. The camera traps were fixed under mango trees 20-30 cm from the ground to capture images of both domestic and wild animals. They were active 24 h a day from 18th December 2017 to 2nd January 2018. Because the bats were originally tracked in November 2016 during the mango harvesting season, we waited for the next mango season (December 2017) to set up the camera traps under circumstances similar to the tracking period.

Results
GPS logger data, foraging areas/feeding roost locations, and new day roosts Twenty five E. helvum bats were tagged across the two study sites: 15 bats in Kilombero (10 e-obs tags and five Argos satellite tags), and 10 bats in Morogoro (all Argos    30 g). The bats were predominantly males (84%) because their larger body mass kept the percent tag weight low and facilitated their inclusion in the study (males: 277 ± 25.8 g, females: 240 ± 34.8 g). The tag affixed with a collar stayed on the bat for the longest time period (14 days compared to a maximum of six days for tags attached by adhesives). We collected data for 1-6 foraging nights per bat; even though the collared bat (M166367) was tracked for 14 days, its GPS fixes corresponded to 6 foraging nights. The number of GPS fixes from e-obs tags (n: 31662) vastly outnumbered those by the Argos satellite tags (n: 156). We identified 505 foraging areas/feeding roost locations and three new day roost locations from the high-resolution e-obs data ( Table 2 and Fig. 2). In contrast to the e-obs tag data, we were unable to classify Argos satellite tag fixes as foraging or not because of the inability to assess time spent at each location and whether the bat was in flight or not. Nonetheless, they still provided information on areas frequented by bats ( Fig. 3 and Additional file 3: Figure S3). The mean cumulative nightly flight distance observed for e-obs tagged bats was 26.14 km (min: 0.33, max: 97.57). One male bat, K5310, flew cumulative nightly  Table S4 and Figure S4). He foraged in urban, semi-urban, and intact forest landscapes along his flight path. Bat K5310 also had the maximum hourly distance flown of any bat: 38.08 km flown between 1 and 2 am on the third tracked night (Additional file 5: Table S5.1 and Figure S5). The   (Table 1 and Additional file 6: Figure S6). The bats tended to visit the same sites on subsequent nights (Additional file 7: Figure S7), and while all male e-obs tagged bats flew in a direction south of their roosting colony, the only female e-obs tagged bat (K5317) flew in the northern direction (Fig. 2).

Distance of foraging areas/feeding roost locations from urban built-up and protected areas
Of the 505 identified foraging areas/feeding roost locations obtained from E. helvum bats tagged with e-obs loggers in Kilombero, approximately 25% (126/505) were in areas classified as built-up by the Global Urban Footprint while 46% (232/505) were within 100 m of built-up areas ( Table 2) [54]. Even though we could not ascertain foraging areas from the Argos satellite tags, distances of GPS fixes from urban areas excluding the tagging location revealed that approximately 60% (84/139) of these locations were within urban areas, and 77% (107/139) were in proximity (within 100 m) to built-up landscapes.
In the case of protected areas, approximately 32% (164/505) of the Kilombero E. helvum foraging points were in or within 100 m of a national park or reserve, the majority (93%) of them in Udzungwa Mountains National Park, followed by Mikumi National Park (Table  2). Eleven GPS fixes from the Morogoro bats were within 100 m of protected areas, all in the Nyandira Reserve.

Site characterization and environmental surveillance
We visited nine sites for habitat characterization and while we did not hone in directly on GPS fixes to identify the exact trees visited by bats, we observed the general diversity of trees present at these sites. Sixteen species of trees (Table 3) were identified and conversations with community members at these sites revealed that mangoes were a favorite of E. helvum. During mango season (November-December), the bats were reported to feed on mango fruit and in addition to the noise they made while feeding at night, they also caused destruction of the mango harvest, scattering chewedupon mangoes on the ground. Five of the sites visited by us corresponded to newly identified day roost locations. We did not observe any new roosting colony, although community members at the Ifakara site pointed out palm trees that served as colony roost locations in the previous year (2015).
At the horticulture garden in Sokoine University of Agriculture (SUA) in Morogoro, we directly observed discarded mangoes that had been chewed and left behind by fruit bats (Fig. 6). Camera-traps set up at this orchard showed visits by five mammals: vervet monkeys (Chlorocebus pygerythrus), domestic cat (Felis catus), common dwarf mongoose (Helogale parvula), Rattus rattus, and humans (Fig. 7). Vervet monkeys at the site were observed handling fallen or discarded mangoes and putting them in their mouths ( Fig. 7 and Additional file 8). The people observed at the camera trapping site were predominantly adults, including farm workers, security guards, and visitors who were there to collect fallen mangoes.

Discussion
Our study takes an integrative approach to better understand E. helvum ecology in Tanzania, looking at both their movement patterns and identification of potential pathways of pathogen transmission among bats and other species. In the first work of this kind in Tanzania, results reveal that one straw-colored fruit bat, E. helvum, These locations were also newly identified day roost sites flew up to 97 km over a single foraging night, frequenting both urban built-up and protected areas. With maximum straight-line distances of 62 km from day roosts to foraging areas/feeding roost locations, their foraging distances enable E. helvum to connect land fragmented by human activities and provide valuable seed dispersal and pollination services across vast distances [16][17][18]. Seed dispersal by E. helvum can help regenerate forests in degraded landscapes and can also contribute to the local economy through support for the propagation of economically important plants and trees, e.g., African teak, a valuable source of timber [74]. The African teak tree (Milicia excelsa) was among several tree species identified by us at sites visited by tracked bats, along with mango and cashew trees, which are important fruit trees grown in Tanzania [74][75][76]. Protected areas like Udzungwa Mountains National Park (UMNP) are important for E. helvum for both foraging and roosting activities. They provide native sources of food to fruit bats, rather than introduced agricultural fruit species like mangoes. In return, bats can help maintain forest tree composition and propagation in a mutually beneficial relationship [3]. Eidolon helvum bats in the Morogoro site were tracked to within 100 m of the Nyandira reserve, part of the Uluguru Mountain range. Both UMNP and the Uluguru mountains are important areas for endemic species of vertebrates and trees in Africa [77], and therefore the seed dispersal services offered by fruit bats in these regions potentially also support the biodiversity contained within them. Forests no longer exist in the Nyandira reserve, due primarily to clearance for new farmland, and it is possible that tracked bats were foraging on human-cultivated plants or trees [78,79]. The loss of natural food sources due to clearing of native forest and the subsequent shift by bats to feeding on agricultural fruit crops can lead to a conflict between bats and fruit-growers [80]. Perceptions of E. helvum shared by community members at our study sites and identified foraging areas/feeding roost locations revealed that bats were disliked and harassed by some community members. Farmers growing mangoes complained of fruit bats negatively impacting their mango harvest, while also being highly-vocal at night and disrupting village households. Some community members drove the bats away with smoke from fires lit under tree roosts or by cutting down the trees themselves. Our findings support the suggestion that preserving natural forests or strategically planting native trees specifically for bat roosts could encourage and draw bats away from fruit crops and human dwellings, thus lessening the potential for bat-human conflict [81].
Camera-traps set up at locations identified by tracking data helped us elucidate species interactions and provide evidence that bats, non-human primates, and other species may have common or shared virus exposure pathways. Due to viral shedding through oral secretions, fruits discarded by bats could serve as vectors for disease transmission to other animals coming into contact with the discarded chewed fruit, e.g. the vervet monkeys pictured handling discarded mangoes [82], and people, especially children who eat discarded fruits from bats because of their sweetness and them having already been spoiled for sale at markets. Similarly, contaminated fecal droppings at foraging and roosting locations could be another route of infection to humans and other species. Bats connecting different landscapes might possibly act as bridge species between relatively undisturbed regions and urban areas, potentially transmitting novel pathogens to other species either directly or indirectly [83]. Currently, there are no known human outbreaks associated with bats in Tanzania, in contrast with bat-associated zoonotic disease outbreaks occuring elsewhere in east-central Africa [84,85]. However, factors such as roosting in urban colonies (our study sites, [16,17]), foraging in both urban and protected areas, and the possibility of being transmission hosts of human diseases [32,33] make tracking E. helvum movements important for understanding further exposure pathways and species interactions.
As a pilot study, we successfully explored new ways to collect data that can be used to inform disease surveillance. Increasing the numbers and species of animals tracked, as well as determining differences due to sex, season, or migratory patterns could further help our understanding of bat movement patterns and factors affecting disease transmission and spread between bats and other species [9,17,23].

Conclusions
Eidolon helvum bats play an important role in the pollination and seed dispersal of plants within both protected and urban areas in Tanzania; and the conservation of bats and protected areas is thus interdependent. Their movement patterns also suggest the possibility of indirect contact among bats, non-human primates, livestock, and humans in bat foraging areas near human dwellings and in horticulture, as well as an opportunity for virus sharing to occur between bats and other wild species in protected areas. Ensuring that bats have access to wild food sources away from human dwellings or crop plantations should be explored as a potential intervention to mitigate conflict between fruit bats and humans, reduce potential virus sharing, and foster a more amicable coexistence between species.