Rates of hematophagous ectoparasite consumption during grooming by an endemic Madagascar fruit bat

Rates of hematophagous ectoparasite consumption during grooming by an endemic Madagascar fruit bat Background: Few details are available on the consumption of ectoparasites, specifically bat flies (Diptera: Nycteribiidae and Streblidae), by their chiropteran hosts while grooming. Such details are important to document consumption rates of ectoparasites by their bat host provide details on the dynamics of host-parasite interactions. We present data on ectoparasite consumption rates for an endemic Malagasy fruit bat (Pteropodidae: Rousettus madagascariensis) occupying a cave day roost colony in northern Madagascar. Using quantified behavioral analyses, grooming and associated ingestion rates were measured from infrared videos taken in close proximity to day- roosting bats. The recorded individual bats could be visually identified to age (adult, juvenile) and sex (male, female), allowing analyses of the proportion of time these different classes allocated to consuming ectoparasites via auto-grooming (self) or allo-grooming (intraspecific) per 10 min video recording session. These figures could then be extrapolated to estimates of individual daily consumption rates. Results: Based on video recordings, adults spent significantly more time auto-grooming and allo-grooming than juveniles. The latter group was not observed consuming ectoparasites. Grooming rates and the average number of ectoparasites consumed per day did not differ between adult males and females. The mean extrapolated number consumed on a daily basis for individual adults was 37 ectoparasites. When these figures are overlaid on the estimated number of adult Rousettus occurring at the roost site during the dry season, the projected daily consumption rate was 57,905 ectoparasites. Conclusions: The details presented here represent the first quantified data on bat consumption rates of their ectoparasites, specifically dipterans. These results provide new insights in host-parasite predation dynamics. More research is needed to explore the mechanism zoonotic diseases isolated from bat flies might be transmitted to their bat hosts, specifically those pathogens that can be communicated via an oral route. Keywords: Diptera, Nycteribiidae, Pteropodidae, Rousettus madagascariensis, Madagascar, Host-parasite interactions Background such as rhabdovirus [6, 7]; and protozoans including the The past decade has seen an important increase in stud- hematoparasite Polychromophilus [8, 9]. Hence, there is ies of bats as reservoirs of diverse diseases [1–3]. An- indirect evidence that bat ectoparasites may be respon- other facet of this research has shown that bat sible for the transmission of infectious diseases to their ectoparasites, specifically hematophagous bat flies of the hosts, although it remains to be demonstrated that this families Streblidae and Nycteribiidae, are possible reser- could be via an oral route, specifically direct consump- voirs and vectors for different zoonotic diseases, e.g. bac- tion of ectoparasites by bats. teria such as Bartonella and Rickettsia [3–5]; viruses Although there are growing literature data on the im- portance of ectoparasites as prey and the associated epi- demiological implications [10, 11], only a few studies * Correspondence: rianaval@yahoo.fr Mention Zoologie et Biodiversité Animale, Université d’Antananarivo, BP that have documented ingestion rates by bats of their ec- 906, (101) Antananarivo, Madagascar toparasites. A project conducted on New World stre- Association Vahatra, BP 3972, (101) Antananarivo, Madagascar blids found evidence that the fruit bat Artibeus 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. Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 2 of 8 jamaicensis (Phyllostomidae) may actively feed on its bat recordings were made in an area of the cave outside the flies of the genus Megistopoda [12]. Further, captive A. typical visitor circuit. jamaicensis readily accepted and consumed Megistopoda Each day of behavioral recordings made in the cave, bat flies and the remains of streblids were identified in sessions commenced at 6:15 h, 7:15 h, 8:15 h, 13:15 h, the stomachs of different species of Costa Rican bats 14:15 h, and 15:15 h. Recordings were made for 10 min [12]. Further, in Malaysia, the bat Megaderma spasma per session, which on a daily basis resulted in one hour (Megadermatidae) feeds actively on Eoctenes spasmae of recording (six sessions × 10 min). The sessions com- (Cimicidae) ectoparasites [13]. Hence, there is indeed mencing at 6:15 h and 13:15 h, when the recording ma- evidence that bats feed on their dipteran parasites, but terial was installed during a period of about 30 min, quantitative details on occurrence and frequency are were followed by 15 min of no activity near the roosting lacking in the literature. bats, to allow them to calm down after being disturbed. The purpose of this paper is to present quantified be- The start and termination of the video recording for the havioral observations on the proportion of time while in other sessions were controlled with the remote device. the day roost site an endemic Malagasy fruit bat grooms The methods for data sampling and processing and actively consumes ectoparasites overlaid on possible followed a previous behavioral study on R. madagascar- age and sex differences. Extrapolations are made as to iensis in the same cave [17]. Video recordings were the average daily ectoparasite consumption rates per in- reviewed on a computer screen measuring 40.5 × 16.2 dividual bat. These data provide new insights into cm, and scored based on behavioral variables using a host-ectoparasite predation dynamics, as well as a pos- scan sampling method [18, 19]. An ethogram (Table 1), sible means zoonotic diseases isolated from bat flies, derived from previous studies of non-Malagasy bat spe- specifically those communicated through oral means, cies [20, 21], was created to categorize and quantify the might be transmitted to their hosts through the con- behavior of R. madagascariensis. sumption of ectoparasites. Scan sampling Methods A scan sampling technique was used to document different Study area behavioral states of target individuals within a group and to Data were collected between 29 June to 10 July 2017 at calculate an individual's time activity budget (i.e. the propor- a day roost site of Rousettus madagascariensis (Chir- tion of time spent in an activity) [17]. One scan sampling optera: Pteropodidae) in the Grotte des Chauves-souris consisted of following five individual bats based on the re- (12°57.4'S, 49°7.1'E), Parc National d’Ankarana in the far corded video, each independently for three seconds and for north of Madagascar. Ankarana is characterized by lime- 10 rounds of observation. Given that in certain cases, an in- stone karst formations and the natural habitat is dry de- dividual disappeared from or others flew into view, two ciduous forest [14]. The study was conducted during the other groups of five individuals were also followed during dry season, and estimates are available for this period on the 10 min period. The proportion of time each individual the number and age ratio of R. madagascariensis occu- performed each of the following behavioral activities was pying the day roost site in the same cave [15, 16]. calculated: rest, crawling into or outside of the focal cluster, inter-bat aggression (“fighting”), auto-grooming Video recording (self-grooming), allo-grooming (associated with the body Data on grooming behavior, specifically bat feeding rates of a neighboring animal), and consuming ectoparasites on on ectoparasites, were calculated via the analysis of re- cordings made with an infrared video camcorder [1080p Table 1 Ethogram of behavior for the scan sampling methods HD Infrared Night Vision and Full Spectrum Camcorder Behavior categories Description (Cleveland Paranormal Supply Co., Mentor, OH, USA)] Rest Not moving any part of the body employing an infrared light for supplementary illumin- Crawl Alternate placement of feet or/and ation [Evolva Future Technology T38 IR 38 mm lens in- thumb claws to shift frared flashlight light night vision torch (Shenzhen position on the rock face Dikesai International Trade Co., Ltd, Guangdong)]. The Groom Scratching or licking the peak intensity wavelengths of these apparatus are 810 body or/and wing phalanges nm. The camcorder, which was connected to a remote Fight Biting or hooking control, was placed at a fixed place within the cave, ap- (rapid swiping with thumb claw) the opponent proximately 4 m from the target Rousettus madagascar- iensis group, with the intent in following individual bat Consume ectoparasites Taking ectoparasites into the mouth, masticating, and no activities. Tourists visit the cave, which disrupts the nat- evidence of rejecting them ural behavior of this species [17], and the video Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 3 of 8 its own body via auto-grooming (Additional file 1: Video to examine possible differences in grooming behavior S1) or a neighboring animal via allo-grooming (Additional and ectoparasites consumption between age and sex file 2: Video S2). classes. The Kruskall-Wallis test with post-hoc Dunn On the basis of recorded video, the sequence of steps tests and the Wilcoxon rank test with R 3.4 [27], were that bats seized ectoparasites and consumed them is as both employed to investigate the relation between ses- follows: detainment with their teeth, tongue, or lips; sion periods and the consumption of ectoparasites. mastication; and then swallowing individual bat flies Based on these calculations, it was possible to estimate (Additional file 1: Video S1, Additional file 2: Video S2). the number of ectoparasites consumed by individual bats Based on analysis of 10 hours of recordings, we have no per day. In turn, these data were superimposed on an es- evidence of bats spitting out invertebrates or consume timate of the number of Rousettus occupying the cave, more than one individual per feeding bout. taking into account the proportion of adults in relation to the juveniles during the same seasonal period as this Rousettus ectoparasites study [15, 16], to estimate the number of ectoparasites Based on 3577 bat flies collected from 639 individual R. consumed per day within the colony. madagascariensis in the same cave, two species were identified [22, 23]: one streblid (Megastrebla wenzeli) Results and one nycteribiid (Eucampsipoda madagascariensis). Time activity budget for each behavior category The streblid, capable of flight, represented 9.8% of the In total, 68 sessions of 10 min were performed, giving bat flies identified, and the wingless nycteribiid, the rise to approximately 11 hours of video recordings. Only remaining 90.2%. Both of these ectoparasites are 20 min of recording were carried out on 29 June 2017 host-specific to R. madagascariensis with a prevalence of and 60 min each day thereafter until 10 July 2017. As 97.1%, a mean intensity of 8.7 bat flies per host individ- the data were collected during the dry season, about five ual, and a mean abundance of 5.5 and 7.9 bat flies for to six months after the birthing season, the proportion adult female and male of R. madagascariensis, respect- of juveniles was high, but less than adults (Table 2). The ively [22, 24]. Apart from these two dipteran families, bat sex ratio of filmed and analyzed individuals was the only other ectoparasites found on R. madagascarien- biased in favor of males represented by the two age clas- sis were minute Acari, but no quantified information is ses. The number of females with neonates excluded from available on levels of parasitism [25]. the dataset (n = 14 individuals) was insufficient to ac- count for the skewed sex-ratio. Determination of Rousettus age and sex classes On the basis of identification of bat flies occurring on In the context of the recordings made of roosting bats, Rousettus madagascariensis in the same cave [22, 23], it two age classes were established, largely based on body is presumed that the vast majority of bat flies captured size: smaller juveniles and distinctly larger adults. The and consumed by the host were nycteribiids, specifically sex of an individual was determined based on its general Eucampsipoda madagascariensis. Further, no evidence body form and associated external sexual organs, with was found of invertebrates flying-off during a bat groom- males having a visible scrotum-like structure, with either ing bout, which in this case would be the streblid Mega- abdominal or descended testicles, and females with evi- strebla wenzeli; however, it is uncertain if such a detail dence of an indented pelvis and in some cases enlarged would have been observable based on the resolution of mammae. In 14 cases, lactating females with neonates the video recordings. attached to their breast were within the framed area of The most common behavioral activity of the sampled video recording. However, as these neonates were hid- bats was rest, which represented more than 70% of the den under the wings of their mothers, no behavior infor- time spent for diurnal within the day-roost site, followed mation was recorded for either individual. by auto-grooming at 15%. No juvenile was observed to engage in a fight or in any form of ectoparasite con- Data analysis sumption. For sampled adults, the mean proportion of For each 10 min scan sample, the mean proportion of time allocated to consuming ectoparasites via time the different age classes (adult and juvenile) and auto-grooming or allo-grooming was 3.3 ± 4.4% (n = 84) sex classes (male and female) spent conducting the five and 0.6 ± 0.9% (n = 84), respectively (Table 2). The mean types of behavior (Table 1) were calculated. The Spear- proportion of time allotted to consuming ectoparasites man’s rank correlation for data with non-normal distri- via auto-grooming for adults females and males was 2.4 butions was employed using SPSS version 21 [26], to ± 3.9% (n = 26) and 4.1 ± 5.8% (n = 58), respectively, examine the correlation between grooming and ectopar- and in the case of the consumption via allo-grooming asites consumption, stratified by age and sex classes. The 0.4 ± 1.0% (n = 26) and 0.8 ± 1.6% (n = 58), respectively. Wilcoxon rank sum test was performed using R 3.4 [27], Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 4 of 8 Table 2 The proportion of time spent by Rousettus madagascariensis associated with each type of behavior. Mean ± standard deviation (minimum-maximum values) of the proportion of time spent by R. madagascariensis, separated into different age and sex classes, associated with each type of behavior. These values are based on six recording sessions per day, each 10 min, for a daily total of 60 min, and over nine days Age and No. of Rest (%) Crawl (%) Fight (%) Auto-grooming Allo-grooming Consuming ectoparasites Consuming ectoparasites sex classes observations (%) (%) via auto-grooming (%) via allo-grooming (%) Juvenile 48 93.6 ± 2.3 ± 2.7 (0– 0 3.3 ± 10.4 0.8 ± 2.0 00 17.5 10) (0–1.4) (0–8.2) (0–100) Adult 84 73.4 ± 0.7 ± 1.1 (0–5) 3.2 ± 2.6 14.2 ± 10.0 4.7 ± 5.1 3.3 ± 4.4 0.6 ± 0.9 14.3 (0–30) (0–83.1) (0–51.6) (0–26.9) (0–7.7) (20–100) Juvenile 36 93.6 ± 1.1 ± 2.1 (0– 0 4.1 ± 7.0 1.2 ± 2.3 00 female 21.0 10) (0–71.4) (0–8.2) (61.5–100) Adult female 26 73.4 ± 0.7 ± 4.8 (0– 5.1 ± 14.4 (0– 13.7 ± 20.4 4.3 ± 4.1 2.4 ± 3.9 0.4 ± 1.0 12.3 7.1) 70) (0–48.9) (0–16.1) (0–13.3) (0–3.9) (28.8–100) Juvenile male 12 93.6 ± 3.5 ± 2.8 (0– 0 2.5 ± 13.8 0.4 ± 1.7 00 34.8 10) 0–18.0 (0–3.2) (0–100) Adult male 58 73.3 ± 0.7 ± 1.7 (0– 1.3 ± 4.3 14.7 ± 16.2 5.1 ± 9.4 4.1 ± 5.8 0.8 ± 1.6 10.2 3.7) (0–30) (0–83.1) (0–51.6) (0–26.9) (0–7.7) (20–100) Correlation between bat grooming behavior and difference in the proportion of time apportioned to the ectoparasites consumption consumption of ectoparasites via auto-grooming be- The mean proportion of time spent by Rousettus mada- tween adult females and males was not significant (Wil- gascariensis auto-grooming and consuming ectoparasites coxon rank sum test: W= 591, P = 0.101, n = 84; Fig. were significantly correlated for adults (Spearman’s rank 2a), as was the case via allo-grooming (Wilcoxon rank correlation: r = 0.552, P < 0.001, two-tailed, n = 84), as sum test: W= 660, P = 0.226, n = 84; Fig. 2b). well as the mean proportion of time allocated to allo-grooming and consuming ectoparasites (r = 0.353, Differences in ectoparasite consumption in the day roost P = 0.004, two-tailed, n = 84). The correlation between site the mean proportion of time allotted to auto-grooming As mentioned in the methods section, daily data record- and consuming ectoparasites in both adult females (r = ing sessions were divided into six different 10 min pe- 0.715, P < 0.001, two-tailed, n = 26) and adult males (r riods. The consumption of ectoparasites by adult = 0.802, P < 0.001, two-tailed, n = 58) were significant. It Rousettus madagascariensis via auto-grooming showed was the same case for the relation between the mean no significant differences across the six recording ses- proportion of time allocated to allo-grooming and con- sions (Kruskall-Wallis test: χ = 8.5, P = 0.134, n = 84). (5) suming ectoparasites in both adult females (r = 0.401, P In comparison, the amount of time allocated to ingesting = 0.042, two-tailed, n = 26) and adult males (r = 0.447, ectoparasites via allo-grooming during the 18:15 h ses- P < 0.001, two-tailed, n =58). sion was significantly greater than the other five sessions [Kruskall-Wallis test: χ = 13.2, P = 0.022, n = 84, (5) Comparison of proportion of time allocated to grooming Dunn post-hoc test: 18:15 vs 7:15 h (Z = 2.4, P= 0.019, n and consuming ectoparasites between the age and sex = 84), 6:15 vs 8:15 h (Z = 2.9, P= 0.003, n = 84), 6:15 vs classes 13:15 h (Z = 2.8, P= 0.004, n = 84), 6:15 vs 14:15 h (Z = The mean proportion of time allocated to 2.4, P= 0.019, n = 84), 6:15 vs 15:15 pm (Z = 1.9, P= auto-grooming in adults was higher than in juveniles 0.040, n = 84)]. (Wilcoxon rank sum test: W= 2420, P < 0.001, n = 132), We make the assumption that measures of individual as well as the time allocated to allo-grooming (Wilcoxon bat ectoparasite consumption after returning to the cave rank sum test: W= 2541, P < 0.001, n = 132). Only (approximately 5:00 h) until the start of the 7:15 h ses- adults consumed ectoparasites (Table 2, Fig. 1). There sion, are represented from data gathered during the 6:15 was no significant difference in periods spent in h session; this period is referred to herein as roosting auto-grooming between adult females and males (Wil- period 1. The data obtained for ectoparasite consump- coxon rank sum test: W= 736, P = 0.291, n = 84), as tion from the other five sessions (7:15 h, 8:15 h, 13:15 h, well as the amount of time allo-grooming (Wilcoxon 14:15 h, and 15:15 h) is assumed to average rates during rank sum test: W= 726, P = 0.119, n = 84). The the balance of the period in the roost and is referred to Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 5 of 8 Fig. 1 Number of adult and juvenile Rousettus madagascariensis consuming ectoparasites via auto-grooming (a) and allo-grooming (b) based on the different daily recording sessions. The X-axis is scored in the following manner: 0 = no ectoparasite was consumed and 1 = at least a single ectoparasite was consumed. Gray coloration is for adults and black for juveniles Fig. 2 Number of adult female and male Rousettus madagascariensis consuming ectoparasites via auto-grooming (a) and allo-grooming (b) based on the different daily recording sessions. The X-axis is scored in the following manner: 0 = no ectoparasite was consumed and 1 = at least a single ectoparasite was consumed. Gray coloration is for females and black for males Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 6 of 8 as roosting period 2. The difference in the time allocated Proportion of time spent grooming and consuming to consuming ectoparasites via auto-grooming between ectoparasites roosting period 1 and period 2 was significantly different In the present study, the correlation between the time (Wilcoxon rank sum test: W = 799, P = 0.008, n = 84), spent by male and female adults grooming and consum- as was also found for the consumption of ectoparasites ing ectoparasites was significant. Bat flies seem to prefer via allo-grooming (Wilcoxon rank sum test: W = 780, P parasitizing adults, as compared to juveniles, which for = 0.002, n = 84). Estimates of ectoparasites consumption Rousettus madagascariensis might in part be associated per day was calculated with the average ingestion rates with fur density providing greater protection from host for the two roosting periods. grooming [12, 22]; however, given that juveniles were not observed feeding on ectoparasites, this supposition is called into question. Most filmed individuals were Estimation of ectoparasite consumption by Rousettus clustered in close groups and often in direct body con- The number of ectoparasites consumed via tact, which would allow direct dispersal of bat flies be- auto-grooming and allo-grooming was assumed to be tween them. Although adult males have heavier streblid constant during roosting period 1 (from 5:00 to 7:14 h) and nycteribiid parasite loads than adult females and during the roosting period 2 (7:15 to 18:00 h). Dur- throughout the year [22], the amount of time in the day ing the nine days of behavioral recording, the extrapo- roost associated with the removal of ectoparasites is not lated mean total number of ectoparasites that an adult significantly different between the sex classes. consumed during the roosting period 1 was 14 ± 23 ec- In a sort of axiom manner, it would be assumed that toparasites (range 2–67) or about on average 6.2 ecto- ectoparasite consumption by a host animal is a manner to parasites per hour, as compared to the roosting period 2, reduce individual ectoparasite loads, although there is an which was 23 ± 7 ectoparasites (range 10–30) or about energetic cost to grooming [28, 29]. Another advantage of on average 2.1 ectoparasites per hour. The estimated this behavior would be nutritional, in that about 37 ecto- mean number of ectoparasites consumed on a daily basis parasites being consumed by individual adult Rousettus (about 13 hours) by an individual adult R. madagascar- per day would be a measurable energetic supplement, par- iensis while in the roost site was 37 ± 16 ectoparasites ticularly gravid female bat flies, supplementing protein in (range 2–67) or 2.8 ectoparasites per hour. a frugivorous species. In general, fruits are rich in carbo- The study period was during the dry season and the hydrates and generally low in fats and proteins [31]. This number of R. madagascariensis occupying the day-roost is an interesting aspect to be examined in future research, site in September 2016, the same seasonal period, using including nutritional analyses of adult females (with and a capture-mark-recapture technique was estimated as without pupae) and adult males, to evaluate the potential 1908 individuals (95% CI: 675–3517 individuals) [15], contribution of ectoparasite consumption in the dietary and 18% were juveniles [16]. With juveniles removed needs of a frugivorous bat species. from the calculations and based on a population of 1565 adults within the day-roost site, using the 37 ectopara- The periods and rates of daily ectoparasite consumption sites ingested per day by R. madagascariensis, the mean Previous studies on non-Malagasy bats [20, 21, 32] number of ectoparasites consumed was 57,905 ± 25,040 showed that bat-grooming activities are more common ectoparasites (range 18,780–104,855). However, given in the period immediately after bats return to their different aspects of the reproductive strategies of bat day-roost sites, as well as before their dusk exit. The flies [28], combined with a prevalence of 97.1% and the filming sessions in the context of the current study do mean intensity of 6.6 bat flies per adult host parasitized not allow late afternoon activities to be addressed as the [24], our inference of daily consumption is certainly last session was at 15:15 h. However, our data indicates overestimated as the estimated bat fly population in the a higher rate of grooming and ectoparasite removal in cave is estimated at 10,025 ectoparasites associated with the early morning period. the R. madagascariensis colony. Previous research on Rousettus madagascariensis at the same study site found seasonal differences in the average number of parasites per host, with higher infest- Discussion ation rates during the dry season (June and October) Diurnal activities of Rousettus madagascariensis [24], which coincides with the data reported herein. Fur- Among the different behavioral activities, adults spent ther, in this bat species, ectoparasite loads are correlated more than 70% of their time at rest, followed by approxi- with host sex and age, with adult males having the high- mately 14% in auto-grooming, and 4% in allo-grooming est rates [23]. Hence, the data presented herein presum- (Table 2). These proportions are similar to those for ably represent the period with the highest levels of bat other non-Malagasy species of bats [20, 29, 30]. fly parasitism rates in R. madagascariensis. Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 7 of 8 As mentioned above, the daily estimated consump- Additional files tion by Rousettus in the Grotte des Chauves-souris Additional file 1: Video S1. Consumption of ectoparasites by adult male day roost colony of close to 58,000 ectoparasites is Rousettus madagascariensis via auto-grooming. (WMV 10725 kb) simply overestimated. This is presumably related to Additional file 2: Video S2. Consumption of ectoparasites by adult male our extrapolations of consumption rates during pe- Rousettus madagascariensis via allo-grooming. (AVI 7806 kb) riods video recordings were not made and certain cases of recorded mastication that were not associ- Acknowledgements ated with the ingestion of bat flies. However, in any We are grateful to the Mention Biologie Zoologie et Biodiversité Animale at the Université d’Antananarivo, Madagascar National Parks (MNP), and case, this study provides new insight into Direction Générale des Forêts for administrative aid and permits (no. 055/17/ host-ectoparasite interactions and population dynam- MEEF/SG/DGF/DSAP/SCB.Re) to conduct this study. We thank the Director of ics of bat flies. These high levels of ectoparasite in- the Ankarana National Park, M. Nicolas Salo, who gave us permission to work in the Ankarana caves. M. Romialy (Baban’i Rapila) kindly assisted the first gestion underscores new questions if bat fly author in the field. consumption by the bat host is associated with regu- lation of their population size associated with body Funding hygiene, a defense mechanism against parasites and/ The study was supported by the grant from the Helmsley Charitable Trust to the Association Vahatra. or as a dietary supplement; further, there is the po- tential role of this behavior in the transmission of Availability of data and materials zoonotic pathogens to the host. Bacteria of the Data supporting the conclusion of this article are included within the article genus Bartonella have been identified in Eidolon and in its additional files. The datasets used are available from the corresponding author upon reasonable request. dupreanum, another endemic Malagasy Pteropodidae and cave-dwelling fruit bat, and from its Authors’ contributions hematophagous nycteribiid Cyclopodia dubia [4]. We RVR and SMG conceived the study, performed the experiment, and wrote the manuscript. SMG supervised the fieldwork. RVR and AA performed the suspect that with testing, R. madagascariensis bat data analysis. All authors edited different versions of the manuscript. All flies will also be found to be positive for Bartonella. authors read and approved the final manuscript. Further, previous research has shown that bats are more vulnerable to bacterial infections [33], as com- Ethics approval and consent to participate All video recordings which were authorized by the Mention Biologie pared to viral infections, and bacterial pathogens Zoologie et Biodiversité Animale at the Université d’Antananarivo, have been isolated from bat flies [4]. Madagascar National Parks (MNP), and Direction Générale des Forêts (authorization no. 055/17/MEEF/SG/DGF/DSAP/SCB.Re). Competing interests Conclusions The authors declare that they have no competing interests. Only adult Rousettus madagascariensis were found to consume ectoparasites via both auto-grooming and Publisher’sNote allo-grooming and a significant positive correlation Springer Nature remains neutral with regard to jurisdictional claims in was found between the proportion of time allocated published maps and institutional affiliations. to grooming and ectoparasite consumption in both Author details sexes of adult bats. The considerable estimate of bat Mention Zoologie et Biodiversité Animale, Université d’Antananarivo, BP fly consumption by their hosts provides a new dimen- 906, (101) Antananarivo, Madagascar. Association Vahatra, BP 3972, (101) Antananarivo, Madagascar. Field Museum of Natural History, 1400 South sion to population dynamics and reproductive strat- Lake Shore Drive, Chicago, Illinois 60605, USA. egies in bat flies. The results presented here will need to be further tested, for example, with a video cam- Received: 14 March 2018 Accepted: 25 May 2018 corder apparatus with high resolution to better docu- ment physical consumption of ectoparasites and References extended across the complete period bats are occupy- 1. Brook CE, Dobson AP. Bats as “special” reservoirs for emerging zoonotic ing their day roost. While to our knowledge, rates of pathogens. Trends Microbiol. 2015;23:172–80. 2. Wilkinson DA, Mélade J, Dietrich M, Ramasindrazana B, Soarimalala V, bat ingestion of their dipteran ectoparasites has not Lagadec E, et al. Highly diverse morbillivirus-related paramyxoviruses in wild been previously estimated, the results presented fauna of the southwestern Indian Ocean islands: evidence of exchange herein lead to a series of questions ranging from the between introduced and endemic small mammals. J Virol. 2014;88:8268–77. 3. Wilkinson DA, Duron O, Cordonin C, Gomard Y, Ramasindrazana B, Mavingui nutritional advantages to the host bat in ectoparasite P, et al. The bacteriome of bat flies (Nycteribiidae) from the Malagasy consumption to possible mechanisms for the trans- region: a community shaped by host ecology, bacterial transmission mode, mission of zoonotic diseases via an oral route be- and host-vector specificity. Appl Environ Microbiol. 2016; https://doi.org/10. 1128/AEM.03505-15. tween ectoparasite-host, as compared to blood meals 4. Brook CE, Bai Y, Dobson AP, Osikowicz LM, Ranaivoson HC, Zhu Q, et al. by these hematophagous ectoparasites. These are as- Bartonella spp. in fruit bats and blood-feeding ectoparasites in Madagascar. pects need to be addressed by future research. PLoS Negl Trop Dis. 2015;9:e0003532. Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 8 of 8 5. Morse SF, Olival KJ, Kosoy M, Billeter S, Patterson BD, Dick CW, et al. Global 28. Marshall AG. Ecology of insects ectoparasitic on bats. In: Kunz TH, editor. distribution and genetic diversity of Bartonella in bat flies (Hippoboscoidea, Ecology of bats. New York: Plenum Publishing Corp; 1982. p. 369–401. Streblidae, Nycteribiidae). Infect Genet Evol. 2012;12:1717–23. 29. Burnett CD, August PV. Time and energy budgets for day roosting in a 6. Aznar-Lopez C, Vazquez-Moron S, Marston DA, Juste J, Ibáñez C, Berciano maternity colony of Myotis lucifugus. J Mammal. 1981;62:758–66. JM, et al. Detection of rhabdovirus viral RNA in oropharyngeal swabs and 30. Codd JR, Sanderson KJ, Branford AJ. Roosting activity budget of the ectoparasites of Spanish bats. J Gen Virol. 2013;94:69–75. southern bent-wing bat (Miniopterus schreibersii bassanii). Aust J Zool. 2003; 51:307–16. 7. Goldberg TL, Bennett AJ, Kityo R, Kuhn JH, Chapman CA. Kanyawara virus: a novel rhabdovirus infecting newly discovered nycteribiid bat flies infesting 31. Ruby J, Nathan PT, Balasingh J, Kunz TH. 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Johnson PTJ, Dobson A, Lafferty KD, Marcogliese DJ, Memmott J, Orlofske SA, et al. When parasites become prey: ecological and epidemiological significance of eating parasites. Trends Ecol Evol. 2010;25:362–71. 11. Orlofske SA, Jadin RC, Johnson PTJ. It’s a predator-eat-parasite world: how characteristics of predator, parasite and environment affect consumption. Oecologia. 2015;178:537–47. 12. Overal WL. Host-relations of the bat fly Megistopoda aranea (Diptera: Streblidae) in Panama. Univ Kansas Sci Bull. 1980;52:1–20. 13. Marshall AG. The ecology of the bat ectoparasite Eoctenes spasmae (Hemiptera: Polyctenidae) in Malaysia. Biotropica. 1982;14:50–5. 14. Cardiff SG, Befourouack J. La réserve spéciale de l’Ankarana. In: Goodman SM, editor. Paysage naturels et biodiversité de Madagascar. Paris: Publications scientifiques du Muséum; 2008. p. 571–84. 15. Noroalintseheno Lalarivoniaina OS, Rajemison FI, Goodman SM. Survie et variation temporelle de la taille de la population de Rousettus madagascariensis (Chiroptera: Pteropodidae) de la Grotte des Chauves-souris d’Ankarana, nord de Madagascar. Malagasy Nat. 2017;12:68–77. 16. Noroalintseheno Lalarivoniaina OS, Rajemison FI, Andrianarimisa A, Goodman SM. Variation saisonnière de la structure d’âge et de la sex-ratio de la population de Rousettus madagascariensis (Yinpterochiroptera: Pteropodidae) à Ankarana, nord de Madagascar. Rev Ecol (Terre Vie). 2018;73:23–30. 17. Cardiff SG, Ratrimomanarivo FH, Goodman SM. The effect of tourist visits on the behavior of Rousettus madagascariensis (Chiroptera: Pteropodidae) in the caves of Ankarana, northern Madagascar. Acta Chiropt. 2012;14:479–90. 18. Altmann J. Observational study of behavior: sampling methods. Behaviour. 1974;49:227–67. 19. Martin P, Bateson P. Measuring behaviour: an introductory guide. 3rd ed. Cambridge: Cambridge University Press; 2007. 20. Markus N, Blackshaw JK. Behaviour of the black flying fox Pteropus alecto:1. An ethogram of behaviour, and preliminary characterisation of mother- infant interactions. Acta Chiropt. 2002;4:137–52. 21. Winchell JM, Kunz TH. Sampling protocols for estimating time budgets of roosting bats. Can J Zool. 1993;71:2244–9. 22. Rajemison FI, Noroalintseheno Lalarivoniaina OS, Goodman SM. Bat flies (Diptera: Nycteribiidae, Streblidae) parasitising Rousettus madagascariensis (Chiroptera: Pteropodidae) in the Parc National d’Ankarana, Madagascar: species diversity, rates of parasitism and sex ratios. Afr Entomol. 2017;25:72–85. 23. Rajemison FI, Noroalintseheno Lalarivoniaina OS, Goodman SM. Parasitism by Nycteribiidae and Streblidae flies (Diptera) of a Malagasy fruit bat (Pteropodidae): effects of body size and throat gland development on parasite abundance. J Med Entomol. 2017;54:805–11. 24. Rajemison FI. Etude bio-écologique des mouches ectoparasites, Nycteribiidae et Streblidae (Insecta: Diptera), de Rousettus madagascariensis G. Grandidier, 1928 (Chiroptera: Pteropodidae) dans le Parc National d’Ankarana, Madagascar. Madagascar: PhD Thesis. Université d’Antananarivo, Antananarivo; 2017. 25. Rajemison FI, Noroalintseheno Lalarivoniaina OS, Andrianarimisa A, Goodman SM. Host-parasite relationships between a Malagasy fruit bat (Pteropodidae) and associated bat fly (Diptera: Nycteribiidae): seasonal variation of host body condition and the possible impact of parasite abundance. Acta Chiropt. 2017;19:229–38. 26. IBM Corporation. IBM SPSS Statistics for Windows, version 21.0. New York: Armonk; 2012. 27. R Core Team. R: a language and environnement for statistical computing, R foundation for statistical computing. 2017. http://www.R-project.org. Accessed 21 Dec 2017. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Parasites & Vectors Springer Journals

Rates of hematophagous ectoparasite consumption during grooming by an endemic Madagascar fruit bat

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Biomedicine; Parasitology; Entomology; Tropical Medicine; Infectious Diseases; Veterinary Medicine/Veterinary Science; Virology
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Abstract

Background: Few details are available on the consumption of ectoparasites, specifically bat flies (Diptera: Nycteribiidae and Streblidae), by their chiropteran hosts while grooming. Such details are important to document consumption rates of ectoparasites by their bat host provide details on the dynamics of host-parasite interactions. We present data on ectoparasite consumption rates for an endemic Malagasy fruit bat (Pteropodidae: Rousettus madagascariensis) occupying a cave day roost colony in northern Madagascar. Using quantified behavioral analyses, grooming and associated ingestion rates were measured from infrared videos taken in close proximity to day- roosting bats. The recorded individual bats could be visually identified to age (adult, juvenile) and sex (male, female), allowing analyses of the proportion of time these different classes allocated to consuming ectoparasites via auto-grooming (self) or allo-grooming (intraspecific) per 10 min video recording session. These figures could then be extrapolated to estimates of individual daily consumption rates. Results: Based on video recordings, adults spent significantly more time auto-grooming and allo-grooming than juveniles. The latter group was not observed consuming ectoparasites. Grooming rates and the average number of ectoparasites consumed per day did not differ between adult males and females. The mean extrapolated number consumed on a daily basis for individual adults was 37 ectoparasites. When these figures are overlaid on the estimated number of adult Rousettus occurring at the roost site during the dry season, the projected daily consumption rate was 57,905 ectoparasites. Conclusions: The details presented here represent the first quantified data on bat consumption rates of their ectoparasites, specifically dipterans. These results provide new insights in host-parasite predation dynamics. More research is needed to explore the mechanism zoonotic diseases isolated from bat flies might be transmitted to their bat hosts, specifically those pathogens that can be communicated via an oral route. Keywords: Diptera, Nycteribiidae, Pteropodidae, Rousettus madagascariensis, Madagascar, Host-parasite interactions Background such as rhabdovirus [6, 7]; and protozoans including the The past decade has seen an important increase in stud- hematoparasite Polychromophilus [8, 9]. Hence, there is ies of bats as reservoirs of diverse diseases [1–3]. An- indirect evidence that bat ectoparasites may be respon- other facet of this research has shown that bat sible for the transmission of infectious diseases to their ectoparasites, specifically hematophagous bat flies of the hosts, although it remains to be demonstrated that this families Streblidae and Nycteribiidae, are possible reser- could be via an oral route, specifically direct consump- voirs and vectors for different zoonotic diseases, e.g. bac- tion of ectoparasites by bats. teria such as Bartonella and Rickettsia [3–5]; viruses Although there are growing literature data on the im- portance of ectoparasites as prey and the associated epi- demiological implications [10, 11], only a few studies * Correspondence: rianaval@yahoo.fr Mention Zoologie et Biodiversité Animale, Université d’Antananarivo, BP that have documented ingestion rates by bats of their ec- 906, (101) Antananarivo, Madagascar toparasites. A project conducted on New World stre- Association Vahatra, BP 3972, (101) Antananarivo, Madagascar blids found evidence that the fruit bat Artibeus 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. Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 2 of 8 jamaicensis (Phyllostomidae) may actively feed on its bat recordings were made in an area of the cave outside the flies of the genus Megistopoda [12]. Further, captive A. typical visitor circuit. jamaicensis readily accepted and consumed Megistopoda Each day of behavioral recordings made in the cave, bat flies and the remains of streblids were identified in sessions commenced at 6:15 h, 7:15 h, 8:15 h, 13:15 h, the stomachs of different species of Costa Rican bats 14:15 h, and 15:15 h. Recordings were made for 10 min [12]. Further, in Malaysia, the bat Megaderma spasma per session, which on a daily basis resulted in one hour (Megadermatidae) feeds actively on Eoctenes spasmae of recording (six sessions × 10 min). The sessions com- (Cimicidae) ectoparasites [13]. Hence, there is indeed mencing at 6:15 h and 13:15 h, when the recording ma- evidence that bats feed on their dipteran parasites, but terial was installed during a period of about 30 min, quantitative details on occurrence and frequency are were followed by 15 min of no activity near the roosting lacking in the literature. bats, to allow them to calm down after being disturbed. The purpose of this paper is to present quantified be- The start and termination of the video recording for the havioral observations on the proportion of time while in other sessions were controlled with the remote device. the day roost site an endemic Malagasy fruit bat grooms The methods for data sampling and processing and actively consumes ectoparasites overlaid on possible followed a previous behavioral study on R. madagascar- age and sex differences. Extrapolations are made as to iensis in the same cave [17]. Video recordings were the average daily ectoparasite consumption rates per in- reviewed on a computer screen measuring 40.5 × 16.2 dividual bat. These data provide new insights into cm, and scored based on behavioral variables using a host-ectoparasite predation dynamics, as well as a pos- scan sampling method [18, 19]. An ethogram (Table 1), sible means zoonotic diseases isolated from bat flies, derived from previous studies of non-Malagasy bat spe- specifically those communicated through oral means, cies [20, 21], was created to categorize and quantify the might be transmitted to their hosts through the con- behavior of R. madagascariensis. sumption of ectoparasites. Scan sampling Methods A scan sampling technique was used to document different Study area behavioral states of target individuals within a group and to Data were collected between 29 June to 10 July 2017 at calculate an individual's time activity budget (i.e. the propor- a day roost site of Rousettus madagascariensis (Chir- tion of time spent in an activity) [17]. One scan sampling optera: Pteropodidae) in the Grotte des Chauves-souris consisted of following five individual bats based on the re- (12°57.4'S, 49°7.1'E), Parc National d’Ankarana in the far corded video, each independently for three seconds and for north of Madagascar. Ankarana is characterized by lime- 10 rounds of observation. Given that in certain cases, an in- stone karst formations and the natural habitat is dry de- dividual disappeared from or others flew into view, two ciduous forest [14]. The study was conducted during the other groups of five individuals were also followed during dry season, and estimates are available for this period on the 10 min period. The proportion of time each individual the number and age ratio of R. madagascariensis occu- performed each of the following behavioral activities was pying the day roost site in the same cave [15, 16]. calculated: rest, crawling into or outside of the focal cluster, inter-bat aggression (“fighting”), auto-grooming Video recording (self-grooming), allo-grooming (associated with the body Data on grooming behavior, specifically bat feeding rates of a neighboring animal), and consuming ectoparasites on on ectoparasites, were calculated via the analysis of re- cordings made with an infrared video camcorder [1080p Table 1 Ethogram of behavior for the scan sampling methods HD Infrared Night Vision and Full Spectrum Camcorder Behavior categories Description (Cleveland Paranormal Supply Co., Mentor, OH, USA)] Rest Not moving any part of the body employing an infrared light for supplementary illumin- Crawl Alternate placement of feet or/and ation [Evolva Future Technology T38 IR 38 mm lens in- thumb claws to shift frared flashlight light night vision torch (Shenzhen position on the rock face Dikesai International Trade Co., Ltd, Guangdong)]. The Groom Scratching or licking the peak intensity wavelengths of these apparatus are 810 body or/and wing phalanges nm. The camcorder, which was connected to a remote Fight Biting or hooking control, was placed at a fixed place within the cave, ap- (rapid swiping with thumb claw) the opponent proximately 4 m from the target Rousettus madagascar- iensis group, with the intent in following individual bat Consume ectoparasites Taking ectoparasites into the mouth, masticating, and no activities. Tourists visit the cave, which disrupts the nat- evidence of rejecting them ural behavior of this species [17], and the video Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 3 of 8 its own body via auto-grooming (Additional file 1: Video to examine possible differences in grooming behavior S1) or a neighboring animal via allo-grooming (Additional and ectoparasites consumption between age and sex file 2: Video S2). classes. The Kruskall-Wallis test with post-hoc Dunn On the basis of recorded video, the sequence of steps tests and the Wilcoxon rank test with R 3.4 [27], were that bats seized ectoparasites and consumed them is as both employed to investigate the relation between ses- follows: detainment with their teeth, tongue, or lips; sion periods and the consumption of ectoparasites. mastication; and then swallowing individual bat flies Based on these calculations, it was possible to estimate (Additional file 1: Video S1, Additional file 2: Video S2). the number of ectoparasites consumed by individual bats Based on analysis of 10 hours of recordings, we have no per day. In turn, these data were superimposed on an es- evidence of bats spitting out invertebrates or consume timate of the number of Rousettus occupying the cave, more than one individual per feeding bout. taking into account the proportion of adults in relation to the juveniles during the same seasonal period as this Rousettus ectoparasites study [15, 16], to estimate the number of ectoparasites Based on 3577 bat flies collected from 639 individual R. consumed per day within the colony. madagascariensis in the same cave, two species were identified [22, 23]: one streblid (Megastrebla wenzeli) Results and one nycteribiid (Eucampsipoda madagascariensis). Time activity budget for each behavior category The streblid, capable of flight, represented 9.8% of the In total, 68 sessions of 10 min were performed, giving bat flies identified, and the wingless nycteribiid, the rise to approximately 11 hours of video recordings. Only remaining 90.2%. Both of these ectoparasites are 20 min of recording were carried out on 29 June 2017 host-specific to R. madagascariensis with a prevalence of and 60 min each day thereafter until 10 July 2017. As 97.1%, a mean intensity of 8.7 bat flies per host individ- the data were collected during the dry season, about five ual, and a mean abundance of 5.5 and 7.9 bat flies for to six months after the birthing season, the proportion adult female and male of R. madagascariensis, respect- of juveniles was high, but less than adults (Table 2). The ively [22, 24]. Apart from these two dipteran families, bat sex ratio of filmed and analyzed individuals was the only other ectoparasites found on R. madagascarien- biased in favor of males represented by the two age clas- sis were minute Acari, but no quantified information is ses. The number of females with neonates excluded from available on levels of parasitism [25]. the dataset (n = 14 individuals) was insufficient to ac- count for the skewed sex-ratio. Determination of Rousettus age and sex classes On the basis of identification of bat flies occurring on In the context of the recordings made of roosting bats, Rousettus madagascariensis in the same cave [22, 23], it two age classes were established, largely based on body is presumed that the vast majority of bat flies captured size: smaller juveniles and distinctly larger adults. The and consumed by the host were nycteribiids, specifically sex of an individual was determined based on its general Eucampsipoda madagascariensis. Further, no evidence body form and associated external sexual organs, with was found of invertebrates flying-off during a bat groom- males having a visible scrotum-like structure, with either ing bout, which in this case would be the streblid Mega- abdominal or descended testicles, and females with evi- strebla wenzeli; however, it is uncertain if such a detail dence of an indented pelvis and in some cases enlarged would have been observable based on the resolution of mammae. In 14 cases, lactating females with neonates the video recordings. attached to their breast were within the framed area of The most common behavioral activity of the sampled video recording. However, as these neonates were hid- bats was rest, which represented more than 70% of the den under the wings of their mothers, no behavior infor- time spent for diurnal within the day-roost site, followed mation was recorded for either individual. by auto-grooming at 15%. No juvenile was observed to engage in a fight or in any form of ectoparasite con- Data analysis sumption. For sampled adults, the mean proportion of For each 10 min scan sample, the mean proportion of time allocated to consuming ectoparasites via time the different age classes (adult and juvenile) and auto-grooming or allo-grooming was 3.3 ± 4.4% (n = 84) sex classes (male and female) spent conducting the five and 0.6 ± 0.9% (n = 84), respectively (Table 2). The mean types of behavior (Table 1) were calculated. The Spear- proportion of time allotted to consuming ectoparasites man’s rank correlation for data with non-normal distri- via auto-grooming for adults females and males was 2.4 butions was employed using SPSS version 21 [26], to ± 3.9% (n = 26) and 4.1 ± 5.8% (n = 58), respectively, examine the correlation between grooming and ectopar- and in the case of the consumption via allo-grooming asites consumption, stratified by age and sex classes. The 0.4 ± 1.0% (n = 26) and 0.8 ± 1.6% (n = 58), respectively. Wilcoxon rank sum test was performed using R 3.4 [27], Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 4 of 8 Table 2 The proportion of time spent by Rousettus madagascariensis associated with each type of behavior. Mean ± standard deviation (minimum-maximum values) of the proportion of time spent by R. madagascariensis, separated into different age and sex classes, associated with each type of behavior. These values are based on six recording sessions per day, each 10 min, for a daily total of 60 min, and over nine days Age and No. of Rest (%) Crawl (%) Fight (%) Auto-grooming Allo-grooming Consuming ectoparasites Consuming ectoparasites sex classes observations (%) (%) via auto-grooming (%) via allo-grooming (%) Juvenile 48 93.6 ± 2.3 ± 2.7 (0– 0 3.3 ± 10.4 0.8 ± 2.0 00 17.5 10) (0–1.4) (0–8.2) (0–100) Adult 84 73.4 ± 0.7 ± 1.1 (0–5) 3.2 ± 2.6 14.2 ± 10.0 4.7 ± 5.1 3.3 ± 4.4 0.6 ± 0.9 14.3 (0–30) (0–83.1) (0–51.6) (0–26.9) (0–7.7) (20–100) Juvenile 36 93.6 ± 1.1 ± 2.1 (0– 0 4.1 ± 7.0 1.2 ± 2.3 00 female 21.0 10) (0–71.4) (0–8.2) (61.5–100) Adult female 26 73.4 ± 0.7 ± 4.8 (0– 5.1 ± 14.4 (0– 13.7 ± 20.4 4.3 ± 4.1 2.4 ± 3.9 0.4 ± 1.0 12.3 7.1) 70) (0–48.9) (0–16.1) (0–13.3) (0–3.9) (28.8–100) Juvenile male 12 93.6 ± 3.5 ± 2.8 (0– 0 2.5 ± 13.8 0.4 ± 1.7 00 34.8 10) 0–18.0 (0–3.2) (0–100) Adult male 58 73.3 ± 0.7 ± 1.7 (0– 1.3 ± 4.3 14.7 ± 16.2 5.1 ± 9.4 4.1 ± 5.8 0.8 ± 1.6 10.2 3.7) (0–30) (0–83.1) (0–51.6) (0–26.9) (0–7.7) (20–100) Correlation between bat grooming behavior and difference in the proportion of time apportioned to the ectoparasites consumption consumption of ectoparasites via auto-grooming be- The mean proportion of time spent by Rousettus mada- tween adult females and males was not significant (Wil- gascariensis auto-grooming and consuming ectoparasites coxon rank sum test: W= 591, P = 0.101, n = 84; Fig. were significantly correlated for adults (Spearman’s rank 2a), as was the case via allo-grooming (Wilcoxon rank correlation: r = 0.552, P < 0.001, two-tailed, n = 84), as sum test: W= 660, P = 0.226, n = 84; Fig. 2b). well as the mean proportion of time allocated to allo-grooming and consuming ectoparasites (r = 0.353, Differences in ectoparasite consumption in the day roost P = 0.004, two-tailed, n = 84). The correlation between site the mean proportion of time allotted to auto-grooming As mentioned in the methods section, daily data record- and consuming ectoparasites in both adult females (r = ing sessions were divided into six different 10 min pe- 0.715, P < 0.001, two-tailed, n = 26) and adult males (r riods. The consumption of ectoparasites by adult = 0.802, P < 0.001, two-tailed, n = 58) were significant. It Rousettus madagascariensis via auto-grooming showed was the same case for the relation between the mean no significant differences across the six recording ses- proportion of time allocated to allo-grooming and con- sions (Kruskall-Wallis test: χ = 8.5, P = 0.134, n = 84). (5) suming ectoparasites in both adult females (r = 0.401, P In comparison, the amount of time allocated to ingesting = 0.042, two-tailed, n = 26) and adult males (r = 0.447, ectoparasites via allo-grooming during the 18:15 h ses- P < 0.001, two-tailed, n =58). sion was significantly greater than the other five sessions [Kruskall-Wallis test: χ = 13.2, P = 0.022, n = 84, (5) Comparison of proportion of time allocated to grooming Dunn post-hoc test: 18:15 vs 7:15 h (Z = 2.4, P= 0.019, n and consuming ectoparasites between the age and sex = 84), 6:15 vs 8:15 h (Z = 2.9, P= 0.003, n = 84), 6:15 vs classes 13:15 h (Z = 2.8, P= 0.004, n = 84), 6:15 vs 14:15 h (Z = The mean proportion of time allocated to 2.4, P= 0.019, n = 84), 6:15 vs 15:15 pm (Z = 1.9, P= auto-grooming in adults was higher than in juveniles 0.040, n = 84)]. (Wilcoxon rank sum test: W= 2420, P < 0.001, n = 132), We make the assumption that measures of individual as well as the time allocated to allo-grooming (Wilcoxon bat ectoparasite consumption after returning to the cave rank sum test: W= 2541, P < 0.001, n = 132). Only (approximately 5:00 h) until the start of the 7:15 h ses- adults consumed ectoparasites (Table 2, Fig. 1). There sion, are represented from data gathered during the 6:15 was no significant difference in periods spent in h session; this period is referred to herein as roosting auto-grooming between adult females and males (Wil- period 1. The data obtained for ectoparasite consump- coxon rank sum test: W= 736, P = 0.291, n = 84), as tion from the other five sessions (7:15 h, 8:15 h, 13:15 h, well as the amount of time allo-grooming (Wilcoxon 14:15 h, and 15:15 h) is assumed to average rates during rank sum test: W= 726, P = 0.119, n = 84). The the balance of the period in the roost and is referred to Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 5 of 8 Fig. 1 Number of adult and juvenile Rousettus madagascariensis consuming ectoparasites via auto-grooming (a) and allo-grooming (b) based on the different daily recording sessions. The X-axis is scored in the following manner: 0 = no ectoparasite was consumed and 1 = at least a single ectoparasite was consumed. Gray coloration is for adults and black for juveniles Fig. 2 Number of adult female and male Rousettus madagascariensis consuming ectoparasites via auto-grooming (a) and allo-grooming (b) based on the different daily recording sessions. The X-axis is scored in the following manner: 0 = no ectoparasite was consumed and 1 = at least a single ectoparasite was consumed. Gray coloration is for females and black for males Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 6 of 8 as roosting period 2. The difference in the time allocated Proportion of time spent grooming and consuming to consuming ectoparasites via auto-grooming between ectoparasites roosting period 1 and period 2 was significantly different In the present study, the correlation between the time (Wilcoxon rank sum test: W = 799, P = 0.008, n = 84), spent by male and female adults grooming and consum- as was also found for the consumption of ectoparasites ing ectoparasites was significant. Bat flies seem to prefer via allo-grooming (Wilcoxon rank sum test: W = 780, P parasitizing adults, as compared to juveniles, which for = 0.002, n = 84). Estimates of ectoparasites consumption Rousettus madagascariensis might in part be associated per day was calculated with the average ingestion rates with fur density providing greater protection from host for the two roosting periods. grooming [12, 22]; however, given that juveniles were not observed feeding on ectoparasites, this supposition is called into question. Most filmed individuals were Estimation of ectoparasite consumption by Rousettus clustered in close groups and often in direct body con- The number of ectoparasites consumed via tact, which would allow direct dispersal of bat flies be- auto-grooming and allo-grooming was assumed to be tween them. Although adult males have heavier streblid constant during roosting period 1 (from 5:00 to 7:14 h) and nycteribiid parasite loads than adult females and during the roosting period 2 (7:15 to 18:00 h). Dur- throughout the year [22], the amount of time in the day ing the nine days of behavioral recording, the extrapo- roost associated with the removal of ectoparasites is not lated mean total number of ectoparasites that an adult significantly different between the sex classes. consumed during the roosting period 1 was 14 ± 23 ec- In a sort of axiom manner, it would be assumed that toparasites (range 2–67) or about on average 6.2 ecto- ectoparasite consumption by a host animal is a manner to parasites per hour, as compared to the roosting period 2, reduce individual ectoparasite loads, although there is an which was 23 ± 7 ectoparasites (range 10–30) or about energetic cost to grooming [28, 29]. Another advantage of on average 2.1 ectoparasites per hour. The estimated this behavior would be nutritional, in that about 37 ecto- mean number of ectoparasites consumed on a daily basis parasites being consumed by individual adult Rousettus (about 13 hours) by an individual adult R. madagascar- per day would be a measurable energetic supplement, par- iensis while in the roost site was 37 ± 16 ectoparasites ticularly gravid female bat flies, supplementing protein in (range 2–67) or 2.8 ectoparasites per hour. a frugivorous species. In general, fruits are rich in carbo- The study period was during the dry season and the hydrates and generally low in fats and proteins [31]. This number of R. madagascariensis occupying the day-roost is an interesting aspect to be examined in future research, site in September 2016, the same seasonal period, using including nutritional analyses of adult females (with and a capture-mark-recapture technique was estimated as without pupae) and adult males, to evaluate the potential 1908 individuals (95% CI: 675–3517 individuals) [15], contribution of ectoparasite consumption in the dietary and 18% were juveniles [16]. With juveniles removed needs of a frugivorous bat species. from the calculations and based on a population of 1565 adults within the day-roost site, using the 37 ectopara- The periods and rates of daily ectoparasite consumption sites ingested per day by R. madagascariensis, the mean Previous studies on non-Malagasy bats [20, 21, 32] number of ectoparasites consumed was 57,905 ± 25,040 showed that bat-grooming activities are more common ectoparasites (range 18,780–104,855). However, given in the period immediately after bats return to their different aspects of the reproductive strategies of bat day-roost sites, as well as before their dusk exit. The flies [28], combined with a prevalence of 97.1% and the filming sessions in the context of the current study do mean intensity of 6.6 bat flies per adult host parasitized not allow late afternoon activities to be addressed as the [24], our inference of daily consumption is certainly last session was at 15:15 h. However, our data indicates overestimated as the estimated bat fly population in the a higher rate of grooming and ectoparasite removal in cave is estimated at 10,025 ectoparasites associated with the early morning period. the R. madagascariensis colony. Previous research on Rousettus madagascariensis at the same study site found seasonal differences in the average number of parasites per host, with higher infest- Discussion ation rates during the dry season (June and October) Diurnal activities of Rousettus madagascariensis [24], which coincides with the data reported herein. Fur- Among the different behavioral activities, adults spent ther, in this bat species, ectoparasite loads are correlated more than 70% of their time at rest, followed by approxi- with host sex and age, with adult males having the high- mately 14% in auto-grooming, and 4% in allo-grooming est rates [23]. Hence, the data presented herein presum- (Table 2). These proportions are similar to those for ably represent the period with the highest levels of bat other non-Malagasy species of bats [20, 29, 30]. fly parasitism rates in R. madagascariensis. Ramanantsalama et al. Parasites & Vectors (2018) 11:330 Page 7 of 8 As mentioned above, the daily estimated consump- Additional files tion by Rousettus in the Grotte des Chauves-souris Additional file 1: Video S1. Consumption of ectoparasites by adult male day roost colony of close to 58,000 ectoparasites is Rousettus madagascariensis via auto-grooming. (WMV 10725 kb) simply overestimated. This is presumably related to Additional file 2: Video S2. Consumption of ectoparasites by adult male our extrapolations of consumption rates during pe- Rousettus madagascariensis via allo-grooming. (AVI 7806 kb) riods video recordings were not made and certain cases of recorded mastication that were not associ- Acknowledgements ated with the ingestion of bat flies. However, in any We are grateful to the Mention Biologie Zoologie et Biodiversité Animale at the Université d’Antananarivo, Madagascar National Parks (MNP), and case, this study provides new insight into Direction Générale des Forêts for administrative aid and permits (no. 055/17/ host-ectoparasite interactions and population dynam- MEEF/SG/DGF/DSAP/SCB.Re) to conduct this study. We thank the Director of ics of bat flies. These high levels of ectoparasite in- the Ankarana National Park, M. Nicolas Salo, who gave us permission to work in the Ankarana caves. M. Romialy (Baban’i Rapila) kindly assisted the first gestion underscores new questions if bat fly author in the field. consumption by the bat host is associated with regu- lation of their population size associated with body Funding hygiene, a defense mechanism against parasites and/ The study was supported by the grant from the Helmsley Charitable Trust to the Association Vahatra. or as a dietary supplement; further, there is the po- tential role of this behavior in the transmission of Availability of data and materials zoonotic pathogens to the host. Bacteria of the Data supporting the conclusion of this article are included within the article genus Bartonella have been identified in Eidolon and in its additional files. The datasets used are available from the corresponding author upon reasonable request. dupreanum, another endemic Malagasy Pteropodidae and cave-dwelling fruit bat, and from its Authors’ contributions hematophagous nycteribiid Cyclopodia dubia [4]. We RVR and SMG conceived the study, performed the experiment, and wrote the manuscript. SMG supervised the fieldwork. RVR and AA performed the suspect that with testing, R. madagascariensis bat data analysis. All authors edited different versions of the manuscript. All flies will also be found to be positive for Bartonella. authors read and approved the final manuscript. Further, previous research has shown that bats are more vulnerable to bacterial infections [33], as com- Ethics approval and consent to participate All video recordings which were authorized by the Mention Biologie pared to viral infections, and bacterial pathogens Zoologie et Biodiversité Animale at the Université d’Antananarivo, have been isolated from bat flies [4]. Madagascar National Parks (MNP), and Direction Générale des Forêts (authorization no. 055/17/MEEF/SG/DGF/DSAP/SCB.Re). Competing interests Conclusions The authors declare that they have no competing interests. Only adult Rousettus madagascariensis were found to consume ectoparasites via both auto-grooming and Publisher’sNote allo-grooming and a significant positive correlation Springer Nature remains neutral with regard to jurisdictional claims in was found between the proportion of time allocated published maps and institutional affiliations. to grooming and ectoparasite consumption in both Author details sexes of adult bats. The considerable estimate of bat Mention Zoologie et Biodiversité Animale, Université d’Antananarivo, BP fly consumption by their hosts provides a new dimen- 906, (101) Antananarivo, Madagascar. Association Vahatra, BP 3972, (101) Antananarivo, Madagascar. Field Museum of Natural History, 1400 South sion to population dynamics and reproductive strat- Lake Shore Drive, Chicago, Illinois 60605, USA. egies in bat flies. 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