DOI: 10.5061/dryad.6hdr7sr8c
title: Navigating the landscape of fear: Fruit flies exhibit distinct anti-predator and anti-parasite defensive behaviours

description: Most organisms are at risk of being consumed by a predator or infected by
 a parasite at some point in their life. Theoretical constructs such as the
 landscape of fear (perception of risk) and non-consumptive effects (NCEs,
 costly responses sans predation or infection) have been proposed to
 describe and quantify anti-predator and anti-parasite responses. How
 prey/host species identify and respond to these risks determines their
 survival, reproductive success and, ultimately, fitness. Most studies to
 date have focused on either predator-prey or parasite-host interactions,
 yet habitats and ecosystems contain both parasitic and/or predatory
 species that represent a complex and heterogenous mosaic of risk factors.
 Here, we experimentally investigated the behavioural responses of a
 cactophilic fruit fly, Drosophila nigrospiracula, exposed to a range of
 species that include parasites (ectoparasitic mite), predators (jumping
 spiders), as well harmless heterospecifics (non-parasitic mites, ants and
 weevils). We demonstrate that D. nigrospiracula can differentiate between
 threat and non-threat species, increase erratic movements and decrease
 velocity in the presence of parasites, but decrease erratic movements and
 time spent grooming in the presence of predators. Of particular
 importance, flies could distinguish between parasitic female mites and
 non-parasitic male mites of the same species, and respond accordingly. We
 also show that the direction of these non-consumptive effects differ when
 exposed to parasitic mites (i.e., risk of infection) versus spiders (i.e.,
 risk of predation). Given the opposing effects of predation versus
 infection risk on fly behaviour, we discuss potential trade-offs between
 parasite and predator avoidance behaviours. Our findings illustrate the
 complexity of risk assessment in a landscape of fear and the fine-tuned
 non-consumptive effects that arise in response. Moreover, this study is
 the first to examine these behavioural NCEs in a terrestrial system.
description: <strong>Video analysis</strong>: behavioural assays
 were documented using digital video recorded in a
 DanioVision<sup>TM</sup> observation chamber (Noldus
 Information Technology, Leesburg, VA, USA) and response variables were
 measured with EthoVision<sup>TM</sup> computer tracking
 software (Noldus Information Technology, Leesburg, VA, USA). In total, we
 generated 286 videos of fly behaviour in response to male mites (n=30),
 female mites (n=30), ant (n=30), weevils (n=30), spider (n=22), control
 (n=144); flies were only tested once. Once the arenas were placed in the
 DanioVision<sup>TM</sup> chamber, 5 minutes of unused video
 was recorded before the five-minute experimental recording period. The
 experimental five-minute video was analysed using
 Ethovision<sup>TM</sup> software that recorded all response
 variables 12.5 times per second. Response variables reported were velocity
 (mm/s), distance travelled (mm), ambulatory movement (yes/no), distance
 between fly and cage (mm), and meander (the number of degrees through
 which the fly changed direction for each mm traveled - °/mm) where an
 increase in the meander value corresponds to an increase in erratic
 movement or path complexity (Richardson et al. 2018).
 We manually calibrated the raw data such that all samples
 displaying velocity below 1.5mm/s, and all corresponding response
 variables (distance, ambulatory movement, meander), were counted as zero
 (distance and meander) or no movement because below this threshold
 EthoVision<sup>TM</sup> often registered false positives.
 Average velocity and meander values for each fly were recalculated
 excluding all zero scores, essentially recreating the variables as
 velocity or meander while mobile. These adjustments were made because most
 flies spent the majority of the five-minute observational period immobile,
 and the large number of zeros in velocity or meander caused unwanted
 smoothing in the data and masked actual differences among treatment
 groups. Some flies remained immobile for the entire observation period and
 so were removed from analysis; these flies were all active and groomed
 periodically, but none moved sufficiently to record velocity or meander
 measurements. A generalised linear model with a binomial error
 distribution and a logit link function was used to test for any effect of
 stimuli species on the proportion of immobile flies, but none was found
 (see Appendix1: Table S2). Time spent grooming was recorded via direct
 observation by a single observer who noted time spent grooming with a
 stopwatch; any flies that spent &gt;15s (5% of video) obscured from
 observer were excluded from analysis. Unfortunately, our stimuli species
 were sporadically available during the 20 weeks we ran video analysis:
 male and female mites were maintained in-lab, although availability was
 highly variable; weevils were provided by an outside lab on an
 <em>ad hoc</em> basis; while ants and spiders were collected
 from the field. Consequently, stimuli species were not evenly distributed
 throughout the trials (see Appendix S1: Table S3). To ensure that small
 variations in RH and temperature during acclimation and recording did not
 have a confounding effect on fly behaviour, we ran a series of models on
 all response variables using only the control video data, as no other
 treatment was present in all trials (see Statistical Analysis section and
 Appendix S1: Table S2).
description: # Navigating the landscape of fear: fruit flies exhibit distinct
 anti-predator and anti-parasite defensive behaviours
 [https://doi.org/10.5061/dryad.6hdr7sr8c](https://doi.org/10.5061/dryad.6hdr7sr8c) This data set contains measurements of fruit fly behavior, specifically: distance travelled in millimeters (abbreviated as 'Dist' in data file), Velocity while moving in millimeters per second ('Vel_mov' in data file), proportion of time spend moving ('Mov_pr' in data file), distance to stimuli species in millimeters ('Dist_Z' in data file), Meander while moving in degrees per millimeter traveled ('Meand_mov' in data file), and time spent grooming in seconds ('Grm_s' in data file). These behaviors were responses to exposure to a variety of other species: mites, weevils, spiders and ants. The column 'Treatment' contains the specifics of which species the fruit flies were exposed to. Of note, mites were further broken down into three additional categories: 10 male mites ('MaleMite' in data file), 10 female mites ('FemaleMite' in data file)  and 30 female mites ('30mite' in data file).  In addition to 'Treatment', 'Dist', 'Vel_mov', 'Mov_pr', 'Dist_Z', 'Meand_mov', and 'Grm_s' headings, we also include 'FileID' which gives the date trials were completed, 'VideoID' which gives the number of each video, and three more columns that are centered Velocity, Meander, and Grooming data. By this we mean that these three columns are generated by centering the data of the named variable around the average of the control data for that variable - e.g. , in cell K,63 the value of -3.23 is the difference between the average of control velocity and the velocity of the fly exposed to the spider in video 2 on the 1st of July in Trial 5. These centered data are used only in Figure 4 in the manuscript that looked specifically at 'Spider' and '30mite' treatments; hence there are no "centered data" entries for the treatments 'MaleMite', 'FemaleMite', 'Ant', 'Weevil' or 'Control'.  # Description of the data and file structure The data is a single csv file. Each cell represents a single measurement for a single fly (other than centered data described above).  NA denotes data that is not available due to (1) measurement error (2) in the case of meander and velocity data, the flies did not move during the trial or (3) in the case of grooming data the flies were not visible long enough to gauge grooming status (see Methods section of MS).  Empty cells indicate that no data was used for a given response variable, e.g. we did not calculate 'centered data' for the treatments  'MaleMite', 'FemaleMite', 'Ant', 'Weevil' or 'Control'.  ## Sharing/Access information Data is not available elsewhere.  ## Code/Software We used R code for all analyses. All packages are readily available through the R website. All packages and relevant functions are listed in the manuscript.