DOI: 10.5061/dryad.m905qfvb0
title: Seasonal bait uptake by individual grey squirrels measured by PIT-tags and Rhodamine B

description: Oral contraceptives are being developed to manage the impacts of the grey
 squirrel. To be effective, contraceptives will need to be deployed at a
 landscape scale and will require a delivery system that is practical and
 economically viable. Understanding grey squirrel feeding behaviour is
 important so that delivery methods can be designed to ensure enough target
 individuals receive an effective contraceptive dose at a time of year that
 will ensure they will remain infertile throughout peak times of breeding.
 The main aims of this study were to assess how sex, season, squirrel
 density and bait point density influenced; 1) the probability of a
 squirrel visiting a feeder and 2) the amount of bait consumed from
 feeders. Field trials were conducted on six woodland populations of
 squirrels in three seasons, with four days of bait deployment via
 purpose-designed squirrel-specific bait hoppers with integrated PIT-tag
 readers.
description: Trials were conducted in five independent woods that were located
 at least 3.5 km apart and within 25 km of York, England
 (53.96<sup>o</sup>N, -1.09<sup>o</sup>E; Figure
 1). Populations of grey squirrels were sampled from woods HW and SC in
 winter 2017/2018, woods SC and PW in summer 2022 and woods GE and BW in
 spring 2023. Autumn was not trialed due to reduced squirrel breeding
 activity and high natural food availability, which could potentially deter
 squirrels from using feeders. To provide two suitable replicates for each
 season, woods were selected that were of a similar size (between 7 and 8
 ha) and structure (mature broadleaf or mature broadleaf conifer mixed tree
 species), that had high levels of grey squirrel activity reported by
 landowners and established squirrel populations, with minimal squirrel
 control conducted within the previous 12 months. At the end of each trial,
 the squirrel population was trapped and removed from each wood as part of
 other related studies. Therefore, wood could not be re-used in trials that
 were within 12 months of each other. The squirrel population at Wood SC
 was removed in January 2018 and the wood was recolonized by squirrels,
 confirmed by the trap results in this study, so was reused in Summer 2022.
 Thus, it was considered that six independent populations of squirrels were
 assessed.  Grey squirrel trapping and sampling methods
 were approved by the joint Animal and Plant Health Agency (APHA) and Fera
 Science Ltd Animal Welfare and Ethical Review Body (AWERB). All animal
 studies were conducted in accordance with the UK Guidance on the operation
 of the Animals (Scientific Procedures) Act 1986. Field trials were
 conducted from December 2017 to February 2018 in woods HW and SC, from
 June to August 2022 in woods PW and SC, and from April to June 2023 in
 woods BW and GE. Single-catch squirrel cage traps (density = 3/ha, n =
 21-24) were deployed across the whole area of each wood on 1-meter-high
 wooden stands and pre-baited with a combination of maize, peanuts, a small
 number of whole hazelnuts and approximately 2 grams of 100% hazelnut paste
 (Bulk<sup>TM</sup>, UK) every 2 to 4 days for between 3 and 13
 days, dictated by human resource availability. For animal welfare and
 health and safety reasons, traps were not set if heavy rain, high winds
 (&gt; 30 mph) or high (&gt; 30<sup>o</sup>c) or low
 (&lt; 2<sup>o</sup>c) temperatures were forecast. Each
 trap was partly covered with a waterproof sheet to provide animals with
 shelter. Traps were either set early in the morning and checked in the
 afternoon or set late afternoon and checked the following
 morning. Trapped squirrels were anaesthetised on site
 using isoflurane via a mask and a PIT-tag
 (Identichip<sup>®</sup>, York, UK) implanted subcutaneously in
 the scruff of the neck. Squirrels were then sexed, weighed (to the nearest
 gram) and recorded as adult or sub-adult (&lt; 450 g, slim, soft
 pelage, body lacking muscle, head large in proportion to body and female
 nipples or male testes not visible). Females were assessed for evidence of
 recent breeding (extended or lactating nipples and palpable foetuses).
 Hair was clipped from the tail for visual identification. Once recovered
 from anaesthesia, squirrels were released under a Natural England licence
 in the location at which they had been trapped. Trapping was conducted for
 3 to 4 days, completed within 18 days, and then the traps
 removed. In spring and summer, within two days of
 PIT-tagging, hoppers were deployed at each wood, while in winter, hoppers
 were deployed within four weeks of PIT-tagging. Hoppers were deployed
 evenly across the whole area of each wood, with locations guided by a 1 ha
 grid generated in ArcGIS (version 10.7.1) overlayed onto a satellite map
 using the ArcGIS Collector mobile phone application. Locations were,
 however, adjusted according to accessibility; for example, steep slopes or
 thick vegetation were avoided. Each hopper was fixed to a 1-metre-high
 wooden stand to reduce non-target access. In winter and summer, hoppers
 were deployed at a density of 3/ha (n = 21-24). Squirrel hopper use data
 from these studies suggested that lowering the hopper density to 2/ha
 could be more cost-effective for bait delivery in terms of field hours and
 bait quantity required. This was tested in spring 2023, using a
 Before-After-Control-Impact (BACI) study design. At woods GE and BW,
 hoppers were deployed at a density of 2/ha (N = 14) and baited for four
 days, then increased to 3/ha (N = 21) at BW and maintained at 2/ha at GE
 for an additional four days each. Each hopper was then baited daily with
 40 g of hazelnut paste mixed with the bait marker dye Rhodamine
 B<strong> (</strong>Merck Life Sciences UK Limited, Dorset,
 UK) at a concentration of 0.18% for four consecutive trial days in winter
 and summer and eight consecutive trials days in spring. The bait was
 manually weighed in and out of the hoppers each day, and the hopper
 entrance and stand checked for evidence of bait spillage, easily
 identified due to the pink colouration of the bait. Any spillage found was
 weighed.Hoppers recorded the date and time at which an individual
 PIT-tagged squirrel was detected and the date and time at which the bait
 door was opened, through the engagement and disengagement of a magnetic
 door switch. In the summer and spring trials, hoppers were also fitted
 with a strain gauge, which recorded the weight of the bait taken for each
 squirrel visit. Within 4 days of hopper deployment in
 spring and summer, and within 21 days of hopper deployment in winter,
 squirrel live-capture cage traps were installed in each wood at a density
 of 2-3 traps/ha. Traps were secured to the same 1-metre-high wooden stands
 used in the hopper trials. Trapping was conducted using the same protocol
 as the trapping for the PIT-tagging, though for this session, traps were
 set and checked at least once every 24 hours where possible. If weather
 conditions did not permit trapping overnight, traps were set early morning
 and checked late afternoon the same day. Trapping was conducted within a
 four-week period, typically Monday to Friday, for a minimum of 5 days and
 for a maximum of 14 days, or until squirrel capture rates were reduced to
 an average of less than one per day over three consecutive days. Squirrels
 that were trapped were humanely dispatched using a UK Home Office-approved
 (Schedule 1) method, by a trained and competent person, and the PIT-tag
 ID, sex and tail clip (if present) were recorded and at least 20 hairs
 were taken from the flank, placed in a plastic sample bag, to be later
 analysed using a Leica DMLB ultra-violet microscope (Leica Microsystems UK
 Ltd) at ×4 magnification for the presence or absence of RB
 fluorescence.
description: # Seasonal bait uptake by individual grey squirrels measured by PIT-tags
 and Rhodamine B
 [https://doi.org/10.5061/dryad.m905qfvb0](https://doi.org/10.5061/dryad.m905qfvb0) ### **Description of the data and file structure** The purpose of this study was to measure factors affecting individual bait uptake by grey squirrels (*Sciurus carolinensis*), to inform the future delivery of an oral contraceptive. The Excel file contains PIT-tag data from grey squirrels trapped in six independent UK woodlands between 2017 and 2023. 202 grey squirrels were anaesthised, PIT-tagged and released in 2 woods in winter 2017/2018, 2 woods in summer 2022 and 2 woods in spring 2023. Bait hoppers with integrated PIT-tag readers were deployed evenly across each wood on wooden stands and baited for up to 2 weeks (density 2/ha or 3/ha) and feeding visits and amount of bait per visit were recorded for 4 to 8 days. For 4 to 8 days the bait was hazelnut paste containing 0.18% Rhodamine B bait marker. When consumed, the bait marker can be detected in squirrel hair under UV light.  At the end of the study, squirrels were trapped and dispatched at each wood under Home Office License for 2-3 weeks, until there were less than 1 capture per day average over 3 consecutive days. A hair sample was taken from each squirrel as an alternative measure of bait uptake. For description of the hopper design used see Beatham, S. E., Goodwin, D., Coats, J., Stephens, P. A., & Massei, G. (2021). A PIT-tag–based method for measuring individual bait uptake in small mammals. *Ecological Solutions and Evidence*, 2, e12081. [https://doi.org/10.1002/2688-8319.12081](https://doi.org/10.1002/2688-8319.12081) In the data file the null values are represented as nn and Excel file includes the following tabs: **Summary**  Information about the squirrels PIT-tagged in each wood and summary for the 4 day bait deployment. Includes: Site (wood Id), Season (Spring, Winter or Summer), Trapped (Number of squirrels trapped and removed in each wood), Squirrel_hopper (squirrels/hopper based on numbers trapped), PIT_tag Id, Sex, Bodyweight of squirrel (g), Hoppers (number of hoppers visited in 4 days), Visits (total number of hopper visits in 4 days), Duration (total time spent visiting hoppers), Weight (weight of bait taken (g) from hoppers in 4 days), Dates (number of dates squirrel visited hoppers in 4 x 24 hours) and Dispatched (Y/N). **Visit_data** PIT-tag data from each squirrel organised into visits. Defined as multiple PIT-tag reads from the same squirrel at the same hopper. A visit for a squirrel was deemed complete once there was at least two minutes between PIT-tag reads from the same squirrel, or another squirrel's PIT-tag was recorded at the same hopper. Included are the headings: Site (wood Id), Season, Trial (time period, defined as 1 (days 1-4) or 2 (days 5-8), Hopper_density (2/ha or 3/ha),  Hopper Id, Date and time, Duration (time from first PIT-tag read to final PIT-tag read), TagId (PIT-tag number/squirrel ID) and Bait_taken (weight of bait taken (g) for each visit recorded by a strain gauge under the hopper bait tray). **Squirrel_trapping** Squirrel trap and dispatch data for each wood. Includes: Season, Site (wood Id), Trap days (total number of trap days), Capture/day for the last 3 days, % squirrel caught in first 5 days of trapping, Total number of squirrels caught and % squirrels that tested positive for Rhodamine B from the hair samples analysed under a UV microscope.