DOI: 10.5061/dryad.866t1g1xz
title: Data from: An assessment of whether age, sex, and reproductive status affect bait uptake by grey squirrels

description: The grey squirrel is an invasive alien species in the UK. The economic
 impact of grey squirrels on England and Wales' forestry is estimated
 to be around £37 million per year, and the presence of this species has
 caused the decline of the native red squirrel due to interspecific
 competition and transmission of diseases. Oral contraceptives, delivered
 in baits, are being developed to decrease the number of grey squirrels.
 Bait uptake can be affected by many factors, which must be understood to
 maximise the effects of oral contraceptives on grey squirrel populations.
 This study investigated the effect of sex, age, and reproductive status on
 bait uptake by grey squirrels. Squirrels were pit-tagged and bait uptake
 measured using squirrel-specific bait dispensers (hoppers) fitted with
 pit-tag readers and baited with hazelnut paste and the biomarker Rhodamine
 B for 5 days. For each visit to the hoppers, the identity of the squirrel,
 date, time, duration, and ID of the hopper visited were recorded. One to 3
 weeks later, as many squirrels as possible were recaptured and assigned
 categories related to sex, age (immature, adult), and reproductive status
 (reproductively active or non-active). The presence of Rhodamine B in the
 hair was also recorded in all these animals as an indicator of bait
 uptake. Overall, 49 % of squirrels were positive for Rhodamine B, with
 sexually active females showing the highest bait uptake. Adult squirrels
 made fewer daily visits to hoppers and visited fewer hoppers per day than
 immature squirrels, though both of these parameters showed greater
 variability in immature animals than in adults. Sex, age, and reproductive
 status did not affect bait uptake in grey squirrels at the density of grey
 squirrels (3.9 – 6.3 squirrels/ha) and bait hoppers (3/ha) tested in this
 study. As several doses of an oral contraceptive are likely to be required
 to effectively suppress reproduction, this study confirmed that it should
 be possible for contraceptives to have an equal chance of affecting each
 category of squirrels within a relatively short period of time. As
 sexually mature females drive population growth, targeting these females
 with oral contraceptives should be a priority.  These results
 could be used to inform targeted interventions aimed at delivering baits
 containing contraceptives or vaccines to grey squirrels and for population
 management.
description: All field work was conducted by trained APHA operatives. Trapping
 was conducted over a 2-to-3-week period for 3-4 trap days per week per
 wood. Grey squirrels were captured using single-capture cage traps
 deployed at a density of 3/ha across each wood. Trapped squirrels were
 anaesthetised on site with isoflurane via a portable anaesthetic machine
 and a mask. Under anaesthesia, squirrels were health checked and equipped
 with a subcutaneous PIT-tag (Identichip®, York, UK) inserted in the scruff
 of the neck, as a unique identifier. Within 2 weeks of the last animal
 equipped with a PIT-tag, traps were removed and squirrel bait dispensers,
 hereafter referred to as hoppers, were deployed across each wood at a
 density of 3/ha (21-24 in total). A hopper consisted of a bait compartment
 within a 10 × 10 × 55 cm length of aluminium tubing and a door that
 squirrels had to lift to access the bait. Each hopper was equipped with a
 PIT-tag reader to record the date and time that each tagged animal entered
 the hopper (Beatham et al. 2021). The hopper trial was divided into a
 pre-baiting and baiting phase. During a ‘prebaiting’ phase, lasting one
 week, hoppers were baited 3 times, every other day for the first 5 days,
 and the hopper door was left open to encourage the squirrels to use these
 devices. The bait consisted of 40 g of hazelnut paste per hopper, as in
 Beatham et al. (2021). During the baiting phase, which
 started the following week, each hopper was set so that squirrels had to
 lift the door to access the bait. Data collection on squirrel visits to
 hoppers began during the baiting phase and lasted 5 days. During the
 baiting phase, 0.18% (weight/weight), Rhodamine B (CAS number 81-88-9,
 Sigma-Aldrich) bait marker was added to the hazelnut paste to monitor
 population bait uptake. Within 4 weeks after the end of the baiting phase,
 a trap and remove operation was carried out in each wood to recapture as
 many squirrels as possible. All trapped squirrels were humanely dispatched
 according to Schedule 1 of the Animals (Scientific Procedures) Act 1986
 (UK) and scanned to assess whether they had a PIT-tag. Hair samples were
 collected from the flank and analysed using an Ultra-Violet microscope, to
 test for Rhodamine B presence (Fisher 1973, Beatham et al. 2024). The
 carcasses were placed in a -20 <sup>o</sup>C freezer until
 post-mortem examinations. During post-mortem
 examinations, squirrels were assigned a sex, age, and reproductive status.
 The age of each squirrel was assessed through tooth eruption, replacement,
 and wear following the method by Hench et al. (1984). Based on this
 method, 4 age classes were identified: A) &lt; 6 months; B) between 6
 months and 14 months; C1) between 14 and 36 months; C2) &gt; 36
 months. For female squirrels, reproductive status was assessed based on a
 combination of features of the vulva and uterus. Vulva scores were defined
 as: 1 = width &lt; 5mm, no noticeable swelling, pale pink; 2 = width
 5mm – 7mm, some swelling, pink; 3 = &gt; 7mm, very swollen, darker
 pink. Uterus status was defined as small, ribbon-like, pale pin, or
 oedematous, or oedematous with embryonic vesicles, embryos, or foetuses
 (Maranesi et al. 2020). Uterine horn (width measured as a cross-section of
 the middle point of a uterine horn) was defined as non-reproductively
 active if ≤ 2.5 mm, or reproductively active if &gt; 2.5 mm.  The
 vesicles, also referred to as “vitelline vesicles,” are the first
 structures that can be observed inside the gestational sac; they have a
 roundish cystic shape with a fine wall (Luis Alcázar and Alcázar, 2008).
 Based on a combination of the above variables, females were assigned to
 the following categories: 1 - Immature - The squirrel
 has not reached sexual maturity. The animal presents tooth wear A/B, vulva
 score 1-2, uterus horn width &lt; 2.5 mm, ribbon-like and pale pink
 uterus. 2 - Adult, not reproductively active - The
 animal presents tooth wear C1/C2, vulva score 1-2, uterus horn width ≤ 2.5
 mm, uterus is small, dark-pink and ribbon-like, and absence of
 oedema.  3 - Adult, reproductively active - The animal
 presents tooth wear C1/C2, vulva score 1-2-3, the uterus is round,
 oedematous, and blood-red, characteristic of a sexually active animal.
 Sometimes vesicles, embryos, and foetuses show ongoing pregnancy. Uterus
 horn width &gt;2.5 mm. For male squirrels,
 reproductive status was assessed based on the method developed by Ferryman
 et al. (2006) as follows: testicle position (abdominal/scrotal); scrotal
 area colour (pink, light grey, dark grey, or black); scrotal and
 periscretal hair/stain; external paired testicle length. These 4 variables
 were used to determine the Visual Breeding Score (VBS, Figure 2). Based on
 the above variables, males were assigned to the following
 categories:  1 – Immature - The squirrel has not
 reached sexual maturity. The animal presents tooth wear A/B. Testes can be
 abdominal or scrotal. Length of testicles (when scrotal) is approximately
 1 cm, and colour is usually pink/light grey and completely covered in fur.
 The VBS value is 0/1/2. 2 – Adult, not reproductively
 active - Male squirrels generally have a period of sexual quiescence in
 summer and sexual redevelopment (recrudescence) in autumn. If
 recrudescence is not prolonged as a single peak of sexual activity, males
 become sexually active again in winter until May (Dubock 1979). The
 recrudescence and quiescence are characterised by low sperm production
 with a reduced testicular size. During this period, squirrels’ testicles
 are scrotal or abdominal, the colour of the scrotum is dark with a grey
 tone, the length of the external testicles is between 0 cm and 4.4 cm, and
 the presence or absence of scrotal hair. The animal presents tooth wear
 C1/C2. The VBS value is 2-5. 3 - Adult, reproductively
 active - Testicles are scrotal, the scrotum is dark in colour with a dark
 grey/black hue, and scrotal hair is absent. The VBS value is ≥ 6,
 confirming a progressive increase in the concentration of spermatozoa and
 reproductive capacity, and the length of the testicles is between 2.3 cm
 and 6.5 cm (Ferryman et al. 2006).
 <strong>Statistical analyses</strong>
 Analyses were carried out on 2 datasets. The first dataset
 (squirrel_nimble_rhod.csv) contained the records of age, gender, and
 reproductive status of each squirrel and whether it tested positive for
 Rhodamine B. The second dataset (squirrel_nimble_time.csv) contained the
 age, gender, and reproductive status of each squirrel and the hopper
 PIT-tag records associated with that squirrel. PIT-tag records were
 aggregated into 12-hour periods over the course of 5 days, resulting in 8
 periods (with recording starting at 12.00 on the first day and ending at
 12.00 on the fifth day). The number of hoppers visited,
 the number of feeding visits, and the total time spent at any hopper were
 calculated for each squirrel. A feeding visit was defined as all
 PIT-tagged animals detected within two consecutive minutes for the same
 squirrel by the same hopper. A two-minute interval was used to standardise
 feeding visits, as squirrels would frequently move in and out of hoppers
 within one visit, and the hoppers used in different years had different
 levels of sensitivity regarding PIT-tag detection. The total time spent at
 any hopper was calculated from the time of the first PIT-tag record until
 the time of the last PIT-tag record for that visit. In instances where a
 squirrel was detected at the hopper only once, so the duration could not
 be calculated, a minimum time of 1 second was assigned.
description: # An assessment of whether age, sex, and reproductive status affect bait
 uptake by grey squirrels #### **Description of the data and file
 structure** **squirrel_nimble_rhod.csv** Contains data on the age, sex,
 and reproductive status of a grey squirrel and whether it tested positive
 for Rhodamine B. A total of 174 squirrels were captured and sampled in
 winter 2018 and summer 2022 in three different woods. The dataset is made
 up of 6 columns: * ID = squirrel ID - Loc = wood Id (PW-HW-SC) * Season = 
 season the squirrel was sampled (Winter/Summer) - Sex = Female/Male * Rho
 = presence or absence of rhodamine in the hair (Yes/No) - Cat = squirrel
 reproductive category assigned (1 immature/ 2 no reproductive adult/ 3
 reproductive adult) **squirrel_nimble_time.csv** Contains data on the age,
 gender, and reproductive status of PIT-tagged squirrel and the hopper
 PIT-tag records associated with that squirrel. PIT-tag records were
 aggregated into feeding visits within 12-hour periods over the course of 5
 days, resulting in 8 periods (with recording starting at 12.00 on the
 first day and ending at 12.00 on the fifth day). The number of hoppers
 visited, the number of feeding visits, and the total time spent at any
 hopper were calculated for each squirrel. A total of 138 squirrels were
 PIT-tagged, and 94 of these were recaptured and assessed for reproductive
 status. In total, 24 females and 21 males were recaptured in winter 2018,
 and 18 females and 31 males were recaptured in summer 2022. The dataset is
 made up of 10 columns: * ID = squirrel ID - Sex = Female/Male * Cat =
 squirrel reproductive category assigned (1 immature/ 2 no reproductive
 adult/ 3 reproductive adult) - Loc = wood Id (PW/HW/SC) * Season = is the
 season the squirrel was captured (Winter/Summer) - Day = trial day (1-5) *
 Time = AM (00:00 - 11:59) or  PM (12:00 - 23:59) - n_visits = number of
 visits made by the squirrels to the hoppers * time 1 = total time the
 squirrel spent visiting hoppers - n_hopper = number of hoppers visited