DOI: 10.5061/dryad.qjq2bvqq5
title: Taste aversion training can educate free-ranging crocodiles against toxic invaders

description: Apex predators play critical ecological roles, making their conservation a
 high priority. In tropical Australia, some populations of freshwater
 crocodiles (Crocodylus johnstoni) have plummeted by &gt;70% due to
 lethal ingestion of toxic invasive cane toads (Rhinella marina).
 Laboratory-based research has identified conditioned taste aversion (CTA)
 as a way to discourage consumption of toads. To translate those ideas into
 landscape-scale management, we deployed 2,395 baits (toad carcasses with
 toxin removed and containing a nausea-inducing chemical) across four gorge
 systems in north-western Australia and monitored bait uptake with remote
 cameras. Crocodile abundance was quantified with surveys. Free-ranging
 crocodiles rapidly learned to avoid toad baits but continued to consume
 control (chicken) baits. Toad invasion at our sites was followed by high
 rates of crocodile mortality (especially for small individuals) at a
 control site but not at nearby treatment sites. In areas with high
 connectivity to other waterbodies, repeated baiting over successive years
 had continuing positive impacts on crocodile survival. In summary, we
 succeeded in buffering the often-catastrophic impact of invasive cane
 toads on apex predators.
description: This data was recorded during baiting trials run in the wild to
 test the effectiveness of taste aversion training with freshwater
 crocodiles. The data has had minimal processing but was collected and
 analysed as below. <strong>Eliciting taste
 aversion</strong> Baiting trials were conducted
 all sites in September and October of 2021. We ran a repeat baiting at
 Bandiln͟gan sites 1 and 2 in September 2022 (but not other sites), to
 explore the longevity of our intervention in a large population
 experiencing toads for the first time. We also wanted to clarify
 population dynamics between years at dry-season waterbodies in this
 important gorge ecosystem, which would allow us to design future
 conservation protocols. Trials were timed to coincide with late dry-season
 peak overlap between crocodiles and toads, when pools were relatively
 isolated. Based on our spotlight counts just prior to trials, we
 calculated the number of bait stations required in each area to keep the
 ratio of crocodiles to bait stations below 2:1, while maintaining 50–100 m
 distance between stations along both sides of the waterbodies. Our
 rationale for these calculations was twofold and based on the spatial
 ecology detected during spotlight surveys. Pragmatically, we aimed to
 maximise the number of bait stations available to crocodiles while
 limiting competition between animals, to ensure wide application of the
 baiting strategy. Ethically, we aimed to minimise the chance of single
 animals consuming too many baits and becoming overly ill.
 At each bait station, two 90-cm metal stakes were placed 2 m
 apart, angled outwards at 30<sup>o</sup>, with their bases in
 water to prevent ant attack (following methods in Aiyer <em>et
 al</em>. [35]). Baits swung freely 100 mm above the surface of the
 water to be visible to crocodiles while discouraging consumption by
 freshwater prawns or fish. Baits were suspended by bulldog clips, allowing
 easy release when pulled (figure 1<em>b</em>).
 To induce generalised aversion to cane toads, each bait consisted
 of the fresh carcass of an adult toad (~100 g, 70 mm) with internal
 organs, head and parotid glands removed to render it non-lethal [36]. To
 ensure the baits would elicit taste aversion, we added 5 mL of lithium
 chloride (LiCl; concentration 0.5 Mol [37]) via intramuscular injection
 into both back legs of the cane toad carcass. We honed the dosage rate
 through pilot studies with captive crocodiles. Generally, chemicals used
 to elicit taste aversion should be odourless and tasteless, such that the
 target aversive ‘cue’ (in this case, the scent or taste of cane toads)
 remains untainted [10].  Because Lithium Chloride (LiCL) is salty, it is
 often (in lab studies at least) administered via injection or drenching
 into the target animal. This was not possible in the context of our
 in-situ conservation study, with large crocodilians. However, crocodiles
 were unlikely to detect the LiCl in baits: a) because they are ambush
 predators, initiating fast attacks from the water then consuming
 toad-sized prey whole and b) they spend time in seasonally brackish
 environments and are accustomed to consuming prey within saline water
 [23]. A paired control bait (chicken neck ~40 g, 50 mm) was hung on one
 arm at each station on Day 1 and Day 5 of trials, so that visiting animals
 could choose between both bait types. This design allowed us to
 distinguish between satiation and taste aversion at the end of the
 trial. To identify consumers of bait, we set up a
 remotely triggered wildlife camera (Little Acorn; model Ltl6310WmC) on the
 riverbank behind every second or third bait station (depending on the
 number of stations at each site). Cameras were set to high sensitivity,
 and recorded a burst of three photos every time they were triggered by
 motion or a thermal differential. We later reviewed photographs using
 IrfanView v4.62 and scored the taxa to species level if
 possible. Baits were deployed from canoes for five
 successive nights, between 1500 and 1700h, and checked between 0600 and
 0800 h the next morning. We recorded whether or not baits were eaten, and
 then removed all remaining baits and replaced them that afternoon (see
 Aiyer <em>et al</em>. [35] for details).
 <strong>Analysis of data</strong>
 For all data from baiting trials, we ran binomial Generalised
 Linear Mixed Models with a logit link function, using the GLIMMIX
 procedure in SAS v9.4 M8 (SAS Institute, Cary, NC). When comparing
 multiple sites, we incorporated ‘bait station ID’ nested within ‘site ID’
 as a random factor in the analyses, to account for pseudo-replication and
 repeated measures. To assess whether rates of toad bait
 consumption declined over the five-day baiting period (demonstrating
 aversion), our dependent variable was bait ‘Eaten/Remain’ and we ran an
 analysis including the independent variables of trial day (1–5), crocodile
 density and population connectivity (open/closed). We included interaction
 terms which might change patterns of bait offtake from those expected with
 the development of learned aversion: trial day * crocodile density
 (increased competition throughout the trial), trial day * population
 connectivity (immigration of naïve individuals during the trial), and
 crocodile density * population connectivity (to compare general levels of
 activity and bait offtake). To compare the offtake of
 toad vs. control chicken baits on Day 1 and Day 5 of trials (further
 demonstrating learned aversion), our dependent variable was bait
 ‘Eaten/Remain’ and we ran an analysis including the independent variables
 of trial day (1–5), treatment (toad/chicken), crocodile density and
 population connectivity (open/closed). We included interaction terms which
 might influence whether baits were differentially consumed during the
 trial: trial day * treatment (choice over time: satiation [neither bait
 consumed on Day 5] vs. learned avoidance [decreased consumption of toad
 baits only on Day 5]), trial day*treatment*crocodile density (increased
 competition overriding learning on Day 5), and trial
 day*treatment*population connectivity (immigration of naïve individuals
 during the trial eating equal numbers of baits on Day 5). We clarified
 whether offtake of both bait types declined over time by conducting GLMMs
 as above, with the independent variable of trial day, analysing chicken
 and toad datasets separately. To compare the response
 of crocodiles in Bandiln͟gan (Windjana Gorge) National Park to baiting
 trials conducted in 2021 vs. 2022, we pooled data together for each year
 (i.e., sites 1 and 2, because sites were not as discrete in 2022). Our
 dependent variable was bait ‘Eaten/Remain’ and we ran a full factorial
 analysis including the independent variables of trial day (1–5), year
 (2021/2022) and the interaction between trial day and year. ‘Bait station
 ID’ was included as a random factor in this analysis.
 To investigate the effect of baiting on crocodile deaths in
 Dan͟ggu Geikie Gorge National Park (where we began to witness crocodile
 mortalities in 2018), we ran a logistic regression analysis with a
 binomial distribution and logit link function, to model the relative
 numbers of dead crocodiles (counts of carcasses) compared to live
 crocodiles (from population spotlighting counts; i.e. the trials/events
 ratio) in each site (treatment vs control) and time period. The
 independent variables were treatment (baited vs. unbaited control),
 intervention status (pre-intervention – 2018-2020/post-intervention –
 2021-2022) and the interaction between the two.  Analyses were conducted
 in SAS 9.4 (SAS Institute, Cary NC). To determine
 whether the cane toad invasion imposed size-selective mortality, we
 compared mean SVLs of dead crocodiles to live crocodiles. Data from live
 animals (n=209) were collected during monitoring programs at Bandiln͟gan
 (Windjana Gorge) National Park over four years pre-cane toad invasion
 (2014–2018). In that catch-and-release program, the crocodile population
 was sampled from the lower sites in the gorge extensively each year via
 seine netting of dry-season pools (selecting a random sample of
 individuals), with morphological measures taken prior to marking and
 release. Throughout that period the population was stable and displayed a
 normal size distribution (Department of Biodiversity Conservation and
 Attractions (DBCA), unpublished data, 2023). We compared the means using a
 one-way ANOVA in JMP v16 (SAS Institute, Cary, NC).
description: # Freshwater Crocodile CTA trial Data
 [https://doi.org/10.5061/dryad.qjq2bvqq5](https://doi.org/10.5061/dryad.qjq2bvqq5) For the most part, this is a dataset of bait offtake records by freshwater crocodiles over a five day trial period. Baits were replenished each day and we checked offtake each morning. We offered a choice of chicken or cane toad baits on day 1 and 5, otherwise only toad baits offered at each station every day. This enabled us to test whether crocodiles were eating less cane toad baits over time and also whether this was an active avoidance of cane toad baits (versus control chicken baits). Trials were run in multiple, independent gorge systems. Crocodiles did show avoidance of cane toad baits by the end of the trials, however the strength of this was associated with how 'open' the environment was and the density of crocodiles within it. We also monitored death rates pre- and post- baiting. Within this dataset are the records of crocodile mortality which showed that baiting significantly decreased rates of mortality. I.e. wild crocodiles can be taught taste aversion with unpalatable baits, and this increases their survival in the face of the cane toad invasion. ## Description of the data and file structure This dataset is presented in an excel file with a series tabs gooding pertinent data for different analyses. We describe the data variables below and following that, our analyses of the data. DATASET LAYOUT: **Tab BAITING TRIALS:** This tab holds all of the data for the CTA baiting trials which ran over 5 days, as well as variables about the crocodile populations at each site, such as density. *Year*: the year of baiting trials (all trials were conducted in 2021 but there was an additional baiting session run at one site in 2022) *Trial day*: the day of the baiting trial in order from 1-5. *Site ID*: the location at which the baiting trial was being carried out. *Site station ID*: the ID of the baiting station within each site. (consisting of two poles overhanging the water so a possibility of two baits hanging at each station). *Treatment*: the bait that was delivered at that bait station (toad CTA bait or chicken control bait). Note: on day one and five of trials there was both a toad and a chicken bait present at each station, but only toad for the rest of the time. *Bait response*: the status of the bait the morning after each trail day. The bait could have been eaten or it had not been eaten and remained on the station. *Density*: the density of crocodiles at each site during the trial period, based on population counts divided by the size of the water body in square metres (as calculated from surface polygons on google earth). The sites surveyed in 2022 did not have this variable and should not be included in initial analyses on baiting efficacy - any blank cell is 'not applicable' and should be excluded. *High/Low density*: Categorical variable of high or low density, relative to other sites. The sites surveyed in 2022 did not have this variable and should not be included in initial analyses on baiting efficacy  - any blank cell is 'not applicable' and should be excluded. *Population open/closed*: whether a population could possibly have emigration through the trial period, based on its connectivity to continuous river systems or other locations that we could not survey. The sites surveyed in 2022 did not have this variable and should not be included in initial analyses on baiting efficacy - any blank cell is 'not applicable' and should be excluded. *Yr1 and 2 comparison*: a sorting variable for the site that was baited and surveyed in both 2021 and 2022, which relates to a specific analysis in the published paper. All other sites/data say ‘not applicable' and should be excluded when comparing this one site in detail.  This analysis focused on the efficacy of baiting over multiple years. **Tab ALL DEATHS** This tab holds all the mortality data from counts of crocodile carcasses during surveys, across all sites from 2018 to 2022. *Site location*: the name of the site where the mortality was detected. *Month*: The month of the year that the mortality was detected *Year*: The year that the mortality was detected. **Tab BODY SIZE LIVE VS. DEAD** This tab holds all the morphology data for the crocodiles who died from cane toad poisoning, vs. a live local population of animals. Ultimately our analyses showed that the live population showed a normal distribution, whereas the crocodiles that had died were all relatively small i.e. toads induced ‘size selective mortality’. *Animal status*: Dead = crocodile carcasses for toad induced mortality detected and measured in the study; Live = morphological measurements during previous surveys of a local healthy population. *SVL*: Snout to Vent Length (in millimetres); the measurement between the tip of the snout and the urogenital opening in front of the hips. This is a standard and reliable measure for many reptiles as the tail can often be lost or damaged and may not truly reflect the body size/age of an individual. *Group*: The stratified group that an animals SVL fell into, for the purposes of graphing and analysing size distribution. **Tab DEATHS TREATMENT VS. CONTROL** This tab holds the data for comparisons of mortality rates between our treatment (CTA baiting intervention) and control site (no CTA baiting intervention) across many years (pre and post CTA intervention). This data was used to show that our interventions stopped the mass mortality events that were happening whereas they did not stop at the control site. *Site*: the name of the site where the mortality was detected. *Treatment*: Treatment = treated with CTA baits; Control = not treated with CTA baits. *Year*: the year of survey, from 2018 – 2022. *Intervention status*: Pre-intervention = prior to CTA baiting occurring; After intervention = post CTA baiting intervention. *Total population*: the number of crocodiles in these sites during the late dry season when populations are static as animals are locate in drying pools. *Deaths*: the number of crocodile carcases detected during surveys each year. *Proportion*: the rate of death as expressed as the proportion of animals within the population that died (deaths/total population = proportion). **Analysis of data** For all data from baiting trials, we ran binomial Generalised Linear Mixed Models with a logit link function, using the GLIMMIX procedure in SAS v9.4 M8 (SAS Institute, Cary, NC). When comparing multiple sites, we incorporated ‘bait station ID’ nested within ‘site ID’ as a random factor in the analyses, to account for pseudo-replication and repeated measures. To assess whether rates of toad bait consumption declined over the five-day baiting period (demonstrating aversion), our dependent variable was bait ‘Eaten/Remain’ and we ran an analysis including the independent variables of trial day (1–5), crocodile density and population connectivity (open/closed). We included interaction terms which might change patterns of bait offtake from those expected with the development of learned aversion: trial day * crocodile density (increased competition throughout the trial), trial day * population connectivity (immigration of naïve individuals during the trial), and crocodile density * population connectivity (to compare general levels of activity and bait offtake). To compare the offtake of toad vs. control chicken baits on Day 1 and Day 5 of trials (further demonstrating learned aversion), our dependent variable was bait ‘Eaten/Remain’ and we ran an analysis including the independent variables of trial day (1–5), treatment (toad/chicken), crocodile density and population connectivity (open/closed). We included interaction terms which might influence whether baits were differentially consumed during the trial: trial day * treatment (choice over time: satiation [neither bait consumed on Day 5] vs. learned avoidance [decreased consumption of toad baits only on Day 5]), trial day*treatment*crocodile density (increased competition overriding learning on Day 5), and trial day*treatment*population connectivity (immigration of naïve individuals during the trial eating equal numbers of baits on Day 5). We clarified whether offtake of both bait types declined over time by conducting GLMMs as above, with the independent variable of trial day, analysing chicken and toad datasets separately. To compare the response of crocodiles in Bandiln͟gan (Windjana Gorge) National Park to baiting trials conducted in 2021 vs. 2022, we pooled data together for each year (i.e., sites 1 and 2, because sites were not as discrete in 2022). Our dependent variable was bait ‘Eaten/Remain’ and we ran a full factorial analysis including the independent variables of trial day (1–5), year (2021/2022) and the interaction between trial day and year. ‘Bait station ID’ was included as a random factor in this analysis. To investigate the effect of baiting on crocodile deaths in Dan͟ggu Geikie Gorge National Park (where we began to witness crocodile mortalities in 2018), we ran a logistic regression analysis with a binomial distribution and logit link function, to model the relative numbers of dead crocodiles (counts of carcasses) compared to live crocodiles (from population spotlighting counts; i.e. the trials/events ratio) in each site (treatment vs control) and time period. The independent variables were treatment (baited vs. unbaited control), intervention status (pre-intervention – 2018-2020/post-intervention – 2021-2022) and the interaction between the two.  Analyses were conducted in SAS 9.4 (SAS Institute, Cary NC). To determine whether the cane toad invasion imposed size-selective mortality, we compared mean SVLs of dead crocodiles to live crocodiles. Data from live animals (n=209) were collected during monitoring programs at Bandiln͟gan (Windjana Gorge) National Park over four years pre-cane toad invasion (2014–2018). In that catch-and-release program, the crocodile population was sampled from the lower sites in the gorge extensively each year via seine netting of dry-season pools (selecting a random sample of individuals), with morphological measures taken prior to marking and release. Throughout that period the population was stable and displayed a normal size distribution (Department of Biodiversity Conservation and Attractions (DBCA), unpublished data, 2023). We compared the means using a one-way ANOVA in JMP v16 (SAS Institute, Cary, NC).