DOI: 10.5061/dryad.0k6djhb6k
title: Chemosensory behaviour of juvenile crown-of-thorns sea stars (Acanthaster sp.), attraction to algal and coral food, and avoidance of adult conspecifics

description: Intraspecific and habitat-mediated responses to chemical cues play key
 roles in structuring populations of marine species. We investigated the
 behaviour of herbivorous-stage juvenile crown-of-thorns sea stars (COTS:
 Acanthaster sp.) in flow-through choice chambers to determine if chemical
 cues from their habitat influence movement and their transition to
 becoming coral predators. Juveniles at the diet transition stage were
 exposed to cues from their nursery habitat (coral rubble-crustose
 coralline algae -CCA), live coral, and adult COTS to determine if
 waterborne cues influence movement. In response to CCA and coral as sole
 cues juveniles moved toward the cue source and when these cues were
 presented in combination, they exhibited a preference for coral. Juveniles
 moved away from adult COTS cues. Exposure to food cues (coral, CCA) in the
 presence of adult cues resulted in variable responses. Our results suggest
 a feedback mechanism whereby juvenile behaviour is mediated by adult
 chemical cues. Cues from the adult population may deter juveniles from the
 switch to corallivory. As outbreaks wane, juveniles released from
 competition may serve as a proximate source of outbreaks, supporting the
 juveniles-in-waiting hypothesis. The accumulation of juveniles within the
 reef infrastructure is an underappreciated potential source of COTS
 outbreaks that devastate coral reefs.
description: <strong>Specimens and rearing
 condition</strong> As the taxonomy of the Pacific
 species of <em>Acanthaster</em> is uncertain (1), the species
 investigated here is referred to as <em>Acanthaster</em> sp.
 or COTS. Adults were collected near Cairns (northern GBR) by COTS control
 divers and shipped to the Sydney Institute of Marine Science where they
 were maintained in flow-through aquaria (filtered sea water, FSW 5 µm) at
 26 °C, the temperature of their habitat
 (http://data.aims.gov.au/aimsrtds/yearlytrends.xhtml). For each of the
 three fertilisations, the gametes of two different males and two females
 were used, and the larvae were reared to the settled juvenile stage
 following routine methods (2). Larvae with a well-developed juvenile
 rudiment (14–16 days post-fertilization) were induced to settle using
 coralline algae (<em>Amphiroa
 </em>sp<em>.</em>).<strong> </strong>The
 juveniles were maintained in a constant temperature room (26 °C, 12:12 hr
 light cycle) in UV sterilised 1 µm FSW with ambient salinity of ~ 34 %
 (Merck salinity probe) and fed small fronds of <em>Amphiroa
 </em>sp. Juveniles, up to 3mm in diameter, were placed in 12 well
 dishes (5 ml wells). Food and water were renewed every 3 days. As the
 juveniles grew, (&gt;3 mm diameter) they were transferred to 6 well
 (10 ml well) dishes and when they reached 10 mm diameter were transferred
 to large culture dishes (100-200 ml). For the large juveniles, culture
 dishes, food, and FSW were renewed twice a week.
 <strong>Behaviour experiments</strong>
 The behaviour experiments were conducted using a suite of cues
 including live coral, coral rubble encrusted with crustose coralline algae
 (CCA), and adult COTS. The CCA-covered rubble was collected from the
 shallow reefs (&lt; 2m depth) at One Tree Island, GBR (23.5076° S,
 152.0916° E) and maintained in a tropical aquarium at 26 °C. Corals
 (<em>Acropora </em>sp.) that are the preferred prey of COTS
 (3) were sourced from aquarium suppliers. The CCA and live corals were
 maintained in separate 20 L closed system aquaria at 26 °C. Adult COTS
 were maintained in a 160 L tropical aquarium system at 26 °C. Before the
 start of the coral and CCA rubble experiments these cues were maintained
 for 2h in the header tank (25 L) source water before turning on the flow.
 The amount of rubble and live coral used covered the base of the header
 tank as similarly as possible between trials to emulate a layer of habitat
 in nature. Adult COTS were placed in the header source for 30 min before
 the start of the experiment. During this time water circulation was
 maintained using a water pump. The behavioural
 responses of the juveniles were tested in a constant temperature room (26
 °C) using a two-channel choice flume as used in behavioural studies of
 fish larvae and sea urchin juveniles (4, 5). Steady gravity-driven flow
 (8.3 cm<sup>3</sup>/s per channel) in the flume was controlled
 by flow meters (Dwyer MMA series) and laminar flow was established at the
 inflow point by course filter pad lined with a mesh screen. To ensure that
 behaviour was not influenced by vision (6), the experiments were done
 under red light 625 λ (measured by an Ocean Optics, Spectrophotometer
 USB4000; Ocean View 2.0 Software) which exceeds the photoreception range
 of COTS eyes (7). Before use in trials, the juveniles were placed in clean
 containers of 1.0µm UV FSW with no food for 24 hrs. These are also the
 conditions they experienced when first placed in the choice flume before
 the turn-on of flow. All experiments were conducted during the
 day. The water flow on each side of the flume was
 segregated by a divider which ensured parallel flow into the main chamber
 supporting a distinct distribution between the cue source flows and
 eliminated backflow. Each side of the flume was supplied with water from a
 25L header tank, that contained a treatment cue. At the start of each
 experimental run, a single juvenile was placed in the centre of the main
 chamber where it would receive even flow from both sides of the flume. At
 the point of water outflow, a gate was installed to allow the juvenile to
 acclimate for one minute in static water. The gate was then removed, and
 the flow was turned on. Although the exact position of the juveniles when
 the gates were lifted, varied somewhat, we did not adjust to avoid a
 physical disturbance. None of the juveniles were in a location where they
 would only experience flow from one side of the flume.
 Juveniles were allocated 20 min to respond as demonstrated by
 upstream movement towards the cue source or downstream movement. The
 treatment cues, live coral, CCA reef rubble, and COTS were tested
 individually against the no cue FSW only flow and against each other
 (CCA/live coral; CCA/COTS; COTS/live coral). The same juvenile was not
 used more than once per treatment. Treatment cues were rotated between the
 left and right sides of the flume to remove a side bias. Images taken at
 20-second intervals after the acclimation period were captured with an
 Olympus Tough TG-6 (Olympus) camera and were then autogenerated into
 time-lapse videos. The camera was mounted above the choice flume with the
 entire arena and scale in view. Three juveniles climbed the chamber wall
 and “surfed’ at the air-water interface oral side up and so were not used
 for the experiment. Juvenile size (diameter mm) was
 similar, but number of replicates varied somewhat among treatments (FSW x̄
 = 10.32, SE = 0.40, n = 42; COTS x̄ = 10.24, SE = 0.42, n = 50; Coral x̄ =
 9.67, SE = 0.21, n = 30; Coral/COTS x̄ = 9.67, SE = 0.21, n = 41; CCA x̄ =
 9.40, SE = 0.19, n = 27; CCA/Coral x̄ = 12.97, SE = 0.25, n = 35; CCA/COTS
 x̄ = 12.77, SE = 0.36, n = 22). We conducted more runs for the adult COTS
 cue to facilitate the interpretation of juvenile responses because this
 treatment was the most variable. Juvenile movement in
 the time-lapse files was analysed using Tracker (Version 6.0.1). Each file
 was calibrated in Tracker to display at a framerate of 1.5/s. Path length
 (m) was manually calculated from the starting point until the juvenile
 moved towards or away from the direction of flow. Tracking was used for
 single and two-cue treatments for the juveniles that remained in the field
 of view for the entire time and so could be completely followed (n: coral
 = 11, CCA = 14, COTS = 20, FSW = 8, CCA/Coral = 13, CCA/COTS = 12,
 Coral/COTS = 12). The paths taken by the juveniles were traced and
 illustrated as in a previous study of sea star behaviour (8).
 <strong>Statistical analyses  </strong> 
 All analyses were performed using R (v1.2.1335) (9). Chi-squared
 goodness of fit test was used to analyse the percentage frequency of the
 juvenile choice responses with equal expected proportions. Choice response
 data were analysed with a 25%:25%:25%:25% probability of upstream movement
 to either treatment cue, downstream, or no movement. For the FSW only
 (control) the data were analysed with a 33%:33%:33% probability of
 upstream movement, downstream movement, or no movement. The choice data
 were illustrated as the percentage of individuals in each response (e.g.,
 to cue, downstream, no movement). Tracking data on
 speed (mm/min), distance moved (mm) and time to make a choice (min) were
 analysed. For the single cue experiments, only the significant responses
 (to or away from cue) as indicated by Chi-squared analysis were tracked.
 The speed data were analysed by one-way analysis of variance (ANOVA) with
 treatment as a fixed factor with four levels (attraction to coral,
 attraction to CCA, upstream movement in FSW only, avoidance to adult COTS)
 with the FSW as the control. Homogeneity of variance (HOV) and normality
 were confirmed by Levene’s and Shapiro-Wilk tests, respectively
 (significance α = p &lt; 0.05). Tukey’s HSD post hoc-pairwise
 comparison was performed to identify treatments that differed. Data on
 distance moved and time to make a choice did not meet the assumption of
 HOV, and so these data were analysed using Kruskal-Wallis non-parametric
 ANOVA with treatment type as a fixed factor. Dunn Test (R Package FSA)
 with Bonferroni corrected p-values were used post hoc to identify
 significant pairwise treatment effects. For the two-cue
 experiments, data on distance moved, time to make a choice and speed were
 analysed by one-way analysis of variance (ANOVA) with treatment as a fixed
 factor with three levels (CCA/live coral, CCA/COTS, live coral/COTS).
 Assumptions of HOV and normality were confirmed as above, except for the
 normality of the distance moved data. As ANOVA is robust to this (10) the
 analysis was undertaken and a Tukey HSD post hoc-pairwise comparison was
 performed.
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description: # Chemosensory behaviour of juvenile crown-of-thorns sea star
 (*Acanthaster* sp.), attraction to algal and coral food, and avoidance of
 adult conspecifics.
 [https://doi.org/10.5061/dryad.0k6djhb6k](https://doi.org/10.5061/dryad.0k6djhb6k) We have submitted our raw data **(Webb_et.al_2024.CSV)** on juvenile behaviour  ### Choice Response Data (Sheet 1): Choice response of juvenile Crown of Thorns Sea Stars (COTS; *Acanthaster sp*.) when exposed to cues from crustose coralline algae (CCA) rubble, live coral, and adult conspecifics. * *Treatment:* The combination of cues exposed to a juvenile COTS * *Juvenile Choice Response:* The direction of movement or no movement made by the juvenile COTS. * *Count:* The number of juveniles that made each choice response.  *Abbreviations included ### Chi-squared analysis (Sheet 2): Chi-squared goodness of fit analysis of the choice response data in sheet 1. Analyses were conducted in R (version 1.2.1335) using Pearson's Chi-squared test for count data. * *Treatment:* The combination of cues exposed to a juvenile COTS * *Expected probability:* The probability assumed for the Chi-squared goodness of fit test * *Total Count:* The total number of juvenile responses recorded in each treatment with equal expected proportions * *p-value:* The p-value result from the Chi-squared goodness of fit test for each treatment with equal expected proportions * *Chi sq:* The Chi-squared value result from the Chi-squared goodness of fit test for each treatment with equal expected proportions * *df:* The degrees of freedom value from the Chi-squared goodness of fit test for each treatment with equal expected proportions * *Significance (&lt;0.05)*: Significance yes or no p-value less than 0.05. *Abbreviations included ### Single Cue Tracking Data (Sheet 3):  The data output of the single cue videos from Tracker of time and distance per treatment. Speed (mm/min) was calculated from this. * *Treatment:* The cue exposed to a juvenile COTS * *Time (min):* The time taken in minutes for juvenile COTS to choose by moving upstream to FSW or cues from CCA rubble, coral; or downstream away from adult COTS.  * *Distance Moved (mm):* The distance moved in mm by juvenile COTS to choose by moving upstream to FSW or cues from CCA rubble, coral; or downstream away from adult COTS.  * *Average Speed (mm/min):* The speed (mm/min) of juvenile COTS, calculated from *distance moved* and *time* for juveniles to make a choice.  *Abbreviations included ### Pairwise Cue Tracking Data (Sheet 4):  The data output of the two (pairwise) cue videos from Tracker of time and distance per treatment. Speed was calculated from this. * *Treatment:* The cues exposed to a juvenile COTS * *Choice:* The direction choice of movement made by the juvenile COTS  * *Time (min):* The time taken in minutes for juvenile COTS to choose by moving upstream to FSW or cues from CCA rubble, coral; or downstream away from adult COTS.  * *Distance Moved (mm):* The distance moved in mm by juvenile COTS to choose by moving upstream to FSW or cues from CCA rubble, coral; or downstream away from adult COTS.  * *Average Speed (mm/min):* The speed (mm/min) of juvenile COTS, calculated from *distance moved* and *time* for juveniles to make a choice.  *Abbreviations included ## **Code/Software** Tracker (Version 6.0.1) was used to manually track the movement of juvenile COTS using the videos recorded. Each file was calibrated in Tracker to display at a framerate of 1.5/s. All analyses were performed in R (v1.2.1335). Pearson's Chi-squared test for count data (*chisq.test*) from Package *stats* version 4.0.4; *anova* from Package *stats* version 4.0.4