Pictures of the eyes were taken in vivo using a Nikon D300 camera equipped with a 105-mm Micro-Nikkor lens and also with a Leica dissection microscope equipped with a digital camera (Evolution MP 5.0, Media Cybernetics, MD, USA).
The 12 animals used for the eye size measurements were collected from several reefs near Cairns, Australia, in May 2015 and transported to the Australian Institute of Marine Science in Townsville by the Australian Marine Parks Tourist Operators crown-of-thorns starfish control programme. They were kept in holding tanks supplied with running, filtered sea water at a temperature of 27° C. In captivity, the starfish were not fed and were used within 3 weeks of collection. Eye dimensions were measured, and ommatidia were counted manually from images taken through a stereo microscope. For this experiment, the animals ranged in diameter from 1.5 to 38 cm, measured under water.
Three eyes from three separate specimens collected off the coast of Cairns, Australia (16°50′50.82″S, 146°13′37.92″E), in April 2014, were used for transmission electron microscopy. Eyes were fixed in 2.5% glutaraldehyde, 1.5% paraformaldehyde and 3% sucrose in 0.1 M phosphate-buffered saline (PBS) buffer overnight. The eyes were post-fixed in 1% osmium tetroxide in 0.1 M PBS overnight at 4 °C, dehydrated in a series of ethanol and acetone, embedded in Epon 812 resin following standard procedures and contrasted with lead citrate and uranyl acetate. The 70-nm-thick sections were observed in a JEOL 1010 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a SC1000 Gatan digital camera (Gatan, Pleasanton, CA, USA). Longitudinal sections of five randomly chosen, fully developed ommatidia were used to measure ommatidial size and receptive fields.
The spectral properties of the red screening pigment were measured on a squash preparation of an eye using a spectroradiometer (ILT900 W, International Light Technologies Inc., Peabody, MA, USA) and a compound microscope. A fresh eye was used from a specimen collected off the coast of Cairns, Australia, and shipped to Denmark in January 2016. After arrival, the starfish was kept at the Danish National Aquarium, The Blue Planet, in a 12,000-L tank with recycling sea water with a salinity of 33 ‰ and a temperature 26.5 °C. The squash preparation was viewed in a compound microscope and arranged such that the red pigment took up most of the visual field. The relative absorption curve was calculated from the difference in the spectra of the unfiltered background light compared to the light that travelled through the red screening pigment, measured with the spectroradiometer.
In the compound eye of the crown-of-thorns starfish, each eyelet (ommatidium) collects light from different areas in space; these differences in viewing direction can be expressed in angles and measured by a goniometer (angle measurement tool). Five eyes of three individuals collected in Cairns in April 2014 and fixed in 4% paraformaldehyde in 0.15 M PBS buffer were used for the goniometry measurements. The eye of the crown-of-thorns starfish is bilaterally symmetrical, as is that of L. laevigata; therefore, only one of the two halves of each eye was measured. The optical axes of six evenly spaced ommatidia around the perimeter of the eye-half were measured to determine the edges of the visual field. To measure the angles between ommatidia, or interommatidial angle, two ommatidia were randomly picked from each eye and the angles between the optical axis of each chosen ommatidium, as well as the optical axes of its nearest neighbours, were measured. Seventy-three angles were measured, and seven outliers were removed that had interommatidial angles greater than 20°.
Animals for the physiological experiments were collected either on coral reefs in Indonesia or on the Great Barrier Reef near Cairns, Australia. The animals from Australia were flown to Denmark in April 2014 and February 2015. The animals from Indonesia were transported to Denmark in May 2015. The starfish varied in size between 10 and 40 cm in diameter, measured underwater, from arm tip to arm tip, not including the spines. The animals were fed a fish-based gel every second day and were used for experiments within 6 months.
The starfish were taken from the holding tank at The Blue Planet in the morning and transported for approximately 30 min to the University of Copenhagen in a styrofoam box containing sea water. After the experiments, the animals were returned to The Blue Planet. The electrophysiology experiments were performed between November 2014 and July 2015. The ambient temperature of the set-up was 25 °C. For the experiments with flash stimulation, seven eyes from four animals were used. For the flicker fusion frequency experiments, eight eyes from four different animals were used.
Three electrophysiological experiments were performed. First, the absolute sensitivity of the eye to light was tested by stimulating the eye with 100 ms flashes of light of varying intensity. Second, as a part of the previous testing sequence, the colour of the light flashes was changed to test the eye’s spectral sensitivity. Third, the temporal resolution was determined by means of a flicker fusion experiment. In this experiment, a light was presented to the eye with a sinusoidally modulated intensity. The photoreceptors can perceive the changes in light intensity up to the so-called flicker fusion frequency.
Eyes were removed from the animals using fine scissors and placed into a Petri dish containing sea water. The extracellular electrode was attached to an ommatidium using suction, and the reference electrode was placed in the bath of sea water. All electrophysiological measurements were taken with 90–120 s rest between stimulation to prevent photoreceptor adaptation.
Prior to the flicker fusion experiments, the eye was adapted to mid-intensity (1.3 × 102 W sr−1 m−2) white light for 15 min after which the eye was exposed to light with a sinusoidally modulated light intensity for 60 s when data were recorded.
For experiments using flash stimulation, the eye was dark adapted for 30 min before the experiment and the experiments were performed in darkness. The duration of the light flashes used was 100 ms, and data were recorded for 5 s.
All recordings were amplified a thousand times, filtered with a 50-Hz notch filter and a 0.1-Hz high-pass filter together with a 1-kHz low-pass filter on a DC1700 differential amplifier (AM Systems Inc., WA, USA). The signals were digitised at 1 kHz using a NI USB-6229 DAQ card (National Instruments, TX, USA), which was controlled by a custom-made LabVIEW program (National Instruments, TX, USA).
As a stimulus, a Luxeon LXHL-LW6C LED (Philips, San Jose, CA, USA) was used for the flicker fusion frequency experiments and a Luminus CBT-90 LED (Luminus, Sunnyvale, CA, USA) for the flash stimulation. Light intensity was controlled with neutral density filters in steps of 0.3 or 0.7 log units, and the wavelength was controlled using interference colour filters (half width = 12 nm, CVI Laser, Bensheim, Germany) in steps of 10 or 20 nm. Light was transferred to the eye using a 1-mm light guide ensuring a close-to-even illumination of the entire eye. At full power, the light intensity at the exit of the light guide during the flicker fusion experiments was 2.5 × 102 W sr−1 m−2. For the spectral sensitivity experiments, the eyes were stimulated with equal quanta light pulses at 1.7 × 1019 photons s−1 sr−1 m−2.
The flash response recordings were analysed manually in the program Igor Pro 7 (Wavemetrics, Lake Oswego, OR, USA). In contrast to linear models where the R
2 is commonly used, the goodness of fit for nonlinear models is best described by Akaike’s information criterion (AIC) (Spiess and Neumeyer 2010). This measure was used to determine the goodness of fit of the opsin template to the spectral sensitivity curve.
The flicker fusion frequency was calculated from the frequency composition of the signal using a fast Fourier transform in R, version 3.2.1 (R Development Core Team 2012) on RStudio (version 0.98.1103). Care was taken that for each experiment the analysed fragment of the recording contained an equal number of six cycles. Therefore, the length of the analysed fragments varied with stimulation frequency; however, they were always chosen to start at the onset of stimulation. The strength of the response was taken as the power of the Fourier transform at the principle frequency of stimulation.
To determine whether the crown-of-thorns starfish uses vision to locate coral reefs, intact, blinded and sham-operated starfish were placed approximately 1 m away from a reef off the coast of Cairns, Australia (16°50′50.82″S, 146°13′37.92″E). The animals were blinded by removing the eye. Note that this does not affect the chemosensory sense since no other tube feet were affected. The sham-operated animals functioned as a control experiment. These animals received a surgical treatment, just like the blinded animals, but two tube feet from the middle of each arm were removed. The putatively olfactory tube feet at the arm tips were unaffected. The starfish eye is a modified tube foot, so removing tube feet is surgically a similar procedure to removing eyes. Initially, ten intact animals were tested; the same ten animals were used for experiments with blinded animals, making these two experiments paired. After the surgery, the animals were allowed to recover for one day. Seven new individuals were used for the experiments with sham-operated animals. All test animals were collected on the test reef the day before their initial experiments, and they varied between 10 and 17 cm in diameter, measured underwater.
The starfish were tested in groups of up to five individuals. The position of the animals was recorded every min for 5–8 min from digital images (GoPro Hero 2, San Mateo, California, USA). The angle between the reef, the starting position and the final position of the animal was taken as the overall direction of movement. The speed of locomotion was calculated from the displacement of the animal between images. If an animal reached the reef, the point where the animal contacted the reef was used as the final position of the animal. In all experiments, the direction perpendicular to the reef front was set to 0°. The walking trajectories obtained were also used to determine the walking speed for each animal. For statistical analysis, the walking directions were tested with circular statistics (Rayleigh test) and the walking speeds were compared using an analysis of variance (ANOVA), treating the data for the intact and blinded treatment group as unpaired.