Fig. 2 a A frontal photo of a fresh rhopalium of Chiropsella bronzie. The upper lens-eye (ULE), lower lens-eye (LLE) and the statolith (St) lie on the medial plane and the pit eyes (PE) and slit eyes (SE) are located laterally. White pigment (arrows) can be seen outside the dark pigment-layer in both lens eyes. b Frontal view of a fresh lens of the lower lens-eye of C. bronzie. c Side view of the same lens. Scale bars 100 lm

Histology

Histological preparations were made from rhopalia fixed in a solution of 2% glutaraldehyde, 2.5% paraformaldehyde, and 3% sucrose in 0.15 M sodium cacodylate buffer. After 2 h in fixative, the rhopalia were washed in buffer, dehydrated with ethanol and acetone, and embedded in Epon 812 resin. Specimens for transmission electron microscopy (TEM) were post-fixed with 1% osmium tetraoxide in 0.15 M buffer for 1 h at room temperature, then dehydrated in a series of ethanol before transferring specimens into pure acetone and then embedding them in Epon 812

resin. Ultrathin sections were cut on a Leica ultra-micro- tome, mounted on single-slot grids and then contrasted with uranyl acetate for 20 min at room temperature, and lead citrate for 3 min at 5LC. Sections were observed on a JEOL 1240 microscope. Sections for light microscopy were cut with a Leica ultra-microtome, 1–2 lm thick and then stained with methylene blue.

To investigate the histological changes on light/dark adaptation, rhopalia were cut from four different medusae, each contributing two rhopalia for light-adaptation and two for dark-adaptation. Electrophysiological experiments have demonstrated clearly that isolated rhopalia, when kept in sea water, are fully functional for several hours and able to undergo light and dark adaptation (Coates et al. 2006) For light-adaptation, rhopalia were placed in a dish of seawater under an incandescent lamp producing 8,000 lux for 40 min while dark adaptation was obtained by placing rhopalia in total darkness for 40 min. Thereafter, the rhopalia were prepared for light microscopy as described above. Fixation for histology was performed in the afternoon or early evening. Earlier pilot studies had indicated that the time of day has no impact on the pupil response.

Measurements of pupil dynamics

Rhopalia were cut off from animals during the day and further light-adapted (8,000 lux) for approximately 10 min. After this light-adaptation, the rhopalium was photographed, and dark-adapted for 15 min and photographed again. The rhopalium was then light-adapted again until the pupil was observed to contract, and finally dark-adapted for at least 1 h before the last photograph was taken.

Focal length measurements in the lower lens-eye

Method 1: point source imaging by isolated lenses

Fresh lenses from the lower eye were excised from the eye by tearing the retinal cup with two needles, one on either side of the lens. More than 50% of the attempts delivered seemingly intact lenses. The isolated lenses were placed in seawater on a microscope slide and covered by a coverslip, which was supported to form a 1 mm deep cavity. The lens was arranged such that the shortest axis was perpendicular to the incoming light, as would be the case in an intact eye. The microscope condenser was removed and a 1 mm pinhole was placed 11 cm below the preparation to create the ‘‘point source’’. Photographs were then taken using a 109 water-immersion lens, focused at different planes from the eye-lens equator to the focal plane and slightly beyond. The focal point was determined to be where the half-width of the blur spot reached a minimum. Focal length was then plotted against lens diameter (range