Ocular and extraocular expression of opsins in the rhopalium of Tripedalia cystophora (Cnidaria: Cubozoa)

Abstract

A growing body of work on the neuroethology of cubozoans is based largely on the capabilities of the photoreceptive tissues, and it is important to determine the molecular basis of their light sensitivity. The cubozoans rely on 24 special purpose eyes to extract specific information from a complex visual scene to guide their behavior in the habitat. The lens eyes are the most studied photoreceptive structures, and the phototransduction in the photoreceptor cells is based on light sensitive opsin molecules. Opsins are photosensitive transmembrane proteins associated with photoreceptors in eyes, and the amino acid sequence of the opsins determines the spectral properties of the photoreceptors. Here we show that two distinct opsins (Tripedalia cystophora-lens eye expressed opsin and Tripedalia cystophora-neuropil expressed opsin, or Tc-leo and Tc-neo) are expressed in the Tripedalia cystophora rhopalium. Quantitative PCR determined the level of expression of the two opsins, and we found Tc-leo to have a higher amount of expression than Tc-neo. In situ hybridization located Tc-leo expression in the retinal photoreceptors of the lens eyes where the opsin is involved in image formation. Tc-neo is expressed in a confined part of the neuropil and is probably involved in extraocular light sensation, presumably in relation to diurnal activity.

Figure 1. Cubozoan visual system.

The visual system of the cubozoan Tripedalia cystophora (A) comprises four sensory structures called rhopalia (B). Each rhopalium carries six eyes of four morphological types (lower lens eye LLE, upper lens eye ULE, pit eye PE and slit eye SE) and a light sensitive neuropil (NP, red broken line). The eyes are responsible for the image formation in the animal and the light sensitive neuropil is thought to be involved in diurnal activity.

Figure 2. Graphical representation of the quantitative amplification of leo and neo.

Example of a qPCR amplification run of Tc-leo and Tc-neo, showing the relative relationship between Tc-leo and Tc-neo expression in the Tripedalia cystophora rhopalium. It is evident that Tc-leo is expressed at higher levels than Tc-neo (see also Table 2). The two red shades each represent one replicate of Tc-leo and the two blue shades correspond to Tc-neo replicates. Green horizontal line depicts the threshold value, and the threshold cycle (C_T) is determined by the cycle number, at which the concentration exceeds the threshold. The lowest C_T value for a replicate was chosen for each opsin in each run. Eight runs were performed.

Figure 3. Opsin expression in the rhopalium of Tripedalia cystophora.

In situ hybridization colorimetric staining places Tc-leo mRNA expression in the cell bodies of the retinal photoreceptors of the lens eyes (upper lens eye ULE and lower lens eye LLE) (A,B). The control with the sense probe (C) is devoid of colorimetric staining validating the positive results in A and B. Tc-neo mRNA is expressed in part of the neuropil (D,E), which is also known to have photosensitive properties . Tc-neo sense control is seen in F. None of the opsins are expressed in the lesser eyes (pit eyes PE and slit eyes SE), suggesting that other opsins could be expressed in these eye types.

Figure 4. Graphical representation of expression of Tc-leo and Tc-neo.

While the Tc-leo is expressed in the retinal photoreceptors of the lens eyes, Tc-neo is expressed in the neuropil. The green areas depict the rhopalial in situ hybridization colorimetric staining pattern of the Tc-leo and blue areas represent Tc-neo (A, side view and B, top view). Upper lens eye (ULE), lower lens eye (LLE), slit eye (SE) and pit eye (PE).

Figure 5. Cnidops phylogenetic tree.

Maximum likelihood phylogenetic analysis including representative animal opsins from the “O&O” data set of Feuda et al. plus additional Cnidarian opsins indicates that Tc-leo and Tc-neo are distantly related opsins whereas Tc-leo and Cr-leo are closely related to each other. Illustrated here is a subset of all the genes analyzed, focusing on Cnidarian opsins; Cladonema radiatum (Cr), Hydra magnipillata (Hm) and Nematostella vectensis (Nv). The colors of the branches correspond to their phylogenetic placement in the analysis of Feuda et al. . The full phylogeny, showing all genes analyzed is included in Figure S2. Numbers at nodes are bootstrap values based on 100 pseudoreplicated datasets, implemented in RAxML , assuming a GTR plus gamma model of protein evolution.

Figure 6. Microscopy of the eyes of Tripedalia cystophora.

Light microscopy of the upper and lower lens eyes (A) and pit and slit eyes (B) show that the nuclei (+) are located in the cell bodies outside the zone of pigment granules (*). Transmission electron micrograph (C) shows the cell membrane (arrowhead) and numerous mitochondria (¤) that are located between the pigment granules and the nucleus suggesting the area of protein translation to be adjacent to the nucleus. The folded membranes of the cilium (○) are evident in the outer segments of the photoreceptor cells (C). Scale bar in (C) 2 µm.