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First published online January 18, 2008
Journal of Experimental Biology 211, 292-299 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012864
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Shadow response in the blind cavefish Astyanax reveals conservation of a functional pineal eye

Masato Yoshizawa* and William R. Jeffery

Department of Biology, University of Maryland, College Park, MD 20742, USA


Figure 1
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  		Fig. 1. The shadow response is elicited by shading 1.5 d.p.f. surface fish (A–D), Pachón cavefish (E–H), and Tinaja cavefish (I–L) larvae. Each frame shows an individual assay chamber. Video recordings were made through the side of the chambers at 0.05 s intervals for a total of 5 s. Examples of frames are shown at 0 s (A,E,I), 1.0 s (B,F,J), and 5.0 s (C,G,K). (D,H,L) Merged frames of all images from 0 to 5.0 s. Before shading (0 s), larvae were located at the bottom of the assay chambers. During shading (1.0 to 5.0 s) most larvae swam upwards until they reached the surface of the water. Scale bar, 1 cm.

 

Figure 2
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  		Fig. 2. Gradual reduction of the shadow response during surface fish, Pachón cavefish and Tinaja cavefish development. Values are means ± s.e.m., N=320.

 

Figure 3
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  		Fig. 3. Rhodopsin-like immunoreactivity in the pineal eye of 1.5 d.p.f., 2.5 d.p.f., 3.5 d.p.f. and 4.5 d.p.f. surface fish (A–H), Pachón cavefish (I–P), and Tinaja cavefish (Q–X) larvae. (A,C,E,G,I,K,M,O,Q,S,U,W) Bright field images with focus on the pineal eye. (B,D,F,H,J,L,N,P,R,T,V,X) Fluorescence images of the same specimens as those shown in bright field. Larvae are shown from the left side with the head at the top. Arrowheads mark pineal eye; arrows mark bilateral eye. Scale bar in X, 100 µm; magnification is the same in each frame.

 

Figure 4
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  		Fig. 4. The shadow response of surface fish (A,C,E,G) and Pachón cavefish (B,D,F,H) after pinealectomy or removal of one or both bilateral eyes. The frames show merged images at 0.05 s intervals from 0 to 5.0 s after the assay chambers were shaded. A normal shadow response is observed in sham-operated surface fish and cavefish controls (A,B) and after removal of one (E,F) or both (G,H) bilateral eyes from surface fish or cavefish larvae. In contrast, the shadow response is absent in most surface fish or cavefish larvae after pinealectomy (C,D). Scale bar, 1 cm. Other details are the same as in Fig. 1.

 

Figure 5
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  		Fig. 5. Quantification of the shadow response after pinealectomy or bilateral eye removal in 1.5 d.p.f. surface fish and cavefish larvae. Values are means ± s.e.m., N=392.

 

Figure 6
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  		Fig. 6. Rhodopsin-like immunoreactivity in the pineal eye of 1.5 d.p.f. surface fish (A,B,E,F,I,J,M,N,Q,R) and Pachón cavefish (C,D,G,H,K,L,O,P,S,T) after pinealectomy or removal of bilateral eyes. (A–D) Sham operated controls. (E–H) Pinealectomy. (I–L) Removal of one bilateral eye. (M–P) Removal of both bilateral eyes. (Q–T) Failed pinealectomy. Image pairs show DIC image (left) and fluorescence images of the same specimens (right). Larvae are shown from the rostral (A–P) or left (Q–T) side. Arrowheads mark pineal eye or deleted pineal eye; arrows, deleted bilateral eye. Asterisks (far left) indicate fish of each class that exhibited a positive shadow response. Scale bar in T, 100 µm; magnification is the same in each frame.

 

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© The Company of Biologists Ltd 2008