FISHERY BULLETIN: VOL. 80. NO. 2 



standard tests and models as described in Sokal 

 and Rohlf (1969). 



Removal, Preparation, and 

 Inspection of Otoliths 



Otoliths were removed from embryos, larvae, 

 and juveniles with fine insect needles mounted 

 on wood rods. The larvae are transparent and the 

 otoliths are birefringent under polarized light, 

 so it is possible to view the sagitta during the 

 dissection. The sagittae were washed with dis- 

 tilled water, dried, and mounted on glass slides 

 with Euparol 5 mounting medium, and viewed 

 with a compound light microscope. 



Photomicrographs were made of each otolith 

 for counts of increments and measurement of 

 otolith diameters. (Thelof the outside edge of the 

 sagitta was considered as a portion of the last in- 

 crement.) To make increment counts, the back of 

 each photograph was marked and the photo- 

 graphs were shuffled. The counting process was 

 performed three times, which gave three un- 

 biased readings for each otolith. If two of the 

 counts were identical, that value was accepted as 

 the increment count for a particular otolith. In 

 cases where all counts differed, the middle count 

 was chosen unless all counts varied more than 

 two increments from each other, in which case 

 that otolith was disqualified and not used in the 

 final tabulation. Sagitta were measured at the 

 widest diameter on the photographs using a cali- 

 per calibrated on a photographed micrometer. 



Sagittae viewed with light microscopy showed 

 fine lines in the I that were concentric with the D; 

 these fine lines have been referred to as "sub- 

 units." In the otoliths of young mummichogs the 

 so-called subunits could not be observed in decal- 

 cified sections with light microscopy or SEM 

 (Fig. 1). The D and I compose an increment and 

 are readily differentiated with light microscopy 

 in Fundulus sagittae (Fig. 2). 



Whole sagittae used for SEM studies were 

 attached to viewing stubs in 5-min epoxy resin. 

 The sagittae were ground to the core in the trans- 

 verse plane on graded grinding stones, polished 

 with diamond-polishing compound, and cleaned 

 with 95% ethanol. The polished surface was de- 

 calcified with 7% EDTA (pH 7.4)(disodium ethy- 

 lenediaminetertacetate) for 1 to 5 min. The speci- 

 mens were coated with gold ( 150A) and observed 

 with a SEM. 



5 Reference to trade names does not imply endorsement by 

 the National Marine Fisheries Service, NOAA. 



Embryological Formation of 

 Otoliths 



Fertilized eggs were kept in light 12 h and in 

 the dark 12 h (L12:D12) at24°C in hatching jars 

 with recirculating seawater (30 ..). A sample of 

 10 eggs was collected each day until hatching 

 and viewed under polarized light (120X) to de- 

 termine when calcification was initiated. The 

 embryos were classified according to Armstrong 

 and Child (1965) with the number of embryos 

 with calcified sagitta noted in each stage. 



Calcified sagittae were removed from the em- 

 bryos and mounted for examination with light 

 microscopy to determine the time of increment 

 formation. Ten- and 14-d embryological sagittae 

 were viewed using SEM to confirm the light 

 microscope observations. 



Effect of Light on 



Increment Formation in 



Embryos and Larvae 



To determine the influence of light on incre- 

 ment formation, developing embryos and larvae 

 were subject to the following conditions: 



EMBRYOS: 



Group ED24— Embryo-dark-24 h, fertilized in 

 the dark, kept in constant darkness until sam- 

 pled 3 d after hatching. 



Group EL24— Embryo-light-24 h, fertilized in 

 the light, kept in constant light until sampled 

 3 d after hatching. 



Group ED24+L— Embryo-dark-24+L, fertilized 

 in the dark, kept in constant darkness except 

 for 1 min of light exposure 10 d after fertiliza- 

 tion. Sampled 3 d after hatching. 



Group EL12:D12— Embryo-light-12 h:dark-12 h, 

 fertilized, placed in L12:D12 and sampled 

 daily. 



All groups were maintained at 24°C and the 

 water (30 '/..) was changed daily. The water was 

 changed in the ED24 group and ED24+L group 

 by pouring the eggs onto a 505 jjl mesh net 

 mounted on the end of 10 cm plastic tubing. The 

 eggs were then washed off the netting with a 

 wash bottle and the entire exercise was per- 

 formed in total darkness. Hatching in the ED24 

 group and ED24+L group was determined by 

 touch, because embryos are hard and easily dis- 

 tinguished when they have hatched. A daily 



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