736 



Fishery Bulletin 91(4). 1993 



Second experiment A second group of 10 juvenile 

 Dover sole ranging from 55.3 to 118.3 mm was col- 

 lected on 20 March 1991 at 77 m off Cape Foulweather, 

 Oregon, and injected the following day. The fish were 

 held in a flow-through filtered seawater tank. Ambient 

 nearshore oceanic water entering the tank averaged 

 10.7°C (range: 8.7-14.6°C) during the experiment. Fish 

 were exposed to diffused natural light and fed as in 

 the first experiment. Seven days after injection, four 

 fish, 55.3-63.9 mm, were sacrificed. The remaining six 

 fish were individually marked with fin clips on 27 April. 

 They were re-injected on 11 September, 174 days after 

 first injection, and sacrificed 19 days later. Lengths 

 ranged from 88.2 to 152.2 mm. 



Otoliths from both groups of fish were prepared as 

 described above and examined at 100-1000 x on a Zeiss 

 microscope equipped with a IV Fl epifluorescence con- 

 denser. The preparation was examined under full spec- 

 trum light to locate areas with distinct growth incre- 

 ments and the OTC band was then observed under 

 ultraviolet light. 



At least two documentary 35-mm slides of each 

 otolith were taken: one showing the fluorescent band 

 as well as the otolith edge and one showing incre- 

 ments under full-spectrum illumination. Each slide 

 was projected and traced onto the same sheet of pa- 

 per, resulting in a diagram that showed the location 

 of the fluorescent band in relation to increments and 

 other landmarks. The diagram served as documen- 

 tation for increment counts as well as a guide for 

 further examination with light microscopy. Photo- 

 graphs and counts were made at one to four loca- 

 tions on each otolith, usually at the rostrum, anti- 

 rostrum, or post-rostrum (Fig. 1). Increment counts 

 were generally made on right otoliths; left otoliths 

 were used only if right otoliths were unavailable or 

 illegible. All otoliths were read at least twice on sepa- 

 rate dates by the same reader and the average was 

 used in subsequent analyses. The range of estimates 

 for each otolith and standard deviation of mean 

 counts were also determined. 



Results were analyzed by regressing number of in- 

 crements between the proximal edge of the innermost 

 fluorescent band and edge of the otolith against num- 

 ber of days since OTC injection. When a second 

 fluorescent band was obvious and the area distal to 

 the second band was illegible, counts were made be- 

 tween bands. To determine if all experimental groups 

 could be combined into one regression, multiple re- 

 gressions, including "dummy variables" corresponding 

 to experimental groups, were analyzed with partial 

 F-tests (Neter et al, 1989:364-370). The slope of the 

 final regression model was compared to a slope of 1.0 

 (Neter et al., 1989). 



Otolith preparation and analysis for 

 microchemistry 



Otoliths from two Stage-3 larvae (51.3 and 61.9mm), 

 three Stage-4 larvae (54.6, 57.1, and 65.7mm), and 

 two Stage-5 juvenile Dover sole (88.6 and 172.2mm) 

 were examined with a wavelength dispersive electron 

 microprobe to determine if microchemical changes were 

 associated with microstructural changes. Preparation 

 and analytical techniques followed Toole and Nielsen 

 (1992). Beam diameter was 5|.im, counting time for 

 each element was 20 seconds, accelerating voltage was 

 15 kV, and current was 20 nA. 



Results 



Validation of increment deposition rate 



First experiment Otoliths from uninjected control fish 

 exhibited bright yellow-green fluorescence around the 

 edge of the otolith and weaker fluorescence associated 

 with strong checks and scratches. However, fluorescent 

 marks observed on injected fish differed markedly in 

 appearance. Twenty-seven injected fish had at least 

 one otolith with one or two distinct fluorescent bands 

 located inside the otolith edge (Fig. 2, A and B). Fluo- 

 rescent bands extended over two to six increments. 



Increments formed after capture were often less dis- 

 tinct than those formed in nature (Fig. 3), and four of 

 27 injected fish with fluorescent bands (14.8%) had 

 otoliths with such poorly defined increments that they 

 were considered unreadable and were eliminated from 

 further analysis. 



Both daily and subdaily increments (Campana and 

 Neilson, 1985) were observed. For those otoliths in 

 which both subdaily and larger increments could be 

 counted in the same area, the ratio of small to large 

 increments averaged 2.78 (rc = 10 fish, range 1.36-3.48). 

 In most cases, larger growth increments could easily 

 be distinguished from subdaily increments by adjust- 

 ing the focus. However, in otoliths of two fish (7.4% of 

 those with fluorescent bands), only subdaily increments 

 could be observed. These fish were also eliminated from 

 further analysis. 



Second experiment Four Dover sole sacrificed 7 days 

 after first injection had not fed. The remaining six fish 

 began feeding approximately two weeks after the first 

 OTC injection. Two of these fish grew rapidly through- 

 out the experiment, whereas the other four fish ceased 

 growing after about 20 weeks (Fig. 4). 



Two of four Dover sole sacrificed 7 days after first 

 injection and five of six fish sacrificed 19 days after 

 second injection had at least one otolith with a 



