Toole et al Otolith microstructure, microchemistry. and early life history of Microstomus pacificus 



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sagittal plane to the level of the central primordium. 

 If resolution was not sufficient, the medial face was 

 also ground to the central primordium. Otoliths from 

 Dover sole >75 mm were ground and read progressively 

 because all increments could not be observed in a single 

 plane in sagittal section. 



In order to examine otolith morphology in other 

 planes, a subsample of otoliths from 17 Stage 3-5 Do- 

 ver sole were embedded in low viscosity Spurr blocks 

 (Haake et al., 1982) and ground to a level even with 

 the central primordium in frontal or transverse sec- 

 tions. Ground sections were covered with a mounting 

 medium and cover slip. 



Additional otolith measurements were obtained from 

 subsamples of Stage-3 and Stage-4 larvae (Table 1) to 

 determine growth and development of features associ- 

 ated with metamorphic stages. The diameter of the 

 clear central growth area (Fig. 1 and Results section 

 below) of 88 right otoliths was measured to the near- 

 est 0.02 mm on a black background at 50x with a 

 dissecting scope and reflected light. Otoliths were then 

 examined at 100, 400. and 1000 x with a compound 

 microscope and video monitor. At 100 x we measured 

 length of the otolith anterior and posterior to the cen- 

 tral primordium to the nearest 0.01 mm in paired left 

 and right otoliths from 93 larvae. 



The number and position of accessory primordia, 

 whether or not central primordium growth was com- 

 pletely enclosed by growth from accessory primordia, 

 number of increments from accessory primordia to 

 otolith edge, and number of increments from enclosure 

 of central primordium growth to otolith edge were de- 

 termined for all Stage-3 and Stage-4 larval otoliths in 

 which the features were legible (Table 1). Similar in- 

 formation was obtained from 6 Stage-5 juveniles. Most 

 counts and determinations were based on right otoliths; 

 however, a subsample of left otoliths was also exam- 

 ined for paired comparisons (Table 1). 



Counts of all growth increments from the first count- 

 able increment outside the central primordium to the 

 otolith edge were made for five of six Stage- 1 larval 

 otoliths and for four Stage-3 larval otoliths exhibiting 

 a variety of patterns of accessory primordium forma- 

 tion. Counts near the central primordium were made 

 with 1000 x light microscopy. 



In all otoliths for which total increment counts were 

 made and in approximately 20% of otoliths for which 

 counts from accessory primordia were made, there were 

 areas where accurate increment counts were not pos- 

 sible. In these areas, the number of increments was 

 interpolated by linear approximation (Methot, 1983; 

 Butler, 1989), based on the average widths of 5-20 

 increments on the distal or, preferably, both the proxi- 

 mal and distal sides of the uncountable area. Stage 3- 

 5 larval otoliths with >5% interpolated increments were 



excluded from further analysis. We present the per- 

 centage of interpolated increments with total increment 

 estimates for the few Stage- 1 specimens examined. 



Validation of increment deposition rate 



Two groups of juvenile Dover sole were given inter- 

 peritoneal injections with a solution of 0.01 g oxy- 

 tetracycline hydrochloride (OTC)/mL distilled water at 

 a dose of 0.1 g OTC/kg fish weight (Campana and 

 Neilson, 1982) to produce fluorescent marks on their 

 otoliths. Dover sole in the first experimental group 

 were held at three constant temperatures to evaluate 

 temperature effects on increment formation. Those in 

 the second experimental group were exposed to identi- 

 cal temperatures and were injected once in March and 

 again in September to evaluate seasonal effects on in- 

 crement formation. 



First experiment An initial group of 48 juvenile Do- 

 ver sole was collected at four stations off Cape Foul- 

 weather, Oregon, on 17 March 1990 (Table 2). Thirty- 

 five fish were injected on that day and 13 served as 

 uninjected controls to check for naturally occurring 

 fluorescence. The fish were held in aquaria contain- 

 ing artificial seawater (Instant Ocean) and fed to sa- 

 tiation with Tiibifex worms once per day in the morn- 

 ing. Fluorescent lights in the room were set to natural 

 cycles, and aquaria were partially covered to reduce 

 incoming light. Rearing conditions are summarized 

 in Table 2. 



Fish were sacrificed 21 («=6), 26 (n=4) or 29 (ra=9) 

 days following initial injection. The remaining 29 fish 

 were given a second injection of OTC 36 days after 

 capture and were sacrificed 5 (n=6), 8 (ra=10), or 12 

 (« = 13) days after the second injection (i.e., 41, 44, or 

 48 days after initial injection). 



