FISHERY BULLETIN: VOL. 80, NO. 2 



vided and subjected to the following conditions: 



Group LaD24— Larvae-dark-24 h, constant 



darkness. 

 Group LaL24— Larvae-light-24 h, constant 



light. 

 Group LaL6:D6— Larvae-light-6 h:dark-6 h. 

 Group LaL12:D12— Larvae-light-12 h:dark- 



12 h. 



All groups were fed newly hatched brine 

 shrimp ad libitum and kept at 24°C with daily 

 changes of water at 30V... Samples of 10 larvae 

 from each group were taken at days 0,6,9, and 16 

 except the L12:D12 group, which was sampled 

 daily. Sagitta were removed from each sample 

 and photomicrographed. Scanning electron mi- 

 crographs were made of samples for comparison 

 with the light micrographs. 



RESULTS 



Formation of Otoliths in 

 Embryos 



The sagittae were the first tissues to calcify 

 and were discernible on days 3 and 4 at embry- 

 onic developmental stages 24-28 (Armstrong and 

 Child 1965). An amorphous mass was discernible 

 in the labyrinth region of the larva before calcifi- 

 cation was initiated. This mass, the core organic 

 matrix, had a gellike consistency and could be 

 dissected. Calcification was initiated in the core 

 of the sagitta of 30% of the embryos on day 3 and 

 100% of the cores showed calcification by day 4 

 (Fig. 3). Increment formation began on day 12, 

 and 20% had one increment. On day 13, 80% had 

 one increment and 20% had two increments. On 

 day 14, the day of hatch, 20% had one increment, 

 70% had two increments (Fig. 2), and 10% had 

 three increments. 



Calcification began with formation of crystals 

 which extended to the edge of the core matrix. 

 Histochemical analyses have shown that calcifi- 

 cation begins in the core at the same time that the 

 core becomes birefringent (J. Yamada 6 ). Multi- 

 ple spherules (Fig. 4a, b) are common in the calci- 

 fied core but their origin and sequence of devel- 

 opment is unknown. The newly formed sagittae 

 had a mean diameter of 0.024±0.004 mm. Calci- 

 fication continued and additional crystals ex- 



tended beyond the original boundary in an in- 

 terlocking fashion until the diameter reached 

 0.048+0.008 mm at day 9 and developmental 

 stage 36. At this time only the core region could 

 be observed, with no increments (Fig. 3). Two 

 days later (on day 11 postfertilization), incre- 

 ment formation was initiated around the core, 

 and the mean sagitta diameter had reached 

 0.074±0.008 mm. When viewed with trans- 

 mitted light, the concentric increments consisted 

 of alternate narrow, dark discontinuous zones 

 (D) and wider, lighter, incremental zones (I) 

 (Fig. 3). The D intersected the I at right angles 

 and were concentric with the core and outer sur- 

 face of the otolith. Upon hatching at day 14, post- 

 fertilization, two or three increments were 

 readily discernible as daily increments started 

 forming 2-3 d before hatching. 



Otoliths examined with the SEM confirmed 

 the increment counts determined under trans- 

 mitted light and showed the orientation of the 

 crystals (Fig. 1). 



Effect of Light on 



Increment Formation in 



Embryos and Larvae 



The light cycle to which an embryo or larva 

 was exposed had an effect on increment forma- 

 tion and hatching time. Embryos in the L12:D12 

 cycle had two or three increments prior to hatch- 

 ing and one increment per day after hatching 

 (Table 1, Fig. 3). Embryos kept in L12:D12 

 hatched at 14 d while those exposed to other light 

 cycles had longer incubation times and a differ- 

 ence in increment formation during incubation 

 and after hatching was apparent in the other 

 groups (Table 2). Embryos incubated inconstant 

 dark (ED24)had a delayed hatch, suppressed in- 

 crement formation (Fig. 5), and a smaller otolith 



Table 1. — Sagitta were from Fundulusheteroclitus 

 embryos and larvaeincubatedonaL12:D12eycleat 

 24°C (N = 10/d). 



6 Faculty of Fisheries, Hokkaido University, Hakodate. 

 Hokkaido, Japan, pers. commun. 



206 



