Blood et al.. Embryonic development of Theragra chalcogramma 



209 



Methods 



Incubation 



Adult walleye pollock were collected with a rope 

 trawl off Cape Kekurnoi (57°42.5'N, 155°16.2'W) in 

 Shelikof Strait, Alaska, on 7 April 1989 from the 

 NOAA research vessel Miller Freeman. Eggs from 

 one female and milt from three or four males were 

 hand stripped into glass petri dishes, gently mixed, 

 and left undisturbed for one minute. Eggs were then 

 rinsed, transferred to 3°C (surface water tempera- 

 ture) seawater in glass jars (3.8 L), and held two 

 hours. Floating eggs with a perivitelline space were 

 assumed to be fertilized (Blaxter, 1969; Alderdice, 

 1988). Viable eggs were poured into eighteen 0.5-L 

 jars filled with 3°C seawater. Eggs were not counted 

 but apportioned similarly among the jars at a con- 

 centration of about one egg/mL. Six capped jars were 

 held in each of three water bath incubators onboard 

 the Miller Freeman. Initial incubation temperatures 

 were set to include the range of temperatures in the 

 area. Mean water temperature at depths of 150-200 

 m in Shelikof Strait, where most eggs are found 

 (March-May) (Kendall and Kim, 1989), is 5°C; ex- 

 tremes of 3.6° and 5.9°C have been reported (Reed 

 and Schumacher, 1989). Incubators were sealed to 

 minimize light and movement and placed in sepa- 

 rate refrigerators adjusted to 3°, 5°, and 7 C. One- 

 half of the water in the jars was replaced every day 

 with seawater of the same temperature. Eggs were 

 preserved in phosphate buffered formalin (5%) 2 or 

 Stockard's solution 3 (Velsen, 1980). Stockard's solu- 

 tion cleared the chorion and darkened embryonic 

 tissue, easing examination of embryonic develop- 

 ment. Phosphate buffered formalin did not darken 

 embryonic tissue as much as Stockard's solution, 

 yielding better definition of some structures like 

 somites and otic capsules. Live, newly hatched lar- 

 vae were measured (standard length in millimeters) 

 and preserved (5% buffered formalin). Detailed exami- 

 nation and morphological description of embryos were 

 completed after eggs were returned to the laboratory. 

 During the first 24 hours after fertilization, eggs 

 were sampled at 2-3 hour intervals. After 24 hours, 

 intervals were increased to about 6 hours. When an 

 interval was less or greater than 6 hours, the sub- 

 sequent sampling time was adjusted to return to the 

 original 6-hour schedule. Data were not recorded for 

 three sampling times late in development because 



intervals were inadvertently extended to 12 hours 

 (236, 258, and 282 hours). 



At each interval, 10 to 50 eggs were sampled from 

 one jar per incubator; only one jar was sampled to 

 ensure there would be enough eggs and larvae left 

 to sample near the end of the incubation period. Jars 

 were sampled in rotation throughout the duration 

 of the experiment until no eggs remained. When 

 eggs began to hatch, all jars were checked and newly 

 hatched larvae were removed in addition to eggs 

 scheduled to be sampled. Dead eggs were removed 

 from the designated sample jar at each interval. 

 Water bath temperatures were recorded for every 

 sampling interval. Frequent opening of refrigerators 

 during the initial short sampling intervals increased 

 temperatures in the refrigerators despite thermostat 

 adjustments. Water bath temperatures stabilized 

 after 48 hours to 3.8°, 5.7°, and 7.7°C. 



Morphological descriptions 



Eggs were examined with the aid of a dissecting 

 microscope (6-50x magnification) and described ac- 

 cording to a 21-stage scheme adapted from Naplin 

 and Obenchain (1980) (Table 2). Morphological 

 terms follow Trinkaus ( 1951) with one exception: the 

 term "blastodisc," in this paper, includes the germi- 

 nal area from the time of cytoplasm polarization 

 until embryonic shield formation (Markle and 



Table 2 



Stages of embryonic development of Theragra 

 chalcogramma (adapted from Naplin and 

 Obenchain, 1980). 



Stage 



Developmental stage 



2 50 mL 37% formaldehyde, 4.0 g sodium phosphate monobasic, 

 6.5 g sodium phosphate dibasic, made up to 1 L with distilled 

 water. 



3 50 mL 371 formaldehyde, 40 mL glacial acetic acid, 60 mL 

 glycerin, and 850 mL distilled water. 



