EMBRYOLOGY OF THE SEA LAMPREY 



113 



action of the thermal units was transmitted witli 

 little time lag to the inner chamber wliich held tlie 

 container of eggs. 



Heat for the high temperatures was provided 

 hy Bronwil circulators and heaters used in aquaria 

 filled with lake water and provided with a con- 

 trollable air bubbler. Bowls and pans were used 

 to contain the eggs within the aquaria as in the 

 troughs. Air bubblers and water circulation by 

 the Bronwil circulators provided air for the 

 aquaria. The circulators equipped with a contact- 

 tiiermostat and thermometer can be utilized for 

 any temperature setting from room temperature 

 to boiling. In order to attain the maxiiuum 

 range from this piece of apparatus the aquaria 

 were located within a cool room where it was 

 expected tiiat the ambient temperature would not 

 rise above the desired temperature. Tiie temper- 

 ature variation for the circulator was advertised as 

 ±0.18° F. In actual practice, however, temper- 

 ature variation could not be noticed. The appara- 

 tus just described was utilized for experiments at 

 temperatures of 65° to 80° F. ; the troughs were 

 used for temperatures from 45° to 70° F. 



Prior to each experiment, troughs and aquaria 

 were washed, air-dried, and refilled with lake 

 water; the thermostat was then set at the desired 

 temperature. Observations of the temperature 

 at 5-minute intervals for a period of 4 to 8 hours 

 and occasional readings in the remainder of a 24- 

 hour interval, assured stabilization at the correct 

 temperature. After the desired temperature was 

 established, Taylor thermographs and hourly 

 readings of total-immersion thermometers (placed 

 on submerged rubber stoppers grooved to receive 

 them) gave a further check. The thermometers 

 were set in such a manner as to be readily visible 

 without liandling. Thermographs were not used 

 with the circulator but tlie temperature was 

 watched for 4 to 12 hours prior to the initiation 

 of the experiment. Because of the small varia- 

 tion in temperature delivered by the circulator, a 

 thermograph record was considered unnecessary 

 except when the ambient temperature was ex- 

 pected to rise above that desired. Furthermore, 

 a submerged thermometer was compared period- 

 ically witli the contact-thermostat and thermom- 

 eter. 



All sampling was random throughout the experi- 

 nuMits, and in general, tiie procedure varied only 

 slightly from that outhned below. Fertilization 



was considered zero time; the first sample was 

 taken 20 to 30 minutes after fertilization. There- 

 after, samples were taken at the following hours: 

 1, 2, 3, 4, .._ 12, 14, 16, 18, 20, 24, 28, 32, 40, 



48, 72. After the 72d hour samples were 



taken at 12-hour intervals until the end of the 

 experiment. In some of the longer experiments, 

 samples were taken each 24 hours after about the 

 18th day. In addition to the samples taken 

 during the run, all remaining eggs and larvae were 

 kept as a final sample. 



Samples of specimens were placed in SjTacuse 

 dishes and the gross morphological characteris- 

 tics of the embryos were observed under a binoc- 

 ular dissecting microscope. The microscope was 

 equipped witii a calibrated ocular micrometer with 

 which all measurements were made. Immediately 

 after these observations all samples were placed 

 in Smith's fixative' for 12 to 24 hours, washed in 

 several changes of water during a 24-hour period, 

 and preserved in a 4-percent solution of formalin. 

 This method of fixation and preservation was 

 most satisfactory as judged by the pliabilitj' of 

 the heavily yolk-laden eggs after 3 years' pre- 

 servation. 



Specimens to be sectioned for microscopic exam- 

 ination were washed overniglit in running tap 

 water, stained in alum-cochineal 16 to 24 hours, 

 then dehydrated and embedded in paraffin. Sec- 

 tions cut at 5 to 10 micra were mounted and 

 counterstained in a 0.5-percent solution of fast 

 green in 95-percent ethyl alcohol and covered 

 permanently. 



DESCRIPTION OF STAGES 



The following description of stages was based 

 on materials which were taken from all of the 

 batches of the different temperature series; but 

 greatest emphasis was placed on observations of 

 the 65° F. batches. Most characteristics of 

 embryos of the other batciies were itlentical with 

 those reared at 65° F. Those differences which 

 were observed are pointed out in the discussion 

 of the particular stage. The time intervals listed 

 for each stage include tiie time between the first 

 and last appearance of the stage in tlie samples 

 reared at 65° F. 



The end-points selected for each stage were 

 established after numerous observations of both 



'.Solution A: PotiiSSium hlchronuile 0.5 grn., wutor 87.5 cc. .Solution B 

 Formalin 10 cc, glacial acetic acid 2.5 cc. MK solutions .1 and B ImmcJl- 

 atcly before using. 



