112 



FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE 



of the Hammond Bay Laboratory, and especially 

 John Howell, for courtesies shown me and the 

 time spent in my behalf. 



STAGES IN NORMAL DEVELOPMENT 

 MATERIALS AND METHODS 



Eggs of the sea lamprey were taken from mature, 

 nest-building or spawning lampreys found in either 

 the Trout River or the Ocqueoc River, both tribu- 

 tary to Lake Huron, Presque Isle County, 

 Michigan. 



Spawning lampreys were seized in a forked grip 

 which placed the lamprey between the forefinger 

 and the middle finger while the thumb anchored 

 the animal. Capture by hand, in this way, 

 minimized the loss of specimens by injury. The 

 lampreys were kept in stream water throughout 

 the transfer from the stream to the central collect- 

 ing point, and to the laboratory where they were 

 tempered and stripped in preparation for fertiliza- 

 tion. To condition the eggs and sperm adequately 

 to the test temperatures the lampreys were 

 tempered in running lake water 2 to 3 hours prior 

 to the initiation of the experiments. 



Eggs were removed rapidly from female lam- 

 preys, held by two people. One held the anterior 

 end by placing a thumb within the oral disc, thus 

 taking advantage of the cusps to prevent slippage. 

 The other held the tail by means of a pair of pliers. 

 While the lamprey was thus outstretched, a 

 transverse slit was made with scissors in the mid- 

 ventral body wall at a point behind the heart and 

 the liver, i.e., at the anterior level of the ovaries. 

 If the females were ripe, eggs began to extrude 

 from this initial opening, whereupon the lamprey 

 was slit to the vent by a rapid stroke with half-open 

 scissors. Since the eggs are completely free in the 

 coelom of a ripe female, they were allowed merely 

 to flow into a 10-liter battery jar which contained 

 a few liters of lake water from the trough where 

 the animals were tempered. Any eggs entrapped 

 within the coelom or folds of the ovary were 

 removed rapidly by dipping the lamprey into the 

 water with the slit open. This method of remov- 

 ing the eggs was quicker and far superior to the 

 milking procedure used by McClure (1893) and 

 others (including the author) because the time 

 required to clear the female of eggs was limited 

 only by the dexterity of the operators. Further- 

 more, the eggs are not distorted or damaged. 

 Blood was not introduced into the fertilization jar 



since the slit produced no blood providing neither 

 the liver nor the heart was pierced. 



The males were grasped in the manner described 

 for the females. The individual who was holding 

 the lamprey by the oral disc forced the milt from 

 within the coelom through the genital papilla in a 

 stream directed over the eggs in the battery jar. 

 Two males were used for each female; four males 

 and two females constituted the usual batch. 



Immediately after the addition of sperm, the jar 

 was provided with an air bubbler and placed in a 

 trough previously brought to the desired tempera- 

 ture. The entire operation from stripping to 

 placing in the jar was completed within 60 seconds. 

 Twenty to 30 minutes after fertilization the eggs 

 were washed. Washing was repeated at least two 

 more times dm-ing the first hour to insure complete 

 removal of excess sperm. After the fii-st hour the 

 eggs were apportioned among enamel pans (8" 

 X 12" X 2") or, on occasion, 4-inch glass bowls. 

 These containers were covered with glass plates 

 to minimize the accumulation of water-borne 

 debris, and submerged. Circulation of water 

 beneath the glass covers and over the developing 

 embryos was insured by allowing a small area of 

 the pans and bowls to remain uncovered. Care 

 was taken not to crowd the eggs in the containers. 

 Preliminary work had shown that eggs arranged in 

 more than a single layer were highly susceptible 

 to attack by fungus. 



Development of the embryos took place in the 

 pans or bowls which were held at the desired tem- 

 perature in either constant-temperature troughs 

 or in 20-gallon aquariums. Each insulated trough 

 measured 12 feet by 2 feet by 9 inches and con- 

 tained an inner-water chamber surrounded by a 

 3-inch outer-water chamber. Spaces beneath the 

 metal divider provided free access between the 

 inner and outer cliambers. Water within the 

 inner chamber was provided with air from con- 

 trollable bubblers. 



The troughs were equipped with thermostati- 

 cally controlled iieating units and refrigeration 

 units whicii provided temperature control within 

 ±0.5° F. The refrigeration tubing, tlie heating 

 elements, and the thermostat bulbs lay within the 

 3-inch outer chamber. 



Water was circulated over the thermal units by 

 a continuously revolving 6-vane water wheel 

 driven by a Ratiomotor. Since water flowed 

 freely between tlie outer and inner chambers, the 



