This work was carried out during the seasons 1911 and 1912 at the laboratory of the Marine Biological Association, Plymouth. The experiments were made in order to obtain a method that could be applied to the sea-urchins of the English coast, and in the hope of raising some of the parthenogenetic larvæ to maturity.
We have been successful in rearing the plutei through the late stages, and large numbers in our cultures formed Echinus-rudiments. A few of the larvaa completed their metamorphosis, but we have not succeeded in getting the young urchins to live for more than a few weeks. In all about fifteen plutei underwent metamorphosis.
The methods used were those elaborated by Loeb and by Delage, and a new method which combined certain features from each of these. Loeb's method consists in treating the unfertilised eggs with butyric acid to cause membrane-formation, and subsequently with hypertonic sea-water.
Owing to the condition of the sea-water at Plymouth it is necessary, after membrane formation, to place the eggs in water of raised OH ion concentration before treating them with hypertonic sea-water. The modification of Loeb's method which we employed is as follows. The unfertilised eggs were placed in--
(1) 3 c.c.N/10 butyric acid + 50 c.c. sea-water for 1.5 minutes.
(2) 0.2 c.c. N/10 NaOH + 50 c.c. sea-water for 6 minutes.
(3) 8 c.c. 2.5 M/NaCl + 5 c.c. sea-water for .75-1 hour.
The best experiments with this method gave 60 per cent. blastulæ. The larvæ were healthy and grew well. All those plutei that underwent metamorphosis were obtained by this method.
Delage's method consists in treating the unfertilised eggs with tanuic acid and ammonia in a mixture of sea-water and cane-sugar. The proportions we found most favourable for B. esculentus at Plymouth were 10 c.c. sea-water + 40 c.c. cane-sugar solution (strength, 388 grms. to a litre) + 1.4 c.c. M/60 tannic acid. The unfertilised eggs were placed in this for six minutes, and then 1.5 c.c. M/10 ammonia was added for one hour. We have obtained 80 percent, blastulæ by this method, but they were not healthy, and died off rapidly during the first week. There is no membrane formation by this method.
The method which we have found most successful consisted in treating the unfertilised eggs first with butyric acid to cause membrane formation, as in Loeb's method, and then, by Delage's method, as described above. In this way we have obtained as many as 90 per cent, blastulse. The larvas are vigorous, and grow for the first three weeks more rapidly than larvae from fertilised eggs. We were, however, unable to get any of these larvae to successfully metamorphose. Given in detail, this method consists in treating the eggs as follows :
(1) 3 c.c. N/10 butyric acid + 50 c.c. sea-water for 1.5 minutes.
(2) Wash in two or three changes of sea-water and transfer to--
(3) 10 c.c. sea-water + 40 c.c. 1.13 M sucrose solution + 1.4 c.c. M/60 tanic acid for 6 minutes.
(4) 1.5 c.c. N/10 ammonia is added to (3) for 1 hour.
(5) Wash in three or four changes of sea-water and transfer to normal sea-water.
Finally, there is always a slight difference distinguishable between all parthenogenetic larvæ as compared with normal sperm-fertilised ones. This is noticeable in length of arms, pigmentation, and rate of growth.