292 EYE FIELD OPERATIONS 



There has been considerable discussion, in recent years, about the possibility and the 

 method of lens regeneration in later larval stages (Schotte and Hummel, 1939 and Stone 

 and Sapir, 1940). The latter authors investigated Urodeles, Anura, and Fish, all of 

 which were at least 25 and many as much as 80 mm. in length, and they came to the con- 

 clusion that at these advanced stages the rim of the iris does not possess the power to 

 regenerate a new lens. In fact, there is evidence of species variation at the earlier 

 larval stages for Stone and Dinnean (1940) found no Wolffian regeneration in A. punctatum 

 at any time. Stone and Sapir (1940) state: "Among the Triturus this unique phenomenon 

 has been proved beyond all doubt. It opens up an interesting field of study to determine 

 more clearly what factors inhibit and release the regeneration of a lens from the rim of 

 the iris in this group of salamanders. " 



ENUCLEATION AND PIGMENTARY RESPONSES 



Scharrer (1932) in reference to salamander larvae, stated that ". . . . in addition to 

 sight and smell, the lateral line sense organs may play a role in obtaining food" and 

 Nicholas (1922) claimed that in the absence of the eyes, the sense of smell became para- 

 mount in the feeding reaction as evidenced by the animal's positive response to substances 

 diffusing in the water. Detwiler and Copenhaver (1940) state: "We wish to emphasize the 

 fact that in the absence of both the eyes and the nasal placodes the larvae feed as well as 

 do the normal animals. " Utilizing these facts, it is possible to enucleate (i. e. , remove 

 the eyes) Amblystoma larvae (stages #25 to #27) and rear them on Enchytrea (white worms) 

 in light, darkness, and even under various concentrations of monochromatic lighting, to 

 determine the relation of the eyes to both growth and pigmentary responses of the skin. 



1. Enucleate Amblystoma larvae (stages #25 to #27) and keep them in adequate 

 aquaria for 3 to 4 days in order to select those individuals which survived the 

 operation most satisfactorily. If necessary, anesthetize them, before enuclea- 

 tion, in 1/3, 000 MS 222. 



2. Prepare a completely darkened environinent (e. g. , photographic dark room) but 

 one in which the temperature does not vary from that of the light environment. 

 This may require the circulation of air with a fan, for any dark cover will absorb 

 radiant energy more rapidly than a light colored cover. The temperature must 

 be checked at least once daily. Another and most satisfactory method is to coat 

 the outside (sides and bottom) of finger bowls with flat black paint, and provide 

 an overlapping black-painted cover. These darkened finger bowls, along with 

 the controls (unpainted finger bowls), can then be kept together in a constant- 

 temperature water bath. Daylight (but not sunlight) should be provided. If this 



is not possible, controlled artificial light (without heat) should be provided. 



3. Place in each of 10 blackened finger bowls a single enucleated Amblystoma with 

 50 cc. of growing medium. Place in each of 10 more blackened finger bowls a 

 single normal (unoperated) Amblystoma. 



4. In a similar manner prepare 10 unpainted finger bowls, placing in each a single 

 enucleated Amblystoma and in each of 10 :Tiore unpainted finger bowls, place a 

 single normal (unoperated) Amblystoma. 



5. All 40 finger bowls should be kept at the same temperature. The water should 

 be changed on alternate days. The young larvae may be fed first on small 

 Daphnia and, as they grow, on small and finally on large Enchytreid worms. 

 The feeding should be identical for all larvae, experimentals and controls, in 

 dark and in light. 



6. The darkened animals may be changed and fed in a photographic darkroom, with 

 a very din:i red (photographic) light, in minimum time. The quickest procedure 

 is to pour the larva and medium through a coarse sieve, to eliminate faecal ma- 

 terial. 



6. Keep the larvae to the time of metamorphosis (50 to 60 days), using this change 

 as one criterion of growth rate. Measurements of all larvae should be taken at 



