190^] Davis. — Life-historu of DoUchoglossus. 189 



D. i)t(.'>iUt(.s has its burrow near the surface, but there are no 

 external indications of its presence except occasionally when the 

 animal extends its proboscis from the burrow (Ritter '02). A 

 certain amount of prospecting is therefore necessary before the 

 animals are found in large numbers. 



When such a place is found, a spadeful of mud is dug up 

 and the burrow of each animal carefully examined for eggs. By 

 breaking one side of the burrow and gently lifting the animal out 

 or piLshing it aside, the eggs, if present, may be seen clinging to 

 the unbroken side. They are usually closely packed and some- 

 times extend aver an area of several square millimeters. The 

 position of the eggs in a burrow is shown in plate 4. Here the 

 animal has been lifted up and to one side, leaving the eggs ex- 

 posed. The eggs are somewhat flattened, whereas in water they 

 are perfectly round. They also appear to be of a darker tint of 

 yellow ochre than when seen in water. This is due to the dark 

 background of the mud. 



From the burrow the eggs are removed by means of a fine 

 pipette to a shallow dish filled with clear water. By holding the 

 dish against a ^^•hite background, the eggs may be easily sepa- 

 rated from the particles of sand, and then transferred to small 

 vials of sea-water for transportation. As eggs or larvae of a 

 single burrow are usually in nearly the same stage of develop- 

 ment, when a large number in one burrow is found, they are kept 

 in a separate vial. On reaching the laboratory the material is 

 transferred from vials to small dishes filled with fresh sea-water. 

 An occasional change of water is all that is necessary to keep the 

 animals alive. 



Zenker's fluid, corrosive-acetic mixture, Lo Bianco 's chrom- 

 osmic mixture, and osmic acid were used as killing and fixing 

 agents. After fixation and washing the specimens were preserved 

 in eighty per cent, alcohol. Eggs or larvae from single burrows 

 were kept in separate dishes. Some were killed and preserved 

 from time to time, the intervals depending upon stage of develop- 

 ment. Fifteen such series were made. 



Various stains were used, including Mallory's connective 

 tissue stain, Meyer's acid haemalum, Benda's iron haematoxjdin, 

 Heidcnhain's iron haematoxylin, Delafield's haematoxylin, and 



