for the presence of dead individuals or evidence 

 of shedding, and periodic counts were made of 

 the shrimp remaining in each experiment. Ab- 

 dominal and cephalothoracic exoskeletons were 

 removed whenever found. Shedding shrimp 

 were observed to eat the branchial cast and 

 associated structures, and these were conse- 

 quently left in the tanks . An indication of the 

 number of shedding individuals was obtained by 

 counting the cepholothoracic exoskeletons found 

 in each tank. Since the entire exoskeleton as 

 well as the newly shed shrimp may be cannibal- 

 ized, this provided only a minimum figure. 

 When bodies were found, shrimp which had died 

 from the effects of experimental procedures or 

 other causes were differentiated from those 

 eaten by their fellows during or immediately 

 after ecdysis by the presence or absence of a 

 firm exoskeleton. 



Stains 



Stains were selected which complied 

 with one or more of the following criteria: 

 solubility in water or other nonlethal solvent, 

 previous use as a vital stain, and a color so 

 opposed to the natural coloration of shrimp as 

 to be readily detected by the casual observer. 



necessary to first dissolve the stain in a small 

 quantity of distilled water when preparing a 

 sea -water solution. When a stain proved difficult 

 to dissolve, the mixture was warmed and filtered 

 through Whatman No. 1 filter paper. The per- 

 centage of stain solution indicated for each 

 experiment is based on the weight of dry stain 

 to 100 milliliters of solvent. As no advantage 

 was found in aging solutions, stains were usual- 

 ly prepared just before use. 



EXPERIMENTAL METHODS 



The marking qualities of each stain were 

 tested by one or more of three methods: immer- 

 sion, injection, and feeding. After preliminary 

 tests the laboratory procedure for each method 

 was standardized as follows: 



A. Immersion: The shrimp sample was 

 placed in an aerated stain solution for a prede- 

 termined number of minutes. The shrimp were 

 then transferred through two changes of fresh 

 sea water at 5-minute intervals so as to remove 

 excess stain adhering to the exterior of the 

 animals or trapped within the branchial chamber. 

 A final transfer was then made to an appropriate 

 storage tank for observation. 



With a single exception all stains were 

 obtained from the Hartman-Leddon Company 

 (Harleco) or the National Aniline Division of 

 the Allied Chemical and Dye Corporation . Wide 

 variability in the characteristics of stains mar- 

 keted by different manufacturers is common, 

 and it was considered most practical to limit 

 tests to material readily available from a few 

 American producers . Stains certified by the 

 Biological Stain Commission at Geneva, New 

 York, were used when available. 



Pertinent data on the source and quality 

 of each stain are shown in table 1 . The total 

 dye content refers to the percentage of dye in 

 the dry stain. The lot number is the manufac- 

 turer's identification number of the dry stain. 

 Complete information on each stain may be 

 found in Conn (1953). 



Distilled water, filtered sea water, 

 glycerine, mineral oil, and alcohol were used 

 as solvents. Most of the dry stains were rela- 

 tively insoluble in sea water, and it was usually 



B. Injection: A 1-cc. tuberculin syringe 

 equipped with a No. 25 by 1/2 -inch needle was 

 most practical for laboratory use. Holding the 

 syringe in the right hand, a shrimp was grasped 

 with the left so that its head was pointed toward 

 the left wrist and with its abdomen held in a 

 flexed position by the left thumb and forefinger 

 (fig. 1). The needle was then introduced through 

 the articular membrane of the sixth abdominal 

 joint slightly to the left of the middorsal line and 

 at an angle approximating 45 degrees. The needle 

 was inserted to a depth of from 2 to 4 millimeters 

 until stain was visibly entering the blood-vascular 

 system through the dorsal abdominal artery. 

 After injection the shrimp were examined for 

 signs of physical damage or excessive weakness. 

 Shrimp killed or incapacitated by overinjection 

 or obvious physical damage were replaced be- 

 fore transfer to final observation tanks. Volume 

 of individual injections generally ranged from 

 0.01 cc. to 0.05 cc. and averaged about 0.03 cc . 

 Injections of greater volume frequently resulted 

 in rapid death and did not produce more vivid or 

 durable staining. The 1/2 -inch No. 25 needle 



