tested as primary marks. They were applied 

 by injecting from 0.03 to 0.08 ml. of marking 

 material between the shrimp's fifth and sixth 

 abdominal segnnents following the method de- 

 scribed by Costello (1964). Of the 65 dyes and 

 inks so tested (table 1), none was found to be 

 fully suitable for use in growth and mortality 

 studies according to my definition, and only 

 four were deemed satisfactory for use as 

 nnarking agents in experiments dealing solely 

 with shrinnp movements. 



The latter included green and blue Bates^ 

 machine inks, both of which concentrated in 

 the branchia within 24 hours after injection 

 and could be visually differentiated from fast 

 green and Trypan blue. The green ink produced 

 a vivid mark that could be identified for up to 

 40 days following application, but thereafter 

 began to fade. The blue ink was not sufficiently 

 striking in color to be distinguishable as a 

 mark by casual observers, but could be de- 

 tected by trained observers. It also began to 

 fade after about 40 days. 



Various combinations of Trypan red and 

 Trypan blue (0.25 percent solutions) were 

 tested as nnarks. Two of these combinations, 

 2 to 1 and 5 to 1 Trypan red to Trypan blue, 

 produced nnarks that were permanent but 

 rather dull and difficult to see. Consequently, 

 shrimp stained with these marks would not be 

 easily detected by fishermen or shrimp proc- 

 essors, and their use would have to be linnited 

 to laboratory studies or to field experiments 

 in which commercial catches are exannined by 

 trained observers. 



SECONDARY MARKS 



A secondary nnark is a spot of color used to 

 code and differentiate a primary mark. Such a 

 mark nnay or nnay not be apparent to the casual 

 observer, but can be distinguished by careful 

 visual or fluorometric exannination. A sec- 

 ondary nnark, since it is not necessarily ob- 

 vious, must be used with a primary nnark. 

 Various inks and fluorescent materials were 

 tested as secondary nnarks (table 2) and were 

 applied together with a primary stain to fornn 

 a double nnark by either the single-injection, 

 double-injection, or tattooing nnethod of mark- 

 ing. 



Double-Injection Method of Marking 



The double- injection nnethod consists of two 

 separate injections, first with a prinnary stain 

 and subsequently with a secondary pignnent. 

 An aqueous solution of a primary stain, fast 

 green or Trypan blue, was injected as de- 

 scribed previously, and a secondary material 

 was applied by means of a 1- or 2-cc. tuber- 



^Trade names referred to in this publication do not 

 imply endorsement of commercial products. 



culin syringe with a 25- or 27-gage needle^ 

 From 0.003 to 0.010 ml. of the secondary 

 nnarking nnaterial was injected through the 

 articular nnennbrane of either the sixth ab- 

 donninal joint to the left of the nniddorsal line, 

 or to one side of the nnidventral line between 

 the second and third abdonninal segnnents. In 

 the latter instance, the needle was directed 

 anteriorly just beneath the cuticle of the exo- 

 skeleton. The location of prinnary and second- 

 ary nnarks is illustrated in figure 1. 



Inks . --For any ink to be suitable as a 

 secondary nnark, most of it nnust rennainatthe 

 injection site. The prinnary nnark is obscured 

 if too nnuch ink migrates into the branchial 

 region, thus preventing differentiation of the 

 nnarked individuals. Red, blue, and black check- 

 writer inks (Sanford), diluted with castor oil 

 or undiluted, were tested for use as secondary 

 nnarks. The results fronn these tests are listed 

 in table 3. In each case, the condition of the 

 secondary mark was recorded at the time of 

 death as good, poor, or unsatisfactory if no 

 nnark was apparent. Neither the diluted nor 

 undiluted inks produced wholly satisfactory 

 secondary nnarks because ink nnoved fronn the 

 injection site into the gills and frequently 

 masked the prinnary marks. Of the inks tested, 

 the undiluted red and blue inks produced the 

 best marks. These two inks are suitable for 

 use as nnarks in field studies designed to 

 obtain general infornnation on movement. 



Fluorescent pigments. --A series of seven 

 laboratory experiments (table 4) was nnade to 

 determine the suitability of fluorescent pig- 

 ments for use as secondary nnarks. The pig- 

 ments Day-Glo neon red A- 12, blaze orange 

 A- 15, arc yellow A-l6, Saturn yellow A- 17, 

 and resoform fluorescent yellow 1 0-2001 (Gen- 

 eral Dyestuff) could be readily distinguished 

 fronn one another. These five pigments were 

 found to provide suitable secondary marks that 

 could be used with primary stains of estab- 

 lished reliability. Mixtures of 1,5 to 4.0 per- 

 cent fluorescent pigment in petroleum jelly 

 produced secondary nnarks that were detect- 

 able under ultraviolet light. 



The fluorescent marking nnaterial was most 

 easily injected into the experinnental aninnals 

 at tennperatures ranging fronn 70° to 90 F. 

 Lower tennperatures increased the viscosity 

 of the petroleunn jelly and made it difficult to 

 eject the mixture fronn the syringe. Con- 

 versely, at tennperatures greater than 90° F., 

 the viscosity of the petroleum jelly was de- 

 creased and more material was often injected 

 into the animals than was necessary for iden- 

 tification purposes. 



In contrast to the primary marking mate- 

 rials, the fluorescent pigment usually re- 

 mained at the injection site, although in some 

 cases nninute announts could be found in the 



