511 Evaluation of Biological Stains, Inks, and Fluorescent Pigments as Marks for Shrimp By Edward F. Klima SPECIAL SCiENTIFiC REPORT-FISHERIES Na 511 UNITED STATES DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE BUR E AtrBTcOMMERciAr^lSHERiEF UNITED STATES DEPARTMENT OF THE INTERIOR Stewart L. Udall, Secretary FISH AND WILDLIFE SERVICE, Clarence F. Pautzke, Commissioner Bureau of Commercial Fishehies, Donald L. McKernan, Director Evaluation of Biological Stains, Ink, and Fluorescent Pigments as Marks for Shrimp By EDWARD F. KLIMA United States Fish and Wildlife Service Special Scientific Report- -Fisheries No. 511 Washington, D.C. May 1965 CONTENTS TABLES No. Page Introduction 1 Types of marks 1 Primary marks 1 Secondary nnarks 2 Double- injection method of marking 2 Single -injection nnethod of marking , 6 Tattooing 7 Summary 7 Acknowledgn-ient 8 Literature cited 8 FIGURE No. 1. Location of nnarking nnaterials in double-marked shrimp 5 1. Inks and dyes tested and found to be unsatisfactory for use as primary marks 3 2. Trade nannes and manufacturers of secondary marking materials 5 3. Results of experiments to determine the suitability of checkwriter inks used with fast green FCF 6 4. Summary of experiments involving fluorescent marks applied by the double-injection technique of marking 6 5. Comparative survival of shrimp receiving no mark, injected with distilled water, or marked with a mixture of two dyes 7 Evaluation of Biological Stains, Inks, and Fluorescent Pigments as Marks for Shrimp by EDWARD F. KLIMA, Fishery Biologist (Research) ABSTRACT Studies were made in the laboratory to find marking materials that can be used to distinguish groups of shrimp in mark- recapture experiments designed to estinnate rates of population growth and mortality. A series of combination n-iarks, formed by injecting various biological stains and fluorescent pigments, was developed. These marks are permanent and do not affect survival. INTRODUCTION Mark-recapture experiments provide a sim- ple and direct nneans for estin-iating growth and mortality in fish and shellfish populations. Recent developnnent of the stain- injection tech- nique of marking has greatly improved our ability to estin-iate these vital paranneters in stocks of penaeid shrinnp. Petersen disk tags, which have been used to mark shrimp, nnay affect survival and growth, whereas nnarking with stains has no discernible effect. Until recently, the staining technique has been sonnewhat limited by the small number of suitable stains available. Of those tested by Dawson (1 957) and Costello ( 1 964), only Trypan blue, Niagara sky blue 6B, Trypan red, and fast green FCF were retained pernnanently in the gills of shrimp. Trypan red is not suitable for use as a nnarking agent, however, because it does not contrast sufficiently with the shrimp's natural coloration to permit shrimp so marked to be easily identified by fishernnen. Shrinnp nnarked with Trypan blue and Niagara sky blue 6B cannot be differentiated from one another and therefore cannot be used to iden- tify groups that n-iight overlap spatially during a particular study. The objective of this in- vestigation was to increase the number of distinguishable marks that can be used sinnul- taneously in mark-recapture experiments. Accordingly, laboratory tests were under- taken to find additional materials for marking shrimp and to deternnine the effect of these marks on shrinnp survival. In the first few experinnents, shrimp were held in groups of 2 0 or more in large tanks supplied with either fresh or recirculating filtered sea water. In later experiments, they were kept in small individual connpartments. The experimental shrinnp ranged in total length from 55 to 179 nnm. --sizes that would nornnally be involved in mark- recapture studies. They were caught in Galveston Bay and included the three species fished commercially in the northern Gulf of Mexico, namely, the white, brown, and pink shrimps, Penaeus setiferus, P^. aztecus, and P. duorarum, respectively. TYPES OF MARKS Permanent, nonlethal nnarks that can be easily detected by casual observers are de- sirable for quantitative mark- recapture studies designed to provide estimates of nnor- tality and growth rates. Marks that do not fulfill all of these qualifications may satisfy the less demanding requirements of studies undertaken to obtain general infornnation on shrinnp movements. PRIMARY MARKS Primary marks are formed upon injection of pignnents (e.g., fast green FCF, Niagara sky blue 6B, and Trypan blue) that rennain in the gills of shrimp and differ sufficiently fronn the animal's natural color to be easily detected by fishermen. Aqueous solutions of biological stains, commercial food colors, and inks were ■"■Contribution No. 200, Bureau of Commercial Fisheries Biological Laboratory, Galveston, Tex. 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 Tatle 1. — Inks and dyes tested and found to be unsatisfactory for vise as primary marks Manufacturer and trade name Concentration of dye Experimental shrimp Remarks Dyes Arthur H. Thomas Co., Philadelphia, Pa. Fluorescelne Baltimore Biological, Baltimore, Mi.: Brom cresol purple Resazurln Fisher Scientific, New York, N.Y. : Brom phenol blue French Food Co., Rochester, N.Y.: Blue liquid food colors Green liquid food colors Red liquid food colors Yellow liquid food colors Harleco, Philadelphia, Pa.: Acid alizarin blue Acid violet lOB Acridin orange Aniline blue Blue 2B Brilliant alizarin blue Brilliant blue FCF Brilliant milUng green Brilliant milling green B Brilliant violet Brom cresol green WS Brom cresol purple WS Brom cresol thymo blue WS CarmLn alum Mayer Clayton yellow Diamond blue 3-B Direct green B Erlochrome violet BA Fuchsln acid Hofman violet Jenner stain Light green SF Malachite green Meldola blue Naphthol Naphthol green B Neutral violet Phosphine Primullne Rhodamin B Rhodamin 6B Saf arlne Percent 1.0 1.0 1.0 1.0 100.0 100.0 100.0 100.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 .5 1.0 Number 10 10 10 10 10 10 10 5 15 15 10 10 15 10 10 10 10 10 15 15 15 10 10 10 15 15 10 10 10 15 10 10 10 15 15 10 10 10 10 10 Faded within 1 week. Do. Do. Do. Do. Do. Do. Do. Do. Do. Faded and/or lethal within 1 week. Faded within 1 week. Do. Do. Do. Faded and/or lethal within 1 week. Faded within 1 week. Do. Do. Do. Do. Do. Faded and/or lethal within 1 week. Faded within 1 week. Do. Do. Faded and/or lethal within 1 week. Faded within 1 week. Do. Do. Do. Do. Do. Do. Do. Faded and/or lethal within 1 week. Lethal within 1 week. Faded within 1 week. Do. Do. Table 1. — IiJss and dyes tested and found to be unsatisfactory for use as primary marks — Continued Manufacturer and trade name Solray purple Tanus green B Thiof lavine S Toluidin blue 0 Uranln Victoria blue B Victoria green K&K Laboratories, Inc., Jamaica, N.Y.: Pontamine fast pink Rivanal Thionine National Aniline, New York, N.Y.: Acrif lavin neutral NF Crystal violet DuPont dearmoior blue Indigo carmine Methylene blue chloride W. H. Curtin & Co., Houston, Tex.: Methyl violet 2-B Rose Bengal. INKS Bates Mfg. Co., Orange, N.J.: Purple Black Harshaw Chemical Co., Cleveland, Ohio: Millioni blue Higgins India Ink Co., Brooklyn, N.Y.: Purple Black National Cash Register Co., Dayton, Ohio: Volatile blue Volatile green Volatile purple Concentration Experimental of dye shrimp Percent Number 1.0 10 1.0 15 .5 20 1.0 10 1.0 10 1.0 10 1.0 10 1.0 10 .5 10 1.0 10 1.0 1.0 1.0 1.0 1.0 1.0 .5 100.0 100.0 1.0 100.0 100.0 100.0 100.0 100.0 10 10 10 10 10 10 10 20 30 10 10 10 10 10 10 Remarks Do. Do. Do. Do. Do. Do. Do. Faded within 1 week. Do. Do. Lethal within 1 week. Faded within 1 week. Lethal within 1 week. Faded within 1 week. Do. Do. Do. Faded and/or lethal within 1 week. Lethal within 1 week. Faded and/or lethal within 1 week. Faded and/or lethal within 1 week. Do. Do. Do. Do. Table 2. --Trade names and manufacturers of secondary marking materials Fluorescent Manufacturer and trade names color Inks: Sanford Ink Co. Bellwood, 111. Black cheokwriter ink No. 638 None Blue checkwriter ink No. 635 Do. Red checkwriter ink No. 638 Do. Pigments : General IJyestuff Co. New York, N.Y. Resoform Fluorescent yellow 10-2001 Yellow Switzer Bros., Inc. Cleveland, Ohio Arc yellow A- 16 Yellow Blaze orange A-15 Orange Neon red A- 12 Red Saturn yellow A-17 Yellow Solutions : Printing Arts Research Laboratory, Santa Barbara, Calif. Fluorographic Chali white ventral sinuses or in the gills (table 4). In no instance, however, did these secondary pig- ments obsucre the primary marks. Both the primary and secondary marks could easily be identified up to 276 days after marking. The marks did not appear to fade, nor were they lost during nnolting. A laboratory experiment was conducted to determine whether it would be possible to localize the secondary pigments in nnore than one area of the shrimp's body in order to in- crease the number of different marks that could be used simultaneously in a mark- recapture experinnent. It was found that the secondary pigments could be confined to the middorsal surface at the joint of the sixth abdominal segment and to the ventral surface between the second and third abdominal seg- ment, thereby effectively doubling the number of distinguishable secondary marks. The possibility that the fluorescent material may be lethal to shrimp was also investigated. The first few experiments provided only pre- liminary information because mortality was confounded by cannibalism or escapement of shrimp. However, the results from these ex- periments suggested that there was no dif- ference in survival between groups of shrimp injected with fast green and any of the five fluorescent marks. In later experiments, it was therefore deenned necessary to determine the effect on survival of only one of the fluorescent marks used with fast green or Trypan blue. The sur- vival of shrimp in this series of experiments (table 5) was not affected by escapement or cannibalism, because each experimental ani- nnal was held in a separate compartnnent. Each experiment included three groups of shrimp, of which one was unmarked, another was injected with distilled water, and a third was injected with a combination mark. The first group served as a control and was conn- pared to the second group to determine the effect on survival of the treatnnent associated with marking. The second group, in turn, acted as a control for the third group to ascertain the influence of the combination mark on sur- vival. Mortality was generally most variable during the first few days after marking and thereafter remained relatively constant. Fewer shrimp died in the marked groups than in the control groups; therefore, neither the marking materials nor the method of marking has any lasting effect on survival. The five fluorescent PRIMARY MARK (Biological Stain) SECONDARY MARK (Fluorescent Pigment) Figure 1. — Location of marking materials in double-marked shrimp. 5 Table 3. — Results of experiments to determine the suitability of checkwriter inks used with fast green FCF [Determination of the condition and location of secondary marks was made after death. Results frcan shrimp that died within 10 days are not included] Ejcperi- Secondary mark Length of experi- ment Condition of secondary marks at injection site Specimens which had ink in gills and at ment Ini Base Dilution Good Poor Unsatis- factory injection site Red Blue Black. . . Red Blue Black. . . Castor oil. . do do None do do 1:2 1:2 1:2 None do do Days 61 61 61 Si, 37 Number Percent 1 2 20 25 17 22 16 6 A 2 1 0 0 12 0 0 2 1 0 6 83 100 100 77 69 89 Table 4. — Summary of experiments involving fluorescent marks applied by the double-injection technique of marking [Determination of the location of the seoonlary marks was made after death. Results do not include shrimp that died within 10 days of marking] Exper- iment Primary mark Secondary mark Experi- mental shrimp Length of ex- peri- ment Specimens which had fluorescent material in gills and at injection site Pigment Base Dilution Number Days Percent 1 Fast green do Blaze orange A-15 Neon red A-12 Castor oil do 1:50 1:50 50 50 61 61 100 100 2 do do do Resoform yellow 10-2001. Neon red A-12 Blaze orange A-15 Petroleum jelly — .do do 1:25 1:25 1:25 50 50 50 84 84 84 52 43 53 3 do do Fast green do do do do Neon red A-12 do do do Petroleum jelly ... .do. •••■•••• ....do ... .do do 3:100 3:100 1:150 1:150 1:150 1:150 1:150 50 50 120 120 120 120 120 55 72 276 276 192 188 276 16 36 Neon red A-12 95 A Blaze orange A-15 Arc yellow A- 16 Saturn yellow A-17 Resoform yellow 10-2001 82 88 85 93 5 do Saturn yeUow A-17 do 1:150 78 24 2 6 do Trypan blue do do do 1:150 1:150 78 78 48 56 12 7 do 2 pigments appear to provide marks that meet the requirements of field mark- recapture studies. Single-Injection Method of Marking This method consists of injecting a mixture of primary and secondary marking materials in a single operation. Various connbinations of fast green and the fluorescent solvent. Fluorographic, were tested as combination marks. The mixture of these materials ap- peared green in color under white light and chalk white under ultraviolet light. After in- jection, the fast green concentrated in the gills within 24 hours, whereas the fluorescent sol- vent remained diffused throughout the body for 2 days and then began to concentrate in the gills. Mixtures of 0.05 percent fast green and Fluorographic in ratios of 7:1 or 8:1 produced Table 5. --Comparative survival of shrimp receiving no mark, Injected with distilled water, or marked with a mixture of two dyes Material Experiment 5 Experiment 6 Experiment 7 Days None Dis- tilled water Fast green and Saturn yellow None Distilled water Fast green and Saturn yellow None Distilled water Tivpan blue and Saturn yellow Number Number Number Number Number Number Number Number Number 0... 78 78 78 78 73 78 78 78 78 1... 72 76 78 78 78 78 77 68 67 2... 68 73 73 76 78 78 73 66 64 4... 67 72 68 76 78 78 71 63 62 8... 65 71 65 76 78 78 68 62 59 12... 57 67 57 76 77 77 64 61 55 16... 54 60 53 75 76 77 56 57 54 20... 45 44 41 74 75 77 39 49 47 2^... 32 34 36 66 69 74 37 44 42 28... _ _ - 62 68 73 30 39 40 32... _ _ - 61 67 72 25 36 34 36... _ _ _ 61 65 66 23 33 31 ^0... _ _ _ 54 58 60 15 26 26 lA... _ _ _ 46 48 58 13 18 19 48... _ _ - 41 27 44 8 15 12 52... _ _ _ - - - 5 11 12 56... - - - - - - 3 5 8 suitable marks. Higher concentrations of the fluorescent solvent tended to mask the green dye. The fluorescent secondary mark could be detected under ultraviolet light for up to 45 days after marking, but soon thereafter began to fade. Consequently, this combination mark is suitable only for short-ternn studies. Tattooing A secondary mark of ink or fluorescent pigment was also applied by tattooing in the manner described by Dunstan and Bostick (1956). A 1-percent suspension of a fluores- cent pignnent in castor oil, or one part Sanford's ink to three parts castor oil, was tattooed into shrimp between the second and third abdominal segments. Within a short tinne, these secondary marks were lost and an infection was observed at the injection site; therefore, this method of marking is not suitable for shrinnp. SUMMARY Prior to this study, fast green FCF, Niagara sky blue 6B, and Trypan blue were the only stains proved suitable for nnarking shrimp in studies of growth and mortality. Laboratory experinnents were therefore made to find addi- tional materials that could be used as shrimp- marking agents. None of the 65 staining nnaterials tested was found to provide fully satisfactory primary marks. Many of the stains were either lethal or faded within 1 week. Combinations of Trypan red and Trypan blue produced marks in the gills that could only be identified by trained observers and would therefore have limited practical use. Green and blue machine inks produced marks that faded but could still be used in short-term marking studies designed to provide general infornnation on shrimp movements. Various inks and fluorescent materials were tested for use as secondary marks in combina- tion with the suitable biological stains. Red and blue checkwriter inks were found to be satisfactory for possible use as secondary marks in field studies designed to obtain gen- eral information on movements. Although the inks remained visible up to 84 days after marking, they frequently migrated into the gills and masked the primary stain. A com- bination of fast green FCF and the fluorescent solvent Fluorographic produced a combination mark adequate for short-term studies. Five fluorescent pigments provided suitable secondary marks which could be used with fast green as a combination nnark in experi- ments designed to estimate rates of growth and mortality. It was possible to localize the fluorescent material in two areas of the shrimp's body, thereby increasing the nunnber of connbination nnarks for shrimp. These marks were permanent and did not affect survival. ACKNOWLEDGMENT Acknowledgment is extended to Charles Knight and Kenneth Osborn for advice and assistance rendered during this study. Clinton E. Atkinson, BCF Biological Laboratory, Seattle, Wash., furnished a tattooing machine. Fred C. June, BSFW, Pierre, S.D., and Thonnas J. Costello and Donald Allen, BCF, Miami, Fla., provided advice on fluorescent pigments and biological stains. Switzer Brothers, Inc., Cleveland, Ohio, and General Dye stuff Com- pany, New York, N.Y., furnished samples of fluorescent pigments. LITERATURE CITED COSTELLO, THOMAS J. 1964. Field techniques for staining- recap- ture experiments with commercial shrimp. U.S. Fish Wild. Serv., Spec. Sci. Rep. --Fish. 484, 13 p. DAWSON, C. E. 1957. Studies on the nnarking of commercial shrimp with biological stains. U.S. Fish Wild. Serv., Spec. Sci. Rep. --Fish. 231 , 24 p. DUNSTAN, WILLIAM A., and WALLACE E. BOS TICK. 1956. New tattooing devices for marking juvenile salmon. Wash. Dep. Fish., Fish. Res. Pap. 1 (4):70-79. MS. #1454 GPO 889 48 1 MBL WHOI Library Serials Hill!!! Iiniiiliiiiiili! 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