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Apparent Control of Sexual Differentiation of Freshwater Prawn, Macrobrachium rosenbergii, Through Dietary Administration of Dopamine Hydrochloride Cortney L. Ohs Louis R. D’Abramo Lora Petrie-Hanson Anita M. Kelly ABSTRACT. Dopamine at concentrations of 0.15, 1.5, and 15.0 mg/kg was included in two different formulated diets that were fed to recently metamorphosed postlarvae of the freshwater prawn, Macrobrachium rosenbergii, for 60 days. Dopamine, in the form of dopamine hydrochlo- ride, was incorporated into the formulated diets by either solubilizing it in alcohol, followed by submersion of diet and evaporation, or inclusion in a lipid premix ingredient. The alcohol evaporation method at 0.15, 1.5, and 15.0 mg/kg significantly increased the percentage of individuals without external male characteristics. This research is the first reported use of dietary administration of dopamine to crustaceans, and the first Cortney L. Ohs, University of Florida, Institute of Food and Agricultural Sciences, Indian River Research and Education Center, 2199 South Rock Road, Fort Pierce, FL 34945. Louis R. D’Abramo, Mississippi State University, Department of Wildlife and Fisheries, 100 Stone Boulevard, Mississippi, MS 39762. Lora Petrie-Hanson, Mississippi State University, College of Veterinary Medicine, Department of Basic Sciences, P.O. Box 6100, Mississippi, MS 39762. Anita M. Kelly, Southern Illinois University, Fisheries and Illinois Aquaculture Center, Mailcode 6511, Carbondale, IL 62901. Journal of Applied Aquaculture, Vol. 18(4) 2006 Available online at http://jaa.haworthpress.com © 2006 by The Haworth Press, Inc. All rights reserved. doi:10.1300/J028v18n04_02 19
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Apparent Control of Sexual Differentiation of Freshwater Prawn, Macrobrachium rosenbergii , Through Dietary Administration of Dopamine Hydrochloride

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Page 1: Apparent Control of Sexual Differentiation of Freshwater Prawn, Macrobrachium rosenbergii , Through Dietary Administration of Dopamine Hydrochloride

Apparent Control of SexualDifferentiation of Freshwater Prawn,

Macrobrachium rosenbergii,Through Dietary Administration

of Dopamine Hydrochloride

Cortney L. OhsLouis R. D’AbramoLora Petrie-Hanson

Anita M. Kelly

ABSTRACT. Dopamine at concentrations of 0.15, 1.5, and 15.0 mg/kgwas included in two different formulated diets that were fed to recentlymetamorphosed postlarvae of the freshwater prawn, Macrobrachiumrosenbergii, for 60 days. Dopamine, in the form of dopamine hydrochlo-ride, was incorporated into the formulated diets by either solubilizing itin alcohol, followed by submersion of diet and evaporation, or inclusionin a lipid premix ingredient. The alcohol evaporation method at 0.15,1.5, and 15.0 mg/kg significantly increased the percentage of individualswithout external male characteristics. This research is the first reporteduse of dietary administration of dopamine to crustaceans, and the first

Cortney L. Ohs, University of Florida, Institute of Food and Agricultural Sciences,Indian River Research and Education Center, 2199 South Rock Road, Fort Pierce, FL34945.

Louis R. D’Abramo, Mississippi State University, Department of Wildlife andFisheries, 100 Stone Boulevard, Mississippi, MS 39762.

Lora Petrie-Hanson, Mississippi State University, College of Veterinary Medicine,Department of Basic Sciences, P.O. Box 6100, Mississippi, MS 39762.

Anita M. Kelly, Southern Illinois University, Fisheries and Illinois AquacultureCenter, Mailcode 6511, Carbondale, IL 62901.

Journal of Applied Aquaculture, Vol. 18(4) 2006Available online at http://jaa.haworthpress.com

© 2006 by The Haworth Press, Inc. All rights reserved.doi:10.1300/J028v18n04_02 19

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documentation of the apparent dietary control of gonadal developmentand sex differentiation in crustaceans. Replication of results, a histologicalexamination of a large sample of individuals, and continued monitoringof those individuals fed the dietary dopamine are needed to verify theapparent effect. doi:10.1300/J028v18n04_02 [Article copies available for a feefrom The Haworth Document Delivery Service: 1-800-HAWORTH. E-mail ad-dress: <[email protected]> Website: <http://www.HaworthPress.com> © 2006 by The Haworth Press, Inc. All rights reserved.]

KEYWORDS. Freshwater prawn, Macrobrachium rosenbergii, sexdifferentiation, sex reversal, dopamine hydrochloride

INTRODUCTION

Sex of crustaceans is determined by presence or absence of an an-drogenic gland and the release of androgenic gland hormone. All crusta-ceans have androgenic glands during early stages of development and ageneticcue is believed to signaldevelopmentof theandrogenicglandandthe subsequent development of male secondary sex characteristics. Theabsence of this genetic cue causes the androgenic gland to atrophy,leading to the development of female secondary sex characteristics(Charniaux-Cotton 1962).

Male freshwater prawn, Macrobrachium rosenbergii, display differ-ential growth rates that contribute to the wide variation of size withintheir populations (Ra’anan and Cohen 1984), while females tend to growat more uniform rates (Daniels 1993). The fastest growing males (“jump-ers”) initially become blue claw morphotypes and numerically representapproximately 20% of the male population. Approximately 60% of malesgrow without a corresponding development of gonadal tissue and aretermed orange claw morphotypes. The other 20% of the male population,termed small males, develop mature gonads but grow at a comparativelyslower rate (Daniels 1993). The small male morphotype is theorized to bethe product of a combination of social dominance (Karplus et al. 1992a)and food deprivation (Karplus et al. 1992b) exerted by the blue clawmales. The resulting wide size variation combined with aggressive be-havior often result in cannibalism and reduced survival within popula-tions. This wide range of sizes within a population encountered at harvestof commercial production enterprises creates problems for marketing.The industry would benefit from a more uniform weight distribution ofmarketable prawns. Reduction of size variation and a corresponding in-

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crease in production have been achieved through size grading of juvenilepopulations prior to stocking into production ponds (Karplus et al. 1986;Daniels and D’Abramo 1994). Production of an all female population ortheeliminationof malemorphotypesalso maycontribute to the reductionin the magnitude of size variation and corresponding greater yields.

Steroids have been used to achieve phenotypical sex reversal in nu-merous fish species through dietary administration, injection, and im-mersion prior to sexual differentiation (Pandian and Sheela 1995). Sexreversal in crustaceans may also be realized through hormone adminis-tration. Dietary administration of hormones to crustaceans appears to bethe most practical approach on a commercial scale. However, this meth-odology offers significant challenges because of the potential for lossarising from leaching from the feed pellets or digestive breakdown. Lossof hormones is exacerbated by the breaking up of formulated feed beforeconsumption.

The endocrine system of crustaceans consists partially of a sinus glandand an x-organ located at the base of the stalked eyes (Chang 1992). Thex-organ produces gonad-inhibiting hormone (GIH) and molt-inhibitinghormone (MIH) which are stored until secretion in the sinus gland, aneurohemal organ. Crustaceans possess a y-organ that is located withinthe central portion of the cephalothorax and produces gonad-stimulatinghormone (GSH) and ecdysone which regulates the molting process. GIHand GSH act directly on the ovary and indirectly on the testes via theandrogenicgland through the releaseofAGH. Crustaceanshavemandib-ular organs (MO), neurohemal organs that are the sites of synthesis andrelease of juvenile hormone, methyl farnosoate (MF). Methyl farnosoateappears to play a role in gonadal maturation in male and females, specifi-cally development of the reproductive system, vitellogenesis, morphotypicdifferentiation, and mating behavior (Laufer et al. 1993).

Crustaceans uniquely possess glandular tissue attached to the vasdeferens (Faxon1884).Theseglandular tissueswere termed theandrogenicgland (AG) by Charniaux-Cotton (1953) and found to produce androgenicgland hormone (AGH) which signals the initiation of all necessary stepsin the sexual differentiation and development of males (Charniaux-Cot-ton 1962). If the AG and the corresponding production of AGH is lack-ing, then female secondary sex characteristics develop. Development ofthe AG is believed to be signaled by a genetic cue, but the nature of thiscue is unknown. Once the AG develops, AGH is produced and signals theinitiation of sexual differentiation that is followed by the development ofmale secondary sexual characteristics (Hasegawa et al. 1993).

Ohs et al. 21

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The sex of M. rosenbergii can be externally identified by the presenceor absenceof themalegonopore complex, a projectionof theexoskeletonsurrounding and covering the external opening of the vas deferens on themesal surface of the coxopodite of the fifth pereiopod. Even in juvenileprawns, the gonopore complex appears as an undifferentiated bud undera dissecting microscope. In females, a slit covered by an operculum onthe medial surface of the coxopodite of the third pereiopod is present butis not a reliable anatomical indicator of sex until late juvenile or earlyadult stages (Malecha et al. 1992).

Surgical implantation of a portion of the AG from an adult male isopodinto a female isopod caused sex reversal after the initiation of morphologi-cal sex differentiation (Suzuki 1999). All male populations of the freshwa-ter prawn, M. rosenbergii, have been created by surgical implantation ofa portion of an adult male’s AG into a juvenile female prawn (Nagamineet al. 1980a; Malecha et al. 1992). To achieve successful sex reversal, im-plantation of androgenic tissue must occur before the length of the cara-pace of the female prawn becomes 7.5 mm, a length that appears tocoincidewith theonsetofovarianfunction(Malechaetal.1992).Althoughthe ability to effect sex reversal in M. rosenbergii through this method wasestablished, survival following surgical implantation was always poor.Recently, apopulationof redclawcrawfish,Cheraxquadricarinatus, con-sistingof91.6% males was produced through implantationof androgenicgland tissue under the carapace of the cephalothorax region, but survivalwas low (25%) (Khalaila et al. 2001). In contrast, removal of theandrogenic gland of male prawns during early stages of development re-sulted in feminization (Nagamine et al. 1980b).

Information about when sexual differentiation occurs in M. rosenbergiiis lacking. Juvenile hormone, methyl farnesoate (MF), administered tolarvae of M. rosenbergii via an Artemia sp. prey, retarded larval develop-ment (Abdu et al. 1998). Malecha et al. (1992) stated that ovarian func-tion apparently begins at a carapace length of 7.5 mm. Therefore, dietaryadministration of hormones to either male or female post-larval prawnsprior to this length-associated stage of development may successfullycontrol sex differentiation.

Zou and Fingerman (1997) added two strong estrogens, endosulfanand diethylstilbesterol, to the culture water of a cladoceran, Daphniamagna, to evaluate the effects on sexual differentiation. No significantdifferences in sex ratios of populations of offspring produced by exposedand control individuals were observed. An increase in molt frequencywas observed and thought to be caused by the binding of estrogenic com-pounds, structurally similar to ecdysteroids, to ecdysteroid receptors.

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Fingerman (1997) reviewed the current knowledge of the role ofneurotransmitters in regulation of the release of reproductive hormonesthatcontrolgonadalmaturation indecapodcrustaceans.Androgenicglandhormone (AGH) controls differentiationof the male reproductivesystemand development of secondary sex characteristics in crustaceans. Secre-tion of GSH and GIH are believed to be regulated by several neuro-transmitters and act directly on the ovary of females. In males, GIH actson the testes indirectly by downregulation of the androgenic glands and asubsequent decrease in production of AGH. Gonad stimulating hormoneis necessary for spermatogenesis to occur in the testes. Dopamine hydro-chloride and methionine enkephalin stimulate the release of GIH whichdirectly inhibitsdevelopmentof theovary and theAG. Lackof AG devel-opment inhibits production of AGH leading to arrested development ofthe testes. Dopamine hydrochloride and methionine enkephalin inhibitrelease of GSH, whereas 5-hydroxytryptamine and red pigment-concen-trating hormone stimulate release of GSH and inhibit release of GIH(Fingerman 1997). This complex system of regulation of GIH and GSHby neurotransmitters may offer a way to control the production of AGHby preventing production of GSH and stimulating the production of GIH.Dopaminehydrochloride isnaturallypresent incrustaceansandadminis-trationviaeitherdietor injectionmayprovideameans tocontrolmalesexdifferentiation and create all female populations.

The objective of this experiment was to determine if dopamine hydro-chloride contained in formulated diets fed to post-larval M. rosenbergiicommunally cultured in aquaria significantly affects survival, weight,length, and sex ratio.

MATERIALS AND METHODS

Preparation and Feeding of Diets

Prawns were fed either a formulated commercial diet, Zeigler (ZeiglerBrothers Inc., Gardners, Pennsylvania;1 Finfish Starter #2, 55% crudeprotein and 15% crude fat), or a formulated experimental diet, BML (Reedand D’Abramo 1989). Each of these diets contained dopamine hydro-chloride (3-Hydroxytyramine,EC 200-529-8, Sigma-Aldrich,St. Louis,Missouri) at 0.15, 1.5, and 15.0 mg/kg (experimental treatments). Con-trol diets contained no dopamine hydrochloride.

Ohs et al. 23

1. Use of trade or manufacturer’s name does not imply endorsement.

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Addition of dopamine hydrochloride to the commercial diet was per-formed similarly to that described by Guerrero (1975) for addition ofandrogens. The calculated amount of dopamine hydrochloride wasweighedanddissolved intoavolumeof ethylalcohol (90%) equivalent to80% (volume/weight) of the weight of feed, that is, if 100 g of diet wasprepared, the amount of desired dopamine hydrochloride was dissolvedinto 80 mL of ethyl alcohol. The diet was then submersed within the ethylalcohol contained in a beaker and placed under a ventilated hood where itwas protected from light. After approximately 15 minutes, the ethyl alco-hol in the beaker evaporated. Under protection from light, diet was thenspread into a single layer on aluminum foil trays to achieve completeevaporation of the remaining ethyl alcohol. This process was terminatedwhen the smell of alcohol could no longer be detected. The control wasprepared similarly, except no dopamine hydrochloride was added to thealcohol before submersion of the diet and evaporation.

The BML diet was prepared according to Reed and D’Abramo (1989)and the necessary amount of dopamine hydrochloride in each diet wasadded to the triglyceride (oil) mixture, the final ingredient added to themixture of dietary ingredients prior to the addition of water and cold ex-trusion. For the control diet, no dopamine hydrochloride was added to thetriglyceride mixture. Following extrusion, diets were protected fromlight while air dried (20�C) for 30 minutes under a ventilated laboratoryhood. All diets were placed in sealedplasticcontainers, stored in a freezer(�80�C) and removed only to obtain the appropriate amount of diet foreach feeding.

The amount of diet fed was based upon rates recommended byD’Abramo et al. (2003), for the nursery phase of culture. The initial feed-ing rate was 15% of the estimated body weight per day and was dividedintoequivalentamounts fed twicedaily.Theamountof feedprovidedasapercentage of the body weight was decreased by 1% each week. Eachweek, the mean individual weight of a sample of prawns (10-20) re-moved from aquaria representing different treatments was determined.The mortality rate was estimated to be 1.5% per week. Daily amounts offeed fed were adjusted to compensate for growth and survival.

Experimental Culture System

The culture system consisted of 33 aquaria (90 L each) provided withflow-through well water at a rate that achieved total exchange of volumeevery6hours.Submersible thermostaticallycontrolledheaters (Visitherm150W, Aquarium Systems Inc., Mentor, Ohio) were used to maintain a

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constant temperatureof 27 � 2�C. A blower provided air that was distrib-uted to each aquarium through a ceramic airstone. Stand pipe drains werecovered with 500-µm diameter nylon mesh, and the top of each aquariumwas covered with clear plexiglass to prevent escape of prawns.

Substrate in the form of high densitypolyethylenemesh (InterNet Inc.,Minneapolis, Minnesota) of three sizes, 0.635, 1.27, 3.175 cm diametersquare mesh, was provided within the water column of each aquarium toreduce the incidenceof cannibalism.The actual surface area of both sidesof the substrate was calculated for the different types of substrate. Thesecalculations yielded an area of substrate that was 230% greater than thatof the surface area of the bottom of each aquarium.

Uneaten diet was manually siphoned from the bottoms of each aquariumonceaweekthroughout theentirecultureperiodtopreventaccumulation.

Experimental Animals and Design

Postlarval prawns were purchased from a commercial supplier (LaurenFarms, Leland, Mississippi). All prawns had metamorphosed to post-larvae by June 15, 2002 and were stocked into 90-L experimental aquariaon June 17, 2002 at a density of 2.5/L. Mean individual weight of prawnsat stocking was 0.015 g.

The design of the experiment was a 4 � 2 factorial with four levels ofdopamine hydrochloride (0, 0.15, 1.5, and 15.0 mg/kg feed) added to twodifferent diets (Commercial and BML). The experiment was completelyrandomized with a factorial arrangement of treatments and treatmentswererandomlyassignedtoeachaquariumcontainingstockedpostlarvae.

Data Collection

Prawns from all treatments were harvested over a range of 51-60 daysbecause of the time required for harvesting and data collection of prawnsfrom all of the aquaria. The feeding of experimental diets continued dur-ing this 10-day period. All prawns were removed from each replicateaquarium, placed into labeled plastic buckets, and counted. All prawnswere individuallymeasured to the nearest mm, from the posterior portionof the eye socket to the end of the telson, and then weighed to the nearestmg. Each prawn was sexed by first positioning the ventral side up under adissecting microscope at a magnification of 40�. Then, the fifth pereiopodswere spread apart with a dissecting probe to locate and identify externalgenital papillae. The observed presence of external genital papillae iden-

Ohs et al. 25

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tified the prawn to be male and the absence to be female. The mean percentsurvival, mean individual wet weight, mean individual length, and meanpercentageofeachsexwas thencalculatedforeachreplicateaquarium.

Statistical Analysis

A two-way analysis of variance (ANOVA), using the general linearmodel procedure (PROC GLM) of SAS (SAS Institute 1988) was used todetermine whether differences in mean percentage of females in the pop-ulation existed between diets fed (Commercial and BML) and amongdietary concentrations (0, 0.15, 1.5, and 15.0 mg/kg) of dopamine hydro-chloride and whether a significant interaction between these factorsexisted. The diet type and the dietary concentration of dopamine hydro-chloride were the independent variables and the percentage of the popu-lation that was anatomically female (without external genital papillae)was the dependent variable. Percentage data were arcsin square roottransformed prior to statistical analysis. All differences were consideredsignificant at P < 0.05.

PROC MIXED procedure of SAS (SAS Institute 1988) was used todetermine if significant differences existed in mean final individual wetweight and mean final individual length among prawns fed the differentdiets, among different dietary concentrations of dopamine hydrochlo-ride, between sexes, and the combinations of these response variables.PROC MIXED was also used to determine if mean percent survival dif-fered significantly among the different diets, the different dietary con-centrations of dopamine hydrochloride, and a combination of theseresponse variables.

RESULTS

Mean weight, mean length, mean percentage survival, and meanpercentage of prawns lacking external genital papillae (from now ontermed females) for each treatment are summarized in Tables 1 and 2, andFigure 1.

There was significant interaction (P = 0.029) between diets fed and di-etary concentrations of dopamine hydrochloride for the mean percentageof females in the population. Therefore, the percentage female was com-pared within concentrations and diets independently. The percentage offemales did not differ significantly among populations fed the BML dietcontaining different concentrations of dopamine hydrochloride (P =

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0.115), but did differ significantly (P = 0.002) among populations fed thecommercial diet containing different concentrations of dopamine hydro-chloride. The percentage of females in the populations fed the commer-cial diet containing 0.15, 1.5, and 15.0 ppm dopamine hydrochloride didnot differ significantly. However, the percentage of the population thatwas female for the diets lacking dopamine hydrochloride (62.6%) wassignificantly less than that of each of the three diets which containeddopaminehydrochloride,80.9,88.0, and71.3%, respectively(Table2).

Survival of prawns in aquaria where the commercial diet was fed(52.2%) was significantly greater (P = 0.026) than that of prawns fed theBML diets (40.2%). Survival percentage did not differ significantlyamong the dopamine hydrochloride concentrations tested for each diet(P = 0.81).

Ohs et al. 27

TABLE 1. Mean±SE percentage survival, individual wet weight (g), and length(mm) of juvenile freshwater prawns fed two types of diets (BML or Commer-cial), each containing different concentrations of dopamine hydrochloride (0,0.15, 1.5, and 15.0 mg/kg).

Diet concentration Survival (%) Weight (g) Length (mm)

BML 0 35.0�3.70 0.29�0.01 24.7�0.28

BML 0.15 43.9�5.47 0.29�0.01 24.9�0.28

BML 1.5 41.3�2.71 0.31�0.01 25.3�0.31

BML 15.0 41.2�9.48 0.26�0.01 24.1�0.26

Commercial 0 54.7�8.69 0.22�0.01 23.0�0.24

Commercial 0.15 56.5�8.15 0.16�0.01 20.4�0.24

Commercial 1.5 45.6�7.95 0.12�0.01 19.0�0.22

Commercial 15.0 52.9�6.67 0.18�0.01 21.6�0.23

TABLE 2. Mean±SE percentage of the female population fed each diet (BMLand Commercial) and concentration (0, 0.15, 1.5, 15.0 mg/kg) of dopaminehydrochloride.

Dopamine concentration (mg/kg) BML (% Female) Commercial (% Female)

0 65.4�1.5 A a 62.6�3.0 A a

0.15 74.0�5.6 A a 80.9�3.2 A bc

1.5 70.9�5.9 A a 88.0�2.9 B b

15.0 79.0�3.4 A a 71.3�2.4 A c

Statistical significance is indicated by different lower case letters within columns and upper case letterswithin rows at (P � 0.05).

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The mean individual wet weight of prawns did not differ significantlyamong dopamine hydrochloride concentrations for each diet (P = 0.814).The overall mean weight of prawns fed the BML diet (0.32 g) was signifi-cantly greater (P < 0.001) than that of prawns fed the commercial diet(0.2 g). However, this difference may be due to significantly lower sur-vival of prawns fed the BML diet. Growth rates decline as biomass perunit volume increases (D’Abramo et al. 2000). The responses to the com-bination of diet and dopamine hydrochloride concentration did not differsignificantly (P = 0.253). The overall mean individual weight of maleprawns (0.31 g) was significantly greater (P < 0.001) than that of femaleprawns (0.21 g).

No significant differences in mean length existed among prawns fedeither diet containing different levels of dopamine hydrochloride (P =0.79).Themean lengthofprawns fed theBMLdiet (25.6mm)was signif-icantly greater (P < 0.001) than that of prawns fed the commercial diet(22.2 mm). Mean length relative to the combination of diet and dopaminehydrochloride concentration did not differ significantly (P = 0.134). Theoverall length of male prawns (25.8 mm) was significantly greater (P <0.001) than that of female prawns (22.1 mm).

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FIGURE 1. Percentage increase of the proportion of females relative to thecontrols in populations of juvenile prawns fed two types of diets (BML andCommercial) containing different concentrations of dopamine hydrochloride(0, 0.15, 1.5, 15.0 mg/kg) for 51-60 days.

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DISCUSSION

Dietary provision of dopamine hydrochloride to postlarvae increasedthe percentage of females within the population as determined by exami-nation of external genital papillae in all treatments. This increase was sta-tistically greater than the control in all treatments receiving dopaminehydrochloride via the commercial diet. This is the first reported use of di-etary administration of dopamine to crustaceans and the first evidencesuggesting that development of gonads and possibly sex differentiationof crustaceans can be affected by dietary administration of this com-pound. Although the dietary administration of dopamine hydrochloridedid not produce 100% females, these data suggest a consistent and signif-icant alteration in the percentages of females in a population when acommercial diet is fed.

Females comprised 65% of the control treatment population, a per-centage slightly higher than the 50-55% commonly observed for juvenileand adult populations. Sex at stocking could not be determined becausetheexternalgenitalpapillaeof malesare not decipherableon newly meta-morphosed postlarvae. Populations of juvenile M. rosenbergii have beenreported to commonly have a greater proportion of females at the termi-nation of the nursery phase of culture (Karplus et al. 1986). The mostplausible explanation for populations composed 65% females in the con-trol treatment at the end of the experiment is differential survival of thesexes. Even with the control population having a higher than expectedpercentage of females, the percentages of the populations that were fe-male in the dietary treatments receiving dopamine hydrochloride via thecommercial diet were all significantly greater than the control. The con-sistency within experimental treatments is notable and suggests that theproportion of prawns without external papillae in the populations wasmodified through dietary administration of dopamine hydrochloride.The possibility of a selective increase in mortality of males within thepopulations provided dopamine hydrochloride, however, cannot beruled out.

The commercial diet was more effective than the experimental diet inincreasing the percentage of the population that was female. The absenceof a significant difference in the percentage of females in the populationbetween the commercial and experimental diets at all dopamine concen-trations except 1.5 mg/kg, suggests that the commercial diet was moreeffective at eliciting a change in the percentage of females in the popula-tion. This difference was likely due to the methodology of adding dopa-

Ohs et al. 29

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mine to the diets rather than the diet itself; therefore it is recommended toadd dopamine hydrochloride via the alcohol evaporation method.

It is unknown whether sex reversal or suppression in development ofmale gonads was achieved. Dopamine hydrochloride may have increasedGIH and decreased GSH release. As a result, release of AGH from theAG would have been halted, leading to suppression in the developmentof testes.

The effect of submersion of diet in water on the loss of dopamine hy-drochloride was not evaluated. Dopamine hydrochloride is a water solu-ble catecholamineand likely leaches from the diet to the water. However,dopamine hydrochloride concentrations selected for treatments werebased upon an exponential increase in levels to compensate for substan-tial loss due to leaching. The effect of digestive processes of prawns onalteration of the chemical structure of dopamine hydrochloride is alsounknown. Nonetheless, dietary addition through use of ethanol in thecommercial diets may be the most effective approach. The variation inresponse within a treatment may be a manifestation of differentialleaching.

The data suggest that dopamine hydrochloride added to the diet via thealcohol evaporation method at a concentration of 0.15, 1.5, and 15.0 mg/kg can effectively increase the percentage of the population that is fe-male. The present study does not include a measure of leaching of dopa-minehydrochloride fromthediet so the level isprobablyanoverestimate.Given that dopamine hydrochloride is a water soluble catecholamine,methods to reduce leaching should be investigated. One possibility maybe toencase thedietparticles inalginateor another substanceafter addingthe dopamine hydrochloride.

Further evaluation of the dietary administration of dopamine hydro-chloride is warranted to determine whether the results can be replicatedand whether females can be exclusively produced. In addition, popula-tionswillneed tobegrown to larger individual sizes todocumentwhetherthe effect of dopamine is permanent or whether the process of sex differ-entiationhas justbeenhalted.Acomplimentaryhistologicalexaminationofa largesampleof individualsofvariousagesshouldbeconducted tocon-firm presence and developmental stages of internal ovaries and testes.

ACKNOWLEDGMENT

This Research was funded by the Mississippi Agricultural and For-estry Experiment Station.

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Chang, E.S. 1992. Endocrinology. Pages 53-91 in A.W. Fast and L.J. Lester, eds. Ma-rine Shrimp Culture: Principles and Practices. Elsevier Scientific, Amsterdam, TheNetherlands.

Charniaux-Cotton, H. 1953. Etude du determinisme des caracteres sexuels secondairesparcastration chirurgiale et implantation diovaire chez un crustacé amphipode(Orchestia gammarella). Comptes Rendus Académie des Science Paris 236:141-143.

Charniaux-Cotton, H. 1962. Androgenic gland of crustaceans. General and Compara-tive Endocrinology Supplement 1: 241-247.

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