GENETICS OF TIGER SHRIMP Penaeus rnonodon : DESCRIPTION OF ELECTROPHORETIC DETECTABLE LOCI

As part of a search for biochemical genetic markers for tiger shrimp, Penuue morndon, wild shrimp were collected from the coastal waters of Aceh, Bali and Sumbawa, (Nusa Tenggara Barat). Tissues samples of hepatopancreas and abdominal muscle from these were analyzed by starch gel electrophoresis. Specific staining for 13 enzymes resolved 21 loci, 6 of which were polymorphic (i. e., EST.2, GPI, IDH, LDH-1, MDH-l and c-GPD). The observed number of phenotypes for respective loci agreed well with HardyWeinberg expectations. The genetic basis of each polymorphism is discussed. Most of the polymorphic loci were dimeric (GPI, IDH, MDH and c-GPD) except for EST-2 was monomeric and LDH-I was tetrameric. The genetic variability in Penaeus monodon wae indicated by the number of polymorphic loci, heterozygosity (I[) and number of alleles per locus (N) was relatively low compared to other marine organisms. The values of H and N for this species ranged from 0.025 to 0.047 and 1.381 to 1.528 respectively. This study suggest the use of GPI, IDH and MDH loci as genetic markers in any genetic improvement program.


INTRODUCTION
Tiger shrimp, Penaeus tnonodon, is an im- portant species in both capture fisheries and coastal aquaculture in Indonesia.An estimated 300.000 ha. of brackishwater pond are in use, mostly converted from mangrove area (Cham-  berlain, 1991).Approximately 90% of the Penaeus monodon fry required for pond stock- ing originate from hatcheries.In 1994, 142 hatcheries were operating in Indonesia.
Although Penaeus ntonodon farms the basic of the largest penaeid aquaculture industry in Indonesia, little is known of it genetics.Some work has been carried out on the hybridization between P. stylostris and P. monodon (Benzie et  aI., 1975) and some quantitative genetic data of P. marguiensis (Goswani et ol., 1986) and P. rnonodon (Sugama et aI., 1993) are currently available, but virtually no biochemical genetic research has been done on P. rnonodon.
Recently, electrophoreticallydetectable enzyme or protein variants (biochemical genctic uariants or genetic uariant) have been used as biochemical genetic markers in biological research.Many workers have shown that en- zymes and proteins are useful genetic markers for fisheries biology, aquaculture and breeding studies (Sbordoni et a1.,1986;Busack, 1988;  Taniguchi and Sugama, 1990;Sugama et ol., 1992;Taniguchi et al., 7994).Sbordoni et al. (1986) have summarized the importance of detecting genetic variance in P. japonicue in hatchery using electrophoretic markers in order to avoid inbreeding.Wohlforth and Hulata (1983) have obtained genetic improvement in tilapia using electrophoretic markers.
Jhe objective of this study is to provide genetic interpretation for the variability pat.terns of electrophoretically detectable enzymes and proteins of wild stock tiger shrimp, Penocuc monodon.The technique will be useful fot monitoring the genetic variability and popula- tion structure of wild stocks, genetic change and inbreeding in hatchery stocks and as biochemi- cal markers in selective breeding for genetic improvement programmes.

Sample Preporation
Shrimp with body length fuom 22,4 to 26.8 cm, were collected from Cempi Bay Dompu, Sumbawa, (Nusa Tenggara Barat), from the coastal waters of Aceh, and Negara (Bali).In each case 100 specimens were taken.The shrimps were transported alive to Gondol Research Station, killed and immediately stored at-20'C until used.Ihe maximum storage time was 2 weeks.Small portions of abdominal muscle and hepatopancreas tissues were dis- sected and placed in individual plastic tubes or rirulti-well disposable trays (Wako-Lab).The tissues were extracted with mixed MgCl, 0.005 M and KCN 0.005 M. Filter paper (5 x 10 mm) were eaturated with extract, lightly blotted and applied to the gel.
Electrophoresis was carried out for 4 hours at 4 MA,/cm2.The enzymes and proteins examined, loci detected and the appropriate buffer system and the tissue giving best resolution are given in Table 1.
Interpretotion of B-and.ingpattern Iocus and allele nomenclature followed the basic method of Alledorf and Utter (1929).
Multiple loci encoding a single enzyme or pro- 20 tein were designated with a hyphenated num- ber indicating the relative migration of their product, the least anodal being designated 1. Alleles designated by the proportional migra- tion distances of their products relative to that of the common allele, which was arbitrarilv designated 100, and other alleles were given numbers that indicated their mobility relative to that of the common allele.

Interpretation of Data
Alleles frequencies (p) were derived from the number of genotypes observed from electropho- retic analysis using the expression: p=(2 Ho + He)/2 N, where, Ho= Number of homozygotes; He= Number of heterozygotes and N= Number of individuals examined.
In the randomly mating population contain- ing known allele frequencies, the expected proportion of each genotype (homo and hetero_ zygotes) is predicted by the Hardy-Weinberg expectation.Deviation from the expected pro_ portion generally indicates the presence of a mixed population which is not the product of random mating.Deviation from Hardv-Wein- berg expectation at each locus was tested for significance using the log-likelihood Chi-square test (X2) for goodness of fit (Sokal and Rohll  1981) with degree of freedom equal to the number of expected genotypes minus the num- ber of alleles.
Heterozygosity estimates were calculated both as the observed proportion of heterozy- g-otes and as the proportion expected through the Hardy-Weinberg expectation.
A locus was considered to be polymorphic if the most common allele was equal to or less than 0.99.

RESULT AND DISCUSSION
Enzymes and proteins tested, loci detected, tissues assayed and buffer systems giving the clearest resolution are shown in Table 1.The enzymes or proteins were located as stained zones of activity after electrophoretic separation in gel media (Fig. l).Each stained zone was attributed to a genetic locus.
Enzymes appearing as a single zone of activity are assumed to represent the product of single genetic loci.Those with two or more well separated zones of activity are assumed to be the products of two or more loci.
Multiple loci have been numbered with a suffix in order of decreasing anodic mobility.The locus with the most anodal migration is designated number one, the next two and so on.The most common allele is designated 100 and other alleles are given numbers that indicate their mobility relative to that of the common IFR Journal Vol.II No.I.1996 allele (Table 2 and Fig. 1).Cathodal systems are designated in similar way but are given a negative sign.Loci in which all samples tested appeared as a single band have been classified as monomorphic.Those loci revealing electro- phoretic variants have been classified as polv- morphic.

Poly-morphismS)
Alcohol dehydogenase (ADH) Aspartate dehydrogenase (AAT)   Esterase (EST)   Glucosephosphate isomerase (GPI) *-Glycerophosphate dehydrogenase (*- (, Glucoeephonp hote leomerose (GPI)   A single anodal zone was contained for thie enzyme in abdominal muscle extracts.It was presumed as a single locus and was polymor- phic.Heterozygous individuals showed a three- banded genotype while homozygous individuals showed a single-banded genotype indicating a dimeric structure for this enzyme which was presumed to be controlled by three alleles (Fig. 1).

Ieocitrote Dehydrogenaee ( I DH)
This enzyme appeared in the anodal zone and was polymorphic in both samples exam- ined.A single locus was detected, presumed to be controlled by three alleles.Heterozygous individuals showed a three-banded genotype.
Homozygous individuals showed a single banded genotype indicating a probable dimeric structure to the enzyme (Fig. l).

Lactate Dehydrogenaee ( LDH)
This enzyme was represented by two zones of activity with different tissue predominance assumed to represent the expression of two loci.
LDH-I was muscle specific and LDH-2 was hepatopancreas spesific.Both appeared in the anodal zone.LDH-1 locus was polymorphic controlled by three alleles and was heterozy- gous.It was not clearly resolved under the electrophoretic conditions used, but it indicates a probable tetrameric structure for the enzyme (Fig. 1).This agrees with the findings of Sugama e, ol. (1988).

Molote dehydrogenaee (MDH)
Two loci were detected in this enzyme MDH-I in the anodal zone and MDH-2 in the cathodal zone.MDH-I was polymorphic and heterozy- gous.Individuals showed three-banded pheno- types indicating a probable dimeric structure of this enzyme (Fig 1 ).
a-Glycerop hoephate dehydrogenaee ( d- GPD) Polymorphic c-GPD was detected in the muscle tissue.The band number of the hetero- zygous type was not clearly resolved under the electrophoretic conditions used but it suggests indicates a probable dimeric structure for the enzyme (Fig. l).This agree with the findings of Sbordoni et ol. (1986).
-Geneticvarlability..The observed number of phenotypes and their Hardy-Weinberg expectations are given in Table 2. Result shows that the differences between observed numbers and their Hardy- Weinberg expectations were not significant at P<0.05 for the respective polymorphic loci examined here (see Xz values in Table 2).These support the validity of the genetic models pro- posed for each polymorphism and indicate that the population samples of Penacua monodon were collected from a single Mendelian popula- tion.
The average observed (Ho) and expected (He) heterozygosities for Aceh, Bali and NTB (Ho/He) were 0.04710.047,0.026|0.026end 0.028/0.026respectively.The number of allele per locus for Aceh, Bali and NTB were l.b28, 1.381 and 1.428 repectively.The value of het- erozygosities (H) and Number of allele per locus (N) of the Aceh sample were slighly higher than the other two samples.Average heterozygoeity (Ho) observed is this study ranged from 0.02b -O.O47, were comparable or slighly lower than those found by other researchers working with penaeid shrimp, such as Penaeus vannamei, H=0.02 (Lester, 1983), Penaeus stylirostris H= 0.06 (Lester, 1983) and Penaeus japonicue (Sbordoni et a,1.,1986),H= 0.039.More compre' hensive reviews reported hetero-zygosities of 0.048 for decapods (Hedgecock el ol., 1982), and 0.082 for crustaceans (Nevo, 1984).Thus deca' pods and crustaceans including Penaeus monodon generally have a low level of genetic variability, as also found in the present study.
The allele frequency differences among localities were tested by Chi-square (X1 test for heterogeneity (Crow and Kimura, 1970).Signif' icant heterogeneity was observed at GPI-100   locus between Aceh and Bali (X2 = 8,49; P< 0'01) and between Aceh and NTB (X2= 8'49; P < 0.01)   but not between Bali and NTB (X'?= 0.001 F>0.05).Dispite a lack of large allele frequency differences among samples, apparent statistical significances at GPI locus indicated that the sample did not represent a single homogeneous gene pool.
The small allele frequency differences re- sulted in very low estimates of genetic diver- gence as measured by genetic distance (Nei,  L972').Genetic distance (D), i e: D=0.00094 for Aceh vs Bali, D= 0.00077 for Aceh vs NTB and D= 0.00004 for Bali vs NTB.
The prime purpose of the present study has been to describe and interpret genetically a large number of electrophoretically detectable loci for the genetic population structure of shrimp and to find a genetic markers for the hatchery stock.Taniguchi et al. (1994) discussed the potentially valuable role of genetic markers in the development of advanced breeding schemes for fish directed toward genetic im' provement of both wild and domesticated stocks.Recently, Goudie et aL (1995) reported that heterozygosity of the GPI locus was associ- ated with growth in wild and domesticated populations of African catfish.
In the present study, the use of the polymor' phic loci (GPI, IDH and MDH) as a genetic marker has further advantages in that resolu' tion of zymograms for those loci are good (Fig. 1).Therefore, the present findings suggest possibilities for the use of GPI, IDH and MDH as biochemical markers in genetic improvement programmes.
of EST activity were observed in the present study.They were aesumed to repreeent the expression of two loci, designated EST-I and EST-2.EST-I was represented by a single variant zone expressed in hepatopancreas extracts only.Genetic interpretation was not attempted for the EST-l locus, because of poor resolution.EST-2 was polymorphic in population samples and it wag pressumed to be controlled by two alleles.The two-banded heterozygous type suggests a mono- meric structure of the enzyme(FiS.1).

Table 1 .
1. Brief descriptions of the poly.morphic loci are given below.A summary of enzymes and protein examined locus designations, tissue and buffer used in Penaeus monodon.