Biochemical genetics in the Argentinean squid,

The Argentinean squid, Illex argentinus (Castellanos, 1960), is a cephalopod which belongs to the Ommastrephidae family and is one of the most important fishing resources in the South West Atlantic. Its current capture rate is more than 400,000 tons/year in areas off the continental shelf and the South Patagonian slope in the Argentinean Sea (Brunetti et al., 1996). It is a neritic-oceanic species which can be found between 23oS and 53oS, and the most frequented region is that between 35oS and 52oS at depths of between 80 and 400 metres (Castellanos, 1964; Brunetti and Perez Comas, 1989a, 1989b; Otero et al., 1981). An exhaustive biological fishing study of this species has been carried out since 1981, a consequence of which has been the proposal of at least four spawning groups or stocks, based on the size structure, the length of the first maturity, spawning area and period (Brunetti, 1981, 1988; Nigmatullin, 1989). However, to date, the degree of genetic isolation between these groups is unknown. The current study consider the species’ genetic variability and their population genetic structure. SCI. MAR., 62(1-2): 141-149 SCIENTIA MARINA 1998


INTRODUCTION
The Argentinean squid, Illex argentinus (Castellanos, 1960), is a cephalopod which belongs to the Ommastrephidae family and is one of the most important fishing resources in the South West Atlantic.Its current capture rate is more than 400,000 tons/year in areas off the continental shelf and the South Patagonian slope in the Argentinean Sea (Brunetti et al., 1996).It is a neritic-oceanic species which can be found between 23ºS and 53ºS, and the most frequented region is that between 35ºS and 52ºS at depths of between 80 and 400 metres (Castellanos, 1964;Brunetti andPerez Comas, 1989a, 1989b;Otero et al., 1981).
An exhaustive biological fishing study of this species has been carried out since 1981, a consequence of which has been the proposal of at least four spawning groups or stocks, based on the size structure, the length of the first maturity, spawning area and period (Brunetti, 1981(Brunetti, , 1988;;Nigmatullin, 1989).However, to date, the degree of genetic isolation between these groups is unknown.The current study consider the species' genetic variability and their population genetic structure.The application of genetic analysis through the technique of enzymatic protein electrophoresis to the study of marine populations is standard practice nowadays and is extremely valuable in population identification (Allendorf et al., 1987;May and Krueger, 1990;Utter, 1991;Avise, 1994).However, work carried out on squid using this method is still scarce (Garthwaite et al., 1989;Carvalho and Pitcher, 1989;Carvalho et al., 1992), and this last study is the only published work on I. argentinus to date.
This current work presents the results of an extensive search for electrophoretically detectable enzymatic loci in the muscle of the Argentinean squid, which would serve as the basis for a more profound analysis that would provide greater knowledge of the population genetics structure of this species.

MATERIAL AND METHODS
A total of 202 individuals captured by the INI-DEP research vessel "Capitán Oca Balda" during February of 1994 in the Argentinean Sea was analysed.Once each squid had been individualised, a piece of skeletal muscle was extracted from the mantle of each example and frozen on board at -20ºC.Later, the samples were transported to Spain in expanded polystyrene boxes with enough dry ice to ensure that they all arrived at their destination still completely frozen.Once they arrived, they were kept at -80ºC in the laboratory until their later electrophoretic analysis.
The sample analysis was performed using the technique of horizontal starch (11%) gel electrophoresis, following the description given by Aebersold et al. (1987).Nine different buffer systems were used: 1-Continuous buffer AC, modified from Clayton and Tretiak (1972).
With respect to the stains, the ones used in this work corresponded to enzymatic systems which required the use of specific substrates for the reaction, in accordance with the protocols described by the following authors: Aebersold et al. (1987), Allendorf et al. (1977), Harris and Hopkinson (1976), Jorde et al. (1991), Morizot and Schmidt (1990), Pasteur et al. (1988).
Table 1 lists the analysed enzymes and indicates the ones which provided the best electrophoretic and staining conditions.

Genetic description and interpretation of zymograms
Forty-six different stains were tested, of which 13 did not show any activity and two gave a resolution that was insufficient for genetic interpretation.The remaining 31 however, produced consistent enzymatic phenotypes and enabled their genotype identification, producing a total of 45 loci that could be used in population studies.
The most conservative genetic explanation was adopted for estimating the number of loci and alleles that coded an enzyme, both when it was described for the first time and when there were differences with other authors, so that the necessary loci and alleles for their explanation would be minimum.The nomenclature used for the loci and allele designation follows the recent standards described by Shaklee et al. (1990).In those enzymes which presented more than one locus, the Arabic number suffix describes the different loci in increasing order of mobility with respect to the anode.The alleles for each locus were designated by their relative electrophoretic mobility with respect to the most common allele for this locus which was taken as *100.
The zymograms obtained with the different enzymatic stains used together with their genetic interpretation are now described.In all cases, the interpretation which is given agrees with the current subunit structure of each enzyme.In spite of not having previous electrophoretic information for each enzyme, whenever possible, the results obtained in this work were compared to those described for the species earlier by Carvalho et al. (1992).
The enzymes analysed are presented already classified into three groups according to activity and degree of resolution.

Enzymes with little activity and insufficient resolution
Octanol dehydrogenase (ODH).
A cathodal zone of moderate activity, probably variable and with insufficient resolution was detected for this enzyme, but could not be genetically interpreted.

Xanthine dehydrogenase (XDH).
Presented an anodal zone of weak activity, with insufficient resolution that could not be genetically interpreted.

Enzymes with good activity and resolution
A list follows which describes the interpretation of the electrophoretic patterns that were obtained with the 31 remaining enzymatic stains.

Aspartate amino-transferase (AAT)
A total of two moderately active anodal zones were observed for this enzyme and this has been interpreted as the expression of two loci, AAT-1* and AAT-2*, both monomorphic.

Acid phosphatase (ACP)
An intensely active anodal zone, close to the origin, has been observed in this enzyme and it has been identified as the expression of the ACP* locus where two alleles have been detected, *100 and *225.

Adenosine deaminase (ADA)
A single variable anodal zone with moderate activity was observed for this enzyme and has been considered as a locus expression, ADA*, with two alleles: *69 and *100.The homozygotes showed a single band and the heterozygotes three bands, a characteristic pattern of dimeric proteins.

Adenylate kinase (AK)
This enzyme presented three anodal zones of differing intensity activity.The first very close to the origin, is a weak zone, quite unclear and with a very diffused aspect that could not be genetically interpreted.The second and third zones are moderately and intensely active respectively and have been identified as the expression of two loci AK-1* and AK-2*.None of these two loci presented allelic variation.

Creatine kinase (CK)
Three zones of activity with different intensity and resolution have been detected in this enzyme, each one is considered to be the expression of a locus: CK-1*, CK-2* and CK-3*.Loci CK-1* and CK-2* have moderate activity and resolution and did not present any variation whatsoever in all the analysed individuals.Locus CK-3* is variable with good activity and resolution and presented two alleles: *95 and *100.

Diaphorase (DIA)
Two anodal zones of activity very close to the advancing electrophoretic front were observed for this enzyme.The first zone is weakly active and without any variation and has been considered as the expression of a single monomorphic locus DIA-1*.The second zone is variable with intense activity and resolution, it has been identified as locus DIA-2* in which three alleles have been detected: *95 ,*100 and *103.

Esterase-D (ESTD)
A single anodal zone has been observed for this enzyme.It has good resolution without variation and has been interpreted as the expression of a monomorphic locus ESTD*.

Fructose-biphosphatase (FBP)
A single anodal zone of moderate activity without variation was observed for this enzyme and it was interpreted as the expression of a monomorphic locus FBP*.

Fumarate hydratase (FH)
This enzyme presents a single invariant anodal zone of moderate activity and intermediate resolution, that was defined as an FH* locus.

beta-N-Acetylgalactosaminidase (bGALA)
This enzyme presents a single anodal zone of weak activity close to the origin with moderate resolution, which has been interpreted as a monomorphic locus bGALA*.

Glutamate dehydrogenase (GLUDH)
A single anodal zone of weak activity was detected in this enzyme with moderate resolution.It was interpreted as the expression of a monomorphic locus GLUDH*.

Grycerol-3-phosphate dehydrogenase (G3PDH)
A single anodal invariant zone of activity was detected in this enzyme that was assumed to reflect the expression of a single locus G3PDH*.

Glucose-6-phosphate dehydrogenase (G6PDH)
A single variable zone of activity was detected in this enzyme.It has weak resolution and has been interpreted as the expression of a single locus G6PDH*, with two alleles: *100 and *107.The observed phenotypes correspond to a dimeric protein where the heterozygotes show three bands and the homozygotes one.

Glucose-6-phosphate isomerase (GPI)
A single anodal invariable zone with intense activity and good resolution was detected in this enzyme which was identified as the expression of a monomorphic locus GPI*.

L-Iditol dehydrogenase (IDDH)
This enzyme presented a single anodal zone of activity which was variable with good resolution and was interpreted as the expression of a single locus IDDH* with two alleles: *100 and *113.

Isocitrate dehydrogenase (IDHP)
Two anodal zones were detected for this enzyme, both were variable with intense activity and good resolution.They were interpreted as the expression of two loci: IDHP-1* with alleles *68 and *100; and IDHP-2* with alleles *94 and *100.All the homozygotes present a phenotype with a single band, whereas the heterozygotes present three bands which is the pattern of the dimeric proteins.

alfa-Mannosidase (aMAN)
This enzyme presents a single anodal zone of activity.It has good resolution without variation and has been interpreted as a monomorphic locus aMAN*.

Malate dehydrogenase (MDH)
This enzyme's zymograms present four anodal zones with different intensities that were interpreted as being the expression of three loci: MDH-1*, MDH-2* and MDH-3*.The fourth zone corresponds to the interloci heterodimer between the first and second locus.The remaining heterodimers were not seen, possibly because they overlapped the expression of one of the other three loci.The intensely active locus MDH-1* and moderately active locus MDH-2*, did not present any variation in the total of analysed individuals.However, locus MDH-3*, with intense activity and good resolution, presented two alleles *100 and *160.The individual homozygotes presented a single band with three for the heterozygotes, which is typical of the dimeric proteins.
When using buffer number 3 (Table 1), the resulting zymograms for this enzyme are the equivalent, in activity and resolution zones, to those obtained for the malic enzyme (ME).In this case, the two loci detected in ME are identical to those designated MDH-2* and MDH-3*, both in allele quantity and individual variation.

Malic enzyme-NAD (ME)
This enzyme presents two intense anodal zones of activity with good resolution and each one is considered as the expression of a locus: ME-1* and ME-2*.Locus ME-1* did not show any variation.However, two alleles were detected in locus ME-2**100 and *160, in which the individual homozygotes are observed with a single band and the heterozygotes with three, a characteristic of dimeric proteins.A zone of weak activity which is observed between both loci was considered to be the expression of the interloci heterodimers.

Malic enzyme-NADP (MEP)
A single variable anodal zone of intense activity and resolution has been detected for this enzyme, which is interpreted as the expression of the locus MEP* with two alleles: *95 and *100.

Mannose-6-phosphate isomerase (MPI)
Two anodal zones with good activity and resolution have been observed for this enzyme and each is considered to be the expression of loci MPI-1* and MPI-2*.Locus MPI-1* did not show any variation in the total of analysed individuals.Two alleles were detected in locus MPI-2*: *85 and *100.The slow allele showed the same electrophoretic mobility as locus MPI-1*.All heterozygotes present two bands which are characteristic of monomeric proteins.

Dipeptidase (PEPA)
A single variable anodal zone of activity was observed with the glycyl-leucine dipeptide.It was interpreted as a single locus, PEPA*, with three alleles: *95, *100 and *105.

Tripeptide aminopeptidase (PEPB)
A single anodal zone of moderate activity was observed for leucyl-glycyl-glycine tripeptide.There was no variation and it was interpreted as a single monomorphic locus PEPB*.

Proline dipeptidase (PEPD)
The zymograms obtained with the phenylalanine-proline dipeptide presented a single variable anodal zone of activity that was considered to be the expression of a single locus PEPD*, with three alleles: *80, *90 and *100.

6-Phosphogluconate dehydrogenase (PGDH)
This enzyme presents a single anodal zone of intense activity and good resolution which was interpreted as the expression of a locus PGDH*.In the total of analysed examples, only one individual presented a phenotype with three bands which was interpreted as a heterozygote for the rapid variant *105.

Phosphoglucomutase (PGM)
This enzyme presented a single anodal zone of intense activity with good resolution which has been interpreted as the expression of a locus PGM* with three alleles: *64, *100 and *114.The homozygotes present a single band and the heterozygotes two, the typical expression of a monomeric protein.

Pyruvate kinase (PK)
Three anodal zones of activity with different intensities and resolutions were detected in the zymograms obtained for this enzyme.The one closest to the origin had moderate activity without any variation and was interpreted as the expression of a monomorphic locus PK*.The two others presented weak activity and such very deficient resolution that they could not be genetically interpreted.

General protein (PROT)
Four invariant anodal zones of intense activity, with good resolution were detected for the general proteins which were interpreted as the expression of four loci: GP-1*, GP-2*, GP-3* and GP-4*.

Superoxide dismutase (SOD)
A single zone of intense activity and good resolution, without any variation, was observed for this enzyme which was interpreted as a monomorphic locus SOD*.

Tyrosine aminotransferase (TAT)
Two anodal zones of activity were detected in this enzyme.The first had moderate activity and good resolution.It did not present any variation in the analysed individuals and is considered to be the expression of a monomorphic locus TAT-1*.The second had intense activity and was interpreted as the expression of locus TAT-2* with two alleles: *89 and *100.The general pattern detected corresponds to a monomeric protein where the individual homozygotes present a single band and the heterozygotes two.

Selection of the loci for the study of genetic variability
The use of a considerable number of loci has been suggested for estimating the genetic variation in natural populations (Nei, 1973;Nei and Roychoudhury, 1974).After several decades of applying electrophoretic techniques in fish, most authors consider that the use of a large number of loci is more important than the use of a great number of individuals (Gorman and Renzi, 1979).This is due to the variance which exists in the heterozygosis between the different loci in a species (Nei et al., 1976).
With the aim of allowing the obtained loci to be used in later population analyses of the Argentinean squid, a large number of enzymatic stains were used, which in turn, provided a high number of well-resolved loci.At the same time, the sample used was also large (N=200) and this ensured the identification of as large a number as possible of allelic variants present in the species sampled area.If the criterion of at least one locus being detected in those cases where there is activity in spite of insufficient resolution is used, then the total number of loci observed during this work was 52.However, not all these loci can be used in the study of genetic variability of the squid and their selection must be based on the following criteria: a) the enzyme resolution must be sufficiently clear to permit the observed phenotypes to be easily read and to allow their correct genotype interpretation.For these reasons, the stains with poor resolution or those where the observed variation could not be genetically interpreted (ODH, XDH) were rejected; b) one must take into account those loci which were detected in different stains and that were identical in mobility, quantity of alleles and observed individual variation (loci 1*, 2* of ME and 2*, 3* of MDH).Only the ME loci or the MDH loci should be used in population studies; c) all the loci which are going to be used must have been correctly resolved in the total of individuals.Even with these restrictions, a large number (45) of useful loci remain for the variability analysis in the Argentinean squid.Table 2 provides a summary of the resolution type for each of the studied enzymes, together with observed loci and detected alleles.
The principal use of this work compared to that of Carvalho et al. (1992), lies both in the greater number of resolved loci and the more detailed interpretation of the obtained zymograms.The latter is especially important for those research teams who would like to undertake more exhaustive future population studies of the Argentinean squid.There is a great need for understanding the patterns for genetic diversity in this species, both from an evolutionary point of view and for the management of the fishing grounds.It should be made clear that only two of the postulated four spawning groups were studies in this work.Any future studies which include all the known groups will probably reveal polymorphism in some of the loci that were shown to be only monomorphic by this work.Carvalho et al. (1992) reported identical results in the number of loci for the following enzymes: AAT, ADA, CK, DIA, ME, ESTD, FBP, FH, G3PDH, G6PDH, GPI, MPI, PEPB, PGDH and SOD.However, for the AK, IDHP, MDH and PGM enzymes, the number of detected loci in this work differ from those reported by Carvalho et al. (1992), probably due to the differing electrophoretic conditions employed in the two studies.).These values are similar to those encountered by Carvalho et al. (1992), who obtained 12 polymorphic loci from 25 resolved loci of which 7 were polymorphic according to the 95% criterion.

TABLE 2 .
Summary of the resolution for the studied enzymes (46) with observed loci (47) and detected alleles in Illex argentinus.a equivalent to MDH-2*, b equivalent to MDH-3*.