ANNALES • Ser. hist. nat. - 13 • 2003 • 1 original scientific article UDK 595.3:575.17(262-191.2) received: 2003-01-05 ENZYMATIC VARIABILITY OF MEDITERRANEAN SLIPPER LOBSTERS, SCYLLARIDES LATUS, FROM SICILIAN WATERS Marco L. UIANCHIN! CNR and Soc. Porto Romano, I-Rotne, Piazza P. Paoü 3 l':'-ma ii: imaifoot@mcitnk.it Ehud SPANIER Leon Rocanati Institute for Maritime Sludies, Unlversity of Haifa, 11-31905 Haifa, Mount Carmei Sergio RAGONESE Ist. ricerche Risorse -Marine e ¡'Ambiente, IRM/VCNR. 1-9)026 Mazara del Valio (TP), Via Luigi Vaceara 61 ABSTRACT A genetic comparison of slipper lobsters, Scyllarides latus, from different areas in Sicily was earned out, vleclro-phoreticalty checking 22 enzymatic activity zones. The analysis has shown a low degree of variability among the specimens; therefore, non-local breeders might be used for restocking without harming the biodiversity. Key words: Crustacea, Decapoda, Scyllaridae, Scyliarides, genetics, electrophoresis VARIABILITA ENZIMATICA NELLA MACNOSA MEDITERRANEA, SCYLIARIDES LATUS, Dl ACQUE SICfUANE SINTESI L'articoh presents H confronto genetico tra magnose (Scyliarides iaiusi provenienti da diverse aree skiliane, confronto effetluato con it controlb elettroforetico di 22 zone di attivitb enzimatica. L'anaiin ha evidenzisto un basso grado di variability tra gli individui, pertanto gii autori ipotizzano die esemplari allevah in a lire localita po-trebbero venir usati nel ripopolamento, evitando eftetti negativi sulla biodiversity. Parole chiave: Crustacea, Decapoda, Scyllaridae, Scyliarides, genetics, elettroforesi 43 ANNALES • Ser. hist. nat. - 13 • 2003 • 1 Maro» I - BIANCHINI er at.: ENZYMATIC VAUIAfiUflY Of MffJITillRANtAN SlIWiB LOBS11 KS SCYLLARIDFJLATijS. fKOM SiOI.IAN WATERS, 43-SP INTRODUCTION On the Sicilian coasts, restocking of the now rare Mediterranean slipper lobster, Scyllarides latus, could achieve stable results, since the decline of the resource seems due to a shortage in recruitment, not to environmental degradation {Bianchini et al., 1998). Of course, a sound enhancement program should operate without disrupting the existing equilibrium. To preserve the biological diversity, and to reduce the risks related to the introduction in the genetic poof of characteristics different from the local ones, it is necessary to make sure that brooders and seeding animals belong to the autochthonous population, or that their origin is genetically similar. With this in mind, morphological (Bianchini ef a/., 19%) and karyoSogical {Deiana et at., 1997) studies may be used for screening, together with genetic analyses, based on PGR methods or on electrophoretk: tec. h n iques. This last approach is based on the notion that proteins under the effect of an electric field migrate along the medium in accordance to their net charge and their molecular weight; utilizing this phenomenon, the electrophoresis displays molecular differences due to ami-noacidic substitutions or deletions. In fact, these differences arise from nucleotide mutations at DNA level of the structural gene, which produce isozymes (Hunter & Markert, 1957) migrating at different velocities. The isozymes, i.e. the multiple forms that an enzyme could as- sume, may depend on the presence of more than one locus codifying the enzyme (allozymes, following Pra-kash et at., 1969), or on the effect of post-translational modifications on the formed polypeptic chains (Richard son et a/., 1986); a locus is considered polymorphic when the frequency of the most common allele is lower than 95%. The last step in the analysis of enzymatic polymorphisms is the interpretation of the observed electrophoretk: pattern, which requires special care in case of species not extensively studied (Richardson ei al., 1986). MATERIALS AND METHODS Thirty-three Mediterranean slipper lobsters, Scyllarides latus, coming from different Sicilian areas (Fig. 1) were examined; their morphometric data are given in Table 1. The electrophoretic runs were commissioned to an external University laboratory, using funds provided by a national program on slipper lobster restocking (III triennial plan of the former Ministry of Merchant Marine). Pereiopod muscles were used; samples were transported In liquid N? to the laboratory, where they were manually homogenized in water, centrifuged at 3000 rpm for 30 min, and stored at -70 'C. The methodology used to study the Scytlarides latus population was the electrophoretic analysis of the enzymatic polymorphisms. Lower Tyrrhenian Vt /V** Palermo jT X .✓-w/' f. i.Trapam / i X / J „i^'V». \ A \ V v Agrigento ft* Catjania Ionian Sea v^---- \ n u V Nc* V X Strait of Sicily X (x Fig. 1: Areas of collection (crosses) of the Sicilian "population" of slipper lobster (Scyllarides latus). Fig. 1: Območja zbiranja (križci) sicilijanske "populacije" velikega nagajivca (Scytlarides latus). 44 ANN ALES • Ser. hist. nat. • 13 ■ 2003 • t Marco I. BIANCHWl c! at.: FNZYMATIC VARIABUfTY O* .MfOITERRANL AN SUPPER lOfiSTEES. Xru.ARIlXS LAVJS, PROM SICILIAN WATERS, 43-5(1 Cellogel (cellulose acetate) was used as support; the electrophoretic runs were performed in Chemetron tanks filled with 250 ml of bridge buffer, holding 3 cellogel stripes (5.7x14 cm), pre-soaked for 15 min in a gel buffer equal to thai of the run (''continuous buffer"}. Each cellogel strip carrier! 5 samples; to allow comparison, she last sample of each strip was repealed on the next one. At the end of the run, every strip was coloured with techniques specific for each enzyme, using buffered solutions with substrates, coenzymes and/or coupled enzymes and colours that bond with the final products. A thermostat was used for the enzymatic reaction; once the colour appeared, the reaction was stopped immersing the cellogel stripes in acetic acid (10%), and the Stripes were sealed and kept at 4 "C Twenty enzymatic sets, accounting for 22 independently variable activity zones, i.e. accounted as the product of 22 loci, were examined. Table 2 reports the tested enzymes, their abbreviations, and the number of interpreted loci; the allele designation follows the numeric system based on the relative gel mobility of isozymes. Table 3 reports, for each tested enzyme, the run conditions (buffers, times, applied tension in Volt, electrical input in mA, depending on the buffer ionic strength) and the colouring techniques, as well as the respective bibliographic references. Tab. 1: Morphometric characteristics of the slipper lobsters (Scyllarides latus} analysed electrophoretically by sex (F = female> M = male). Tab. 1: Morfomctrične značilnosti velikih nagajivcev (Scyllarides latus), analizirane elektroforetično po spolu (F - moški, M = žensirej. F M number 13 20 [weight (g) mean 440.6 36!.7 S D 191.0 76.7 [min 258.0 237.0 [max 940.0 523.0 Icarapace length (mm) mean 100.4 94.8 Jsi) 13.2 7.0 Imin 88.6 81.7 i max 132.2 106.7 RESULTS The Sicilian slipper lobsters show a very modest electrophoretic variability in the 22 examined loci (Tab. 4): 20 loci arc- completely, or almost completely, fixed in the same allele, and the polymorphism is low in the other 2 loci f|DH and MDH-2) too. Table 5 reports the estimates of the populations I genetic variability, expressed as mean number of alleles per locus (N,), percent of polymorphic ioci (p), observed (I U and expected (H,,) heterozygosity (Nei, 1978). The studied population presents low values of polymorphism, with Ho=0.02Q, H,=0.019, mean number of alleles per locus equal to 1.2 and percent of polymorphic loci equal to 9.1, Moreover, the observed genotypic frequencies were compared with the expected frequencies, to display possible divergences from the Hardy-Weiriberg equilibrium, per population and per locus (Tab. 6). Using the x~ analysis, the studied Sicilian population of 5. latus results in substantial equilibrium in al! the examined loci. Tab. 2: Enzymatic systems analysed electrophoretically in the slipper lobster (Scyllarides latus). Tab. 2: Elektroforetično analizirani encimatski sistemi velikega nagajivca (Scyllarides latus). enzyme (Enzyme Commission number) abbreviation No. of ; loci Alcohol dehydrogenase (1.1.1.1) ADIT 1 1 Adenilate chinase (2.7.4.3) ADK 1 Aldolase (4.1.2.13) AI.DO 1 Creatine chinase (2.7.3.2) CK 1 Esterase (3.1.1.1) EST 1 Fructose-1,6-diphosphatase (3.1.3.11) FDP 1 Fumarase (4.2.1.2) FUM 2 Glyceraldehyde-3-phosphate dehydrogenase (1.2.1.12) GAPD 1 Glucose-phosphate isomerase (5.3.1.9) GPI 1 E'sochinase (2.7.1.1) UK 1 Isocitrate dehydrogenase (1.1.1.42) IDH 1 Lactate dehydrogenase (1.1.1.27) LDH 1 Ma I ate dehydrogenase (1.1.1.37) MDH Malic enzyme (1.1.1.40) ME 1 Mannose-phosphate isomerase (5.3.1.8) MP! 1 Amino-peptidase (3.4.11) PEP 1 6-Phosphogluconate dehydrogenase (1.1.1.44) 6PGD 1 Phosphoglucomutase (2.7.5.1 ) PGM 1 T r ios o • pli osp h a t e i som era se (5.3.1.1) TP! 1 Xantine dehydrogenase {1.2.1,37) XDH 1 Total number of analyzed enzymes - 20 Total number of interpreted loci - 22 119 ANNAlfcS • Ser. hist. nat. ■ 13 • 2003 • 1 Marco L. BIANCHiNI «I a-1.. LNZVMAtfC VARIABILITY OF MEDITERRANEAN SUPPER LOBSTERS, SCYUAPJOtt LAT 'US. I'ROM SICILIAN WATfSS, 43-SO Tab. 3: Electroplwretic methods used for separation of the slipper lobster (Scy Ha rides latus) samples. Tab. 3: Elektroforetične metode, uporabljene za ločevanje primerkov velikega nagajivca (Scyllarides latus). enzyme buffer (conc.) Volt/hour/mA coloring technique (reference) ADH C tlx) 160/2:00/10 Richardson el a!., 1986 ADK B (2 x) 160/2:30/18 Richardson etal., 1986 ALDO TEC 0.075 <1x) 560/2:00/14 Harris & Hopkinson, 1976 CK C(2x) 160/2:00/18 Richardson etal., 1986 EST G (1x) 160/1:00/18 Richardson etal., 1986 FDP B (1 \) 160/2:00/10 Richardson et a/., 1986 FUM A <2x) 160/1:30/8 Richardson ef al., 1986 GAPD C (1x) 160/2:00/10 Richardson etal., 1986 GPI C Ox) 160/2:00/10 Richardson et si., 1986 ¡HK COx) 160/1:15/10 Richardson etal., 1986 IDH A (1x) 160/1:45/10 Ayala eta!., 1972 LDH B0x) 160/1:45/10 Selander ef a/., 1971 MDH COx) 160/1:30/10 Richardson etal.. 1986 ME B (1x) 160/1:30/10 Richardson etal.. 1986 MPI B Ox) 160/1:00/10 Richardson et al., 1986 PEP A (2x) 160/1:45/8 Richardson etal., 1986 6PGD B Ox) 160/1:30/10 Richardson etal., 1986 PCM C Ox) 160/1:45/8 Richardson etal., 1986 TPI BOx) 160/2:00/10 Richardson etal., 1986 XDH F Ox) 160/1:45/18 Richardson et al., 1986 Bibliographic references for the buffers: A.. B, C, F, G: Richardson etal., 1986 TEC 0.075: Meera Khan, 1971 DISCUSSION The eiectrophoretie analysis of the enzymatic polymorphisms has shown low levels of variability in the Sicilian population of Scyllarides latus. This result should not be interpreted as a bottleneck effect of the numerical scarcity of the Sicilian population, but may be structural to the taxon (Hardwick & Cline, 1984, 1985, 1986). In fact, preliminary unpublished results on two other populations, from Israel and from the Azores, seem to confirm a minimal heterozygosity. Moreover, this lack of genetic diversity is in substantial agreement with the pattern observed in other species of large-size decapod crustaceans (Hedgecock, 1987). On the other hand, the apparent resemblance of the slipper lobster samples could be linked to peculiar variability patterns of the gene-enzyrne systems hitherto tested; other kind of loci might display higher levels of variation. The genetic analyses have shown a high similitude among the studied animals, coming from distant geographical locations: this fact suggests that nori-local specimens could be used, as breeders or seeding stuff, in restocking and stock enhancement programs in impoverished areas, without exorbitant risks of genetic contamination and biodiversity reduction. Nevertheless, the seemingly genetic homogeneity of the Mediterranean slipper lobsters needs further testing, perhaps with more sophisticated techniques (e.g. the analysis of microsatellite loci), before the actual introduction of allochthonous material. CONCLUSIONS On the Sicilian coasts, restocking of the now rare Mediterranean slipper lobster, Scyllarides latus, could achieve stable results, since the decline of the resource seems due to a shortage in recruitment, not to environmental degradation. To preserve the biodiversity, and to reduce the risks related to the introduction in the genetic pool of characteristics different from the local ones, it is necessary to make sure that brooders and seeding animals belong to the autochthonous population, or that their origin is genetically similar. With this in rnind, a genetic comparison of slipper lobsters, Scyllarides latus, from different areas in Sicily was carried out, electrophoretically checking 22 enzymatic activity zones (ADH, ADK, ALDO, CK, EST, f DP, FUM-1, FUM-2, GAPD, GPI, HK, !DH, LDH, MDH-1, MDH-2, ME, MP!, PEP-2, 6PGD, PGM, TPt, XDH), 46 ANNALCS • Ser. hist. nal. • 13 • 2003 • 1 Marco L BIAMCHINI a H.' fNZYMATIC VARIA8M.ITY Of MEDITERRANEAN SI.IPPFR LOBSTERS, SCYLLARIOES LATUS. FROM SICH IAN WATERS. 43-50 Tab. 4: Frequencies of alleles for 22 loci in the Sicilian population of slipper lobster (Scyllarides latus). Legend: N _ number of specimens; A = most common/only allele; B - second allele. Tab. 4: Frekvence alel ¿a 22 lokacij sicilijanske populacije velikega nagajivca (Scyllarides latus). Legenda: S - št. primerkov/ A = najpogostejši/edini alel; B = drugi alel. InrllS ADH N 29 A 1.000 ADK N 24 A 1.000 ALDO [n~~ 29 A 1.000 CK iN 24 A 1.000 1 EST IN 29 A 0.983 B 0.017 FDP N 27 A 1.000 i FUM-1 n 29 A 1.000 FUM-2 N 29 A 0.983 ß 0,017 CAPD N 29 A 1.000 CPf n 29 A 1.000 HK n 29 A 1.000 locus 1DH N 29 A 0.948 B 0.052 LDH N 29 A 1.000 MDH-1 N 29 A 0.983 b 0.017 MDH-2 N 29 A 0.879 b 0.121 ME IN 29 [A 1.000 MP1 N 29 A 1.000 PEP-2 N 29 A 1.000 I GPGD j N 29 A 1.000 PCM N 29 A 1.000 TRI N 29 A 1.000 XDH N 29 A 1.000 Tab. 5: Cenetic variability estimates (± standard error) for 22 loci in the Sicilian population of slipper lobster (Scyllarides latus). Tab. 5: Ocene genetske variabilnosti (t standardna napaka) za 22 fokacij sicilijanske populacije velikega nagajivca (Scyllarides latus). mean No. mean No. al- % polymorphic mean heterozygosis (± SD) | ind./populat. (± leles/locus (NJ loci (p) observed (H„) expected (Ha) | SD) 28.5 (0.3) 1.2 (0.1) 9.1 0.020 (0.012) 0-019(0.011) 1 47 ANNALES • Ser. hisl. nat. ■ (3 • 2003 • 1 Marco I. BfANCBINl «r tl.; ENZYMATIC VARIABILITY OF MEDITERRANEAN SI.IPRi'R I OEM IRS, SO variocs laws, [ROM SO.IAN WATERS, 43-50 Tab. 6: test for the rejection of the Hardy-Weinberg equilibrium in the Sicilian population of slipper lobster iScyllarides latus). Tab. 6: Test % za zavrnitev Hardy-Weinbergovega ravnovesja v sicilijanski populaciji velikega nagajivca (Scyi-larides latus). locus/class observed expected X d.o.f. P EST 0.000 1 1 .ooo ! A-A 28 28.000 A-B 1 1.000 j B-B 0 0.000 lfUM-2 0.000 1 1.000 A-A 28 28.000 A-B 1 1.000 B-B 0 0.000 IDH 0.057 1 0.812 A-A 26 26.053 A-B 3 2.895 r 1 B-B 0 0.053 I MDH-1 0.000 1 1.000 A-A 28 28.000 A-B 1 1.000 B-B 0 0.000 MDH-2 0.461 1 0.497 A-A 22 22.368 A-B 7 6.263 B-B 0 0.368 The Sicilian slipper lobsters show a very modest eleclrophoretic variability in the examined loci. 20 foci are completely, or almost completely, fixed in the same allele, and the polymorphism is low in the other 2 loci tIDH and MDH-2) too. The observed heterozygosity (HG) equals 0.020, and the expected heterozygosity (Ha) equals 0.019, the mean number of alleles per locus (NJ is 1.2 and the percent of polymorphic loci is 9.1. Moreover, using the analysis, the studied Sicilian population of S. latus results in substantial equilibrium in all the examined loci. In fact, this lack of genetic diversity is in substantial agreement with the pattern observed in other species of large-size decapod crustaceans. The genetic analyses have shown a high similitude among the studied animals, coming from distant geographical locations: this result suggests that non-local specimens could be used, as breeders or seeding stuff, in restocking and stock enhancement programs in impoverished areas, without exorbitant risks of genetic contamination and biodiver-sitv reduction. 48 ANNALES • Ser. hist. nat. • 13 • 200.3 • 1 M,,.ru i. BIANU nNl7¡7/"~rN7YMAÍ!r VARIABILITY OF MfOITERRANFAN SUIW: LOBSTERS, XTUAMOC: I aTUS. FROM SICIUAN WATttó, «Ü-ST ENCIMATSKA VARIABILNOST VELIKEGA NAGAJIVCA, SCYLLARIDES LATUS, V SICILIJANSKIH VODAH Marco i. BIANCHINI CNR and Soc. Porto Romano, l-Roms, fiazu P. Paoli 3 £-mail: iinarbol@mt ink i? Ehud SPANIER Leon Recanali Inslitule for Mariliine Suiciies, Universi|y of Haifa, U.-31905 Maifa, Moti nt Carine! Sergio RAGONESE Ist. ricercbe Risorse Marine e 1'Ambieme, IRMA/CNR, 1-91026 Mazara det Valjo (TP). Vi.i Luigi Vaccara 6! POVZETEK V sicilijanskih obalnih vodah bi z vlaganjem zdaj redkega velikega nagajive a, Scyllarides latus, dosegli bržkone : dobre rezultate, saj vse kaže, da upadanja njegove populacije ne gre pripisali degradaciji lega morskega okolja. Toda da bi ohranili biodiverziteto v leh vodah in hkrati zmanjšali tveganja, povezana z vlaganjem osebkov v genetski "poo!" z značilnostmi, ki se razlikujejo od lokalnih, bi bito treba zagotoviti, da osebki za razplod teh rakov pripadajo avtohtoni populaciji, ali pa da je njihov izvor genetsko podoben lokalnemu. V ta namen smo opravili genetske primerjave med velikimi nagajivci, Scyllarides latus, iz različnih območij si-cilijanskih obalnih voda z elektroforetičnim pregledovanjem 22 enciinatskih con aktivnosti (ADH. ADK, Al DO, CK, £57. FDP, FUM-h FUM-2, GARD, GPI, HK, IDH, LDH, MDH-1, MDH-2, ME, MPi, PEP-2, 6PGD, PCM. TPI, XDH). Na raziskanih lokalitetah je bila ugotovljena zelo skromna elektroforevčna variabilnost velikih nagajivcev. 20 J lokalitet je docela, ali skoraj docela, ustaljenih v istem afelu, polimorfizem pa je nizek tudi v dveh preostalih lokacijah iIDH in MDH-2). Opazovana heterozigotnost