SCRS/2008/071 Collect. Vol. Sci. Pap. ICCAT, 63: (2009)

SCRS/2008/071 Collect. Vol. Sci. Pap. ICCAT, 63: 161-173 (2009) HISTORICAL LANDING STATISTICS, SIZE, SEX AND MICROSATELLITE ANALYSES OF BLUEFIN TUNA (THUNNUS THYNNUS THYNNUS) IN THE CENTRAL-SOUTHERN MEDITERRANEAN SEA Adriana Vella 1 SUMMARY Bluefin tuna has been caught in the Maltese Islands, in the centre of the Mediterranean Sea, with traps since 1748 which reached regular usage around 1948. However this fishing method was replaced by bluefin tuna catches with long-line, initially as a by-catch in the swordfish fisheries prior to fine-tuning the gear for bluefin tuna in 1995, when the Japanese bought bluefin tuna caught by Maltese fishermen. Total bluefin tuna long-line fisheries landings in the Maltese Islands have followed an overall decline from a peak of 353,014kg in 1995 to 227,008kg in 2006. Close to 400 bluefin tuna specimens caught south of the Maltese Islands, were sampled during six years to study variations in sizes, sex ratios, biogeographic characteristics and molecular genetics using four microsatellite loci. This study results indicate decreasing average bluefin tuna catch body size and an increasing sex bias with females composing the greater percentage of specimens caught. A significant genetic heterogeneity, F ST value of 0.018 (P=0.015), was found among the bluefin tuna sampled in the spawning and fishing region south of the Maltese Islands. RÉSUMÉ Depuis 1748, le thon rouge est capturé dans les îles maltaises, au centre de la Méditerranée, avec des madragues, dont l utilisation s est généralisée aux alentours de 1948. Toutefois, cette modalité de pêche a été remplacée par les prises de thon rouge à la palangre, tout d abord en tant que prises accessoires des pêcheries d espadon avant le perfectionnement de l engin ciblant le thon rouge en 1995, lorsque les japonais achetaient du thon rouge capturé par les pêcheurs maltais. Les débarquements totaux des pêcheries palangrières de thon rouge dans les îles maltaises ont suivi un déclin général, à la suite du chiffre maximum de 353.014 kg atteint en 1995, se situant à 227.008 kg en 2006. Près de 400 spécimens de thon rouge capturés au sud des îles maltaises ont été échantillonnés pendant six ans afin d étudier les variations de tailles, de sex-ratios, les caractéristiques biogéographiques et la génétique moléculaire en utilisant quatre loci par microsatellite. Les résultats de cette étude indiquent une diminution de la taille corporelle moyenne des thons rouges ainsi qu une augmentation des biais sexuels, les femelles représentant le pourcentage le plus élevé des spécimens capturés. Une forte hétérogénéité génétique, valeur de F ST de 0,018 (P=0,015), a été constatée entre les thons rouges échantillonnés dans les zones de frai et les lieux de pêche au sud des îles maltaises. RESUMEN Desde 1748, en las islas de Malta, en el centro del Mediterráneo, el atún rojo se ha capturado con almadrabas alcanzado un uso regular hacia 1948. Sin embargo, este método de pesca fue sustituido por las capturas de atún rojo con palangre, inicialmente como captura fortuita en las pesquerías de pez espada antes de ajustar el arte para el atún rojo en 1995, cuando los japoneses compraban el atún rojo capturado por los pescadores malteses. Los desembarques totales de atún rojo de las pesquerías de palangre en las islas de Malta han sufrido un descenso general desde un pico de 353.014 kg en 1995 hasta 227.008 kg en 2006. Cerca de 400 ejemplares de atún rojo capturados al sur de las islas de Malta fueron muestreados durante seis años para estudiar variaciones en las tallas, sex ratios, características biogeográficas y genética molecular usando cuatro loci microsatelitales. Los resultados de este estudio indican un descenso medio en el tamaño del cuerpo del atún rojo capturado y un sesgo sexual creciente ya que las hembras suponen el mayor porcentaje de los ejemplares capturados. Una 1 Conservation Biology Research Group, Department of Biology, University of Malta, Msida, Malta; avel@cis.um.edu.mt 161

heterogeneidad genética significativa, un valor de F ST de 0.018 (P=0.015), se encontró entre los atunes rojos muestreados en la zona de pesca y de desove de las islas de Malta. KEYWORDS Bluefin tuna fisheries, longlining, trap fisheries, Mediterranean Sea, genetics, microsatellites, Maltese Islands 1. Introduction The Mediterranean is an important sea for bluefin tuna species survival and continuity as much as it is important to various Mediterranean fishermen that have earned a living from fishing this species for hundreds of years (Sara 1998; FAO 2002). However this same sea has on the one hand been considered as a closed and small sea not capable of sustaining bluefin tuna throughout their life history, on the other hand it is found to be heterogeneous through its region from West to East with two main basins considered to split this sea in two. As marine research on the qualities of this sea and its marine organisms expand a realization that regional differences causing separation of populations is possible in the Mediterranean (GFCM 1998; Carlsson et al. 2004), as much as between the Mediterranean and the Atlantic (Chikhi et al. 1997 for sardines; GFCM 1998; Roldan et al. 1998 for hake; Naciri et al. 1999 for sea bass; Natoli et al. 2005 for bottlenose dolphins). Differentiation at population level is usually expected among species with poor migratory behavior or with limited larval/juvenile transportation with currents. Bluefin tuna on the contrary is a well-known migratory species (Mather et al.1995; Block et al. 1998). This species distribution spans the world with spawning sites found in various parts of the world, including the Mediterranean (Block et al. 2001; 2005; De Metrio et al. 2004). To determine the presence of spawning sites in the Mediterranean, there have been several studies ranging from larval surveys to analyses of the reproductive status of the female bluefin tuna. The study conducted by Medina et al., 2002, has shown that the females collected at the Balearic Islands had a five-fold more highly yolked oocytes than samples collected from the Strait of Gibraltar. Moreover specimen from the Balearic Islands also had shown the presence of postovulatory follicles in their ovaries, thus indicating the presence of recent egg release. Another study by Nishida et al., 1998, has shown the presence of high larval concentrations around the Balearic Islands, around the Maltese Islands, eastern Sicily and south Tyrrhenian Sea. Furthermore, two studies by Karakulak et al., 2004, and Oray et al., 2005, focused on whether or not there are any eastern Mediterranean spawning sites apart from those in the western and central Mediterranean. Through the use of histological and larval surveys respectively, these studies have shown the presence of a spawning site in the Levantine Sea, between Cyprus and Turkey. One of the major techniques used to analyze bluefin tuna migratory movements uses electronic tagging. Studies by Block et al. (1998, 2001 & 2005) indicated spawning in the Atlantic and in the Central Mediterranean. A study by De Metrio et al., 2004, showed electronically tagged bluefin tuna did not migrate towards the Strait of Gibraltar after reproduction, but stayed in the eastern Mediterranean. Due to the need to manage in sustainable way the exploitation of a species at stock or population level, several studies have looked at the use of genetic techniques to assess the population structure and subdivisions present in its global and regional distribution. Bluefin tuna is no exception and is of particular interest due to its commercial value. Various genetic studies on bluefin tuna have focused on allozymes (Pujolar et al., 2003), mitochondrial DNA (Ely et al., 2001; Takeyama et al., 2001; Vinas et al., 2003; Manchado et al., 2004); and microsatellites (Takagi et al., 1999; McDowell et al., 2002; Clark et al., 2004; Carlsson et al., 2004). Genetic studies conducted by Takagi et al. 1999, using four microsatellites; Ttho-1, Ttho-4, Ttho-6 and Ttho-7 (all four had dinucleotide motif repeats), have shown that there was no significant difference between the N.W. Atlantic samples and the Mediterranean (Messina) samples (F ST =0.002, P>0.294), though the use of a larger sample size may have been required since the loci used tend to be considerably polymorphic. Vinas et al. (2003) analysed the mitochondrial control region of 22 tunas caught in Libyan waters, 23 in Tunisian waters and 12 in 162

Maltese fishing area. 24 bluefin tunas from the Gulf of Cadiz in the East Atlantic Ocean were also analyzed. The only significant result was obtained between the Maltese samples and other regions sampled (F ST =0.013, P<0.001). This difference was interpreted as being due to small sample size (n=12). The study by Carlsson et al. 2004, has used nine microsatellites; Tth5, Tth8, Tth10, Tth21, Tth34, Ttho-1, Ttho-4, Ttho-6 and Ttho-7 (first five were with tetranucleotide motif repeats; last four had dinucleotide motif repeats). It was observed that there were significant genetic differences between collections from the Tyrrhenian and Ionian Sea (on both sides of the Messina Strait) when using microsatellite and mitochondrial markers (F ST =0.0087, P=0.015 and ST =0.0367, P=0.030, respectively). The latter authors suggested that the results indicated the possibility of a genetically discrete population in the eastern basin of the Mediterranean Sea possibly affected by currents within the Mediterranean Sea which might limit the mixing of larvae and eggs. Tuna has been caught in the Maltese Islands with traps since 1748 reaching regular usage around 1948 (Farrugia Randon 1995). However this fishing method was finally replaced with long-line, initially as a by-catch in the swordfish fisheries prior to fine-tuning gear for bluefin tuna in 1995, when the Japanese bought bluefin tuna caught by Maltese fishermen. Bluefin tuna fishing is now a seasonal activity for Maltese Fishing is undertaken between May and mid-july. Each fishing expedition involves on average 4 days of effort with 40 to 50 miles of long-line, set at least three times, each extending down to 100m with bait which is mostly imported mackerel. Boats utilized for this fishing measure between 10 to 26m. The increase in purse-seine fishing effort in the central and southern Mediterranean Area can jeopardize the survival of the species population and the livelihoods of Maltese long-line tuna fishermen if continued unchecked (Vella 2002). Vessels from Malta and Tunisia found themselves being joined by Italians, Spanish, French, and recently, increasing Libyan fishing vessels. The sudden increase in exclusive fishing zones by Tunisia and Libya further proves the increasing desire to increment the income from this fish species. In order to better understand the bluefin tuna population structure and diversity found south of the Maltese islands, a study was started in 1998 (Vella 2006) and has recently extended its sampling range west ward and east ward. 2. Methods Three hundred and eighty specimens of bluefin tuna caught during the Maltese tuna long-line fishing season between 1998 and 2004 were measured (fork length and weight), sexed, and tissue sampled. The tissues collected were stored in 20% DMSO saturated with Sodium Chloride for subsequent molecular genetic analyses. Sampling occurred throughout the fishing season and encompassed May, June and July of each year. Records of GPS position and sea depth at each location where each specimen was caught were taken. DNA extraction from the stored tissues was carried out using the proteinase K/phenol/chloroform /isoamylalcohol method (Hoelzel 1998). The extracted DNA quality was analysed by using a 0.8% agarose gel. Those samples whose DNA was greater that 300bp, were stored for further analyses. This reduced the effective bluefin tuna sample for the microsatellite study to one hundred and ninety eight specimens. The PCR products were stored at -20 C until further analyses. Sizing of alleles was carried out using ABI***. The fluorescent dyes used were Ttho-1 TET, Ttho-4 FAM, Ttho-6 HEX and Ttho-7 FAM Result files were analyzed using GenescanView1.2_8. Each allele size was identified using the program and they were binned manually to coincide with the repeated nucleotide sequence of their respective microsatellite locus. Allele frequencies were calculated as: number of the allele / total number of alleles (for that particular locus). Heterozygosity observed (Ho) and expected (He) were calculated using Arlequin ver.3: Ho is the number of heterozygotes observed as a ratio of the total number of individuals, while He is the expected number of heterozygotes if the total number of individuals were at Hardy-Weinberg equilibrium (HWE). Fixation index (F ST ) was used to measure bluefin tuna population differentiation based on genetic polymorphism data, such as microsatellites. This statistic compared the genetic variability within and between populations. F ST was calculated using Arlequin ver.3. This analysis was important in order to investigate the presence or absence of any significant differences between the various allele frequencies amongst bluefin tuna from different sampling areas south of the Maltese Islands illustrated in Map. 1. 163

3. Results An overview of reported local bluefin tuna landings between 1957 and 2006 are illustrated as bar graphs in Figures 1 and 2. These show how bluefin tuna landings have increased through the years. However a decline in recent years is also noted though fishing efforts have increased. Pie charts in Figures 3 to 7 illustrate the average % landings per month between 1957 and 2006. Figures 3 and 4 show how trap fishing in Malta seemed to allow for bluefin tuna landings throughout the year, however there was a shift to seasonal bluefin tuna landings during May, Jun and July with the onset of long-lining as may be seen in Figures 5, 6 and 7. In recent years the long-line fishing season somehow shrank to consisting of mostly the month of May and part of June which is spawning time for the bluefin tuna in this region of the Mediterranean. Bar graph in Figure 8 illustrates the % sex composition of the sample each year between 1999 and 2004. On alternative years one may note a progressive increase of % females with the highest (70%) in 2004 showing a high bias toward females. In fact, scatter graphs shown in Figures 9 and 10 illustrate average body size (fork length) per year and the relation between body weight (in kg) and fork length (in meters) respectively. Figure 9 indicates a clear reduction in the average size of the sampled individuals in 2003 and 2004. Figure 10 shows a clear and close positive logarithmic relation between body size (kg) and fork length (m) with a high R 2 value of 0.86. The model equation is included with the scatter plot and line fit. The molecular genetics analyses results for bluefin tuna in this study are included in Tables 1 and 2. Table 1 shows the number of alleles, number of private alleles, allele richness, Ho and He for each microsatellite locus at each location. On investigating the genetic distance between samples caught during the six year study period (1999-2004) from three locations south of the Maltese Islands, genetic diversity between individuals sampled from regions A and C (refer to Map 1) was found to be significant on a pairwise test: F ST = 0.018 (P = 0.015) as shown in Table 2. 4. Discussion An overview of reported local bluefin tuna landings between 1957 and 2006 illustrate how this the bluefin tuna evolved in importance for the Maltese especially when trade with the Japanese became possible. While stimulating greater fishing effort among Maltese fishermen, a decline in the landings of recent years may be indicative of regional over-exploitation in the fishing grounds utilized by the Maltese fishermen too. Equally relevant is the increasing fishing effort placed in a spawning ground which is clearly seen when comparing the pie charts for %monthly landings between 1957 and 2006. While Figures 3 and 4 show how trap fishing in Malta allowed bluefin tuna landings throughout the year, Figures 5 and 6 show how long-lining somehow shifted the effort to May, Jun and July. Intensified fishing effort has annually shrank the bluefin tuna long-line catch, with 2006 registering the shortest fishing season for Maltese long-liners, when most bluefin tunas where caught in May. Percentage sex composition of the sample each year between 1999 and 2004 also indicate a change in the bluefin tuna catch composition. Progressive increase in female percentage composition, with the highest recorded (70%) in 2004 shows a high bias toward females. This could also coincide with the reduction in average size of the tuna caught in 2003 and 2004. In fact, Figure 9 indicates a clear reduction in the average size of the sampled individuals in 2003 and 2004. Figure 10 shows a clear and close positive logarithmic relation between body size (kg) and fork length (m) with a high R 2 value of 0.86. The model equation is included with the scatter plot and line fit. The molecular genetics analyses results for bluefin tuna in this study included in Tables 1 and 2 show the number of alleles, number of private alleles, allele richness, Ho and He for each microsatellite locus at each of the three locations: A, B, and C indicated in Map 1. The number of alleles per microsatellite locus within samples varied from 5 for Ttho1 in the three locations to 13 for Tth07 in location B. Allele richness per locus and sample varied from 3.894 at locus Tth01 in location C to 10.2 at locus Tth07 in location B. Average observed heterozygosities varied from 0.368 in location C at locus Tth01 to 0.925 in location C at locus Tth04. The expected heterozygosities varied from 0.536 in location C at locus Tth01 to 0.885 in location C at locus 164

Tth07 (Table 1). The genotype distribution at loci Tth06 and Tth07 in location C deviated significantly from Hardy-Weinberg expectations (HWE) and thus were removed from subsequent analyses with the consequence that the F ST value increased further in its significance. As locus Tth06 is also probably affected by null-alleles (also found to be so in other studies, such as Carlsson et al. 2004) its removal from the final F ST analyses aided in this as well. Investigating the genetic distance between samples from the different locations South of the Maltese Islands during the six year study period (1999-2004) indicated an F ST which varied from 0.018 (P=0.015) to 0.025 (P=0.012), when removing the two loci in location C found to be significantly deviating from the HWE obtained in this study. This clearly shows a significant difference between bluefin tuna sampled in different these locations in the Maltese Fishermen s fishing grounds. This is a particularly interesting result as the whole study area (illustrated in Map 1) could be considered as relatively small for a highly migratory fish species such as the bluefin tuna. It also suggests that the region studied, south of the Maltese Islands, may have some barrier to geneflow for bluefin tuna coming from the different Mediterranean regions. Such a situation has already been found across the strait of Gibraltar for various marine species as indicated above (Chikhi et al. 1997 for sardines; Roldan et al. 1998 for hake; Naciri et al. 1999 for sea bass), and some other areas in the Mediterranean, including the strait of Messina (Carlsson et al., 2004). However in this study area there does not seem to be any evident physical environmental condition that would encourage such reduction in geneflow and significant population diversity. These results, together with the need to be sampled and analyzed various other regions in the Mediterranean, stimulate more research into the genetics and biogeographic characteristics of bluefin tuna in the Mediterranean. Research on possible causes for reduction in bluefin tuna geneflow in the central and southern Mediterranean should also be considered. The increasing fishing effort and efficiency in bluefin tuna catch in the past ten years by international fishing fleets in this fishing ground may be affecting the Mediterranean population by: reducing the average size of tuna, and also affecting population sex ratio, which can have effects on future recruitment. A greatly reduced spawning stock, a decreasing proportion of older and thus larger bluefin in the catch, and generally very low recruitment year after year are all clear signs of recruitment over-fishing. The realization that there may be distinct bluefin tuna stocks in the Mediterranean would be important to consider when planning and managing fishing activities and quotas, to safeguard and conserve all stocks. As tuna ranching/penning activities in this same region has further increased the effort and disturbance in this spawning and fishing ground, with offshore activities which are rarely monitored for quotas, discards, etc. there is an urgent need to regulate or reduce such exploitation efforts until effective monitoring and enforcement is in place. 5. Acknowledgements Thanks are due to the following contributors: The University of Malta for funding the costs of molecular genetics analyses for this research project; My Research Assistant, Noel Vella for his dedicated contribution; Maltese long-line fishermen assisting in the bluefin tuna sampling phase; Fisheries Department Statistics Officer, Tony Tanti for forwarding statistics on total monthly landings; Dr. Rus Hoelzel for assisting with his expert advice at various stages of this research project. References Block, B.A., Dewar, H., Farwell, C.J, Prince, E.D. 1998. A new satellite technology for tracking the movements of Atlantic bluefin tuna. In Proc. Natl. Acad. Sci., USA Vol. 95 pp9384-9389 Ecology. Block, B.A., Dewar, H., Blackwell. S.B., Williams, T.D., Prince, E.D., Farwell, C.J., Boustany, A., Teo, S.L.H., Seitz, A., Walli, A., Fudge D. 2001. Migratory movements, depth preferences, and thermal biology of Atlantic Bluefin tuna. Science 293, 1310-1314. Block, B.A., Teo, S.L.H., Walli, A., Boustany, A., Stokesbury, M.J.W., Farwell, C.J., Weng, K.C., Dewar, H., Williams, T.D. 2005. Electronic tagging and population structure of Atlantic bluefin tuna in Nature Vol, 434 - letters to nature April 2005. 165

Carlsson, J., McDowell, J.R., Diaz-James, P., Carlsson, J.E.L., Boles, S.B. 2004 Microsatellite and mitochondrial DNA analyses of Atlantic bluefin tuna (Thunnus thynnus thynnus) population structure in the Mediterranean Sea in Molecular Ecology: Vol. 13 Page 3345 - November 2004 Volume 13 Issue 11. Clark, T.B., Ma, L., Saillant, E., Gold, J.R. 2004. Microsatellite DNA markers for population-genetic studies of Atlantic bluefin tuna (Thunnus thynnus thynnus) and other species of genus Thunnus, Molecular Ecology Notes, Volume 4 Page 70 - March 2004doi:10.1046/j.1471-8286.2004.00572.x Volume 4 Issue 1 Primer Note. Chikhi, L, Agnese, J.F, Bonhomme, F. 1997. Strong differences of mitochondrial DNA between Mediterranean Sea and Eastern Atlantic populations of Sardinella aurita. C.R.Acad. SciIII 320:289-297. De Metrio, G., Oray, I., Arnold, G.P., Lutcavage, M., Deflorio, M., Cort, J.L., Karakulak, S., Anbar, N., Ultanur, M. 2004. Joint Turkish-Italian research in the eastern Mediterranean: bluefin tuna tagging with PopUp satellite tags. Collect. Vol. Sci. Pap. ICCAT, 56(3): 1163-1167. Ely, B., Stoner, D.S., Dean, J.M., Alvarado Bremer, J.R., Chow, S., Tsuji, S., Ito, T., Uosaki, K., Addis, P., Cau, A., Thelen, E.J., Jones, W.J., Black, D.E., Smith, L., Scott, K., Naseri, I., Quattro, J.M. 2001. Genetic analysis of Atlantic northern bluefin tuna captured in the northwest Atlantic Ocean and the Mediterranean Sea. Collect. Vol. Sci. Pap. ICCAT, 54(2): 372-376. FAO. 2002. FAO Yearbook. Fisheries Statistics: capture production 2000. FAO Fisheries Series no. 60, Rome, Italy, FAO. Farrugia Randon, S. 1995. The fishing industry in Malta: past, present and future. Pin pub. Independence print, Malta. GFCM. 1998. Studies and Reviews No 70-FAO.: Zoogeographical categories relevant to fisheries management in the Mediterranean. Hoelzel, A. R. 1998. Molecular analysis of populations; a practical approach. Oxford University Press, Oxford, UK. Karakulak, S., Oray, I., Corriero, A., Deflorio, M., Santamaria, N., Desantis, S., De Metrio, G. 2004. Evidence of a spawning area for bluefin tuna (Thunnus thynnus L.) in the eastern Mediterranean. J. Appl. Ichthyol 20, 318-320 et al., 2004. Manchado, M., Catanese, G., Infante C. 2004. Complete mitochondrial DNA sequence of the Atlantic bluefin tuna, Thynnus thynnus. Fish. Sci. 70, 68-73. Mather, F.J, Mason, J.M, Jones, A.C. 1995. Historical Document. Life History and Fisheries of Atlantic Bluefin Tuna. NOAA Technical Memorandum, 370. NMFS-NOAA, Silver Spring, MD. McDowell, J.R., Diaz-Jaimes, P., Graves, J.E. 2002. Isolation and characterization of seven tetranucleotide microsatellite loci from Atlantic northern bluefin tuna Thunnus thynnus thynnus. Molecular Ecology Notes 2, 214-216. Medina A., Abascal F.J., Megina C. and Garcia A. 2002. Stereological assessment of the reproductive status of female Atlantic northern bluefin tuna during migration to Mediterranean spawning grounds through the Strait of Gibraltar. Journal of Fish Biology 60, 203-217. Natoli, A., Birkun, A., Aguilar, A, Lopez A., Hoelzel, R.A. 2005. Habitat structure and the dispersal of male and female bottlenose dolphins (Tursiops truncates) in Proc.Biol. Sci June 22; 272 (1569): 1217-1226. Naciri, M., Lemaire, C., Borsa, P., Bonhomme, F. 1999. Genetic study of the Atlantic/Mediterranean transition of sea buass (Dicentrarchus labrax). J. Hered. 90 591-596. Nishida, T., Tsuji, S., Segawa, K. 1998. Spatial data analyses of Atlantic bluefin tuna larval surveys in the 1994 ICCAT BYP. Collect. Vol. Sci. Pap. ICCAT, 48(1): 107-110. 166

Oray, I.K., Karakulak, F.S., Alichi, Z., Ates, C., Kahraman, A. 2005. First evidence of spawning in the eastern Mediterranean Sea preliminary results of tuna larval survey in 2004. SCRS/2004/144 Col. Vol. Sci. Pap. ICCAT, 58(4): 1341-1347. Pujolar, J.M., Roldan, M.I., Pla, C. 2003. Genetic analysis of tuna populations, Thunnus thynnus thynnus and T. alalunga. Marine Biology 143, 613-621. Roldan, M.I., Garcia-Marin, J.L., Utter, F., Pla, C. 1998. Population genetic structure of European hake, Merluccius merluccius. Heredity. 81:327-334. Sara, R. 1998. Dal Mito all Aliscafo: Storie di tonni e di tonnare, Raimondo Sara, Piazzale De Gasperi, 18-90146 Palermo Italy. Schlitzer, R. 2008. Ocean Data View, http://odv.awi.de Takagi, M., Okamura, T., Chow, S., Taniguchi, N. 1999. PCR primers for microsatellite loci in tuna species of the genus Thunnus and its application for population genetic study. Fisheries Science, 65(4), 571-576. Takeyama, H., Chow, S., Tsuzukim H., Matsunaga T. 2001. Mitochondrial DNA sequence variation within and between Thunnus tuna species and its application to species identification. JFish Biol, 58:1646-1657. Vinas, J., Pla, C., Tawil, N.Y., Hattour, A., Farrugia, A, de la Serna, J.M. 2003. Mitochondrial genetic characterization of bluefin tuna (Thunnus thynnus) from three Mediterranean (Libya, Malta, Tunisia) and one Atlantic locations (Gulf of Cadiz). Collect. Vol. Sci. Pap. ICCAT, 55(3): 1282-1288. Vella, A. 2002. Bluefin tuna fisheries in Central/Southern Mediterranean, around the Maltese Islands, in Proceedings of the International Conference on Mediterranean Fisheries - Naples 2002. Vella, A. 2006. Blue fin tuna conservation in the Mediterranean Sea: ecology and genetic assessment in Central Mediterranean Region. Oral Presentation of Paper at the 1 st European Conservation Biology Society Conference (Hungary - August 2006). (In press). 167

Table 1. Bluefin tuna microsatellite analyses results. Microsatellites Regional samples south of the Maltese Islands location A location B location C (n = 42) (n = 112) (n = 44) Tth01 number of alleles 5 5 5 number of private alleles 1 1 0 allele richness 4.419 3.928 3.894 Ho 0.552 0.507 0.368 He 0.707 0.642 0.537 Tth04 alleles 9 12 10 number of private alleles 0 2 1 allele richness 8.004 9.056 8.098 Ho 0.846 0.829 0.925 He 0.830 0.836 0.830 Tth06 alleles 10 11 8 number of private alleles 2 2 2 allele richness 10.000 9.678 7.740 Ho 0.529 0.630 0.619* He 0.882 0.864 0.822 Tth07 alleles 11 13 11 number of private alleles 2 2 0 allele richness 9.552 10.200 9.969 Ho 0.750 0.754 0.694* He 0.834 0.829 0.886 *Indicates deviation from HWE after Bonferroni correction P<0.0041. Table 2. F ST values for the bluefin tuna microsatellite study between locations are shown below the diagonal, P values are shown above diagonal. A significant F ST value obtained between individuals sampled in location A (West South-West of the Maltese Islands) and location C (South South-East of the Maltese Islands) may be noted. On removing the two loci/locations which deviate from the HWE the F ST between the bluefin tuna samples from the two locations A and C would go up to 0.025 with a significant p value of 0.012. LocationA LocationB LocationC LocationA - 0.802 0.015 LocationB -0.007-0.988 LocationC 0.018-0.021-168

50000 Annual bluefin tuna landings in Kgs 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 1957 1959 1961 1963 1965 1967 1969 1972 1974 1976 1978 1980 1982 1984 1986 Years Figure 1. Bar graph showing the annual landings of bluefin tuna between 1957 and 1986 in Malta. 400000 Annual bluefin tuna landings in Kgs 350000 300000 250000 200000 150000 100000 50000 0 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 Figure 2. Bar graph showing the annual landings of bluefin tuna between 1987 and 2006 in Malta. 1998 Years 1999 1999 2000 2001 2002 2003 2004 2005 2006 169

Dec 7% Jan 9% Nov 10% Feb 8% Oct 5% Mar 4% Apr 11% Sep 21% May 11% Aug 5% Jul 2% Jun 7% Figure 3. Pie chart showing the average % bluefin tuna landings per month between 1957 and 1964 in Malta. Nov 7% Dec 4% Jan 3% Feb 5% Mar 1% Apr 0% Oct 13% May 11% Jun 10% Jul 10% Sep 36% Aug 0% Figure 4. Pie chart showing the average % bluefin tuna landings per month between 1965 and 1974. 170

Aug 2% Sep 2% Jul 25% May 27% Jun 44% Figure 5. Pie chart showing the average % bluefin tuna landings per month between 1975 and 1985 in Malta. Jul 18% May 38% Jun 44% Figure 6. Pie chart showing the average % bluefin tuna landings per month between 1986 and 1999 in Malta. Jul 9% Apr 1% Jun 35% May 55% Figure 7. Pie chart showing the average % bluefin tuna landings per month between 2000 and 2006 in Malta. 171

% Males (dark grey) & % Females (light grey) 80 70 60 50 40 30 20 10 0 1999 2000 2001 2002 2003 2004 Years sampled Figure 8. % Male and female bluefin tuna per sample between 1999 and 2004 in Malta. 2.90 Average Fork length (m) 2.70 2.50 2.30 2.10 1.90 1.70 1.50 1998 1999 2000 2001 2002 2003 2004 2005 Years samples Figure 9. Bluefin tuna fork length in study samples between 1999 and 2004. 3.50 3.00 Fork Lenth (m) 2.50 2.00 1.50 1.00 0.50 0.00 y = 0.6256Ln(x) - 0.8829 R 2 = 0.8653 0 100 200 300 400 Body Weight (kgs) Figure 10. Bluefin tuna body length versus body weight in study sample between 1999 and 2004. 172

Map 1. Locations of all bluefin tuna samples collected for this study indicating the sub-divisions selected for the regional groupings in the microsatellite analyses (Ocean Data view software by Schlitzer 2008). 173