Determination of Atlantic bluefin tuna (Thunnus thynnus) spawning time within a transport cage in the western Mediterranean

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1 2205 Determination of Atlantic bluefin tuna (Thunnus thynnus) spawning time within a transport cage in the western Mediterranean Ana Gordoa, Maria Pilar Olivar, Raquel Arevalo, Jordi Viñas, Balbina Molí, and Xenia Illas Gordoa, A., Olivar, M. P., Arevalo, R., Viñas, J., Molí, B., and Illas, X Determination of Atlantic bluefin tuna (Thunnus thynnus) spawning time within a transport cage in the western Mediterranean. ICES Journal of Marine Science, 66: For the first time, tuna spawning in a transport cage being towed from the western Mediterranean spawning ground to a fattening facility off the coast of northeastern Spain was examined during the 2008 fishing season. Daylight and night surface plankton samples were collected using bongo nets located in front of and behind the transport cage. The results for the different time intervals revealed clear and massive nocturnal spawning from 03:00 to 05:00, when the rear bongo was completely jammed with eggs (up to eggs per 1000 m 3 ). Egg size and morphology were consistent with the features of Thunnus thynnus eggs, and identification was confirmed by genetic analysis. Microscopic examination showed the eggs to be in the very early developmental stages. Spawning took place every night over the entire journey. The study showed that neither captivity nor handling/environmental changes along the route inhibited T. thynnus spawning to a very precise biological clock. The study also revealed the diel temporal concurrence of T. thynnus spawning and jellyfish larvae at the sea surface. Keywords: Atlantic bluefin tuna, eggs, jellyfish larvae, Mediterranean, spawning period, Thunnus thynnus. Received 18 March 2009; accepted 26 June 2009; advance access publication 11 August A. Gordoa, R. Arevalo, and X. Illas: Centro de Estudios Avanzados de Blanes (CSIC), Acc. Cala Sant. Francesc 14, Blanes, Girona, Spain. M. P. Olivar and B. Molí: Institut de Ciències del Mar (CSIC), Passeig Marítim de la Barceloneta 37-49, Barcelona, Spain. J. Viñas: Laboratori d Ictilogia Genètica, Departatment de Biologia, Universitat de Girona, Girona, Spain. Correspondence to A. Gordoa: tel: þ ; fax: þ ; gordoa@ceab.csic.es. Introduction The Atlantic northern bluefin tuna (Thunnus thynnus) has, historically, been targeted by different fisheries and countries along its migratory routes from feeding to spawning grounds. In the Mediterranean Sea, tuna fishing commenced around 7000 BC (Desse and Desse-Berset, 1994). Since the 16th century, the early fishing modalities of handlines and beach-seines have been replaced gradually by traps (Ravier and Fromentin, 2001), and new tuna fisheries and fishing grounds developed during the 20th century. However, the new situation that began with the development of the Japanese sushi sashimi market in the 1980s, with the resulting development and growth of fisheries and fishing areas, resulted in overfishing on both western and eastern Atlantic stocks (Fromentin and Powers, 2005). Atlantic bluefin tuna (ABFT hereafter) inhabit the entire North Atlantic and Mediterranean and are managed as two separate stocks: a West Atlantic stock, and an East Atlantic and Mediterranean stock. The two-stock premise is supported by differences observed between the stocks in age- and size-at-first-maturity (Mather and Shuck, 1960; Mather et al., 1995; Diaz and Turner, 2007) and growth (Cort, 1991; Turner and Restrepo, 1994), and in the separate spawning grounds (Block et al., 2005; Carlsson et al., 2007). Concurrently, tagging with data-storage tags has improved our understanding of ABFT dynamics and revealed high rates of transatlantic migration and spatial overlap (Block et al., 2001), calling into question the suitability of present management boundaries and units (Rooker et al., 2007). The weak condition of the eastern Mediterranean ABFT stock has been highlighted in the latest reports of the ICCAT Standing Committee on Research and Statistics (ICCAT, 2009), which also emphasized data limitations and high levels of underreporting for the eastern stock, especially in the Mediterranean. The last Committee report found difficulty in drawing any clear conclusion from fisheries indicators in the absence of precise information on the catch composition, effort, and spatial distribution of the purseseine fisheries (which produce.60% of the recent total reported catch; ICCAT, 2009). Recent growth in the number of tuna fattening farms in the central and eastern Mediterranean has displaced purse-seine fleets east to an unknown extent. Although the tuna population in the Mediterranean basin as a whole is considered part of the eastern Atlantic stock, the existence of a local or resident eastern Mediterranean subpopulation has been postulated, and recent findings appear to lend credence to this premise (Block et al., 2005; Demetrio et al., 2005). The reproductive biology of bluefin tuna in the Mediterranean is relatively well understood thanks to the extensive research already carried out on the species. Differences in spawning times among Mediterranean regions are known: June July in the western Mediterranean (Sará, 1973; Duclerc et al., 1974; de la Serna and Alot, 1992; Susca et al., 2001; Medina et al., 2002; # 2009 International Council for the Exploration of the Sea. Published by Oxford Journals. All rights reserved. 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2 2206 A. Gordoa et al. Corriero et al., 2003), and 1 month earlier, May June, in the Levantine Sea. These differences have been attributed to temporal differences in water warming between regions (Karakulak et al., 2004; Oray and Karakulak, 2005). Spawning areas in the Mediterranean are well identified (Duclerc et al., 1973; Nishida et al., 1998; García et al., 2003; Karakulak et al., 2004), as are the size- and age-at-sexual-maturity and the reproductive biology (Rodríguez-Roda, 1964; Abascal et al., 2004; Corriero et al., 2005). However, issues such as spawning time and reproductive behaviour are still unknown for ABFT, though for Pacific bluefin tuna (PBFT; Thunnus orientalis), the results of research on cage culture and aquaculture have provided much information on spawning behaviour (Masuma et al., 1993, 2003, 2006). Natural spawning in net cages was first confirmed three decades ago (Harada et al., 1979), and completion of the PBFT life cycle in captivity by spontaneous spawning was achieved in 2002 (Sawada et al., 2005). Histological studies on ABFT in Mediterranean farming cages revealed vitellogenesis and spermatogenesis in both treated and untreated animals, although final maturation was only observed in females induced by hormone treatment (Corriero et al., 2007). Moreover, neither mating nor eggs were observed in cages (Fauvel et al., 2005), and improved handling to reduce mortality and stress was deemed to be necessary for controlled bluefin tuna reproduction. The extreme sensitivity of tuna to stress is presumed based on high rates of mortality after handling and abrupt environmental variation in the cages (Fauvel and Suquet, 2004). This sensitivity has been suggested to result in the cessation of reproductive activity (Corriero et al., 2007). In the Mediterranean, most purseseine catches are transported in towing cages to sea-cage facilities for fattening. The tuna are exposed to the stress associated with different aspects of handling: fishing and transfer from purseseines to transport cages, and the stress related to transport from the fishing grounds to farming facilities. The objective of this study was to examine, for the first time, whether tuna spawning takes place, and if so, when, during the transport from the spawning areas to fattening farms off northeastern Spain. This information is crucial to the optimum development of ABFT aquaculture. Material and methods Tuna caught by two purse-seiners on the spawning grounds around the Balearic Islands in June 2008 were transferred to a transport cage. Waiting time in the transport cage from the first to the last transfer was 3 d. Cage dimensions were 50 m diameter by 30 m depth. Sampling began when transport started in the Ibiza channel on 27 June and ended on 5 July in the proximity of the farming facility (Figure 1). Towing speed during transport was a steady 0.7 knots, typical of transport cages of this kind, to avoid damaging the tuna. Surface plankton samples were collected by bongo gear fitted with nets of mesh size 0.3 mm. Two bongos were used, one in front and one behind the cage, the front bongo as a reference for egg quantities in the field, the rear one to determine the egg production coming from the cage. Distance between the vessel and the cage was 180 m. The front bongo was hung from a wire 100 m long attached to the vessel s stern, and the rear one from a wire attached to the rear of the cage. Both bongos were mounted at a depth of 3 m. Duration of all hauls was 45 min, and vessel speed during sampling was 0.7 knots. Upon retrieval of the gear, plankton samples were immediately preserved in 5% buffered formalin. Samples were collected by both day and night, although sampling was intensified at night on the advice of the fishers, whose experience told them that ABFT spawn at night. Sampling consisted of two hauls in daylight, starting between 12:00 and 16:00, and a minimum of five hauls at night, starting between 22:00 and 04:00. Transport to the fattening facility took 9 d. Plankton samples were collected whenever weather conditions allowed. In all, 47 stations were sampled, 38 in the area where the fish were caged (water depth 1000 m) and during transportation ( m deep), and nine on the continental shelf near the farm ( m deep). Many of the plankton samples collected in offshore waters could not be preserved, however, because the nets were completely clogged with jellyfish larvae. In each sample, the eggs were sorted into three groups by egg diameter: 0.9 mm,.0.9 and 1.1 mm, and.1.1 mm. The intermediate size interval is thought to correspond to the egg size for T. thynnus (Sanzo, 1932; Dicenta, 1975; Richards, 2006). The numbers of eggs in the intermediate size interval present in each sample were counted, except for saturated samples, for which five subsamples each of 3 ml were taken to estimate total abundance. ABFT species validation was carried out for 15 eggs from the intermediate size class by comparing the sequence of the mitochondrial DNA (mtdna) control region with previously published sequences for the eight species of the genus Thunnus (Alvarado Bremer et al., 1997, 2005). Methods of DNA extraction, amplification of the mtdna control region, and sequencing of the amplicon followed the protocol described by Viñas et al. (2004). DNA extraction volumes were modified to adjust the protocol to single eggs. Results Night samples were dominated by jellyfish larvae, as proved by their regular presence in all the front bongo samples. Jellyfish larvae occurrence increased from midday to night, peaking at around 01:00 02:00 (Figure 2), when 100% of the samples taken by both bongos were clogged with jellyfish larvae. These massive, nocturnal surface proliferations of jellyfish larvae were found every day over the entire route, both offshore and over the continental shelf. By day, 10 stations were sampled, but because of the accidental loss of a front bongo, just nine daylight front samples were available for analysis. At the 37 stations sampled at night, 13 rear and 6 front samples were deemed successful; in all other cases, the bongo was jammed with jellyfish larvae, except in one case where the front bongo was broken. At the nine stations sampled during the period 03:00 05:00, the rear bongo was completely filled with eggs in all but one case (in which the bongo was jammed with jellyfish larvae), whereas the front bongo contained just a few eggs on one occasion (in the samples other than the single one when the net was broken, it was jammed with jellyfish larvae). Interestingly, no other plankton component appeared in the night samples filled with eggs. The eggs collected matched the features of T. thynnus eggs in Balearic waters (Dicenta, 1975) in terms of their size, oil-globule diameter, and the narrow perivitelline space and homogeneous yolk. The DNA sequences obtained from individual eggs were compared with the published sequences of the eight Thunnus species (Alvarado Bremer et al., 1997, 2005). Always the eggs were positively identified as ABFT.

3 Atlantic bluefin tuna spawning determined in a transport cage 2207 Figure 1. Map of the study area and estimated egg densities along the sampling transect. Stations labelled with a j denote samples clogged with jellyfish larvae, those with a b a broken bongo net, open triangle the locations of farming facilities, and closed triangle the starting locations. Figure 2. Percentage of samples from the front and rear bongos clogged with jellyfish larvae by period. The estimated T. thynnus egg densities in the hauls performed during the period 03:00 05:00 ( eggs per 1000 m 3 ) were generally several orders of magnitude higher than those collected Figure 3. Estimated egg density (log scale) from the front and rear bongos at each station by time interval. in the rest of the samples. These results are shown in Figure 3, but note the logarithmic scale on the y-axis. In our opinion, estimates of egg density derived from these saturated samples were

4 2208 A. Gordoa et al. likely underestimates of the real concentrations in the sea because of the deteriorating filtering efficiency of the nets as they filled with eggs. Two stations sampled by day also showed sizeable egg densities in the rear bongo ( eggs per 1000 m 3 ) compared with the densities in the front bongo (100 eggs per 1000 m 3 ). The remaining valid hauls from the rear bongo had egg densities similar to those collected in the front bongo. Egg densities (with the same morphological and meristic features) from the valid front bongo samples ranged from 6 to eggs per 1000 m 3, and no day/night abundance pattern could be established from them. The estimated densities of tuna eggs collected during the nighttime hauls stayed high and did not decline throughout the transport to the farm. The results therefore suggest that spawning took place every night over the entire journey (Figure 1). Microscopic examination showed the eggs to be in the very early developmental stages, most showing only 2, 4, or 8 cleavages (Figure 4), indicative of recent fertilization. Discussion The most relevant findings relating to the aim of this study were observed between 03:00 and 05:00. Strikingly then, when the rear bongo was completely filled with a thick broth of eggs, the front bongo was clogged with jellyfish larvae, as it was during the immediately preceding hours of darkness. The difference in the results between the front and rear bongos, saturated with jellyfish larvae and eggs, respectively, suggests that some process responsible for jellyfish dispersal away from the cage was operating when the tuna spawned. Such dispersal could be caused by the hydrodynamic effects generated by the behaviour of the spawners. The conspicuous difference between the front and rear catch components indicated that the eggs came from the cage and were the product of spawning of the tuna in the cage. Another clear difference between rear and front concentrations was observed at midday, and although it was not massive and only at two stations, we believe that it indicated modest, erratic spawning by day. Egg concentrations in the different time intervals revealed a clear pattern of nocturnal spawning from 03:00 to 05:00. Nocturnal spawning appears to be common in other species of tuna, but this is the first study to have established a preferred spawning period for T. thynnus in the eastern Atlantic. Thunnus albacares spawn between 22:00 and 06:00 (Schaefer, 1998), T. obesus from 19:00 to 24:00 (Nikaido et al., 1991), and T. orientalis Figure 4. A Thunnus thynnus egg with eight cells collected from an egg-saturated sample. from 19:00 to 23:00 (Schaefer, 2001). The difference between the spawning periods of T. thynnus and those of other tuna species is its shorter duration (2 h), and although nocturnal as in the other tuna species, it seems to take place shortly before sunrise. In the Mediterranean, a recent study of the reproduction of ABFT in floating cages involved hormone-induced maturation, and although fertilized eggs were collected from the cages, spawning itself was not observed (Mylonas et al., 2007). However, the spawning period recorded in our study could easily explain why spawning may have been missed in earlier investigations, unless there was a monitoring protocol in place between 03:00 and 05:00. The results also revealed spawning every night throughout the journey, first at station 5 and finally at the last station near the fattening facility. The average time between spawning episodes of ABFT has been estimated at 1.2 d (Medina et al., 2002). Similar spawning intervals have been reported for mature and reproductively active female T. maccoyii (1.62 d), T. obesus (1.09 d), and T. albacares ( d; McPherson 1991; Nikaido et al., 1991; Farley and Davis, 1998; Schaefer, 1998). Our observation of many eggs being in the first cleavage stages confirms that the eggs had been fertilized and also signalled the fast cell division displayed by the species, considering that the maximum time between spawning and sampling was,1h. According to Miyashita et al. (2001), egg incubation time for ABFT at 248C is ca. 32 h, and the eggs of other species such as T. albacares (Harada et al., 1980, in Fishbase) also exhibited fast embryonic development. For Auxis rochei, two- and four-cell stages were also observed in the early hours after fertilization (Inoue et al., 1974). Although the eggs collected from the cage had been fertilized, their further development and the subsequent survival of the larvae in the area around the transport route is uncertain, because the environment is different from and may not meet the presumably optimal conditions of the natural spawning grounds around the Balearic Islands (García et al., 2006). Nevertheless, three decades ago, the area between the Balearic Islands and the Iberian Peninsula was identified as a spawning ground, although a minor one (Dicenta, 1977), as was broad larval distribution around the three major Mediterranean spawning grounds (Levantine Sea, central and western Mediterranean; Nishida et al., 1998; García et al., 2005). The tuna observed spawning in captivity during transport passed through areas of differing oceanographic environments. Moreover, they had been subjected to handling: catching, transfer, and waiting for the cage to be filled. Our results showed that neither the different aspects of handling nor environmental changes inhibited T. thynnus spawning to a very precise biological clock. Despite the potential stress factors, the spawners studied proved to be highly resilient, which opens the prospect of using fattening cages as experimental platforms for aquaculture, as has been the case for PBFT without artificial induction (Sawada et al., 2005). To date, PBFT have exhibited a more highly developed immune system than other marine teleosts, so permitting aquaculture without medical treatment (Watts et al., 2003). The daily pattern in the percentage of samples lost to jellyfish clogging was indicative of diel vertical migration by jellyfish, as already reported for different jellyfish taxa (Kaartvedt et al., 2007). This pattern was observed over the entire route, both offshore and inshore, indicative of the massive and widespread presence of jellyfish larvae in the plankton. Moreover, the eggs in several samples were observed inside jellyfish larvae, and although this could be an accidental outcome of the handling, the possibility

5 Atlantic bluefin tuna spawning determined in a transport cage 2209 that eggs may actually have been ingested by jellyfish larvae cannot be dismissed. Our findings contribute new information on the reproductive biology of ABFT relevant to management of the purse-seine fisheries and aquaculture development. Implementation of a management plan to minimize the impact of the purse-seine fisheries on spawning would appear to be a desirable goal, and as a consequence, prolonging the waiting time in the spawning area until the captive fish have finished spawning could be a sensible measure to be considered. Acknowledgements The study was designed and carried out in the framework of a cooperative project between the Centre for Advanced Studies at Blanes (CEAB) of the Spanish National Research Council (CSIC) and Balfegó Tuna S.L. 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