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251 



We fed the broodstock a controlled diet of market 

 squid (Loligo opalescens), Argentine shortfin squid illlex 

 argentinus). Pacific thread herring iOpisthonema spp.). 

 Pacific anchoveta {CetengrauUs mysticetus), and bigscale 

 anchovy (Anchovia macrolepidota). The daily ration was 

 usually divided into 50% squid and 50% fish. 



Nonlinear least-squares procedures were used to ob- 

 tain growth parameters for the yellowfin broodstock 

 {Tomlinson-*) in order to estimate growth rates and 

 sizes at age and length (Wexler et al., 2003). 



Spawning 



During October 1996, the fish in the main broodstock 

 tank began to exhibit courtship behavior in the late 

 afternoons (see subsection "Spawning behavior"). We 

 began monitoring the broodstock tank for egg produc- 

 tion during this time. On 8 October 1996, we collected 

 fertilized eggs in the tank for the first time. The fish 

 spawned sporadically throughout October and Novem- 

 ber 1996, and by December 1996 were spawning daily. 

 Using the estimated ages of wild yellowfin tuna in 

 the eastern Pacific Ocean (Wild, 1986) and applying 

 them to the lengths and weights of our broodstock fish 

 (Wexler et al., 2003), we estimated the average age 

 of the broodstock fish at the time of first spawning. 

 During 1999-2000, we estimated the spawning peri- 

 odicity and age at first-spawning of individual females. 

 We compared the mitochondrial DNA genotypes of the 

 female broodstock with those of their offspring (eggs 

 and yolksac larvae) on a weekly basis from August 1999 

 through August 2000. Mitochondrial DNA analysis was 

 conducted by using polymerase chain reaction/restric- 

 tion fragment length polymorphism (PCR/RFLP) meth- 

 ods. The genetic analysis is described in greater detail 

 in Niwa et al. (2003). 



To determine the time of spawning each day, we 

 checked approximately every 15 minutes for fertilized 

 eggs at the surface of the tank with a hand-held dipnet. 

 usually beginning in mid- afternoon because the fish 

 most often spawned during the late afternoon or eve- 

 ning. Yellowfin tuna eggs, when fertilized, are positively 

 buoyant and rise to the surface in a tank. The time at 

 which eggs were first caught in the dipnet was noted as 

 the "time of spawning." This is a conservative estimate 

 because the fish spawned at different depths and loca- 

 tions in the tank, and we always sampled for eggs at 

 the same location in the tank. We recorded the tempera- 

 ture of the broodstock tank at the time of spawning. 



Although we did not anticipate spawning to occur in 

 the reserve tank, the yellowfin tuna began spawning 

 after 7 to 8 months in captivity in mid-April 1997. After 

 the initial spawning in the reserve tank, we monitored 

 the tank daily for fertilized eggs. In October 1997, we 

 collected eggs spawned by the one remaining pair of 



■* Tomlinson, P. 2001. Personal comniun. Inter-American 

 Tropical Tuna Commission. 8604 La Jolla Shores Drive, La 

 JoUa, CA 92037. 



fish to study Mendelian inheritance of nuclear DNA 

 variants (Chow et al., 2001). We sacrificed the breeding 

 pair after six spawning events and took samples of their 

 muscle tissue for genetic analysis. 



Spawning behavior 



When the fish spawned before sunset, we made visual 

 observations to describe the courtship and spawning 

 behavior. In addition, we used an underwater video 

 camera connected to a surface video recorder to tape the 

 spawning behavior. On several occasions, we positioned 

 the camera at a depth of 1 to 4 m in different locations 

 and at different angles in the main broodstock tank. The 

 camera recorded continuously for approximately 1 h. We 

 viewed the video tapes and chose footage that showed 

 both courtship and spawning behaviors for behavioral 

 analysis. 



Egg collection 



In early 1997, we constructed an egg-collection system 

 with three PVC pipes, placed at different depths between 

 the water surface and 70 cm below the surface, so that 

 eggs were siphoned into a square egg-collection basket 

 (1 mxl m) made of porous fabric (mesh size 200 jim). We 

 also collected eggs with dipnets and with an egg seine 

 that sampled the entire surface of the tank. In early 

 1998, we attached a stationary drift net (trapezoidal 

 opening 70 cm height x 20 cm [top width] and 40 cm 

 [bottom width] x 1.6 m long) to one of the siphons to fur- 

 ther standardize the egg collections. We considered the 

 siphon system (1997) and the drift net-i-siphon system 

 (1998-2000) as equivalent sampling systems because 

 the same area in the water column (70 cmx20 cm) was 

 sampled, siphoned into the same egg collection basket ( 1 

 mxl m), and sampled daily for the same period. 



We collected and counted eggs from each spawning 

 event approximately two hours after the estimated time 

 of spawning. We washed the eggs from the collection 

 basket into a 2G-L container. We then set the egg col- 

 lection basket back into place until the next morning, 

 when we made a second, supplementary collection. We 

 included the second collections in the daily estimates of 

 egg numbers, but these eggs were not used in develop- 

 mental or experimental analyses. 



To determine the number of eggs collected, we brought 

 the egg collection container to 10 L of water volume and 

 lightly agitated the mixture until the eggs were well 

 distributed. We took three 5-mL samples with a wide- 

 mouthed pipette and placed them in three separate, 

 glass counting dishes. We counted the individual eggs in 

 each of the three dishes under a dissecting microscope, 

 calculated the mean, and estimated by extrapolation the 

 total number of eggs in the container. Standardized egg 

 production in the main tank was calculated daily as the 

 number of eggs collected divided by the total biomass 

 of females assumed to be spawning (all those >20 kg) 

 in the tank. 



