650 



Validation of age estimates from otoliths 

 of larval and juvenile spotted seatrout, 

 Cynoscion nebulosus 



Allyn B. Powell 



Elisabeth H. Laban 



Center for Coastal Fisheries and Habitat Research 



National Ocean Service 



National Oceanic and Atmospheric Administration 



101 Pivers Island Road 



Beaufort, North Carolina 28516-9722 



E-mail address (lor A B Powell) allyn powellanoaagov 



Scott A. Holt 



G. Joan Holt 



University of Texas Marine Science Institute 

 750 Channelview Drive 

 Port Aransas, Texas 78373 



Otolith microstructure analysis has 

 been shown to be valuable for relating 

 biotic and abiotic factors to growth and 

 survival (Crecco and Savov, 1985; Thor- 

 rold and WiUiams, 1989; Maillet and 

 Checkley, 1991; Jenkins et al., 1993) 

 and determining size-specific mortality 

 (Gleason and Bengtson, 1996; Sabo and 

 Orth. 1996; Hare and Cowen, 1997) for 

 the early life history stages of fishes. 

 Prior to using otolith microstructure 

 analysis, it is important that otolith 

 validation studies be undertaken to 

 examine the rate of increment forma- 

 tion and the timing of initial increment 

 formation, and to check the interpre- 

 tive skills of the otolith reader (Geffen, 

 1992). 



Our validation study dealt with 

 spotted seatrout, Cynoscion nebulo- 

 sus, an economically important sciae- 

 nid. Otolith increment deposition rates 

 from spotted seatrout have been vali- 

 idated by using tetracycline marking 

 of wild-caught fish larvae and juve- 

 niles collected in Tampa Bay, Florida 

 (McMichael and Peters, 1989). Because 

 known-age larvae were not used, the 

 timing of initial increment formation 

 was not determined. The purpose of 

 our study was to determine the rate 

 of increment formation and timing of 

 initial increment formation as well as 

 to provide confidence to otolith read- 



ers by using known-age larvae and 

 wild-caught juveniles. Our study is 

 ancillary to spotted seatrout early life 

 history studies in Florida Bay, Ever- 

 glades National Park. 



Materials and methods 



Spotted seatrout eggs were obtained 

 from the Texas Parks and Wildlife 

 GCCA/CPL Marine Development Cen- 

 ter hatchery from adults collected from 

 upper Laguna Madre, Texas. Eggs were 

 transported to the University of Texas 

 Marine Science Institute ( UTMSI ), Port 

 Aransas, Texas, in May 1997. Eggs were 

 incubated in 600-L tanks at egg densi- 

 ties of 100 eggs/L. Tanks were held in 

 greenhouses, illuminated with natural 

 sunlight and 40-W overhead floures- 

 cent lights at a 24-h (15L:9Di photo- 

 period. A closed system was used, but 

 water was continuously recirculated 

 through a biofilter During the rearing 

 process, ambient temperatures ranged 

 from 25.0 to 28.0°C; salinities, from 32 

 to 33 ppt. Larvae to age 12 d were fed 

 rotifers (5/mL), which in turn were pro- 

 vided the algae Isochrysis. At age 13 

 d, brine shrimp nauplii, enriched with 

 oil emulsion (Leger et al., 1986), were 

 introduced (4 rotifer.s/mL; 2 nauplii/niL 

 on a daily basis I, and from age 15 d 



until the end of the experiment, 2-3 

 nauplii/niL were maintained on a daily 

 basis. Food density was estimated twice 

 a day and food added to the required 

 density. Frozen adult brine shrimp and 

 red drum, Sciaenops ocellatus. eggs 

 were added on a daily basis from age 23 

 d to the end of the experiment. Otoliths 

 of reared larvae were marked with aliz- 

 arin complexone (ALC) at concentra- 

 tions of 50 mg/L for four hours at age 

 9 and 16 d. Fifty larvae were sacrificed 

 at ages 6, 9, 14, 20, 23, and 32 d and 

 preserved in 959f ethyl alcohol. Larvae 

 sacrificed at ages 6 and 9 d were not 

 immersed in ALC. 



Wild-caught juveniles were collected 

 in Aransas Bay, Texas, in August 1997 

 with a 6-m bag seine (4.5-mm mesh 

 in both the wings and the bag). Juve- 

 niles were placed in 45-L tanks and 

 transported to the rearing facility at 

 UTMSI, Port Aransas, Texas. Juveniles 

 were held in 3-m raceways in 1100-L 

 water at temperatures 27.0-28.0°C and 

 a salinity of 36 ppt. Juveniles were 

 segregated by size to avoid cannibal- 

 ism. Two days after capture, otoliths 

 were marked with ALC ( 100 mg/L for 

 4 h). Otoliths were similarly remarked 

 7 d later (9 d after capture). Mortality 

 associated with the marking process 

 was nil. All fish were sacrificed and 

 preserved in 957r ethyl alcohol at 21 d 

 after capture. 



Otoliths were removed with probes 

 and fine-tipped forceps. Polarized light 

 was used to facilitate location of oto- 

 liths from fish <6 mm standard length 

 (SL). All otoliths, except for the right 

 sagitta, were placed on a slide, covered 

 with mounting media and archived. 

 The right sagittal otolith was embed- 

 ded for transverse sectioning or polish- 

 ing (or for both procedures). The left 

 sagitta was embedded for transverse 

 sectioning if the right was damaged 

 during preparation. 



Sagittae were read with a light micro- 

 scope at lOOOx magnification under 

 oil immersion with blue light epiflu- 

 orescence to detect ALC marks that 

 fluoresce as reddish orange. The first 

 increment was determined as that fol- 

 lowing the core increment, the latter a 

 well-defined dark increment surround- 

 ing the core. Sagittae were read by one 



Manuscript accepted 29 December 1999. 

 Fish. Bull. 98:650-654 (2000). 



