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Fishery Bulletin 92(2). 1994 



rificed and preserved in 5-8% buffered formalin. Iden- 

 tifications of preserved sciaenid larvae from pigment 

 characters were based on Ditty (1989). 



Sciaenid eggs collected in the same area during 

 spring 1991, 1992, and 1993 were sorted from fresh 

 plankton samples. To avoid contamination by the 

 morphologically similar eggs of the cynoglossid 

 Symphurus plagiusa and the soleid Trinectes macula- 

 tus that contain several oil globules and are abundant 

 in lower Chesapeake Bay during the spring, all eggs 

 with >3 oil globules were omitted from the samples. 

 Although eggs of most spring-spawning sciaenids gen- 

 erally possess three or fewer oil globules (usually two) 

 those of Menticirrhus saxatilis may contain from 1 

 to 16 oil globules (Johnson, 1978). After sorting, eggs 

 were measured, placed in scintillation vials with 26 

 ppt seawater, and frozen at -70°C for genetic analy- 

 sis. Individual eggs were thawed and remeasured prior 

 to homogenization to assess shrinkage. 



Sciaenid eggs were genetically typed by compar- 

 ing mtDNA restriction fragment patterns of indi- 

 vidual eggs with those of known adults. To obtain 

 patterns of known adults, mature female sciaenids 

 (B.chrysoura, C. nebulosus, C. regalis, M. saxatilis, 

 and P. cromis) were collected by pound net, trawls, 

 and hook and line in April and May 1990 and 1991. 

 Ovarian tissue was excised and frozen at -70°C. 

 MtDNA was purified from ovarian tissue by cesium 

 chloride equilibrium density gradient ultracentrifu- 

 gation following the protocols of Lansman et al. 

 (1981). To determine a restriction enzyme that un- 

 ambiguously identified the different sciaenid spe- 

 cies, aliquots of mtDNA were individually digested 

 with the following restriction enzymes: Apal, Aval, 

 Banl, Banll, Hindlll used according to manu- 

 facturer's instructions. The resulting fragments 

 were separated electrophoretically on 1.0% agarose 

 mini-gels run at 5 V/cm for four hours and visual- 

 ized with ethidium bromide. 



MtDNA-enriched genomic DNA was isolated from 

 individual eggs following the protocols of Graves et 

 al. (1990). Entire DNA samples were digested with 

 a single discriminating restriction endonuclease, 

 separated electrophoretically, and transferred to a 

 nylon filter (Southern transfer) following standard 

 protocols (Sambrook et al., 1989). Filters were hy- 

 bridized with highly purified black drum mtDNA, 

 nick-translated with biotin-7-dATP, washed, blocked 

 and visualized following the methods of Graves et al. 

 (1990). 



Results 



A total of 10,803 sciaenid eggs was sorted from 

 samples collected in 1990 and 1991. Outside egg 



diameter of all specimens ranged from 0.650 to 1.12 

 mm. Successive blind readings of samples of 75 to 

 100 eggs were used to assess measurement error. No 

 differences were found in the size-frequency distri- 

 butions indicating good agreement within the 0.025- 

 mm size classes (two-sample <-test, P<0.05, n=79). 



Qualitative analysis of culture experiments using 

 the two egg types of Joseph et al. (1964) revealed 

 the presence of three species. Cultures containing 

 eggs designated Type I (<0.80 mm) resulted in lar- 

 vae of B. chrysoura, whereas cultures of eggs desig- 

 nated Type II (>0.85 mm) resulted in larvae of C. 

 regalis and P. cromis. 



Analysis of preserved ichthyoplankton samples 

 from 1990 and 1991 revealed the presence of larvae 

 of B. chrysoura, C. regalis, and P. cromis. No early 

 life history stages of other sciaenids were identified; 

 however, yolk-sac larvae could not be identified to 

 species. Because rearing studies and analysis of 

 field-caught plankton samples revealed the presence 

 of more than two species, we could not rely on the 

 criteria of Joseph et al. (1964) for specific identifi- 

 cation. We therefore examined weekly frequency of 

 occurrence of all sciaenid eggs during 1990 (Fig. 1) 

 and 1991 (Fig. 2). Based on temporal occurrence and 

 size frequency we identified three modes. The larg- 

 est eggs (>0.975 mm), Type C, were most abundant 

 during the period 23 April through 9 May. Type-C 

 eggs declined in abundance throughout May in both 

 years. Mid-sized eggs (0.850-0.950 mm), designated 

 Type B, generally appeared later than Types A and 

 C. Type-B eggs did not exceed 5% of the total fre- 

 quency of sciaenid eggs until 15 May 1990 and 9 

 May 1991. Type-B eggs increased in abundance from 

 mid-May until the end of sampling. The smallest 

 eggs (<0.850 mm), designated Type A, co-occurred 

 with Type-C eggs; however, they did not exceed 5% 

 of the total sciaenid eggs until 8 May 1990 and 9 

 May 1991. In 1990, Type-A eggs peaked in abun- 

 dance on 15 May and gradually declined through- 

 out the sampling period. In 1991, Type-A eggs were 

 most abundant during the last sample on 28 May. 



To test the hypothesis that eggs designated Types 

 A, B, and C were separate species assemblages, the 

 mtDNA restriction fragment patterns of known 

 adult sciaenids were compared with those of fresh 

 egg samples separated into Types A, B, and C. Re- 

 striction fragment length polymorphism analysis of 

 mtDNA, purified from adult B. chrysoura, C. 

 nebulosus, C. regalis, Menticirrhus saxatilis, and P. 

 cromis, revealed species-specific restriction fragment 

 patterns for each of the five enzymes. Of the five en- 

 zymes, Hindlll showed the greatest differences be- 

 tween species, facilitating visualization with the 

 Southern blotting procedure (Table 2). 



