184 
Fishery Bulletin 106(2) 
A 
B 
(A) Five species of Centropristis (sea bass) are currently recognized in the western North Atlantic Ocean. Black 
sea bass ( Centropristis striata ) ranges along the northeast and southeast shelves of the U.S. coast and inhabits the 
Gulf of Mexico on the northwest coast of Florida. Bank sea bass (C. ocyurus ) and rock sea bass (C. philadelphica) 
are found along the southeast U.S. coast and in the Gulf of Mexico. Centropristis rufus is reported off Martinique 
in the southeastern Caribbean Sea. Twospot sea bass ( C . fuscula) is reported from Cuba. (B) Sampling stations are 
marked with an x where Centropristis spp. larvae were collected during three cruises ranging from Chesapeake 
Bay to southern Georgia during September 2000, November 2000, and February through March 2001. During 
each cruise, transects running cross-shelf from 10 m to > 1000 m water depth were sampled. The dashed line 
approximates the 200-m depth contour. 
the southeast U.S. shelf and the Gulf of Mexico where 
C. striata, C. ocyurus, and C. philadelphica cohabit 
(Fig. 1), larvae less than 12 mm cannot be reliably iden- 
tified to species because fin ray formation is not com- 
plete. To identify larvae of these species, we developed 
species-specific polymerase chain reaction (PCR) and 
RFLP assays based on the internal transcribed spacer 
(ITS) regions of the ribosomal gene complex. The ITS 
regions are noncoding spacer regions situated between 
the 18S and 5.8S rRNA genes (ITS1) and between the 
5.8S and 28S rRNA genes (ITS2) (Fig. 2A). The ITS 
regions work well for species identification because in 
most eukaryotic organisms these regions diverge rapidly 
during speciation, enabling even closely related species 
to be distinguished. The ITS sequences have been used 
to successfully identify species in groups as diverse as 
fungi (Lu et al., 2002), plants (Baldwin, 1992), insects 
(Rafferty et al., 2002), and dinoflagellates (Connell, 
2001; Litaker et al., 2003). Other ITS-based assays 
have been developed to help address conservation and 
management problems in the trade of shark products 
(Chapman et al., 2003; Shivji et al., 2005) and for iden- 
tifying wildlife tissues in forensic applications (Sweijd 
et al., 2000; Ambercrombie et al., 2005). 
PCR and RFLP assays offer rapid methods for iden- 
tification of fish larvae and eggs. The PCR assays can 
be performed in a one-step process after DNA extrac- 
tion and they enable species identifications to be made 
even when meristic characters and other identifying 
features have been damaged or are underdeveloped. 
Alternatively, the RFLP assays provide a cost efficient, 
two-step, PCR-based species identification method. In 
the first step, PCR is used to amplify the ITS regions. 
In the second step, restriction enzyme digestion of the 
PCR product is conducted and species-specific frag- 
ments are analyzed. This study describes the develop- 
ment, optimization, and validation of PCR and RFLP 
assays for three species of Centropristis. 
Materials and methods 
Collection and preservation of adult fish 
Tissue samples were obtained from adult and juvenile 
fish and either stored in 95% ethanol or frozen at -80°C. 
Centropi'istis striata were collected from the Atlantic 
coast of the southeast United States, and C. ocyurus and 
C. philadelphica were collected from both the southeast- 
ern U.S Atlantic coast and the Gulf of Mexico. At least 
three individuals from each location were analyzed. Ten 
other co-occurring southeast U.S. reef fishes were also 
collected for sequencing of the ITS regions and served as 
negative controls for the PCR and RFLP assays. Five of 
these species were from the subfamily Serraninae: sand 
perch ( Diplectrum formosum), butter hamlet ( Hypoplec - 
