Vandersea et al.: Identification of larval Centropristis spp using ribosomal DNA-specific molecular assasy 
191 
A 12345678 9 10 11 12 13 14 15 16 17 
B 123 4 5 678 9 10 11 12 13 14 15 16 17 
246 bp. 
123 bp« 
C 1 2 3 4 5 6 7 8 9 10 11 12131415 16 17 
_ . _ , 
246 bp 4 
123 bp 
Figure 5 
Results of species-specific polymerase chain reaction (PCR) assays 
for sea bass (Centroprisitis) larvae (A) black sea bass ( Centropristis 
striata ), (B) bank sea bass (C. ocyurus), and (C) rock sea bass (C. 
philadelphica). In each panel, the gels were loaded in the follow- 
ing manner: lane 1, 123-bp ladder; lane 2, positive controls for A 
(C. striata), B ( C . ocyurus), and C (C. philadelphica)-, lanes 3-13, 
Centropristis larvae, lane 14, negative control; lane 15-16, PCR 
inhibition controls; lane 17, no DNA control. 
PCR versus RFLP assays 
The species-specific PCR and RFLP assays 
can be used to accurately identify larvae. The 
decision of which assay to use could be based 
on labor and reagent costs. The PCR assays are 
attractive because they are rapid single step 
assays, whereas the RFLP assays employ PCR 
in the first step and restriction enzyme diges- 
tion of the PCR products in the second step. In 
most cases, PCR reagents are more expensive 
than those needed for restriction enzyme analy- 
sis. However, PCR assays can be cost effective if 
they are multiplexed. Excellent examples of suc- 
cessful multiplex PCR assays are described by 
Chapman et al. (2003) and Hyde et al. (2005). 
Unfortunately our attempts at multiplexing the 
assays were unsuccessful because of the produc- 
tion of nonspecific PCR products. 
RFLP assays are cost effective because they 
require less expensive reagents than PCR as- 
says. However, the RFLP assays require an 
extra hour or two to process and involve ad- 
ditional labor costs. The RFLP assays devel- 
oped here employed genus-specific primers 
rather than species-specific primers to amplify 
Centropristis genomic DNA from adult fish 
(Table 3). Using genus-specific primers, we 
simplified the PCR reaction mixtures for all 
three species and ensured that only Centro- 
pristis DNA would generate PCR products. 
The resulting PCR products were subsequent- 
ly digested with Alu I enzyme to produce spe- 
cies-specific fragments that were easily distin- 
guished by their unique banding patterns once 
electrophoretically separated on an agrose gel. 
The unique sizes of these fragments were due 
to species-specific differences in the locations 
of the Alu I restriction sites within the ITS1, 
5.8S gene, and ITS2 regions (Fig. 6B). 
Shipboard operational molecular assays 
The PCR and RFLP assays could be conducted at sea 
with a small thermocycler and the other minor equip- 
ment necessary for DNA extraction. If onboard con- 
ditions were favorable, the assays could be analyzed 
immediately after PCR thermocycling by gel electro- 
phoresis and with the use of a digital imaging camera. 
Similar methods described by Hyde et al., (2005) were 
used at sea to identify the eggs and larvae of blue marlin 
( Makaira nigricans), shortbill spearfish ( Tetrapturus 
angustirostris), and wahoo [Acanthocybium solandri). 
Their procedures incorporated a boiling method to 
quickly prepare amplifiable DNA from fresh tissue. If 
this would not be practical, then PCR products could be 
frozen and analyzed at an onshore laboratory. Results 
for 30 larval assays could be attained in four hours or 
less at a cost of ~$5 per sample, excluding the cost of 
equipment. Alternatively, larvae could be collected and 
sorted; all Centropristis larvae would be separated and 
assayed or stored individually in 95% ethanol for pro- 
cessing at a shore-based laboratory. DNA could then be 
extracted from a small tissue sample (e.g., an eye) from 
each specimen. The intact larvae would be returned to 
storage in 95% ethanol in case any additional morpho- 
logical or molecular evaluation was required. If analysis 
of a large number of samples was desired, the individual 
PCR assays could be adapted to a SYBR green quantita- 
tive PCR format. Because this approach eliminates the 
need for gel electrophoresis, it could be used to process 
hundreds of samples provided that sorting could be 
expedited. 
Conclusion 
Both the species-specific PCR and RFLP assays described 
in this article can successfully identify C. striata, C. 
ocyurus, and C. philadelphica. The assays are rapid, 
cost effective, simple to perform, and highly accurate. 
