Vandersea et at: Identification of larval Centropristis spp using ribosomal DNA-specific molecular assasy 
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A 
F1S H5.8SR 
CentropFWl FIS H5 8SF2 CentropREVl 
NSF17877F FISH5.8SF FISHLSU5'REV 
Cphil-fTSR5 Cocy-ITSR7 Cstri-ITSR4 
Figure 2 
Diagram of the small subunit (SSU), internal transcribed spacer regions (ITS), 5.8S, and 
large subunit (LSU) genes. (A) Location of the universal polymerase chain reaction (PCR) 
primers used to amplify the ITS spacer regions, the genus-specific restriction fragment 
length polymorphism (RFLP) primers, and the internal sequencing primers. (B) Location in 
the ITS1 region of the Centropristis PCR assay primers. 
trus unicolor), pygmy sea bass (Serraniculus pumilio), 
tattler ( Serranus phoebe), and belted sandfish {Serranus 
subligarius). Four species were from the family Ser- 
ranidae, red grouper {Epinephelus rnorio ), rock hind 
( Epinephelus adscensionis), speckled hind ( Epinephelus 
drummondhayi) , gag grouper (Mycteroperca microlepis), 
and one from a related perciform family, Sparidae, the 
spottail pinfish ( Diplodus holbrooki). 
Collection and preservation of larvae 
Three cruises were conducted from Chesapeake Bay to 
southern Georgia during early fall (September 2000), 
late fall (November 2000), and late winter (February 
and March 2001). During each cruise, transects run- 
ning cross-shelf from 10 m to >1000 m water depth were 
sampled (Fig. 1). The deepest stations were sampled to 
50 m. Ichthyoplankton were collected with a 1-m Tucker 
trawl fitted with 333-pm mesh nets. The Tucker trawl 
was fitted with three nets. The first net sampled from 
the surface to the deepest deployment depth. For stations 
in <50 m water depth, the lower and upper halves of 
the water column were sampled discretely by using the 
second and third nets during the retrieval of the Tucker 
trawl. For stations in >50 m water depth, the 50-25 m 
depth was sampled discretely with the second net and 
the 25-0 m depth interval was sampled discretely with 
the third net. Depth was determined from the wire angle 
of the gear and length of wire deployed. Tow speed was 
between 1.5 and 2 knots. 
Samples were initially preserved in 95% ethanol. All 
larvae were sorted from these samples, transferred to 
70% ethanol, and identified to the lowest taxonomic level 
possible. Larvae were identified to the genus Centropris- 
tis based on characteristic body shape, pigmentation, 
and when available, dorsal and anal fin meristics (Ken- 
dall, 1972). Seventy-six larvae were chosen for genetic 
analyses. These specimens were selected in an attempt 
to analyze equal numbers of larvae collected from north 
and south of Cape Hatteras, North Carolina. In this 
respect, the larvae were not a random sample of the 
Centropristis larvae identified from the collections. The 
larvae ranged in size from 1.5-11 mm notochord length 
(or standard length). Each individual to be used for PCR 
analyses was rinsed three times with clean 95% ethanol, 
digitally imaged, and stored in a separate sterile vial 
with 95% ethanol. To prevent DNA cross-contamination, 
the forceps used for handling larval fish were decon- 
taminated between specimens with 10% sodium hypo- 
chlorite, rinsed with clean deionized water, and dried 
with a clean Kimwipe (Kimberly-Clark, Roswell, GA). 
Determination of ITS region sequences 
To identify species-specific primer sites it was first 
necessary to amplify and sequence the ITS regions 
from adult Centropristis and other reef fish. To accom- 
plish this, universal PCR primers were designed for the 
amplification of the 3' end of the small subunit (SSU), 
the internal, transcribed spacer region ( ITS 1 ) , 5.8S 
subunit (5.8S), the second internal transcribed spacer 
region (ITS2), and the first ~48 base pairs of the large 
subunit (LSU) rRNA genes (~1300-bp product). The uni- 
versal forward primer NSF1787F (Fig. 2A, Table 1) was 
designed with the reverse complement of the universal 
reverse primer, NSR1787/18, listed on the ribosomal 
RNA database website. The universal reverse primer 
FishLSU5’Rev (Fig. 2A, Table 1) was designed by align- 
ing (CLUSTAL X algorithm; Thompson et ah, 1997) the 
large subunit rDNA sequences of the following fish spe- 
cies that were available in GenBank: European perch 
( Perea fluviatilis Z18686), rainbow trout ( Oncorhynchus 
