Spanik et al.: Using DNA barcoding to improve taxonomic resolution of the diet of Lutjianus campechanus 125 
prey item to qualitatively assess digestion level (Smith 
et al., 2005; Fig. 1). Invertebrate prey were identified on 
the basis of morphology only, and digestion level was not 
assessed. 
Molecular identification of fish prey 
Muscle tissue from fish prey items that could not be 
morphologically identified to species level (digestion 
codes 2-3; Fig. 1) was retained for DNA barcoding. 
Tissue samples were rinsed with distilled water, pre- 
served in a 2.5-mL vial containing 95% ethanol, and 
stored at —20°C. When possible, the most superficial 
layer was discarded to avoid contamination from the 
predator’s stomach lining or other co-occurring prey 
items. Prey assigned a digestion code of 4 (Fig. 1) were 
not used for DNA barcoding because of predator con- 
tamination and low DNA yield in preliminary trials. 
Laboratory utensils were dipped in a 10% bleach solu- 
tion and rinsed with distilled water between stomachs 
and between each prey item to prevent contamination. 
To isolate DNA, muscle tissue (~0.5 g) was blotted dry, 
placed in a microcentrifuge tube, and immersed in a 
200-pL digestion solution, consisting of 145.5 pL nuclei 
lysis solution, 36.36 pL EDTA (0.5 M), 14.5 pL pro- 
teinase K (20 mg/mL), and 3.64 pL milli-Q water, and 
Figure 1 
Images of prey items found in stomachs of red snapper (Lutjanus campecha- 
digested overnight at 55°C. The next morning, 180 mg 
of Promega Wizard SV lysis buffer (Promega Corp., 
Madison, WI) was added, and the sample was vortexed. 
Then DNA was isolated from the supernatant by using 
a Promega Wizard SV spin-column assembly according 
to the manufacturer’s instructions. 
A region of approximately 650 base pairs of the cyto- 
chrome oxidase I gene was amplified by using the univer- 
sal fish primers Fish-F1 and Fish-R2 (Ward et al., 2005). 
A 25-pL polymerase chain reaction (PCR) volume consisted 
of 16.4 pL milli-Q water, 2.5 pL 1x 5PRIME HotMaster 
PCR Buffer (Qiagen Beverly Inc., Beverly, MA), 2.0 pL 
dNTPs, 2.0 pL MgCl, 0.3 pL each primer, 0.1 pL 5PRIME 
HotMaster Tag DNA polymerase (Qiagen Beverly Inc.), and 
1.0 pL DNA template. The thermal cycler profile consisted 
of an initial denaturation at 94°C for 4 min, followed by 
35 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 1 min, 
and a final extension at 72°C for 10 min (modified proce- 
dure from Ward et al., 2005). Each PCR contained a positive 
and negative control. 
Polymerase chain reaction products were subjected to 
electrophoresis on a 1.5% agarose gel stained with ethid- 
ium bromide and then examined under ultraviolet light. 
Products were purified by using Affymetrix ExoSAP-IT 
(Thermo Fisher Scientific Inc., Waltham, MA) following 
the manufacturer’s protocol. Samples were sent to Euro- 
fins Genomics (Louisville, KY) for bidi- 
rectional Sanger sequencing with the 
same primers mentioned previously. 
Bidirectional sequences were aligned 
by using Sequencher, vers. 5.4 (Gene 
Codes, Ann Arbor, MI) and trimmed 
by using default criteria. Trimmed 
sequences were compared to their chro- 
matograms and edited accordingly. 
Edited sequences were queried within 
the GenBank database of the National 
Center for Biotechnology Information 
(available from website) by using the 
Basic Local Alignment Search Tool 
(Altschul et al., 1990). A species was 
assigned to a prey item when a query 
sequence (minimum 300 base pairs) 
and a known reference sequence shared 
298% similarity. If a species-level iden- 
tification could not be assigned by 
using GenBank, the sequence was sub- 
mitted to the Barcode of Life Database 
(available from website). Our edited 
sequences were deposited into Gen- 
Bank (accession numbers MT580050-— 
MT580051, MT582543-MT582580). 
nus) collected along the Atlantic coast of the southeastern United States in 
2017 and 2018, shown as examples of samples assigned to the 4 digestion 
codes used to qualitatively assess the condition of fish prey. The codes are 
Data analysis 
(1) fresh, easy to identify visually; (2) partially digested: some skin or scales 
remain, many identifiable features remain; (3) mostly digested: most identifi- 
able features absent, with some flesh attached to hard parts; and (4) severely 
digested: only hard parts (e.g., otoliths or vertebra) remain. 
The relative contribution of prey items 
to the diet of red snapper examined in 
our study was described by using 3 tra- 
ditional metrics: mean percentage by 
