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Fishery Bulletin 1 14(2) 
A B 
Figure 1 
Depiction of the image analysis process for rapid egg measuring: (A) fish eggs are 
grouped and digitally photographed on a white background; (B) a black and white 
threshold is applied to the image; (C) after applying the Hough transformation to the 
black-and-white image, most eggs are found and accurately measured (mm); and (Di 
measured egg diameters then can be edited manually and oval anchovy egg measure- 
ments can be added. The center and diameters (or long and short axis of ovoid eggs) 
are indicated with the cross hair symbols. Egg numbers on the images are also indi- 
cated and can be linked back to the output text files. 
base pairs (bp) of the mitochondrial gene COI was am- 
plified with primer cocktails C_FishFltl + C„FishRltl 
and AquaF2 + C_VRlLRtl (Ivanova et al., 2012). The 
PCR mix included 6.25 pL of 10% trehalose, 1.25 pL 
lOx PCR buffer, 0.625 pL (2.5 mM) MgCl 2 , 0.125 pL (10 
pM) forward and reverse primer cocktail, 0.625 pL (10 
mM) deoxyribonucleotide triphosphates (dNTPs), 0.625 
pL Platinum Taq polymerase (Thermo Fisher Scientific 
Inc.), 3 ul H 2 O and 1 pL of DNA template. 
The following cycle conditions were used to run 
PCRs: 1 min at 94°C followed by 5 cycles of 30 s at 
94°C; 40 s at 55°C and 1 min at 72°C, followed by 35 
cycles of 30 s at 94°C; 40 s at 55°C, and 1 min at 72°C, 
with a final extension of 10 min at 72°C. PCR products 
were visualized by using agarose gel electrophoresis 
with buffer-less precast E-Gel system (Thermo Fisher 
Scientific Inc.). Successful amplicons were cycle se- 
quenced by using BigDye Terminator vers. 3.1 (Thermo 
Fisher Scientific Inc.) and the manufacturer’s recom- 
mended protocols. Sequencing products with incorpo- 
rated BigDye Terminator were purified with the bead 
cleanup method of solid phase reversible immobiliza- 
tion (SPRI; Agencourt Bioscience Corp., Beverly, MA) 
and were analyzed on an Applied Biosystems 3730x1 
DNA Sequencer (Thermo Fisher Scientific Inc.). 
Applied Biosystems trace files for each specimen 
were assembled into contigs (contiguous sequences of 
DNA) with the use of Codon Code Aligner software (Co- 
donCode Corp., Centerville, MA). Resulting consensus 
sequences and trace files were uploaded to the NIFEB 
project file on the BOLD data system. 
Sequence analysis and species identification 
For a comparison of sequences and species identifi- 
cation, we used the BOLD system and its analytical 
tools. Neighbor-joining taxonomic identification trees of 
Kimura 2-parameter distance were built on the basis of 
COI-5P (5' region) nucleotide sequence data to provide 
a graphic representation of the pattern of divergence 
between species (Saitou and Nei, 1987). Regardless of 
bp length, medium- and high-quality COI sequences 
