153 
NOAA 
National Marine 
Fisheries Service 
Fishery Bulletin 
ftr established 1881 -ds 
Spencer F. Baird 
First U S. Commissioner 
of Fisheries and founder 
of Fishery Bulletin 
Integrating DNA barcoding of fish eggs into 
ichthyoplankton monitoring programs 
Email address for contact author: david.richardson@noaa.gov 
Abstract— The data collected through 
ichthyoplankton monitoring surveys 
provide valuable insight into the 
spawning dynamics of multiple spe- 
cies. Fish eggs, more than larvae, 
offer a more precise evaluation of 
species-specific spawning character- 
istics; however, egg collections are 
greatly underused because of the 
limitations associated with morphol- 
ogy-based identifications. In recent 
years, a new means of molecular 
identification, termed DNA barcod- 
ing, has made species identification 
readily available across a broad 
range of taxa. We used DNA bar- 
coding to identify ethanol-preserved 
fish eggs collected during 2002-2012 
along the northeastern U.S. conti- 
nental shelf. A subsampling protocol 
was used to select 1603 unidentified 
eggs for analysis. DNA sequences 
were successfully obtained from 
1495 (93.26%) of these eggs, repre- 
senting 50 species — many of which 
have either never before been iden- 
tified to the species-level as eggs or 
have been identified previously only 
to a higher taxonomic level or dur- 
ing specific developmental egg stag- 
es. In comparison with past attempts 
at morphological identification, our 
molecular identifications comprise a 
broader diversity of eggs and provide 
a technique with high success rates 
of unambiguous identifications that 
is not sensitive to egg stage. Overall, 
this work shows that DNA barcoding 
of fish eggs is sufficiently advanced 
to be incorporated into long-term, 
regional-scale ichthyoplankton moni- 
toring programs. 
Manuscript submitted 6 April 2015. 
Manuscript accepted: 20 January 2016. 
Fish. Bull. 114:153-165 (2016). 
Online publication date: 5 February 2016. 
doi: 10.7755/FB.114.2.3 
The views and opinions expressed or 
implied in this article are those of the 
author (or authors) and do not necessarily 
reflect the position of the National 
Marine Fisheries Service, NOAA. 
Leah A. Lewis 1 
David E. Richardson (contact author ) 1 
Evgeny V. Zakharov 2 
Robert Hanner 2 
1 Northeast Fisheries Science Center 
National Marine Fisheries Service, NOAA 
28 Tarzwell Drive 
Narragansett, Rhode Island 02882 
2 Biodiversity Institute of Ontario 
University of Guelph 
Guelph, Ontario, Canada NIG 2W1 
Pelagic eggs and larvae, collec- 
tively referred to as ichthyoplank- 
ton, are the life-history stages of 
fish that are most abundant within 
the marine epipelagic zone. Be- 
cause of this prominence, a number 
of long-term ichthyoplankton moni- 
toring programs have been estab- 
lished worldwide. The data collected 
through large-scale surveys provide 
fisheries-independent information on 
the spawning locations, times, and 
intensities of multiple species simul- 
taneously. Ichthyoplankton data have 
also been used to estimate spawning 
stock biomass (Zeldis, 1993) and to 
develop long-term indices of abun- 
dance (Richardson et al., 2010a). In 
contrast to larvae, fish eggs provide 
a more precise means of evaluating 
the distribution and abundance of 
spawning fish populations because of 
the reduced cumulative influence of 
egg transport and mortality (Ouellet 
et al., 1997; Richardson et al., 2009). 
However, the number of identifi- 
able egg characteristics is far fewer 
than the number of morphological 
features available for larval iden- 
tification. Consequently, the major 
prerequisite — accurate species-level 
identification from eggs — remains an 
obstacle for the use of fish eggs in 
long-term monitoring and manage- 
ment programs. 
Historically, the identification of 
fish eggs has been limited; illustra- 
tive guides often reference a small 
subsample of eggs from known par- 
ents or a small quantity of eggs col- 
lected during surveys and reared 
through the larval and juvenile stag- 
es (Colton and Marak 1 ; Ahlstrom and 
Moser, 1980; Berrien and Sibunka 2 ). 
On a broad scale, identifications tra- 
ditionally have been based on mor- 
phological characteristics, including 
egg shape and diameter; number, 
size, and position of oil globules; 
width of the perivitelline space; na- 
ture of the egg yolk and chorion sur- 
face; and embryonic pigmentation. 
1 Colton, J. B., Jr., and R. R. Marak. 1969. 
Guide for identifying the common plank- 
tonic fish eggs and larvae of continental 
shelf waters, Cape Sable to Block Island. 
Bur. Commer. Fish., Biol. Lab. Ref. 69-9, 
43 p. 
2 Berrien, P. L., and J. D. Sibunka. 2006. 
A laboratory guide to the identifica- 
tion of marine fish eggs collected on the 
northeast coast of the United States, 
1977-1994. U.S. Dep. Commer., NOAA, 
Northeast Fish. Sci. Cent. Ref. Doc. 06- 
21, 162 p. 
