Lefebvre and Denson: Inshore spawning of Rachycentron canadum in South Carolina 
401 
maintained for 48 hr before the start of this study. Four 
50-mL aliquots of eggs (~2xl0 4 eggs) were used in the 
trial. Before the addition of eggs to the water baths at 
~11 hr postspawn, digital micrographs of eggs were 
recorded with a Nikon 5 SMZ1500 stereo microscope 
(Nikon Instruments, Inc., Melville, NY) mounted with 
a Micropublisher 3.3 camera (Qlmaging, Surrey, BC, 
Canada). Thereafter, -10 eggs or larvae were collected 
from each tank, and micrographs were recorded every 
4 hr until 61 hr after spawning. After images were re- 
corded, eggs and larvae were preserved in 10% neutral 
buffered formalin. 
To capture earlier stages of egg development and to 
expand the range of experimental temperatures, we ex- 
panded the egg-development study in 2008. Three water 
baths were heated to and maintained at 24.0°C, 26.0°C, 
and 28.0°C in a temperature-controlled laboratory 30 hr 
before the beginning of this second study. Two hours 
after time of spawning, a 25-mL aliquot of cobia eggs 
(~lxl0 4 eggs) was divided between petri dishes in each 
temperature bath. Every hour, until 13 hr after spawn- 
ing, and every other hour thereafter up to 25 hr after 
spawning, a sample of -10 eggs was removed from each 
water bath, digital micrographs were recorded, and eggs 
were preserved in 10% neutral buffered formalin. 
Micrographs of live eggs were taken, and both egg 
diameters and oil-droplet diameters were measured to 
the nearest micrometer by using ImageJ image-analysis 
software (vers. 1.38, ImageJ, Bethesda, MD). To deter- 
mine if damage and diameter changes occurred with 
preservation, changes in appearance were noted, and 
measurements of egg and oil-droplet diameters were 
measured to the nearest micrometer from micrographs 
of preserved eggs taken -1 year from the date of collec- 
tion in 2007 and 2 months from the date of collection in 
2008. Diameters were measured only for undamaged, 
preserved eggs. Percent shrinkage was calculated as 
the change in egg and oil-droplet diameters between 
the live and corresponding preserved eggs multiplied by 
100. To determine if a significant decrease in diameter 
because of preservation had occurred, 2-sample £-tests 
were performed independently for 2007 egg and oil- 
droplet diameters and 2008 egg diameters. Because of 
non-normality of the 2008 oil-droplet-diameter data, a 
Wilcoxon rank test was performed with these data to 
determine if significant shrinkage had occurred after 
preservation. 
With the use of the micrographs of live eggs from the 
egg-development studies, coarse stages of embryologi- 
cal development were described. Because the earliest 
stages of egg development were captured only in the 
2008 study and larvae hatched only in the 2007 study, 
the 2 temperature treatments that were closest in tem- 
perature (26.5°C in 2007 and 26°C in 2008) were used 
for the characterization of egg-development stages from 
the earliest ones to the hatching stage. 
5 Mention of trade names or commercial companies is for 
identification purposes only and does not imply endorsement 
by the National Marine Fisheries Service, NOAA. 
Ichthyoplankton survey 
Field collections Five stations in each of the 2 estuaries 
(Fig. 1), located in known cobia fishing grounds, were 
sampled weekly from 6 May to 15 June 2008 in PRS 
and 8 May to 8 June 2008 in SHS. Collections occurred 
between 0745 and 1945 hr. Survey stations in PRS were 
located 12.1 to 20.6 km inshore from the mouth of that 
estuary. Stations in SHS were located 5.1 tol4.3 km 
inshore from the mouth of that estuary. Stations were 
positioned upriver of particular bathymetric features 
where anglers typically target cobia. These stations were 
adjacent submerged sand bars and banks where water 
depths rose from 10 to 12 m at mean low water to 2 to 
3 m at mean low water. Depths of stations ranged from 
5.5 to 9.6 m at the time of net deployment. Anchored 
plankton nets, made of Nitex nylon mesh (Sefar Holding 
AG, New York, New York), were deployed at slack water 
before the daylight flood tide. These plankton nets had 
a mouth 0.5-m in diameter and were 2.5 m long, with a 
mesh size of 505 pm along the length and 303 pm at the 
codend. Floats attached to circular frames maintained 
the position of these nets at -1 m below the surface. 
Flow meters were mounted in the center of these nets 
at the most seaward and farthest inshore stations. At 
the time of net deployment, ancillary water data were 
collected (temperature, salinity, dissolved oxygen) at the 
surface and bottom of the water column with a handheld 
instrument, YSI 556 MPS (YSI, Inc., Yellow Springs, 
Ohio). Plankton nets were deployed for the length of 
time required to set all nets and return to retrieve the 
first one (70-150 min). The average current speed during 
the time of collection was calculated by using the flow 
meter readings. Volume of water filtered was calculated 
directly from flow meter readings in nets. For nets with- 
out flow meters, current speeds and volumes filtered 
were estimated by averaging the values from the most 
seaward and farthest inshore stations. 
Egg and larval identification Plankton samples were 
rinsed and then sorted under a dissecting microscope. 
When settled plankton volumes were greater than 1 
L, samples were split with a Burrell plankton splitter 
(Burrell et ah, 1974) until a settled plankton volume of 
0.5 L or less was attained. All nonclupeiform larvae were 
removed, identified to lowest possible taxonomic level 
(Moser et ah, 1984; Richards, 2005), and preserved in 
70% isopropyl alcohol. Cobia larvae were identified from 
descriptions of Ditty and Shaw (1992). All eggs measur- 
ing between 1.0 and 1.4 mm with an ocular micrometer 
and having one or more of the morphological character- 
istics corresponding to cobia eggs — single, large (300 
to 600 pm), pigmented oil droplet, heavily pigmented 
embryo, or narrow perivitelline space (Ditty and Shaw, 
1992; Ditty, 2006) — were removed and preserved in 10% 
neutral buffered formalin. 
Digital micrographs of preliminarily identified cobia 
eggs were recorded. Diameters of all eggs and of single, 
intact oil droplets from preserved eggs were measured 
to the nearest micrometer with ImageJ. To aid in iden- 
