68 
Fishery Bulletin 119(1) 
in estuarine waters of LIS, nearshore coastal waters 
(0-5 km [0-3 mi]) of New Jersey and New York, and off- 
shore waters along the Atlantic coast from New Jersey to 
Virginia. Highly valued, the Atlantic surfclam fishery gen- 
erated more than $29.2 million in revenue during 2019 
(National Marine Fisheries Service, commercial fisheries 
landings database, available from website). 
Changes in environmental conditions of the habitat 
of Atlantic surfclam are comparable to changes in the 
open ocean, with seasonal variability in Q,,sgonite Near 
or below the physiological threshold of 1.5 identified for 
other bivalves, yet there have been no studies examin- 
ing how larval Atlantic surfclam respond to increasing 
pCO, levels. On the basis of the RCP 6.0 and RCP 8.5 
scenarios (IPCC, 2014) and current conditions of the hab- 
itat of Atlantic surfclam, our study investigated the effect 
of low, medium, and high levels of pCO, (344, 821, and 
1243 patm) on growth, mortality, and metamorphosis of 
larval Atlantic surfclam. We hypothesized that larvae of 
Atlantic surfclam might have reduced rates of survival 
and growth and experience longer times to metamorpho- 
sis with increased pCO, concentrations. We also hypoth- 
esized that allocation of energetic resources in larval 
Atlantic surfclam, measured by using lipid concentra- 
tions, might change in response to variations in carbon- 
ate chemistry. 
Materials and methods 
Spawning 
Adult brood stock of Atlantic surfclam were collected 
from 2 locations in LIS. In 2014, 32 clam were caught on 
4 November from Stratford, Connecticut, and 75 individ- 
uals were collected on 6 November from Norwalk, Con- 
necticut, from on board a commercial shellfish boat. The 
brood stock acclimated for 169 d at the Milford Laboratory 
(41°12’43.82’N, 73°3’12.96’W) of the Northeast Fisheries 
Science Center in a flow-through seawater system (16°C 
[standard deviation 3]) delivering natural seston that was 
supplemented with a drip of cultured Tetraselmis chui 
(strain PLY429). Brood stock were conditioned for spawn- 
ing following the protocol outlined in Goldberg (1989). On 
22 April 2015, 30 scrubbed Atlantic surfclam from both 
collection sites were placed in a spawning table filled with 
10-pm-filtered seawater. To induce spawning, seawater 
temperature was increased by 2°C every 30 min, to a max- 
imum of 26°C. Upon release of gametes, male and female 
Atlantic surfclam were placed into separate glass dishes. 
Gametes were collected from 7 females and 5 males, and 
then pooled eggs were fertilized with pooled sperm in 
ambient seawater, yielding a total fertilized egg count of 
81.6 million. 
After fertilization, 30 embryos/mL were added to each 
of 9 clean 15-L buckets containing 10 L of 0.35-um-filtered 
seawater that had been pumped directly from Milford 
Harbor and bubbled with CO, to reach target condi- 
tions (described in the “Experimental design” section). 
Larvae were fed after 24 h with cultured Tisochrysis lutea 
(isolator number: T-ISO) at an algal density of 50,000 
cells/mL (Goldberg, 1989). Every Monday, Wednesday, and 
Friday, the larvae in buckets were screened, buckets were 
cleaned, and water in the buckets was replaced with pre- 
conditioned water for each pCO, treatment level. Cleaning 
helped reduce ciliate levels that were present in all treat- 
ments. Larval experiments were conducted from 22 April 
through 20 May 2015. 
Experimental design 
Nine buckets were placed in a temperature-controlled 
water table (Table 1). The target pCO, levels (~400, ~800, 
and ~1200 patm) were chosen on the basis of RCP scenar- 
ios for future CO, concentrations in the region and the 
3 levels identified in the guide to best practices for OA 
research and data reporting (Riebesell et al., 2011). Com- 
pressed air was passed through a PureGas” CO, absorber 
(Altec Air, Broomfield, CO) before being delivered to GFC 
mass flow controllers for air and carbon dioxide (Aalborg 
Instruments and Controls, Orangeburg, NY). The CO.- 
stripped air was mixed with research-grade CO, to cre- 
ate and maintain 3 target pCO, treatment levels and 
was distributed to 3 replicate buckets per treatment at 
a moderate aeration rate (12 L/min) that is routinely 
used in bivalve culture (Loosanoff and Davis, 1963; 
Bayne, 1965). 
Biological sampling 
Larvae samples for examining size, survival, metamor- 
phosis, and lipids were taken on a volume basis weekly 
when buckets were cleaned. Briefly, larvae were screened 
on a nylon mesh sieve (40-150 pm) and concentrated in 
a 100-mL graduated cylinder. A subsample of 1 mL was 
removed from the graduated cylinder and preserved in 
a 1% solution of buffered formalin for measurement of 
shell heights (maximum length, from anterior to posterior, 
parallel to the hinge; number of samples [n]=50 larvae). 
When the experiment began on 22 April 2015, 4 samples 
were taken for initial measurements of size, and triplicate 
samples from each bucket were obtained and preserved 
weekly for size measurements and for determination of 
the percentages of all larvae in the sample that had died 
(percent mortality) and that had undergone metamorpho- 
sis by using an Olympus inverted microscope (Olumpus 
IX51, Olympus Corp., Tokyo, Japan) and the software 
ImageJ, vers. 1.49 (Rasband, 2015). 
Lipid samples were obtained at the end of the experi- 
ment on day 28. Known volumes (0.3-0.5 mL) of concen- 
trated Atlantic surfclam were collected and placed in glass 
tubes that had been prerinsed with methanol and chloro- 
form. Samples were stored in 2 mL of chloroform, purged 
of air with nitrogen gas, and held at —80°C until analysis 
Mention of trade names or commercial companies is for identi- 
fication purposes only and does not imply endorsement by the 
National Marine Fisheries Service, NOAA. 
