Wilborn et al.: First observation of the use of coral habitat by larval Sebastes polyspinis 75 
survival. Several factors have led to difficulty in exploring 
this hypothesis. First, trawling for samples both destroys 
the habitat (Hourigan, 2009; Clark et al., 2016) and inte- 
grates catches over large distances, making fine-scale 
associations difficult to determine. Second, identification 
of the early life stages of rockfishes is difficult prior to 
development of distinguishing characteristics (Matarese 
et al., 1989; Johansson et al., 2018). 
We developed a plankton pump to sample zooplank- 
ton and larval fish in deepwater coral habitat in Alaska 
(Wilborn et al., 2020). The plankton pump provides a 
non-destructive method of sampling that can provide 
fine-scale information on habitat associations. The aim 
of this study was to explore the potential role of deep- 
sea coral habitat in the early life history of rockfishes 
in the North Pacific Ocean without damaging the habi- 
tat. Genetic techniques solved the second issue of species 
identification. The objectives of this note are to report 
the first known instance of a single larval rockfish cap- 
tured directly from deep-sea coral habitat and to discuss 
supplementary evidence from larval rockfish captured 
in association with deep-sea corals during bottom-trawl 
surveys in the Gulf of Alaska. 
Materials and methods 
The method used to capture zooplankton and larval fish 
from coral and sponge habitats in the Gulf of Alaska is 
described in Wilborn et al. (2020). In brief, the plank- 
ton pump is an autonomous sampler that was deployed 
from a contracted research vessel at depths of 80-105 m. 
We deployed the pump 8 times, in areas of known coral 
and sponge habitat, as well as in areas without coral 
and sponge habitat (5 and 3 deployments, respectively). 
It was programmed to sample the seafloor plankton by 
drawing water through a 333-ym-mesh zooplankton net. 
Sampling began after the pump reached the seafloor and 
ended after 15 min, with an automated door closure pre- 
venting contamination of the sample during retrieval. 
The mean volume of water filtered per deployment was 
6.32 m® (standard deviation 2.46). Prior to retrieval of 
the plankton pump, a series of photographs of the sea- 
floor were taken by a camera mounted on the pump to 
document the surrounding habitat. The 8 samples were 
collected in the western Gulf of Alaska between the Shu- 
magin Islands (~158°W) and Samalga Pass (~170°W) in 
2017 (further details on this sampling and the organisms 
captured can be found in Wilborn et al., 2020). All sam- 
ples collected from the plankton pump were individually 
extracted and preserved in plastic containers with a 95% 
solution of ethanol and glycerol for further analysis in 
the laboratory. 
To genetically identify the larval rockfish captured in 
the plankton pump, a 750-base-pair (bp) region of the mito- 
chondrial cytochrome b gene was amplified to identify the 
rockfish to species. This method has been used successfully 
to discriminate among known species of rockfish (Rocha- 
Olivares et al., 1999). For each sampled larva, DNA was 
extracted from the caudal fin by using a QlIAamp' DNA micro 
kit (Qiagen Inc., Hilden, Germany) and eluted in 25 pL of 
buffer. The mitochondrial DNA fragment of the cytochrome 
b gene was amplified through a polymerase chain reaction 
cocktail by using primers GluDG (5’ TGA CTT GAA RAA 
CCA YCG TTG 3’) and CB3R (5 ATA TCA TTC TGG CTT 
AAT GTG 3’) as described in Rocha-Olivares et al. (1999). 
Thermalcycling conditions were 90°C for 2 min, followed 
by 36 cycles of 94°C for 50 s, 51°C for 50 s, and 72°C for 
50 s. Polymerase chain reaction fragments were visualized 
with E-Gel EX Agarose gels (1%; Thermo Fisher Scientific, 
Waltham, MA). Polymerase chain reaction products were 
cleaned and sequenced in forward and reverse directions at 
Molecular Cloning Laboratories in San Francisco, California. 
The resulting data were assembled in Sequencher, vers. 5.0 
(Gene Codes Corp., Ann Arbor, MI). High-quality forward and 
reverse sequences (with quality scores >40) were aligned to 
produce a 655-bp fragment of the mitochondrial cytochrome 
b region in Sequencher. Consensus sequences were aligned 
in the sequence alignment editor in BioKdit (vers. 7.2; Hall, 
1999) to highlight differences, and trimmed sequences were 
assigned GenBank accession numbers (Table 1) (GenBank, 
available from website). 
In addition to analyzing DNA from the single unknown 
rockfish larva, for comparison, we sequenced DNA from 
adults of 4 species of rockfish known to occur in the region 
where the larva was collected (Table 1). The new sequences 
of DNA from these adults were cataloged in the University 
of Washington Fish Collection (available from website). The 
4 species are Pacific ocean perch (Sebastes alutus) (voucher 
no. UW151031), dusky rockfish (S. ciliatus) (UW155786), 
northern rockfish (S. polyspinis) (UW155787 and UW189442), 
and light dusky rockfish (S. variabilis) (UW45510). We also 
compared the DNA from the larva to DNA from another 
available specimen of northern rockfish (UW113251) for 
which DNA had been sequenced during an unrelated study 
in the eastern North Pacific Ocean (at 52°34’N, 170°41’W) 
in 2004. We expected the unknown rockfish larva to be iden- 
tified either through sequences from the 6 samples on hand 
or through sequences available in open-access repositories 
of genetic data (e.g., GenBank). 
Subsequent to the capture of the northern rockfish 
larva in 2017, a reexamination of corals collected during 
the bottom-trawl survey conducted by the NOAA Alaska 
Fisheries Science Center in the Gulf of Alaska in 2017 
was undertaken. These coral specimens were collected as 
part of routine efforts to collect genetic information on 
corals and sponges in the Gulf of Alaska. Additional rock- 
fish larvae were found lodged in specimens of 2 species of 
coral (a Plumarella superba and a Callogorgia compressa; 
Fig. 1). The larvae and corals were collected on 30 May 
2017 from the haul of a bottom-trawl tow conducted at a 
depth of 136 m in the Gulf of Alaska (52°41’N, 169°32’W). 
The DNA from 5 individuals associated with Callogorgia 
compressa and from 6 individuals associated with 
' 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. 
