Introduction:
Within recent years, the breadth and severity of ocean acidification (OA) has begun
to be better understood. As carbon emissions continue to rise, the world’s oceans
act as sink for the carbon dioxide. As these levels rise, the ecological and financial
implications become more severe (Range et al., 2011; Kleypas et al., 2006). While
OA is becoming much more well understood, literature on environmental effects on
bivalve optics is limited.
The current literature on scallop optics tends to concentrates on the
biomechanics of the eye itself (see Land, 1964). Scallops are an ecologically and
economically valuable family of organisms, both as prey species for other aquatic
organisms as well as for scallop aquaculture. Despite the lack of genomic data on the
scallop (family Pectinidae), current research is looking to illuminate the functions of
the various phototransduction pigments and where they stand in genetic
relatedness to each other (see Kojima et al., 1997; Pairett, 2013). We aim to look at
the beginning of this cascade, specifically with the protein opsin, to attempt to draw
out the implications of decreasing ocean pH on scallop vision.
Experimental Procedures:
We collected 43 specimens of pink scallops (Chlamys rubida) off the coast of Friday
Harbor, San Juan Island, Washington. After the collection the specimens
acclimatized in flow tanks for one week before being placed into experimental setup. Initially, a light/no light condition was set up for preliminary data. Then, using a
2X2 experimental design, four tanks were used for four separate conditions—
normal pH/light, low pH/light, normal pH/no light, low pH/no light—the scallops
were allowed to acclimatize to each condition for a period of 3 days. Tissues were
extracted from the gill, mantle, and eye. It should be noted that due to the extraction
methods used and the close adherence of eye to mantle, the eye samples contained
small amounts of mantle tissue. Tissue samples were preserved at -80° C for
processing.
The frozen tissue samples were processed for RNA extraction using the protocol
established for the University of Washington Fisheries 441 course. The tissue was
homogenized with TriReagent® RNA isolation reagent to facilitate the precipitation
of RNA. A sequence of ethanol rinses and centrifuge cycles produced a RNA sample
from each tissue which were each quantified with NanoDrop®. After RNA isolation,
the samples were processed for cDNA again using the University of Washington
FISH 441 protocol.
The preliminary test was run using qPCR using the primers set out by Serb et al.
(2013). As discussed below, the qPCR was ineffective with the primers used
therefore the primary samples were processed with conventional PCR using the
protocol outlined by Serb et al. Reagents used and amounts include
12.5uL green master mix, 1uL primer, 2.5uL MgCl2, 5uL nuclease free water, and
3uL cDNA.
Results:
We did not see proper annealing of primers in either qPCR or our relaxed condition
PCR. After RNA extraction from the whole tissue, the sample was quantified using
NanoDrop® and all samples showed high concentrations and purity (averaging
450.7 ng/uL; 260nm/280nm: 1.89). The primers used were not species specific due
to the lack of diversity in available scallop genomics. When no amplification was
seen in the initial qPCR, we opted to use the less expensive PCR with a more relaxed
temperature setting in an attempt to facilitate annealing. As Fig. 1 indicates, bands
were seen at ~150bp and the negative control showed no bands, however we
expected our gene product to show at ~550bp.
Discussion:
While the results proved less than desirable, they ultimately shed light on the
current state of knowledge on OA and its effects on important oceanic species.
Research continues to build the knowledge base on OA, however there yet remains
many holes in our understanding of the full effects of OA. The primers used were not
species specific thus as new genetic evidence becomes available it may reveal
unique aspects of the scallop genome. With this new evidence, interesting aspects of
the scallops’ ability for cope with environmental stressor may become known. In
turn, these genetic indicators may reveal information on ways to better manage,
from an anthropogenic perspective, the effects of OA on in-danger species.
The margin for error within the complex processes that make up PCR is
relatively high considering the approach we took to processing our samples. The
primers we used were pulled from recently published literature and were not
specific to the species we were testing. As more evidence mounts for genetic
variability among species, the gene specific primers that become available may
prove more effective. The protocol used for the PCR procedure followed that set out
by Serb et al. however we used an available gotaq master mix which had a preset
proportion of taq, taq buffer, and dNTPs. Therefore were unable to perfectly
replicate the procedure as set out by Serb et al. in our experiment. Human error is
certainly a consideration when looking at results like ours. While this is certainly a
possibility, it is unlikely. All our samples showed the same result, i.e. bands at
~150bp, and the negative control showed no bands. This, as well as our NanoDrop
results, lend to concluding there was little contamination in the samples.
We need to spend more time on this project in order to elicit more concrete
results. A larger sample size and more time to process those samples is required for
any hope of significant findings. The appropriate primers and optimization
sequence for PCR need to be teased out through experimentation to determine first
whether opisn is present in our sample tissues, and then to determine the effects of
environmental stressors on opsin levels. Once these conditions are solved then the
bigger picture implications can be better understood. It would be advisable to look
at the effects of OA on developing scallop larvae to further understand the
implications on lowering ocean pH on bivalve organisms.