Putting the year to rest..

And our final installment of highlights from the last year...

3) Anesthesia in mature oysters

In shellfish research, there are many situations in which tissues of individual animals need to be sampled multiple times, necessitating nonlethal sampling methods. In particular, an accurate way to effectively measure fecundity, one of the primary metrics of fitness in our project, would be highly beneficial.  We needed a nonlethal method to remove broods from female Olympia oysters to enable direct measurement of fecundity, and so we explored the possibility of oyster anesthesia.  Although relaxation methods have been developed for a number of oyster species, none were directly applicable or effective for the Olympia oyster, Ostrea lurida. We optimized an anesthetization method to induce gaping in the Olympia oyster.  We tested concentrations of 25-100 g/L MgSO4 and exposure durations of up to three hours on adult Olympia oysters, and measured response, recovery, and survival.  To stimulate opening of the valves to ensure exposure to the anesthetic, we also tested temperature increase and ambient air exposure as pre-treatments. We found the optimal concentration to be 85 g/L in 50% seawater, which resulted in 45% of the animals anaesthetized in two hours; the remaining oysters did not open their shells and were therefore not exposed. We also found pre-treatment with 30 minutes of air exposure and a temperature increase of 10°C to increase the proportion of oysters that opened their shells for exposure to the treatment.  We observed no adverse effects of treatment on subsequent recovery or survival. Future work will assess the influence of microalgae presence on the proportion of open oysters, efficiency of brood removal, and the effect of brood removal on survival. We are planning to use this method to measure fecundity and success in conservation efforts. In addition, this method can accommodate nonlethal tissue sampling.

Genetic Assessment

More research highlights from the past year....

2) Genetic assessment of restoration aquaculture practices

Olympia oyster seed produced from wild broodstock in our restoration hatchery from two different years (2010 and 2011) were compared.  Wild broodstock were collected from five locations in in the south subbasin of Puget Sound in both 2010 and 2011. Each year, approximately 500 broodstock were randomly assigned to each of 20 breeding groups.  In 2010, the larvae produced by each breeding group were collected and reared separately as a seed group (n=20). In 2011, the larvae produced on a given day  from all breeding groups were reared separately as a seed group (n=20).  A tissue subsample from 10 individuals from each seed group was taken in 2013 and stored in 70% EtOH. From 10 randomly selected seed groups from each year, DNA was extracted from each sampled individual following standard protocols, and each individual was genotyped at 7 microsatellite loci.  Raw genotype data, binned into length classes using TANDEM (Matschiner & Salzburger, 2009), were used to calculate pairwise relatedness using the maximum likelihood estimator of Queller and Goodnight (1989) as implemented in GenALEx (Peakall and Smouse 2006, 2012).  Mean pairwise relatedness values within each seed group ranged from 0.07-0.28 (2010) and 0.09-0.33 (2011) (Fig. 3, open circles).   Mean pairwie relatedness among 100 pairs over all seed groups (100 bootstrap replicates) did not differ significantly from zero for 2010 or 2011 (P<0.05; Fig. , solid circles).  This result suggests that the partial matrix breeding design as implemented incorporates sufficient genetic diversity to obviate undo relatedness, assuming equal contribution from each breeding group.  Future work will broaden comparisons to include the restoration seed, commercially produced seed, and wild groups. We will conduct allele count rarefaction (HP-Rare, Kalinowski 2005), apply the more powerful groupwise maximum likelihood relatedness estimator of Jones & Wang (2009), and test for shifts in allele frequency among groups.

Fig. 3. Mean pairwise relatedness values for offspring from ten breeding groups of Olympia oysters, Ostrea lurida, in 2010 and 2011 (open circles) and from pairs randomly sampled from each year (closed circles).

Fig. 3. Mean pairwise relatedness values for offspring from ten breeding groups of Olympia oysters, Ostrea lurida, in 2010 and 2011 (open circles) and from pairs randomly sampled from each year (closed circles).

Deployment and monitoring

1) Deployment and monitoring of oysters in reciprocal transplant experiment

Adult oysters were collected from three locations in Puget Sound (Fidalgo, Dabob, and Oyster Bays) during November and December 2012 (Figure 1). Mass spawning of oysters occurred in June 2013 following several months of conditioning. Larvae were raised in flowing seawater and fed microalgae. Following setting on microcultch, juveniles were cultured in flowing seawater in Port Gamble. In August 2013, 480 oysters (5-10 mm) from each population were planted at Fidalgo, Oyster Bay, Dabob, and Manchester Bays.  At each site, oysters from each population were placed into four 0.61M X 0.61M growout trays (120 each). In each tray, oysters were equally distributed in four 10x7.5cm mesh (1475 micron) bags with 24 oysters glued to ceramic tiles. Trays were  anchored into substrate using using rebar stakes. In late autumn trays at Fidalgo Bay, Oyster Bay, and Manchester were transferred from substrate to a midcolumn hanging system tied to a floating docks. At each site, a HOBOlogger temperature logger (OnSet, USA) was deployed to monitor temperature every 15 minutes.

 

Figure 1. Map of oyster outplanting sites. Field sites include Fidalgo Bay (purple), Dabob Bay (blue), Manchester (red), and Oyster Bay (orange).


Site Monitoring and Growth

Survival and growth were assessed at all sites in December 2013, Dabob in January 2014, and Fidalgo, Manchester, and Oyster Bays in February 2014. Survival was measured by counting all live animals remaining in each tray for each population. Growth was measured via size and weight measurements taken directly from live subsamples collected in the field and measured in the lab. In the lab each of the sampled oysters was measured using calipers for hinge to bill length and weighed with shell to determine whole body weight.


Figure 2. Number of surviving oysters at each field site. Circle color reflects originating broodstock population. Temperature data is displayed to scale with 0C and 5C indicated. Grey arrows indicate when oysters were relocated from substrate to suspension. Asterisk denotes estimate as some trays were inaccessible at time of sampling.



Samples for genetic testing were collected at outplanting in August 2013 for baseline data. Samples were also taken in December 2013. These samples included approximately 32 animals per population at Fidalgo, Manchester, and Oyster Bays. At Dabob we collected 43 from the Fidalgo population, 50 from the Oyster Bay population, and 59 from the Dabob population due to the high mortality. We visited Dabob again in January to collected another 22 from the Fidalgo population, 33 from the Oyster Bay population, and 39 from the Dabob population.


A week of highlights

This week we will be highlighting a few of our accomplishments on our NOAA funded project "Alleviating Regulatory Impediments To Native Shellfish Aquaculture".  The overall goals of this project are to increase our knowledge of local adaptation in Olympia oysters to address concerns that interbreeding between potentially maladapted cultured and wild stocks could negatively impact wild populations. This reporting period marks the deployment and monitoring of oysters in our reciprocal transplant experiment. This field work will be the basis of our characterization for a home field advantage in survival, maturation and growth in Olympia oysters. In addition, over the past year we have continued genetic assessment of restoration aquaculture practices and experimented with anesthesia in mature oysters.

 

Sugartime!

Written by Charles Dueber

Cyclodextrins are composed of sugar molecules bound into a ring called a cyclic oligosaccharide. There are three main types of cyclodextrins: α, β, and γ (shown below). The unique molecular structure of cyclodextrin gives it a hydrophobic interior cavity and hydrophilic exterior, however, the entire molecule is water soluble. This quality has allowed cyclodextrins to be used in the pharmaceutical industry as drug carrier molecules. Recently, it was found that cyclodextrins can be used all by themselves to stimulate the innate immune system. Unpublished data from the University of Wisconsin has shown that zebrafish larvae treated with cyclodextrin and then challenged by lethal concentrations of bacterial LPS had a survival rate of nearly 100% .

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My capstone project is looking at whether β-cyclodextrin can be used to stimulate an immune response in the Pacific oyster. I am interested in this because local shellfish hatcheries have had trouble with outbreaks of the bacteria Vibrio tubiashii which can cause significant mortality to larval and juvenile Pacific oyster production (Elston et al. 2008). Unfortunately, few chemical therapeutants have been approved for use in aquaculture especially for prophylactic use. Cyclodextrin could be a novel immune stimulant that has the potential to enhance shellfish production. I recently performed the experiment for my project which consisted of three treatment groups of oysters: control, β-cyclodextrin exposed, and Vibrio tubiashii exposed. Each group was exposed to their corresponding treatment for 24 hours after which gill and mantle samples were taken. RNA was then isolated from the samples and is currently being used to make cDNA. Expression of defense genes will be analyzed via qPCR to determine if there is a difference between oysters in the control, β-cyclodextrin, and V. tubiashii groups. If you're interested in my project you can take a look at my notebook here.

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