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The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression
Chapters
Summary August 8th, 2018
The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.
Transcript
The culture facility can help answer key questions within larval ecology, biofouling, and evolutionary biology. More specifically, it can be used in studies on receptor-ligand interactions during larval settlement, development of new antifouling technologies, and the evolution and function of genes involved in osmoregulation. The main advantage of this model organism is that the barnacle Balanus improvisus is robust, reproduces all year round, and has a relatively short generation time.
Though this method can provide insight into the culturing of barnacles, it can also be applied to the culturing of other marine organisms. Generally individuals new to this method will struggle because of the large number of details that are important for success, such as the supply of good seawater, robust production of high-quality feed, and a constant attendance to cleaning and feeding the cultures. Demonstrating the procedure will be Martin Ogemark and Anna-Lisa Wrange, technician and researcher working with barnacles.
To begin, set up frames that can hold several transparent thermoplastic panels. Drill two holes in the top corners of each panel and use cable ties to attach the panels to the frames. To collect adult barnacles, deploy the panels vertically at a depth of one to three meters in calm waters.
After allowing barnacles to settle and grow to five millimeters in diameter, bring the panels into the laboratory. Then place the panels vertically in a stand with milled grooves in a polyethylene tray. First, using non-toxic, water-resistant tape, tape five panels together to create a cube that is open at the top.
Place the cube in a small tray and fill it with seawater. Next, add approximately 200 cyprids at the top of the cube. Every other day, feed the juvenile barnacles with 100 milliliters of Skeletonema marinoi.
After two to three weeks, the panels of the cube should contain established juveniles that are at least five millimeters in diameter. Take the cube apart, and move the panels to trays with flow-through seawater. To harvest Artemia nauplii, first turn off the aeration pump and darken the bottle, then illuminate the lower part of the bottle for 10 minutes.
The hatched Artemia nauplii will swim toward the light. The non-hatched cysts will sink to the bottom, while cyst shells will float to the top. Open the clamp at the bottom and collect the intermediate fraction as a dense population of the swimming Artemia nauplii.
To clean the panels with adult barnacles, gently spray them with freshwater and remove any fouling with a soft toothbrush. Clean the trays without barnacles and the tubes with hot freshwater. Next, place a sieve into the polyethylene tray with an overflow port.
Collect the barnacle larvae that settle in the sieve overnight. Then fill a bucket with 20 liters of filtered seawater and one liter of a diatom microalgae mixture to create a density of 50, 000 diatoms per milliliter. Transfer the previously collected barnacle nauplii to a crystallizing dish and illuminate the dish from the side.
Then use a pipette to collect the nauplii that swim towards the light and transfer them to another dish. Transfer the nauplii to a beaker containing one liter of filtered seawater for counting. Next, add one liter of nauplii to the previously-prepared bucket.
After three days, use a 90-micrometer sieve to collect the barnacle nauplii, then clean the bucket, fill it with filtered seawater, and add new diatom feed and the barnacle nauplii. After two to three more days of incubation, use a 320-micrometer sieve on top of a 160-micrometer sieve to separate the non-molted barnacle nauplii from the cyprids. After setting up four four-liter bottles for microalgae culture, place the bottles in light at intensity of 50 to 100 micromoles per square meter.
Harvest the cultures when they are sufficiently dense to be used for feeding. First, select large individuals and withhold food for two days prior to dissection. Then, clean the barnacle shell with a toothbrush and rinse with water to minimize contamination from other species present in the environment.
Place an individual barnacle on an even surface for dissection, then insert the tip of a pair of forceps between the tergal and scutal plates. Take hold of one plate and pull it gently to remove it. Next, use forceps to take hold of the cirri and pull the barnacle straight out.
Finally, place the dissected barnacles directly in 96%ethanol or RNA-stabilizing solution for fixation. With the protocol described here, up to four batches of barnacle nauplius larvae can be produced in one week. Maximally, each batch from the culturing system consists of approximately 12, 000 nauplii, which means that up to 50, 000 nauplii can be cultured per week.
Within a week, 70 to 90%of the collected nauplii will develop into cyprids, yielding roughly 30, 000 cyprids per week. An average of 500 nanograms of high-quality RNA was obtained from as few as 20 individual cyprids regardless of settlement stage. In barnacles, 28S rRNA breaks down when heated.
Homogenization by ceramic beads provides RNA with the highest integrity, thus making it the preferred method for handling barnacle and arthropod tissue. Using qPCR analysis, a two-fold increase was observed for the long variants of NAK1 mRNA and low salinity compared to the short NAK. This indicated that the long form is predominant under low salinity conditions.
A few weeks of training is required to master the different steps involved in the barnacle culture. While attempting this procedure, it is particularly important to minimize contamination in all steps of the culturing workflow. After its development, this culturing technique has been instrumental for giving us a better understanding of barnacle ecology, physiology, and molecular biology.
It is also an important cornerstone in making the barnacle Balanus improvisus into a potent marine model system.
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