The Barnacle Balanus improvisus as a Marine Model – Culturing and Gene Expression

1. Collection of Adult Barnacles in the Field to Start a New Broodstock

1. Deploy thermoplastic (poly-methyl methacrylate) transparent panels (110 x 110 x 1.5 mm³) at ≈ 1–3 m depth in calm waters in order to collect adult barnacles from the field. Use frames that can take several panels (Figure 2A), drill 2 holes (with a diameter of 6 mm) in the top corners of each panel and attachthepanelstotherackusingcableties. Thus, thepanelswillhangverticallyin the water.
   1. When the settlement of barnacles has occurred (identified as small white hard dots on the panels), leave the panels for at least 2–3 weeks to make sure that the barnacles are large enough (5 mm) to be transferred to the lab without getting dried or damaged.
2. Bring the panels into the laboratory when they are covered with settled B. improvisus that have reached a size of ca. 5 mm indiameter.
   NOTE: The time for adults to develop to 5 mm in the field is highly dependent on the availability of food and the temperature. Normally, this takes about 3 weeks at the Tjärnö Marine Research Laboratory (58.87 °N, 11.14 °E). Most commonly, panels for the new broodstock are generally deployed in late June and collected at the end of August.
3. Before introducing the panels into the culture facility, clean other organisms from the panels. It is most important to remove other barnacle species. Please note that it is important to frequently clean the panels to get rid of contaminating species during the cultivation since this will significantly increase the chances of survival of the broodstock.
4. Place the panels vertically on edge in a stand with milled grooves in a polyethylene tray (400 x 400 x 20 mm³) (Figures 2C and 2E). The grooves in the stand are 15 mm apart.
5. In order to prepare the trays, make an entrance port at the base and an exit port at the top on the opposite side of each tray. Connect the entrance port of each tray to a 100 L reservoir containing about 30 L of water with an inlet of open seawater at 20 °C to allow a flow-through at a rate of ca. 2 L per min.
   NOTE: Up to 8 trays were connected to a single 100 L reservoir.
6. Connect the 100 L reservoir to temperature-controlled seawater (20 °C) (e.g., provided by a heat pump exchanging heat from the incoming seawater).
   NOTE: The barnacle B. improvisus can grow and reproduce at a full marine salinity (30–35 psu); however, reducing the salinity to ca. 25 psu by adding freshwater to the 100 L reservoir increases the output of larvae from the culture.

2. Starting New Generations of Adults from Cultured Cyprids

1. Construct cubes of thermoplastic panels open at the top by taping 5 panels together using a non-toxic, water-resistant tape.
2. Place the cube in a small tray (in case of leakage) and fill it with seawater (25 psu). Keep the water at room temperature (ca. 20 °C). Add roughly 200 cyprids at the top of the cube.
3. Feed the juveniles with 100 mL of Skeletonema marinoi every other day (see step 5) while in the cube.
   NOTE: Juvenile barnacles can have difficulties with ingesting Artemia salina (see step 3) since they are big and, hence, difficult to swallow for a newly settled barnacle.
4. Change the water 1x a week.
5. After 2 weeks, when the panels contain enough established juveniles of at least 5 mm in diameter, take the cube apart and move the panels with the juveniles to trays with flow-throughseawater and, thereafter, feed the barnacles with A. salina nauplii.

3. Culture of Artemia salina Nauplii as Feed for Adult Barnacles

1. Set up a culturing system for A. salina by using a 1.5 L plastic bottle where the bottomis cut off and the bottle is placed upside-down in a stand and illuminated from the side. Fit the bottleneck to a silicon tube with a clamp and attach it to an aeration pump (Figure 2D).
2. In order to hatch the eggs, add about 15 mL of dry resting eggs of Artemia to 1 L ofseawater.
   NOTE: Artemia eggs hatch into nauplius larvae after 24–48 h. To delay the hatching of A. salina nauplii (e.g., during weekends), the light can be turned off to reduce the temperature slightly.
3. To harvest Artemia nauplii, turn off the aeration and darken the upper part of the bottle with aluminium foil (or similar e.g., a can).. Illuminate the lower part of the bottle for 10 min. Hatched Artemia nauplii will swim toward the light. Non-hatched cysts will sink to the bottom, and cyst shells will float at the surface.
4. Open the clamp at the bottom. Collect the intermediate fraction as a dense population of the swimming nauplii.
   NOTE: Every day (except on weekends), 1 L of the dense Artemia nauplii suspension was manually added to the 100 L reservoir connecting to the trays with the adult barnacles. Avoid any feeding with empty cyst shells, since they do not provide nutrition and mainly result in needing to clean the culture at more frequent intervals.

4. Collection and Rearing of Barnacle Larvae

1. Clean the panels with adult barnacles by gently spraying them with freshwater and, if necessary, remove any fouling from the barnacle shells and panels using a soft toothbrush. Also, clean the trays (without barnacles) and tubes in hot (75 °C) freshwater.
2. Place a sieve made of a 90 µm plankton-net glued to the end of a cut PVC pipe (16 cm in diameter, 15 cm in height) into a polyethylene tray (30 x 20 x 10 cm³) with an overflow port. Collect the B. improvisus larvae in the sieve overnight (Figure 2C).
   NOTE: The sieve was positioned just below the exit port of the tray of the barnacle panels to receive the outflowing water and to filter out the nauplii. The larvae remained in the sieve while the seawater overflowed the small tray. This small tray ensured that the sieve never driedout.
3. Place a 30 L bucket in a large water bath where the temperature is maintained at 26 °C using aquarium fish tank heaters (Figure 2E). Aeration and agitation are assured by air-bubbling through the system.
4. Fill the bucket with 20 L of filtered seawater (0.2 µm; 25 psu). Add 1 L to the bucket of a 60/40 mix of the 2 diatom microalgae, S. marinoi and C. gracilis (see step 5). This will give an initial density of about 5 x 10⁴ diatoms per mL in the bucket.
   NOTE: Barnacle nauplii larvae are positively phototactic. The buckets were made of opaque white plastic with lids that let through some light. During the winter, the room was lit during the daytime (8:00–17:00), but dark during the nighttime. During the summer, the outside light came into the room during most of the day.
5. Transfer the collected B. improvisus nauplii to a crystallizing dish (300 mL; 90 mm in diameter; 50 mm in height).
6. Illuminate the dish from the side, which will attract the larvae to the light source. Collect the barnacle nauplii that gather at the incoming light with a pipette and transfer them to another crystallizing dish. Remove any remaining Artemia larvae.
7. Transfer the B. improvisus nauplii larvae to a beaker with 1 L of filtered seawater.
8. Count the number of nauplii by stirring the beaker gently to get an even suspension of larvae and then taking five 1 mL samples from different places in the beaker. Transfer each sample into a microplate for a visual inspection with a stereomicroscope. Count the number of nauplii in each of the five samples and add them up.
9. Multiply the counted number by 200 (1,000 mL/5 mL) to estimate how many thousands of larvae are in the whole beaker. If there are too many nauplius larvae in the beaker, dilute the sample until the density is at most 14,000 larvae/L (usually in the range of 11,000–12,000 larvae/L).
10. Add 1 L of B. improvisus nauplii to one bucket, thus adding roughly 11,000–12,000 larvae per bucket.
    NOTE: Make sure not to add more nauplii, since this will result in too little food in relation to the larvae and, hence, increase mortality.
11. After 3 days, collect barnacle nauplii on a 90 µm sieve. Then clean the bucket (with 75 °C freshwater), fill it with filtered seawater, add a new diatom feed (the same amount as at the start), and finally add the nauplii again. Cyprids start to appear after about 6 days (± 1 day).
12. Collect cyprids first with a 90 µm sieve (designed as in point 4.2 above). Then separate non-moulted barnacle nauplii and cyprids with a 320 µm sieve on top of a 160 µm sieve. B. improvisus nauplii will collect in the 320 µm sieve and cyprids in the 160 µm sieve.
    NOTE: The cyprid larvae may be stored at 10 °C in a crystallizing dish in darkness for later use, up to ca. 6 days. However, the storage can affect the quality and performance of the barnacle larvae, so experiments comparing different treatments should ideally use larvae from the same batch (and similar storage time) to avoid confounding effects¹⁶.

5. Culture of Microalgae as Feed for Barnacle Nauplius Larvae

NOTE: Algae were grown in 3 different types of cultures: (i) stock cultures, which are for the long-term maintenance of the strains that were being used for the inoculation of the scaling-up; (ii) start cultures, which are the first step in the scale-up; and finally (iii) the production culture, which is the final production scale of large quantities of algae as barnacle feed.

1. Order diatom species from the Culture Collection of Algae and Protozoa (CCAP) to be used as feed for the barnacle nauplius larvae. The 2 species Skeletonema marinoi (CCAP strain 1077/5) and Chaetoceros simplex var. gracilis (CCAP strain 1085/3) both give good results as feed.
2. Filter all seawater to be used in the algal cultures, using a cartridge filter system with a nominal pore size of 0.2 µm. (The filtered seawater is also to be used for the hatching of A. salina eggs, and the culturing of barnacle nauplius larvae.) Autoclave the filtered seawater for the algal cultures (at 105 °C for 5 min).
3. Prepare a medium for the culture of microalgae, using autoclaved seawater enriched with Guillard's f/2 solution containing inorganic nutrients, trace metals, and vitamins (see Guillard 1975¹⁷ for the detailed recipe). In addition, prepare a solution of silicate (Na₂SiO₃), but keep this separate from the f/2 enrichment to prevent a solid precipitation.
   ​NOTE: The concentrations of the enrichment stock solution and the stock solution of silicate were prepared to be used as a 1 mL addition of each to 1 L of seawater.
4. Autoclave all equipment used in the algal culture at 120 °C for 20 min. Autoclave screw-capped test tubes with 1 mL of f/2 enrichment and 1 mL of silicate solution. Add the enrichment and silicate solutions to theseawater when the autoclaved glassware and liquids have cooled down to room temperature.
5. Grow stock cultures of microalgae in 40 mL test tubes with screw caps.
   1. Fill the test tubes with about 30 mL of the enriched seawater. Inoculate the cultures with a sterile Pasteur pipette using about 1 mL of a 2 week old stock culture. The screw cap is then fitted but not tightened,to allow some gas exchange. If available, the inoculation should be carried out in a laminar flow cabinet to reduce the risk of contamination.
      NOTE: The aim of the stock cultures is to maintain algal cultures on a long-term basis and to serve as inoculum for start cultures. Stock cultures were re-inoculated every 2 weeks.
   2. Expose the new stock culture to a white light with an intensity of ca. 25–50 µmole m^-2s^-1 (with a light-dark cycle of 16:8 h). Move the old stock cultures to a low light intensity as a back-up. Discard any 2 week old stock cultures.
6. For the scaling up to the production culture of algae, inoculate the start cultures from the stock culture and grow them in 500 mL Erlenmeyerflasks.
   1. Autoclave 4 Erlenmeyer flasks, pipettes and cotton stoppers with 300 mL of filtered (0.2 µm) seawater, and 4 test tubes with 0.3 mL of f/2 enrichment and 4 with silicate solutions. Cool down the f/2 enrichment and the silicate to room temperature, and add the solutions to the Erlenmeyer flasks. Inoculate them with ca. 1 mL of the 2-week old stock culture.
      NOTE: Prepare 2 flasks with S. marinoi and 2 with C. simplex.
   2. Put the flasks on a shaking table in a light intensity of 50 µmole m^-2s^-1. When the start cultures have acquired dense algal populations (yellow-brown in color), they are ready to be used as inoculum for the production cultures that will provide the barnacle larvae with food.
7. Grow production cultures in 4 L polycarbonate bottles with silicone stoppers with 2 drilled ports fitted with glass tubes.
   1. Connect 1 glass tube reaching to the bottom of the bottle to an air pump via a silicone tube fitted with a 0.2 µm air filter. Make sure the second glass tube ends just below the silicone stopper and is filled with cotton to allow the exit of injected air.
   2. Each week, fill four 4 L bottles with filtered seawater and autoclave them together with the stoppers. In addition, autoclave 4 test tubes with 4 mL of f/2 enrichment and silicate solutions.
   3. After cooling, add the f/2 enrichment and silicate solutions to the bottles and then inoculate them with half of the volume of the start-up cultures in the Erlenmeyer flasks.
   4. Place the four 4 L bottles at a light intensity of 50–100 µmole m^-2s^-1.
   5. Harvest the production cultures after ca. 1 week; they are then sufficiently dense to be used for feeding the barnacle nauplius larvae.
      NOTE: Production cultures have a lifespan of about 2 weeks, which means that there are 8 active production bottles at any time.

6. Designing Experimental Studies Using Barnacles

1. Place panels with juvenile or adult barnacles in controlled aquaria where they can be grown under identicalconditions. This is called a common-garden experiment, which, for instance, can be used to understand local adaptions or phenotypic plasticity¹⁸, or to study gene expression changes in relation to external factors (e.g., salinity, temperature, or pH).
   ​NOTE: It is also possible to use barnacles directly collected from the field on panels. An advantage of using laboratory-bred individuals is that maternal effects can be avoided when using the next generation of offspring.
   1. Expose barnacles to the specific environmental conditions during a chosentime interval, followed by the harvesting of adults (e.g., for studies on gene expression)².
2. For experiments with cyprid larvae to study gene expression³, place the cyprids in controlled aquaria where larvae can be cultivated under identical conditions.
   1. Harvest the cyprids after specified time periods by filtering with sieves (as described in step 4.7), and extract RNA according to the protocols below (step 8).

7. Dissection of Barnacles

1. Clean the lab space where the dissection and DNA sampling are performed, bothbefore and between individuals, including all dissection tools. This is done using chlorine for the bench (or 96% ethanol for the forceps).
   NOTE: Labelled tubes containing fixation media (ethanol or an RNA stabilization solution) are prepared in advance.
2. Select large and short-term starved individuals (do not feed them for 2 days prior to the dissection). Clean the barnacle shell with a toothbrush to minimize the risk of contamination from other species (e.g., bacteria, algae) and rinse it off with water.
   ​NOTE: The reason for the short-term starvation of individuals before the dissection is to avoid any DNA contamination (e.g., from Artemia cysts in the gut).
3. Place the individual barnacles on an even surface, either attached to a panel or loose in a dish. Dissect the respective tissues from the adult. There are several ways to dissect barnacles, depending on the purpose of the study.
   1. Dissection method A: fixing the whole barnacle as quickly as possible.
      1. Remove the whole barnacle from the panel by using a scalpel, inserting it under the barnacle and close to the panel surface.
         NOTE: In most cases, this manipulation leaves the basal plate almost intact, thus not affecting the barnacle inside.
      2. Carefully crack the outer shell on one side by inserting forceps (Figure 3). This is done to facilitate the intrusion of a fixating medium inside the shell.
         NOTE: If the shell is not broken at all before placing the barnacle in a test tube, it may lead to a slower fixation process and an inferior quality of DNA later.
      3. Put the broken barnacle in the test tube containing either an RNA storage solution or ethanol. Leave the barnacle in the solution for at least 24 h for its fixation. When the barnacle is fixed, the animal can be taken out of the fixation media and placed on a dissection tray. The cirri, mantle, and soma (body) can be separated from each other and placed in separate tubes for further extractions of DNA or RNA.
         NOTE: It is important to observe if egg lamellae with fertilized eggs are present inside the barnacle. If present, these should be removed before proceeding with the DNA extractions to avoid finding multiple genotypes in the sample.
   2. Dissection method B: removing the barnacle from the shell before its fixation.
      ​NOTE: This method may not always result in the mantle being sampled since it is attached to the inside of the calcareousshell. But it can be a quick method for sampling DNA from an individual.
      1. Carefully remove, by using dissection forceps, the tergal and scutal plates (Figure 3) by inserting the tip of the forceps between the tergal and scutal plates, grabbing hold of a plate, and pulling gently to remove them.
      2. Grab hold of the cirri with the forceps and pull the barnacle straight out and place it directly in 96% ethanol or RNA stabilizing solution for fixation..
         NOTE: Sometimes, the mantle will also be sampled at the same time, as seen as a thin epithelium with dark pigmentation (in B. improvisus). Otherwise, the mantle can be removed from the inside of the shell using a scalpel and, thereafter, placed in ethanol or RNA stabilizing solution.
         NOTE: The tergal and scutal plates (if intact) can be dried and saved since they are useful for the species' identification¹⁰. A general recommendation, if there is any doubt of species, is to also photograph the whole barnacle prior to initializing the dissection.

8. RNA Extraction for Quantitative PCR

1. Put adults to be used for RNA extractions into an RNA stabilizing solution, incubate them overnight (up to 24 h) at 4 °C, and then store them at -80 °C.
   NOTE: Cyprid larvae are preferentially dry-frozen, without RNA later directly in -80 °C, by placing them in cryotubes and then submerging the tubes briefly (30 s) in liquid nitrogen before storing them at -80 °C.
2. At the time of the RNA extraction, thaw the barnacles on ice and use them intact or dissect out the tissues to be used (see step 7).
   NOTE: Due to high genetic diversity between individuals (≈ 3–5% variation in coding regions; Alm Rosenblad et al., unpublished data) it is advisable to poola number of adult individuals, which minimizes the effect of sequence variation between individuals in the later qPCR step.
3. For the RNAextraction, add 350 µL of lysis buffer provided with an RNA preparation kit into homogenization tubes containing 2.8 mm of ceramic beads.
   NOTE: Ceramic beads provide a better yield of RNA compared to sonication (Representative Results).
4. Take an adult barnacle (whole or tissues) or collect a minimum of 20 cyprid larvae using forceps and put them into homogenization tubes.
5. Proceed directly to the disruption and homogenization with a bead mill.
6. Put the homogenization tubes with the sample and beads in the bead mill holder. Shake them with a frequency of 4.0 m/s for 20 s. Cool the sample for 1 min on ice. Repeat 2x.
7. Prepare the RNA according to the protocol of the commercially available RNA isolation kit.
   NOTE: A risk assessment (including a reading of the safety data sheets) should be performed prior to using RNA extraction methods, to identify hazardous chemicals that require the use of a fume hood and other protective clothing, including gloves (e.g., the addition of β-mercaptoethanol to the lysis buffer during the RNA extraction should be performed in a fume hood).
8. Quantify the RNA. Prepare a working solution and add a standard and samples to a total volume of 200 µL. Vortex them for 2–3 s and incubate them for 2 min. Insert the tubes into a fluorometer and take readings.
9. Check the RNA samples for any protein contamination with a spectrophotometer, and check their RNA integrity with an automated electrophoresis system (Representative Results).
   NOTE: For good quality preparations, RNA is recommended to have a ratio of 260/280 nm of 2–2.2 and a DNA of around 1.8. Lower values indicate protein contaminations and that the extraction procedure has to be optimized.

9. Gene Expression: cDNA Synthesis and qPCR

1. Treat the obtained RNAwith DNase to remove any remainingDNA before making the cDNA for qPCR.
   1. Check for contaminating genomic DNA by adding an RNA sample but no reverse transcriptase when preparing the cDNA. If there is a PCR amplification of the selected gene on the cDNA made without reverse transcriptase, there is DNA contamination in the RNA.
      NOTE: For samples to be used for RNA-seq (RNA-sequencing using next-generation sequencing technology), the DNase step is less critical. In particular, for samples with low levels of RNA, the DNase step might be omitted in order not to lose too much of the RNA in the process.
2. Perform cDNA synthesis on DNase-treated RNA using an amount of RNA within the range specified in the commercial cDNA synthesis kit, but usually, use at least 50 ng.
3. Design qPCR primers so that they anneal to parts of the gene of interest where the sequence identity between individuals is as high as possible, but the sequence identity between gene paralogs is as low as possible. Please see the corresponding publications for the specific primer-pairs used for gene expression studies of Na⁺/K⁺ ATPase³ and aquaporins² (Figure 5).
   NOTE: For this purpose, RNA-seq sequence data from hundreds of cyprids or several adults can be used.
4. Design primers for a gene to be used for the normalization of the expression levels (e.g., actin). The primers used successfully for actin are, forward: 5'-CATCAAGATCAAGATCATCGC-3', and reverse: 5'-ATCTGCTGGAAGGTGGAC-3'.
   NOTE: Actin is a commonly-used control gene. However, others have also been suggested as a reference and tested out on barnacles¹⁹. Besides actin, the use of RPL8, 36B4, EF1, and NADHd1 as a reference has been tested³. It was concluded that there are no large differences in the expression levels of the respective reference genes between soma, cirri, adult, or cyprids.
5. Optimize the annealing temperature for the designed primers by adding increasing amounts of cDNA (typically in the range of 0.25–50 ng) to a mix containing SYBR Green, dNTP, polymerase, and 0.3 µM each of forward and reverse primer.
6. Run qPCR protocols at different annealing temperatures as follows: an initial denaturation temperature of 95 °C for 3 min, a denaturation step at 95 °C for 20 s, annealing temperatures of 55–63 °C for 20 s, and an elongation at 72 °C for 30 s. In total, run 40 PCR cycles.
   NOTE: Primer efficiencies should lie in the range of 90–105% and are calculated as E = (10^(-1/slope)-1) x 100 where the slope is the slope of the curve obtained when plotting the log value of the cDNA concentrations against their cycle threshold (Ct) values.
7. Perform qPCR using an appropriate amount of cDNA, usually 1–10 ng, using the PCR protocol described in step 3 with the optimum annealing temperature found.
   NOTE: The higher amount of cDNA is only used for genes that are expressed in extremely low amounts.