Name: the barnacle balanus improvisus as a marine model - culturing and gene expression
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Description: Watch this Scientific Journal Video about The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression at JoVE.com

This research was supported by grant 2017-04559 from the Swedish Research Council (VR) and the EU supported project SEAFRONT to Anders Blomberg. In particular, the establishment of the culturing facility has, over the years, been supported by grants to Per R. Jonsson from the following funding agencies: SSF (Swedish Foundation for Strategic Research) through the program Marine Science and Technology and MISTRA through the program Marine Paint. Kent Berntsson was instrumental in the early phases of setting up the culturingfacility. Additional funding for establishing the culturing facility has come from the Centre for Marine Evolutionary Biology (www.cemeb.science.gu.se), which is supported by a Linnaeus grant from the Swedish Research Councils FORMAS and VR.

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	<li>Darwin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A monograph of the sub-class Cirripedia, with figures of all species. The Balanidae (or sessile cirripedes); the Verrucidae, etc., etc., etc. , (1854).</li><li>Lind, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+aquaporins+from+the+euryhaline+barnacle+Balanus+improvisus+reveals+differential+expression+in+response+to+changes+in+salinity.">Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity.</a> PLoS ONE. 12 (7), 0181192 (2017).</li><li>Lind, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+alpha-subunit+of+Na+/K++ATPase+from+the+euryhaline+barnacle+Balanus+improvisus+reveals+multiple+genes+and+differential+expression+of+alternative+splice+variants.">Molecular characterization of the alpha-subunit of Na+/K+ ATPase from the euryhaline barnacle Balanus improvisus reveals multiple genes and differential expression of alternative splice variants.</a> PLoS ONE. 8, 77069 (2013).</li><li>Fyhn, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holeuryhalinity+and+its+mechanisms+in+a+cirriped+crustacean,+Balanus+improvisus.">Holeuryhalinity and its mechanisms in a cirriped crustacean, Balanus improvisus.</a> Comparative Biochemistry and Physiology - Part A: Comparative Physiology. 53 (1), 19-30 (1976).</li><li>Wrange, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+story+of+a+hitchhiker:+population+genetic+patterns+in+the+invasive+barnacle+Balanus+(Amphibalanus)+improvisus+Darwin+1854.">The story of a hitchhiker: population genetic patterns in the invasive barnacle Balanus (Amphibalanus) improvisus Darwin 1854.</a> PLoS ONE. 11 (1), 0147082 (2016).</li><li>Berntsson, K. M., Jonsson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+patterns+in+recruitment+and+succession+of+a+temperate+marine+fouling+assemblage:+a+comparison+of+static+panels+and+boat+hulls+during+the+boating+season.">Temporal and spatial patterns in recruitment and succession of a temperate marine fouling assemblage: a comparison of static panels and boat hulls during the boating season.</a> Biofouling. 19 (3), 187-195 (2003).</li><li>Lind, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine+receptors+from+the+barnacle+Balanus+improvisus.+are+activated+by+the+alpha2-adrenoceptor+agonist+medetomidine.">Octopamine receptors from the barnacle Balanus improvisus. are activated by the alpha2-adrenoceptor agonist medetomidine.</a> Molecular Pharmacology. 78 (2), 237-248 (2010).</li><li>Semmler, H., Hoeg, J. T., Scholtz, G., Wanninger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+the+naupliar+musculature+and+a+scanning+electron+microscopy+atlas+of+nauplius+development+of+Balanus+improvisus+(Crustacea:+Cirripedia:+Thoracica).">Three-dimensional reconstruction of the naupliar musculature and a scanning electron microscopy atlas of nauplius development of Balanus improvisus (Crustacea: Cirripedia: Thoracica).</a> Arthropod Structure &amp; Development. 38 (2), 135-145 (2009).</li><li>Anderson, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Barnacles - structure, function, development and evolution. , (1994).</li><li>Kennedy, V. 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The authors have nothing to declare.

The barnacle culture at Tj&#228;rn&#246; Marine Research Laboratory (Sweden) has been running over 20 years and has been used for studies in many different research areas. Over 30 scientific papers have been published that have utilized the culturing system during the past years, including studies in antifouling13,22, hydrodynamics23, chemical ecology24, climate change16, evolutionary biology5, and molecular biology2.
To avoid the selection of certain individuals that are more adapted to the laboratory environment (individuals that might not be representative of the wild population), it is recommended to collect a new broodstock from the field each year. In addition, it is also good practice to rejuvenate the culture annually, since there is roughly 50 - 80% of mortality in adults during a normal year. However, if the aim is to produce inbred lines or to set up studies of experimental evolution, only laboratory-reared families are to be used.
A good time to collect B. improvisus on panels at the Tj&#228;rn&#246; Marine Research Laboratory is in June&#8211;August because at that time, there is a good supply of cyprid larvae in the sea. Check the panels weekly to see when the barnacle settlement starts and manually remove other settled species than B. improvisus (e.g., mussels, tunicates, bryozoans, hydroids, nemerteans/tubeworms, and other barnacle species) from the panels (e.g., with a toothbrush). Around Tj&#228;rn&#246;, there are three shallow-water barnacle species present (B. improvisus, Semibalanus balanoides, and Balanus crenatus). However, B. improvisus is the dominant fouler of smooth hard surfaces during July&#8211;August. S. balanoides has its settlement period during early spring and prefers mainly natural substrates (e.g., stones). B. crenatus can occur at low numbers on the panels during the summer.
It is also possible to start new adult barnacle generations from cultured cyprids, which would be essential if certain linages with specific traits have been established, or in studies of experimental evolution. The most convenient way to start new generations of adults is to settle cyprids on thermoplastic panels in the laboratory. These panels with newly settled cyprids could also be used in experimental treatments or for exposure in the field. In emergency cases, one can also use adults on boulders from a nearby site (e.g., Idefjorden in the case of the Tj&#228;rn&#246; Marine Research Laboratory) where B. improvisus is common. These already established adults are treated in the same way as the adults on the panels, thus being placed in trays and fed via the flow-through system. Flow cells can also be used to establish panels with barnacles25. These are flow-through chambers with plankton net on the sides on which the cyprids do not settle, with panels as the only settlement surface for the larvae.
There are several steps that are critical for setting up a long-term functioning barnacle culture including all life stages. The methods used to culture B. improvisus are probably, to a great extent, also applicable to other marine invertebrates with free-swimming, planktotrophic larvae. Culturing procedures for some species are already well described (e.g., for blue mussels and different species of oysters)26, while for other marine invertebrates, there are only a few examples of long-term cultures spanning their whole life cycle. One of the first successful attempts to culture barnacles (B. amphitrite) was done by Rittschof et al.15. Long-term financial and personal resources should be in place before considering setting up a barnacle culturing facility. The maintenance of this kind of year-around barnacle culture requires at least one person working half-time. There may be some potential for the future automation of some steps in the production line, mainly the culturing of microalgae27. In addition, in order to succeed, it is essential to have access to large quantities of high-quality seawater. The culturing of microalgae, Artemia, and barnacles does not involve any particular safety procedures. However, tests of some antifouling substances or toxic chemicals may need special precautions.
The panels were checked several times a week for contaminations. The seawater used in the culture was pumped up from a 40 m depth in the Koster Fjord outside the Tj&#228;rn&#246; Marine Research Laboratory and was passed through two sand filters before entering the lab water system. If no filtering of the water had been done, there would be much more contamination in the culture. It is essential to regularly clean the panels in the culture from detritus and other invertebrates (e.g., stolon-building hydroids and predatory nemerteans) that enter the system through the supply of seawater from the field. For example, if no larvae are produced despite the fact that the culture has been well-fed and otherwise seems to be in good condition, the problem might be the presence of nemerteans that appear to inhibit mating. Naturally, many of the contaminating organisms in the culture at the Tj&#228;rn&#246; Marine Research Laboratory were specific for the Swedish west coast, and other types of contaminating organisms will be prevalent and be more of a challenge in other geographic areas. On the west coast of Sweden, it is unusual to find contamination by other barnacle species on the panels. Occasionally, the establishment of S. balanoides has been found, but this is a very marginal problem (at the most, one S. balanoides contaminant for 10,000 B. improvisus samples). The lack of contaminating species was most likely dependent on the regime to establish new cultures during the summer, when larvaefrom B. improvisus were highly dominant. In addition, there was also a clear enrichment of B. improvisus on the panels since this species is selective for smooth surfaces13.
It is essential to remove dead adult barnacles. If the empty shells are left on the panels, they can become a shelter both for Artemia nauplii as well as for various contaminating species. In addition, it has been noticed that dead individuals influence the well-being of neighboring individuals, probably with the release of toxic compounds during the decomposition. An additional consequence of adult mortality is that some individuals will be left alone and too far away from any other adults to allow mating (even though barnacles have the longest penis in the animal world in relation to its size)28. These individuals will survive but are non-productive for larvae. However, these solitary adult individuals can gently be removed without harming the base-plate and be placed horizontally close to others to enable mating. Barnacles can also be mated by placing panels with one adult on each but close enough so that cross-fertilization can occur. In this way, genetic lines can be produced14.
It is critical to produce a feed of high quality and to feed cultures almost every day. Even a few days without food can result in a decreasing release of larvae. Previous tests of diet composition have shown that diatoms are essential for the growth and survival of barnacle nauplii. Several diatom species seem adequate as feed, although small or solitary cells (less than 10 &#181;m in diameter) may be necessary for the nauplii&#39;s ingestion. The species S. marinoi, C. simplex, and T. pseudonana have all proved to be adequate feed for B. improvisus nauplii, as well as easy to cultivate. In addition, the feed quality is generally higher for exponentially growing algae. It has also been reported that diatoms are essential for establishing productive cultures of B. amphitrite15. A theory of the importance of diatoms is that they have a unique fatty acid profile and are particularly rich in the highly polyunsaturated 20:5 fatty acid29. It has been shown that certain fatty acids are important for the successful development of oyster larvae30.
Over the years, there have been no incidences of detrimental diseases in the barnacle culture. In many commercial invertebrate aquacultures, like oysters and mussels, diseases are rather common and may be very harmful. Detrimental effects of viruses have also been reported from wild populations. The native oyster in France was replaced by the Portuguese oyster Crassostrea angulata in 1925, but this species was wiped out by an iridovirus around 197031. More recently, there have been massive mortality events in the Pacific oyster Crassostrea gigas in cultures worldwide, which appears to be associated with the ostreid herpesvirus 132. No reports on pathogens, bacteria, or viruses on barnacles have been published so far. However, in the ongoing genome-project on B. improvisus, virus sequences were found (Alm Rosenblad et al., unpublished data) but with no apparent link to symptoms of diseases. Mixtures of antibiotics have previously been applied to the cultures to minimize the risk of bacterial infections; however, this procedure is currently abandoned and so far, this has not caused any contamination problems.
If seawater is heated (as described above), overheating may be the most serious risk in the culture production line. It is, of course, difficult to safeguard against overheating, although sensors and appropriate alert systems may be used (e.g., sending e-mail or text messages to responsible persons). Incidences of this kind in the past have resulted in the substantial killing of adults in the culture. This can, of course, be devastating and ruin long-term investments of time and money. In particular, this would be catastrophic if inbred genetic lines have been established. To ensure the longevity of such lines and secure them from accidental loss, it would be desirable to develop a cryopreservation methodology for barnacles. It has been reported that larvae from the Pacific oyster can be frozen down and revived with partial success33. Cryobanking has also been a valuable tool to preserve the genetic resources of a wide range of species34. Even nauplii from B. amphitrite are reported to survive freezing35, and it was found that 20% of the frozen-down individuals successfully metamorphosed into cyprids36. However, applying freezing for the long-term sustainability of cultures has so far not been adopted, but this would indeed be needed for the maintenance of selected lines; this would be an essential step to firmly establishing B. improvisus into a potent marine modelsystem.
Here, a protocol was presented for the dissection of various tissues from adults of B. improvisus (i.e., cirri, soma, and mantel). However, it should be stressed that other tissues can also be extracted. For instance, the soft tissue between the exterior and internal mantle of the membranous-base species Tetraclita japonica formosana has been carefully isolated and used for an RNA extraction and RNA-seq analysis of gene expression37. The outlined optimized extraction protocol described here provides sufficient amounts of high-quality RNA for sequencing from a minimal amount of starting material. First, the collection of individual larvae directly into the homogenization tubes minimizes any loss during the transfer from one tube to another. Furthermore, among the different methods tested, the homogenization with ceramic beads proved to be the most efficient in terms of RNA yield and integrity, compared to sonication or pestle homogenization. When planning for gene expression or genomics experiments, one has to keep in mind the challenge of the high genetic variation in barnacles, at least for B. improvisus. Barnacle has a genetic diversity in the range 3&#8211;5%, even in coding regions (Alm Rosenblad et al., unpublished data). This, of course, puts specific demands on the design of primers for qPCR analysis, where more conserved regions should be identified and used as templates for primers in order to get consistent expression results betweenbatches. Conserved regions for target genes, like aquaporins and Na+/K+ ATPases, can be identified by studying the sequence variability of these genes in RNA-seq data obtained from populations of cyprids containing hundreds of individuals. For a genome analysis, DNA will be sampled. However, obtaining high-quality DNA from B. improvisus can be challenging21.
In conclusion, the established barnacle culture has proven to be instrumental in different kinds of experimental studies. In particular, the all-year-around larval production allows us to conduct experiments without being limited to the naturally occurring spawning period (for B. improvisus, this is during the summer). Obtained larvae can be used to perform a wide set of experimental studies, including settlement assays, behavior assays, expression studies on specific genes, as well as genome-wide transcriptome studies.

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. Most of the 1,200 species of barnacles inhabit shallow water and many are often exposed to low salinity. One species, the bay barnacle Balanus (Amphibalanus) improvises (B. improvisus), can tolerate almost freshwater and Charles Darwin described this species from a small stream in the estuary of Rio de la Plata in Uruguay1. The extreme tolerance tolow salinity makes B. improvisus a particularly relevant model for the studies of osmoregulatory mechanisms2,3. This barnacle prefers brackish conditions but is capable of living in waters with salinities from around 1.6 psu to as high as 40 psu4. It is the only barnacle species found in the brackish Baltic Sea. B. improvisus is believed to originate from the east coast of the American continent but today is found worldwide due to dispersal by shipping5. It is a major fouling organism and is commonly found on rocks, jetties, and boat hulls, and is therefore of general interest for understanding the mechanisms of biofouling on constructions in marine and brackish waters6,7.
Similar to most other barnacles, B. improvisus is hermaphroditic with cross-fertilization; reproduction occurs through mating between neighboring individuals using an elongated penis and internal fertilization. The reproductive period is mainly from May to September. B. improvisus has seven pelagic larval stages (six nauplii followed by one cyprid stage8). The fertilized egg hatches into a nauplius larva which is free-swimming and feeds in the water column for up to several weeks before molting into a non-feeding cyprid larva. The cyprid uses multiple cues to find a suitable site to settle and then undergoes metamorphosis into a sessile juvenile barnacle9. The species can be cultured in the laboratory and has a lifespan of 1&#8211;2 years in the sea (2&#8211;3 years in laboratory culture). On average, B. improvisus grows to 10 mm in diameter (with a maximum of around 20 mm) and reaches a maximum height of about 6 mm (although it can grow taller under crowded conditions). The species can be identified by its smooth calcareous even shell (white or greyish), the radially patterned calcareous base of the shell plate, and the shape of the tergal plates1,10.
The barnacle B. improvisus has several advantageous features as a model for studies of osmoregulation, with a focus on molecular and physiological mechanisms as well as ecological interactions and evolutionary consequences. It is also widely used as a model for the investigations of settling biology, in particular in relation to antifouling research and the mechanisms involved7,11,12,13. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. The ability to culture this barnacle through its whole lifecycle all year round is, therefore, a major asset to enable various types of molecular and mechanistic studies. In addition, its presence in marine/brackish waters worldwide allows for a combination of field and experimental studies. Controlled breeding can also produce families of known pedigrees for long-term culturing14, and a generation time of a few months may allow long-term experimental evolution. There is also a draft genome and several transcriptomes available, and these resources have been used for the cloning of several genes (e.g., genes of importance in osmoregulation)2,3.
The aim of this protocol is to describe how to establish and maintain a culture of the barnacle B. improvisus throughout the year in order to perform gene expression studies on adults or larvae of this organism. Rittschof et al.15 briefly described a method for culturing barnacles from the release of nauplii to the settlement of cyprids for the species Balanus amphitrite. The protocol has been adapted for the all-year-round culturing of B. improvisus at Tj&#228;rn&#246; Marine Research Laboratory (Sweden), and a detailed description of all steps in the production line is outlined, including the production and rearing of barnacle larvae, as well as the administration of feed for adults and larvae. For an overview of the complete procedure, see Figure 1. The use of the culturing system is exemplified with some common experimental set-ups and illustrated in functional genomics studies of Na+/K+ ATPase and aquaporins, elucidating their possible functions in osmoregulation2,3. It is sometimes essential to examine the gene expression in specific tissues, and some of the basics of barnacle dissection will be covered. With a good supply of high-quality seawater, the culturing of the barnacle B. improvisus, and potentially many other species, should be possible in marine laboratories throughout the world.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Plexiglas (poly-methyl methacrylate) panels</td>
			<td style="width:364px;">Plastic produkter, Bromma, Sweden</td>
			<td style="width:153px;"></td>
			<td style="width:385px;">transparent glas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5-L PET bottle</td>
			<td style="width:364px;"></td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artemia</td>
			<td>INVE Aquaculture, Belgium</td>
			<td style="width:153px;"></td>
			<td>We have tested different companies; this is really the best one</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Skeletonema marinoi (CCAP strain 1077/5)</td>
			<td style="width:364px;">CCAP (Culture Collection of Algae and Protozoa); Scotland&#160;</td>
			<td style="width:153px;">strain 1077/5</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Chaetoceros simplex var. gracilis (CCAP strain 1085/3)</td>
			<td style="width:364px;">CCAP (Culture Collection of Algae and Protozoa); Scotland&#160;</td>
			<td style="width:153px;">strain 1085/3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millipore cartridge filter system</td>
			<td style="width:364px;">Millipore&#160;</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cartridge with a nominal pore size of 0.2 &#181;m</td>
			<td style="width:364px;">Millipore&#160;</td>
			<td style="width:153px;">cartridge CWSS01S03</td>
			<td>High capacity for large volumes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polycarbonate bottle</td>
			<td style="width:364px;">Nalgene</td>
			<td style="width:153px;"></td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNA later</td>
			<td style="width:364px;">Qiagen</td>
			<td style="width:153px;">76106</td>
			<td>&#160;Fixation solution to preserve RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">TURBO DNA-free Kit</td>
			<td style="width:364px;">Invitrogen/Thermofisher Scientific&#160;&#160;</td>
			<td>AM1907</td>
			<td>DNAse kit to remove DNA from prepared RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iScript cDNA Synthesis Kit</td>
			<td style="width:364px;">Biorad</td>
			<td>1708890</td>
			<td>cDNA synthesis kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR Green supermix</td>
			<td style="width:364px;">Biorad</td>
			<td>1708880</td>
			<td>Dye for QPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">RNeasy minikit</td>
			<td style="width:364px;">Qiagen</td>
			<td>74104</td>
			<td>RNA extraction of adults or many cyprids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Soft tissue homogenising CK 14, 2 ml tubes</td>
			<td style="width:364px;">Precellys</td>
			<td style="width:153px;">KT03961-1-003.2</td>
			<td>Ceramic beads for homogenisation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">RNeasy micro kit</td>
			<td style="width:364px;">Qiagen</td>
			<td style="width:153px;">74004</td>
			<td>RNA extraction of few cyprids</td>
		</tr>
	</tbody>
</table>

the barnacle balanus improvisus as a marine model - culturing and gene expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

With the described procedure for the culturing of adult barnacles of B. improvisus, up to four batches of nauplius larvae can be produced per week. It would be possible to collect nauplius larvae almost every night, but this requires more people and infrastructure (with many barnacles in the broodstock, a culture will release larvae continuously). An additional limiting factor for the larvae production appears to be the availability of feed of high quality, in particular regarding the diatom Skeletonema. Maximally, each batch from the culturing system consists of roughly 12,000 nauplii, so up to 50,000 nauplii per week can be cultured. However, some weeks there may be up to tenfold fewer larvae produced. A single adult can produce up to 7,000 larvae per day14, which means that 1&#8211;2 adults are releasing larvae for each batch. Within a week, about 70&#8211;90% of the collected nauplii will develop into cyprids (yielding roughly 30,000 cyprids per week, maximally) that can be used for settlement assays and molecular studies.
It should be stressed that there are variations in cyprid features between batches, and in general, there are larger variations between batches than within batches. For example, the settling success in settlement assays varies between 30 and 70% for different batches. Most likely, this is caused by the individual genetic variation between the specific pairs of adults releasing larvae during the different sampling periods. It is, of course, recommended that repeated experiments (biological replicates) should include cyprids from a number of batches if more general statements about the results are to be made. The batch-to-batch variation puts demands on the experimental design, where proper controls and normalizations in gene expression studies should be applied. However, even after several statistical normalization procedures have been implemented that considerably reduce between-batch variation, some effects of the batch are usually still apparent (unpublished data).
Following the provided protocol, it is possible to obtain, on average, 500 ng of high-quality RNA from as little as 20 cyprids, irrespective of the stage of barnacle settlement (Table 1). The quality of the RNA is usually measured as the ratio between the 18S and 28S peaks (the expected position of the two peaks are indicated in Figure 4). However, in the case of barnacles and many other arthropods, the 28S rRNA breaks down when heated (as part of the analysis method) and migrates together with the 18S peak20. This is why there is, in principle, one single rRNA peak in this type of analysis for barnacles. It is clear from this test (Figure 4) that a homogenization by ceramic beads provides the RNA with the highest integrity and is, therefore, the method of choice. The RNA is sufficient in amount and quality to generate high-quality sequencing libraries for sequencing, resulting in an average of 70 million reads per sample (the number of reads, of course, depends on the level of multiplexing during the sequencing). The amount of RNA is also sufficient for cDNA synthesis and qPCR expression analysis of a large number of genes.
Figure 5&#160;shows the result from qPCR analyses of aquaporins and of Na+/K+ ATPase (NAK1) splice variants, where expression changes were investigated in response to changes in environmental cues2,3. A comparison of the relative expression of the long and short splice variants of NAK1 shows a twofold increase for the long NAK1 mRNA in low salinity in relation to the short NAK (Figure 5A). Thus, the data indicate that alternative splicing makes the long form predominant under low salinity conditions. In the case of aquaporins, it is apparent that the two water-transporting paralogs AQP1 and AQP2 display differential expression (Figure 5B). In particular, in the mantle tissue, it is apparent that the AQP1 is substantially down-regulated at lower salinities, which is not seen for AQP2. Instead, AQP2 shows a slightly increased expression at lower salinities, but in the soma. These findings provide a base for investigations of the functional roles of the different B. improvisus ion transporters and aquaporins in barnacle osmoregulation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57825/57825fig1.jpg" /> 
Figure 1: Overview of the whole culturing procedure and the RNA extraction for gene expression studies in adults. To initiate a new culture, panels are attached to a frame and deployed in the sea at 1 - 3 m depth. After several weeks, the panels with adults/juveniles are placed vertically in racks in trays in the laboratory. Each tray holds about 40 panels with adults. With roughly 100 adults per panel, in total &#8776; 4,000 adult individuals are cultured per tray. The adult barnacles are fed with Artemia and can be kept year-around. Nauplius larvae are collected several times per week from the trays via a filtering through a sieve. The collected nauplii are transferred to buckets kept at 26 &#176;C in a water bath and fed with microalgae. Nauplii are reared until they molt into non-feeding cyprids, which are collected by filtering. New panels can be established in the laboratory by the settling of cyprids on panels, either to provide new panels for the year-around culture or to be used for specific experimental set-ups with altered external conditions. RNA is then extracted from juveniles/adults at the end of the experiment or at specific time-points. <a href="https://www.jove.com/files/ftp_upload/57825/57825fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57825/57825fig2.jpg" /> 
Figure 2: Images of some important steps in the culturing procedure. (A) This image shows the frames with panels for collecting new populations from the field. (B) This image shows the panels with adult barnacles of B. improvisus in racks that are placed in trays. The panels are placed about 2 cm apart. (C) This image shows the trays with the barnacle panels and the feeding tank to the left. From each tray, there is an outlet where the sieves are placed for the collection of nauplius larvae. (D) This image shows the production of Artemia feed for the adult barnacles. (E) This image shows the rearing of nauplii in buckets placed in a water bath set to 26 &#176;C. <a href="https://www.jove.com/files/ftp_upload/57825/57825fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57825/57825fig3.jpg" /> 
Figure 3: Description of the initial dissection steps of adult barnacles: removing the body from its shell. (A) Grab one of the opercular plates by inserting forceps gently through the aperture. Pull gently to remove the plate and expose the animal. (B) Pull out the animal by grabbing the soma part just below the cirri. (C) This panel shows the overall anatomy of acorn barnacles. The mantle and potentially fertilized eggs (in the ovary) stay in the shell cavity when the body is pulled out. A note on barnacle anatomy (for a more in-depth account, see Anderson)9: the wall plates of a barnacle slope inward, and together, they form a volcano-like cone. An opening, the aperture, is covered by the two opercular plates, which form a door, or operculum, to close the aperture. Acorn barnacles generally have a calcareous basal plate that is glued firmly to the substratum; however, some species of barnacles lack this calcareous plate (e.g., S. balanoides). Barnacles secrete the exoskeleton from the darkly pigmented mantle (the carapace). The outer surface of the double-layered mantle is calcified to become rigid, while the inner surface of the mantle is not calcified and is therefore flexible. Inside the aperture, the cirri are present in a retracted position. These are the thoracic appendages that barnacles use for suspension feeding. The ovaries are located close to the base of the barnacle, while the testes lie in the soma. This figure has been adopted from Panova et al.21 and has been published with permission from Springer. <a href="https://www.jove.com/files/ftp_upload/57825/57825fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57825/57825fig4.jpg" /> 
Figure 4: Determination of rRNA integrity by capillary gel electrophoresis. RNA was prepared by two different homogenization methods: (A) sonication and (B) ceramic beads. The unit on the y-axis, FU, stands for fluorescence units. <a href="https://www.jove.com/files/ftp_upload/57825/57825fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57825/57825fig5.jpg" /> 
Figure 5: Gene expression results from two different studies of genes important for osmoregulation in B. improvisus.&#160;(A) This panel shows the differential expression as measured by qPCR of splice variants of the Na+/K+ ATPase NAK1 in response to various salinities and pCO2 levels3. In the low salinity treatment, the expression of the long isoform (NAK1-L) is increased relative to the short (ANOVA, P &#60; 0.001). (B) This panel shows the expression of aquaporins in adults of B. improvisus during their exposure to various salinities2. qPCR was used to determine the aquaporin expression levels relative to actin. Adult individuals were incubated at 3 different salinities (3, 20, and 33 PSU) for 14 days. For the RNA preparation, the soma, cirri, and mantle of the adults were separated. In both figures, error bars indicate the standard deviation. The ** and *** indicate the level of significance (ANOVA), 0.01 and 0.001, respectively. These figures have been modified from Lind et al.2,3. Both figures are published with permission from PLoS ONE. <a href="https://www.jove.com/files/ftp_upload/57825/57825fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Settlement Phase</td>
			<td>Total amount of RNA (ng)</td>
		</tr>
		<tr>
			<td>Free swimming</td>
			<td>512</td>
		</tr>
		<tr>
			<td>Exploration: Close search</td>
			<td>518</td>
		</tr>
		<tr>
			<td>Attached cyprid</td>
			<td>550</td>
		</tr>
		<tr>
			<td>Newly metamorphosed juvenile</td>
			<td>832</td>
		</tr>
	</tbody>
</table>
Table 1: Yields of RNA. This table shows the RNA quantity extracted with an RNA preparation kit from pools of 20 cyprid individuals collected at different stages during the settlement process.

Watch this Scientific Journal Video about The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression at JoVE.com

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

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.

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

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

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