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.
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.
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 1–2 years in the sea (2–3 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 Tjärnö 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.
The barnacle culture at Tjärnö 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 Tjärnö Marine Research Laboratory is in June–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 Tjärnö, 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–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 Tjärnö 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 Tjärnö 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 Tjärnö 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 µm in diameter) may be necessary for the nauplii'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 3–5%, 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.
The authors have nothing to disclose.
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.
Plexiglas (poly-methyl methacrylate) panels | Plastic produkter, Bromma, Sweden | transparent glas | |
1.5-L PET bottle | |||
Artemia | INVE Aquaculture, Belgium | We have tested different companies; this is really the best one | |
Skeletonema marinoi (CCAP strain 1077/5) | CCAP (Culture Collection of Algae and Protozoa); Scotland | strain 1077/5 | |
Chaetoceros simplex var. gracilis (CCAP strain 1085/3) | CCAP (Culture Collection of Algae and Protozoa); Scotland | strain 1085/3 | |
Millipore cartridge filter system | Millipore | ||
cartridge with a nominal pore size of 0.2 µm | Millipore | cartridge CWSS01S03 | High capacity for large volumes |
polycarbonate bottle | Nalgene | autoclavable | |
RNA later | Qiagen | 76106 | Fixation solution to preserve RNA |
TURBO DNA-free Kit | Invitrogen/Thermofisher Scientific | AM1907 | DNAse kit to remove DNA from prepared RNA |
iScript cDNA Synthesis Kit | Biorad | 1708890 | cDNA synthesis kit |
SYBR Green supermix | Biorad | 1708880 | Dye for QPCR |
RNeasy minikit | Qiagen | 74104 | RNA extraction of adults or many cyprids |
Soft tissue homogenising CK 14, 2 ml tubes | Precellys | KT03961-1-003.2 | Ceramic beads for homogenisation |
RNeasy micro kit | Qiagen | 74004 | RNA extraction of few cyprids |