Table_1_Osmoregulation in Barnacles: An Evolutionary Perspective of Potential Mechanisms and Future Research Directions.DOCX

Barnacles form a globally ubiquitous group of sessile crustaceans that are particularly common in the coastal intertidal. Several barnacle species are described as highly euryhaline and a few species even have the ability to colonize estuarine and brackish habitats below 5 PSU. However, the physiological and/or morphological adaptations that allow barnacles to live at low salinities are poorly understood and current knowledge is largely based on classical eco-physiological studies offering limited insight into the molecular mechanisms. This review provides an overview of available knowledge of salinity tolerance in barnacles and what is currently known about their osmoregulatory strategies. To stimulate future studies on barnacle euryhalinity, we briefly review and compare barnacles to other marine invertebrates with known mechanisms of osmoregulation with focus on crustaceans. Different mechanisms are described based on the current understanding of molecular biology and integrative physiology of osmoregulation. We focus on ion and water transport across epithelial cell layers, including transport mechanisms across cell membranes and paracellular transfer across tight junctions as well as on the use of intra- and extracellular osmolytes. Based on this current knowledge, we discuss the osmoregulatory mechanisms possibly present in barnacles. We further discuss evolutionary consequences of barnacle osmoregulation including invasion-success in new habitats and life-history evolution. Tolerance to low salinities may play a crucial role in determining future distributions of barnacles since forthcoming climate-change scenarios predict decreased salinity in shallow coastal areas. Finally, we outline future research directions to identify osmoregulatory tissues, characterize physiological and molecular mechanisms, and explore ecological and evolutionary implications of osmoregulation in barnacles.</p

Table_2_Osmoregulation in Barnacles: An Evolutionary Perspective of Potential Mechanisms and Future Research Directions.DOCX

Barnacles form a globally ubiquitous group of sessile crustaceans that are particularly common in the coastal intertidal. Several barnacle species are described as highly euryhaline and a few species even have the ability to colonize estuarine and brackish habitats below 5 PSU. However, the physiological and/or morphological adaptations that allow barnacles to live at low salinities are poorly understood and current knowledge is largely based on classical eco-physiological studies offering limited insight into the molecular mechanisms. This review provides an overview of available knowledge of salinity tolerance in barnacles and what is currently known about their osmoregulatory strategies. To stimulate future studies on barnacle euryhalinity, we briefly review and compare barnacles to other marine invertebrates with known mechanisms of osmoregulation with focus on crustaceans. Different mechanisms are described based on the current understanding of molecular biology and integrative physiology of osmoregulation. We focus on ion and water transport across epithelial cell layers, including transport mechanisms across cell membranes and paracellular transfer across tight junctions as well as on the use of intra- and extracellular osmolytes. Based on this current knowledge, we discuss the osmoregulatory mechanisms possibly present in barnacles. We further discuss evolutionary consequences of barnacle osmoregulation including invasion-success in new habitats and life-history evolution. Tolerance to low salinities may play a crucial role in determining future distributions of barnacles since forthcoming climate-change scenarios predict decreased salinity in shallow coastal areas. Finally, we outline future research directions to identify osmoregulatory tissues, characterize physiological and molecular mechanisms, and explore ecological and evolutionary implications of osmoregulation in barnacles.</p

Differential expression of <i>NAK1</i> and <i>NAK2</i> in different life stages and tissues.

<p>QPCR was performed to compare the mRNA expression of the <i>NAK1</i> and <i>NAK2</i> genes between adults (n=10) and cyprids (n=5), and between the soma (n=14) and cirri (n=14) within an adult. For NAK1, primers detecting both the short and long N-terminal variants were used. The expression was normalized to the geometric average of five different reference genes. Error bars show the standard deviation. <b>A</b>) There was no significant change of <i>NAK1</i> expression between cyprids or adults (t-test, <i>P</i>=0.485) or between soma and cirri (paired t-test, <i>P</i>=0.206). <b>B</b>) Comparison of <i>NAK2</i> expression between cyprids and adults showed that NAK2 was very lowly expressed in cyprids compared to the adult barnacles (t-test, <i>P</i>=0.036). <i>NAK2</i> expression was also very low in the cirri compared to the soma in the adult barnacle (paired t-test, <i>P</i>=0.002). The same samples were used for analysis of both <i>NAK1</i> and <i>NAK2</i> expression.</p

Correlating pairwise genetic distances between <i>B</i>. <i>improvisus</i> populations to geographical distance and oceanographic connectivity.

<p>Pairwise genetic distance (Φ_ST, COI) for populations of <i>B</i>. <i>improvisus</i> in the Baltic Sea plotted as a function of (a) closest geographical shipping distance (km) (Mantel test: R = 0.46, p = 0.075); (b) minimum oceanographic connectivity between sampling sites; (c) standardised and null allele corrected pairwise genetic distance ((<i>F</i>_ST/1-<i>F</i>_ST) in microsatellites plotted as a function of minimum oceanographic connectivity between sampling sites.</p

Phylogenetic relationships of the cloned <i>B. improvisus</i> NAKs.

<p>A phylogenetic tree was constructed using N-terminal truncated NAK sequences from <i>B. improvisus</i> and other metazoan organisms, e.g. humans, zebrafish, insects, crustaceans, and other invertebrates, as well as choanoflagellates. As outgroup, Na⁺/K⁺ ATPases from the slime mold <i>Dictyostelium</i> were used. In addition, human H⁺/K⁺ ATPase was added. The <i>B. improvisus</i> Nak1 sequence was found in the same clade as other arthropod Nak1 sequences. However, no clear-cut orthologues to the <i>Balanus</i> Nak2 were found. In general, Nak2 sequences have diverged more rapidly in relation to Nak1. Nak1 branches are shown in red, Nak2 branches in green. The divergent <i>Drosophila</i> NAK (CG3701) branch and the <i>C. elegans</i> catp-4 branch are shown in black. Choanoflagellate NAKs are shown in blue. <i>Balanus</i> Nak1 and Nak2 are marked with arrows. Scale bar shows expected number of changes per site. For abbreviations of species names and accession numbers see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077069#pone.0077069.s009" target="_blank">Table S3</a>.</p

Conservation over wide evolutionary distances of potential 14-3-3 binding sites in the N-terminus of NAKs.

<p><b>A</b>) An alignment of the conserved 27 amino acid sequence in the N-termini of NAKs from a wide range of organisms is shown. <b>B</b>) A sequence logo (<a href="http://weblogo.berkeley.edu" target="_blank">http://weblogo.berkeley.edu</a>) of the conserved 27 amino acid sequence from arthropods is shown and potential 14-3-3 binding sites are marked with brackets. For abbreviations of species names and accession numbers see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077069#pone.0077069.s009" target="_blank">Table S3</a>.</p

mtDNA (COI) and microsatellite data for the barnacle Balanus improvisus

These files contain raw data on mtDNA sequences (694 bp of COI, cytochrome oxidase I) from Balanus improvisus, as well as a microsatellite genotype list based on four loci, for B. improvisus. Data include 13 populations on a global scale, covering most of the species current distribution. For further details about the genetic marker development methods, quality of markers and results, we refer to the research article Wrange et al

Differential expression of the splice variants of <i>NAK1</i> in response to various salinities and pCO₂ levels.

<p>QPCR was performed to measure the mRNA expression of the short (NAK1-S) and long (NAK1-L) NAK1 isoforms in a cyprid population exposed for 24 hours to combinations of salinities of 33 PSU or 6 PSU and pCO₂ levels of 970 or 380 ppm. The experiment was performed on four different batches of cyprids for each treatment. Error bars show the standard deviation of the four repeats. In low salinity treatment the expression of the long isoform is increased relative to the short (ANOVA, <i>P</i><0.001). </p

Sampling information and genetic diversity for mtDNA (COI) and microsatellite markers for the barnacle <i>B</i>. <i>improvisus</i>.

Sampling information and genetic diversity for mtDNA (COI) and microsatellite markers for the barnacle B. improvisus.</p

The 27 amino acid stretch of the longer N-terminus of Nak1 is encoded by a distinct exon.

<p>PCR on genomic DNA from a pool of cyprids was performed to investigate the genomic structure leading to the variable N-terminus of Nak1. Two different products (NAK1a and NAK1b) were obtained of about 4.7 and 7 kb, and revealed that the 81 nucleotide insertion, corresponding to the longer variants of the cDNA, is encoded by a separate exon (exon 2). In the Nak1a clones MD or MSMD were found at the predicted translation start, whereas only MSMD was found in the NAK1b clones. L and S stand for long and short form, respectively. </p