Electronic data caloxin

Electronic data caloxi

DPM ouabain

DPM ouabai

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

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

Wood et al.PRSB2016_Supporting data

Raw dat

Presence or absence of mucin specific binding of pathogens.

Presence or absence of mucin specific binding of pathogens.</p

Standard curves for calculation of adherent bacteria from luminescent signals.

Luminescence produced by A. hydrophila (A), V. harveyi (B), M. viscosa (C) and Y. ruckeri (D) using the Bac Titer-Glo™ reagent. The standard curves allow for transformation of Signal/noise ratios of luminescent signals into number of adhered bacteria/cm2 surface of the plate well using linear regression. The data points are expressed as mean±SEM and are technical replicates (due to low variation between replicates, the error bars are small, and what looks like symbols in the graph are the error bars). CFU = colony forming unit.</p

Effect of fluid velocity on bacterial binding to mucins.

A and B. A. salmonicida binding to Atlantic salmon skin and distal intestinal mucins increased with growing linear velocity of the surrounding liquid. A. salmonicida bound with higher avidity to skin mucins at a fluid velocity of 2 cm/s (p≤0.05; n = 7) and to distal intestinal mucins at 1.5 cm/s and 2 cm/s fluid velocity (p≤0.05 and p≤0.01, respectively; n = 7). C. A. hydrophila binding to skin mucins was reduced at 2 cm/s fluid velocity (p≤0.05; n = 7). D. A. hydrophila binding was higher at 1.5 cm/s and 2 cm/s fluid velocity compared to the static environment (p≤0.05 and p≤0.01, respectively; n = 5). Data points represent mean±SEM of biological replicates. The results were reproduced twice. Statistics: One-Way ANOVA with Dunnet´s post-hoc test (compared to 0 cm/s velocity).</p

Qualitative analysis of binding specificity of pathogens to Atlantic salmon mucins.

A. Mucins were isolated by isopycnic density gradient centrifugation, and fractions were collected from the bottom of the tube. Fractions were analyzed for carbohydrate content (glycan), DNA content and density. The glycan peak at fractions 8–11 corresponds to the mucins, and for the experiments presented in Figs 3 and 4, the fractions were pooled based on the glycan peak for each sample. B.-D. Examples of pathogen binding patterns to gradient fractions. B. Low avidity, mucin-specific binding of M. viscosa to an Atlantic salmon skin sample. The binding signal is low but exceeds that of the control and follows the mucin glycan peak (fractions 9–11 in this sample). C. High avidity, mucin-specific binding of A. hydrophila to Atlantic salmon gill sample. The binding follows the mucin glycan peak (fractions 7–10), although avidity appears stronger to a low density glycoform of the main mucin peak (i.e. the binding curve is shifted slightly to the right of the main mucin peak). D. Absence of Y. ruckeri binding to a pyloric cecal sample. The binding signal to the sample is lower than to the non-mucin control and does not follow the mucin glycan peak (fractions 9–11). Binding is expressed as signal/noise luminescence. The horizontal dashed lines denote the binding signal of bacteria to the plastic well. The results of all these qualitative binding analyses are summarized in Table 1. Abbreviations: EU count = europium count; Lum = luminescence, expressed as signal/noise (S/N).</p

Quantitative analysis of pathogen binding to Atlantic salmon mucins.

Mucin containing fractions from individual fish and tissue sites were pooled according to the method shown in Fig 1A (n = 5 for each tissue). Pathogen binding to each of these 25 samples was analyzed using the Bac Titer-Glo method, and the luminescence signals were transformed to CFU/cm2 according to standard curves for each pathogen (Fig 2) to allow comparison of binding levels between pathogens. A. A. hydrophila binding to gill mucins was higher compared to the proximal intestinal mucins: p≤0.05; n = 5). B. V. harveyi bound with no distinguishable organ preference (p = n.s.). C. The level of M. viscosa binding differed between mucin groups (distal intestine vs. skin and gill: p≤0.05). D. Y. ruckeri bound to proximal and distal intestinal mucins more than to gill mucins (p≤0.01 and p≤0.05). Bars denote median ± interquartile range of biological replicates, after subtracting the background signal. The results were reproduced twice. Statistics: Kruskal-Wallis test by ranks with Dunn´s Post Hoc test to compare binding to mucins from different epithelial sites. The numerical p values on the graphs show the result of the test, without the post hoc test. Abbreviations: Pyloric = pyloric cecal mucins; Proximal = proximal intestinal mucins; Distal = distal intestinal mucins.</p