Should I stay or should I go? The settlement-inducing protein complex guides barnacle settlement decisions

Abstract

Reproduction in barnacles relies on chemical cues that guide their gregarious settlement. These cues have been pinned down to several sources of settlement pheromones, one of which is a protein termed settlement-inducing protein complex (SIPC), a large glycoprotein acting as a pheromone to induce larval settlement and as an adhesive in surface exploration by the cyprids. Settlement assays in laboratory conditions with Amphibalanus (=Balanus) amphitrite cyprids in the presence of SIPC showed that cyprids exhibit settlement preference behaviour at lower concentrations of SIPC [half maximal effective concentration (EC 50)=3.73 nmol l −1 ] and settlement avoidance behaviour at higher concentrations (EC 50 =101 nmol l −1). By using truncated fragments of SIPC in settlement assays, we identify that domains at the N-terminus of SIPC transduce settlement preference cues that mask the settlement avoidance cues transduced by domains at its C-terminus. Removing the N-terminal 600 amino acids from SIPC resulted in truncated fragments that transduced only settlement avoidance cues to the cyprids. From the sexual reproduction point of view, this bimodal response of barnacles to SIPC suggests that barnacles will settle gregariously when conspecific cues are sparse but will not settle if conspecific cues inform of overcrowding that will increase reproductive competition and diminish their reproductive chances.

Evaluation of Antifouling Potential and Ecotoxicity of Secondary Metabolites Derived from Red Algae of the Genus Laurencia

“…Wells were then blocked by incubation with 5% BSA in TBST (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Wells were then incubated for 1 h at room temperature with the mouse anti-SIPC monoclonal antibody [28] at 1:100 dilution of the cloned cell line culture supernatant in TBST with 5% BSA, washed with TBST (3 × 5 min) and incubated with an HRP-conjugated anti-mouse antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Wells were then washed with TBST (3 × 5 min) and immunostaining was detected using the 20 × LumiGLO ® chemiluminescence kit (Cell Signaling Technology, Danvers, MA, USA) in a Bio-Rad ChemiDoc XRS + Gel Photo Documentation System.…”

“…Evaluation of surface exploration activity of A. amphitrite cyprids in the presence of perforenol ( 9 ) and bromosphaerol. ( A ) A mouse anti-SIPC monoclonal antibody that can detect native SIPC deposited by cyprids of A. amphitrite on the surface of polystyrene tissue culture plates [28] was used in Western blots after A. amphitrite settlement bioassays in the presence of perforenol ( 9 ) and bromosphaerol or artificial sea water (ASW, serving as control). Sterile tissue culture plates were incubated at 25 °C without (blank) or with 10 cyprids each in 2 mL sterile ASW for 24 h in the presence or absence (ASW) of the indicated concentrations of perforenol ( 9 ) or bromosphaerol.…”

“…Barnacle species, and especially the cyprids of the model organism Amphibalanus amphitrite , due to the already established protocols for settlement bioassays [21,22,23], have become one of the most frequently used animals to evaluate the antifouling activity of isolated secondary metabolites or synthetic compounds [18,20,23,24,25,26]. To establish a gregarious mode of settlement, A. amphitrite cyprids deposit a protein named Settlement Inducing Protein Complex (SIPC) which acts as a pheromone cue for their conspecifics to settle nearby and sexually reproduce [27,28,29,30,31,32].…”

Inventory of potential new anti-fouling strategies inspired by nature

“…This stimulates a clustered settlement, which increases the reproductive chances of the group. While low concentrations of SCIP stimulate settlement, higher concentrations have the opposite effect to avoid overpopulation of an area (Kotsiri et al, 2018).…”

The complex barnacle perfume: identification of waterborne pheromone homologues inBalanus improvisusand their differential expression during settlement

“…While the exact mechanism remains unknown, it has been suggested that either variable regions of the SIPC or glycosylation patterns might provide species specificity (Yorisue et al 2012). Further research showed that SIPC has a dual role; it works as a settlement-inducing cue by attracting cyprids, but also as a settlement avoidance cue at higher concentrations informing of overcrowding and increased reproductive competition (Kotsiri et al 2018). A SIPC homologue has also been identified and characterised in Balanus glandula (called MULTIFUNCin) where it induces both gregariousness as well as predation by sea snails (Ferrier et al 2016;Zimmer et al 2016).…”