© 2018. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2018) 221, jeb185348. doi:10.1242/jeb.185348 RESEARCH ARTICLE Should I stay or should I go? The settlement-inducing protein complex guides barnacle settlement decisions Manto Kotsiri1, Maria Protopapa1, Sofoklis Mouratidis1, Michael Zachariadis1,2, Demetrios Vassilakos1, Ioannis Kleidas1, Martina Samiotaki3 and Skarlatos G. Dedos1,* ABSTRACT a preferred substrate for settlement, after which it metamorphoses Reproduction in barnacles relies on chemical cues that guide their into a juvenile barnacle, and then the adult stage, in which it is gregarious settlement. These cues have been pinned down to several capable of sexual reproduction (Høeg and Møller, 2006). Early sources of settlement pheromones, one of which is a protein termed observations on the surface exploration behaviour of barnacles settlement-inducing protein complex (SIPC), a large glycoprotein (Yule and Walker, 1985; Yule and Crisp, 1983; Crisp and acting as a pheromone to induce larval settlement and as an adhesive Meadows, 1962; Crisp, 1961) led to the discovery of the in surface exploration by the cyprids. Settlement assays in laboratory proteinaceous nature of the signals that these animals use to conditions with Amphibalanus (=Balanus) amphitrite cyprids in the inform their conspecifics of their presence and thus increase their presence of SIPC showed that cyprids exhibit settlement preference reproductive chances (Matsumura et al., 1998b; Dreanno et al., behaviour at lower concentrations of SIPC [half maximal effective 2006c). Several earlier reports (see Rittschof and Cohen, 2004; −1 Rittschof, 1990) and further research have shown that this concentration (EC50)=3.73 nmol l ] and settlement avoidance −1 reproductive strategy comes with a cost, because the same behaviour at higher concentrations (EC50=101 nmol l ). By using truncated fragments of SIPC in settlement assays, we identify that chemical cue acts as a potent feeding stimulant for a barnacle domains at the N-terminus of SIPC transduce settlement preference predator, the whelk Acanthinucella spirata (Zimmer et al., 2016). cues that mask the settlement avoidance cues transduced by Intense research efforts culminated in the discovery of settlement- domains at its C-terminus. Removing the N-terminal 600 amino inducing protein complex (SIPC), a glycoprotein that induces acids from SIPC resulted in truncated fragments that transduced conspecific larval settlement in Amphibalanus amphitrite (Darwin only settlement avoidance cues to the cyprids. From the sexual 1854) (Matsumura et al., 1998b; Dreanno et al., 2006a,b,c). reproduction point of view, this bimodal response of barnacles Originally shown to comprise 3 subunits of 98, 88 and 76 kDa to SIPC suggests that barnacles will settle gregariously when (Matsumura et al., 1998b), cloning of the cDNA that encodes for conspecific cues are sparse but will not settle if conspecific cues SIPC from A. amphitrite revealed that SIPC is a single protein of inform of overcrowding that will increase reproductive competition and 1547 amino acids with a calculated molecular mass of 171.7 kDa diminish their reproductive chances. (Dreanno et al., 2006c). It is firmly established that SIPC is an N-linked glycan-containing protein and that glycans, such as KEY WORDS: Barnacles, Amphibalanus amphitrite, Settlement- mannose, can induce larval settlement behaviour (Pagett et al., inducing protein complex, Gregarious settlement, Predation, 2012; Dreanno et al., 2006b; Thiyagarajan et al., 2002). Research on Biofouling another barnacle species, Balanus glandula, has identified another orthologous protein termed MULTIFUNCin (Ferrier et al., 2016), INTRODUCTION which shares 78% nucleotide sequence identity with SIPC and is The reproductive behaviour of barnacles has always fascinated expressed as three subunits of 98, 88 and 76 kDa in addition to the scientists, among them Charles Darwin (Darwin, 1854), partly 199 kDa of the full-length protein (Ferrier et al., 2016). Positive- because their sessile trait makes them adopt reproductive strategies settlement-directed purification of MULTIFUNCin and its subunits that rely on proximity to conspecifics (Barnes and Barnes, 1977; indicated that only the 199 kDa and the 98 kDa proteins were Crisp and Meadows, 1962; Crisp, 1961), but also because these inducing settlement of the cyprids of this species (Ferrier et al., animals are model organisms for marine biofouling (Aldred and 2016). These authors reported failure to express MULTIFUNCin in Clare, 2008; Rittschof and Cohen, 2004; Rittschof et al., 1984, Escherichia coli and Pichia pastoris heterologous systems (Ferrier 1992). The life cycle of these animals consists of the nauplius et al., 2016). Another study, however, reported the expression of a (larval) stage, the cyprid stage, in which the barnacle actively seeks recombinant SIPC from A. amphitrite in a baculovirus expression system (Zhang et al., 2016). This recombinant SIPC from A. amphitrite was lacking its N-terminal signal peptide sequence, 1Department of Biology, National and Kapodistrian University of Athens, had an apparent molecular mass of around 200 kDa and did not Athens 157 84, Greece. 2Institute of Biosciences and Applications, National Center for Scientific Research ‘Demokritos’, Agia Paraskevi, Athens 15310, Greece. induce cyprid settlement (Zhang et al., 2016). This inability of 3Biomedical Sciences Research Center ‘Alexander Fleming’, Fleming 34, heterologously expressed A. amphitrite SIPC to induce settlement 16672 Vari, Greece. of the cyprids is in disagreement with results from another *Author for correspondence ([email protected]) study, which showed that native SIPC, purified from A. amphitrite homogenates, induces cyprid settlement with a half maximal M.K., 0000-0001-6949-8490; M.P., 0000-0002-9030-1469; S.M., 0000-0002- effective concentration (EC ) of 102 nmol l−1 (Pagett et al., 2012). 0388-3369; D.V., 0000-0003-2695-0795; I.K., 0000-0003-3232-0167; S.G.D., 0000- 50 0002-0432-338X Throughout the extensive literature pertaining to the identification of SIPC or other orthologous proteins (Dreanno Received 23 May 2018; Accepted 1 October 2018 et al., 2007; Ferrier et al., 2016; Matsumura et al., 1998b), emphasis Journal of Experimental Biology 1 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb185348. doi:10.1242/jeb.185348 is given to positive-settlement-directed purification, i.e. to 5 h), adults were returned to seawater for larval release. Hatched identifying and reporting only subunits that exhibit a settlement- nauplii were attracted to a point light source, collected and placed inducing response; therefore, it is critical to note that such positive- into a beaker containing 2 or 3 l of 0.7 μm GF/F (Whatman)-filtered settlement assay designs may not allow for the identification of any natural seawater at a density of approximately 1–2 nauplii ml−1 with negative settlement cues. Therefore, comparing the results from B. gentle aeration. Nauplii were maintained at 27°C at a 12 h:12 h glandula MULTIFUNCin (Ferrier et al., 2016) and the A. amphitrite light:dark photoperiod on a diet of Chaetoceros gracilis (class: SIPC (Pagett et al., 2012), there is ambiguity as to which of the SIPC Bacillariophyceae) provided at a density of 2×105 cells ml−1 subunits or the full-length protein exert the settlement behaviour. Is (Matsumura et al., 1998a; Thiyagarajan et al., 2002). Cultured in it the N-terminal 98 kDa subunit (Pagett et al., 2012; Ferrier et al., these conditions, nauplii metamorphosed to cyprids in 5 days. 2016; Matsumura et al., 1998b; Dreanno et al., 2006c) that exerts the Aliquots of these cyprids were collected with a wide-mouthed settlement cues? Are the post-translational modifications (PTMs) of Pasteur pipette and aged at 4°C (Phang et al., 2009) for 1 day prior SIPC, which are critical for its activity as a pheromone cue (Aldred to use in settlement assays. Only batches of cyprids that were and Clare, 2008; Matsumura et al., 1998b; Zhang et al., 2016; active and had numerous oil cells, representing energy reserves Dreanno et al., 2007, 2006a), solely responsible for its biological (Lucas et al., 1979), were used in settlement assays (Rittschof et al., −1 activity or is the reported EC50 of 102 nmol l (Pagett et al., 2012) 1984, 1992). of native SIPC a composite effect brought about by the simultaneous presence of all three subunits (Matsumura et al., 1998b)? Construction of recombinant SIPC and truncated Attempting to answer these confounding questions, in this study SIPC fragments we engineered, expressed and purified a recombinant form of SIPC We obtained a plasmid containing the full-length cDNA of the from A. amphitrite as well as a systematic series of SIPC truncated A. amphitrite SIPC gene cloned in pCR4-TOPO (Dreanno et al., fragments to identify whether SIPC, its putative subunits 2006c) (Thermo Fisher Scientific). The full-length cDNA was (Matsumura et al., 1998b; Dreanno et al., 2006a) or its PTMs sequenced and then transferred as an EcoRI (NEB) fragment into (Pagett et al., 2012) are responsible for its biological activity. We pENTR1α (Thermo Fisher Scientific). The sequencing results engineered a recombinant form of SIPC and its truncated fragments revealed differences between the obtained clone and the original to contain an N-terminal myc-tag and a C-terminal 6×His-tag to be cDNA deposited in GenBank (acc. no.: AAR33079.1; see able to detect and purify the full-length protein