An alpha2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite

Abstract

Many benthic marine invertebrates, like barnacles, have a planktonic larval stage whose primary purpose is dispersal. How these species colonize suitable substrata is fundamental to understanding their evolution, population biology, and wider community dynamics. Unlike larval dispersal, settlement occurs on a relatively small spatial scale and involves larval behavior in response to physical and chemical characteristics of the substratum. Biogenic chemical cues have been implicated in this process. Their identification, however, has proven challenging, no more so than for the chemical basis of barnacle gregariousness, which was first described >50 years ago. We now report that a biological cue to gregarious settlement, the settlement-inducing protein complex (SIPC), of the major fouling barnacle Balanus amphitrite is a previously undescribed glycoprotein. The SIPC shares a 30% sequence homology with the thioester-containing family of proteins that includes the alpha(2)-macroglobulins. The cDNA (5.2 kb) of the SIPC encodes a protein precursor comprising 1,547 aa with a 17-residue signal peptide region. A number of structural characteristics and the absence of a thioester bond in the SIPC suggest that this molecule is a previously undescribed protein that may have evolved by duplication from an ancestral alpha(2)-macroglobulin gene. Although the SIPC is regarded as an adult cue that is recognized by the cyprid at settlement, it is also expressed in the juvenile and in larvae, where it may function in larva-larva settlement interactions.

Fig. 1.

Predicted structure of B. amphitrite SIPC and similarity to the thioester region of Limulus sp. and other barnacles. (A) Predicted structure of the SIPC showing the putative disulfide bridge pattern deduced from the conservation of specific cysteine residues between SIPC and A2M. Thick lines indicate disulfide bridges that are conserved in the Limulus and human sequences. Light lines denote the disulfide bridge showing similarity only to Limulus. The 17-aa signal peptide is denoted by the black box. The potential N-linked glycosylation sites (Asn-Xxx-Ser/Thr and Asn-Xxx-Cys) are marked by black diamonds. (B) Amino acid sequence alignment of the thioester-like region from SIPC and A2M. Black and gray shading denotes identical and functionally similar amino acids, respectively. The horizontal lines indicate the amino acid sequences to which the forward and reverse PCR primers were designed. GenBank accession numbers are as follows: Limulus sp., T18544; B. amphitrite, AY423545 and AY781062; B. improvisus, AY781061; and M. rosa, AY781063.

Fig. 2.

Expression of the SIPC transcript. (A) Northern blot. Total RNA extracted from the prosomas of B. amphitrite (Ba) and M. rosa (Mr) was hybridized with two probes corresponding to the N-terminal (Left) and the C-terminal (Right) regions of the protein. The RNA ladder is indicated on the left. (B) Expression of B. amphitrite SIPC and EF1 mRNA in different tissues. Lane 1, molecular weight marker (1 kb plus DNA ladder; Invitrogen); lane 2, epidermis; lane 3, egg mass; lane 4, ovary; lane 5, penis; lane 6, gut; lane 7, muscle; lane 8, cirri; lane 9, seminal vesicles. For the no-template control see lane 3 in C. (C) Expression of B. amphitrite SIPC and EF1 mRNA in different development stages. Lane 1, molecular weight marker (1 kb plus DNA ladder; Invitrogen); lane 2, blank; lane 3, no-template control; lane 4, nauplius stage 1; lane 5, nauplius stage 2; lane 6, cyprid (immediately after metamorphosis from the stage 6 nauplius); lane 7, cyprid 2 days after metamorphosis; lane 8, juvenile; lane 9, adult.

Fig. 3.

Unrooted phylogenetic tree showing the relationship of B. amphitrite SIPC to the TEP. CF indicates the branch leading to the complement factor proteins. The full tree comprising A2M, SIPC, complement factor, and other TEPs is in Fig. 6. Numbers by major nodes represent bootstrap values for 100 replicates. The scale bar indicates the length of each branch.

Fig. 4.

Settlement choice assay of B. amphitrite cyprids with B. amphitrite SIPC and hA2M. B. amphitrite cyprid settlement was significantly higher (P < 0.05) on the B. amphitrite SIPC (applied at 100 ng/0.8 cm²) compared with the control or hA2M (applied at 10, 50, or 100 μg/0.8 cm²), which were not significantly different from each other (P > 0.05).