Gene co-option, duplication and divergence of cement proteins underpin the evolution of bioadhesives across barnacle life histories.

Acquisition of new genes often results in the emergence of novel functions and is a key step in lineage-specific adaptation. As a group of sessile crustaceans, barnacles establish permanent attachment through initial cement secretion at the larval phase followed by continuous cement secretion in juveniles and adults. However, the origins and evolution of barnacle larval and adult cement proteins remain poorly understood. By performing microdissection of larval cement glands, transcriptome and shotgun proteomics and immunohistochemistry validation, we identified 30 larval and 27 adult cement proteins of the epibiotic turtle barnacle Chelonibia testudinaria, of which the majority are stage- and barnacle-specific. While only two proteins, SIPC and CP100K, were expressed in both larvae and adults, detection of protease inhibitors and the cross-linking enzyme lysyl oxidase paralogs in larvae and adult cement. Other barnacle-specific cement proteins such as CP100k and CP52k likely share a common origin dating back at least to the divergence of Rhizocephala and Thoracica. Different CP52k paralogues could be detected in larval and adult cement, suggesting stage-specific cement proteins may arise from duplication followed by changes in expression timing of the duplicates. Interestingly, the biochemical properties of larval- and adult-specific CP52k paralogues exhibited remarkable differences. We conclude that barnacle larval and adult cement systems evolved independently, and both emerged from co-option of existing genes and de novo formation, duplication and functional divergence of lineage-specific cement protein genes. Our findings provide important insights into the evolutionary mechanisms of bioadhesives in sessile marine invertebrates.

Footprints of natural selection at the mannose-6-phosphate isomerase locus in barnacles.

The mannose-6-phosphate isomerase (Mpi) locus in Semibalanus balanoides has been studied as a candidate gene for balancing selection for more than two decades. Previous work has shown that Mpi allozyme genotypes (fast and slow) have different frequencies across Atlantic intertidal zones due to selection on postsettlement survival (i.e., allele zonation). We present the complete gene sequence of the Mpi locus and quantify nucleotide polymorphism in S. balanoides, as well as divergence to its sister taxon Semibalanus cariosus We show that the slow allozyme contains a derived charge-altering amino acid polymorphism, and both allozyme classes correspond to two haplogroups with multiple internal haplotypes. The locus shows several footprints of balancing selection around the fast/slow site: an enrichment of positive Tajima's D for nonsynonymous mutations, an excess of polymorphism, and a spike in the levels of silent polymorphism relative to silent divergence, as well as a site frequency spectrum enriched for midfrequency mutations. We observe other departures from neutrality across the locus in both coding and noncoding regions. These include a nonsynonymous trans-species polymorphism and a recent mutation under selection within the fast haplogroup. The latter suggests ongoing allelic replacement of functionally relevant amino acid variants. Moreover, predicted models of Mpi protein structure provide insight into the functional significance of the putatively selected amino acid polymorphisms. While footprints of selection are widespread across the range of S. balanoides, our data show that intertidal zonation patterns are variable across both spatial and temporal scales. These data provide further evidence for heterogeneous selection on Mpi.

First recorded occurrence of the parasitic barnacle (Anelasma squalicola) on a Greenland shark (Somniosus microcephalus) in the Canadian Arctic.

A solitary Anelasma squalicola specimen was collected from the cloaca of a Greenland shark (Somniosus microcephalus), the first time this association has been recorded. The specimen's identity was confirmed through morphological and genetic assessment (mitochondrial markers: COI and control region). A. squalicola is a species typically associated with deep-sea lantern sharks (Etmopteridae) and, until the present observation, had never been observed at a sexually mature size in the absence of a mating partner. Given the reported negative effects of this parasite on its hosts, monitoring Greenland sharks for additional cases is recommended.

Ecological Load and Balancing Selection in Circumboreal Barnacles.

Acorn barnacle adults experience environmental heterogeneity at various spatial scales of their circumboreal habitat, raising the question of how adaptation to high environmental variability is maintained in the face of strong juvenile dispersal and mortality. Here, we show that 4% of genes in the barnacle genome experience balancing selection across the entire range of the species. Many of these genes harbor mutations maintained across 2 My of evolution between the Pacific and Atlantic oceans. These genes are involved in ion regulation, pain reception, and heat tolerance, functions which are essential in highly variable ecosystems. The data also reveal complex population structure within and between basins, driven by the trans-Arctic interchange and the last glaciation. Divergence between Atlantic and Pacific populations is high, foreshadowing the onset of allopatric speciation, and suggesting that balancing selection is strong enough to maintain functional variation for millions of years in the face of complex demography.

Convergent evolution of barnacles and molluscs sheds lights in origin and diversification of calcareous shell and sessile lifestyle.

The calcareous shell and sessile lifestyle are the representative phenotypes of many molluscs, which happen to be present in barnacles, a group of unique crustaceans. The origin of these phenotypes is unclear, but it may be embodied in the convergent genetics of such distant groups (interphylum). Herein, we perform comprehensive comparative genomics analysis in barnacles and molluscs, and reveal a genome-wide strong convergent molecular evolution between them, including coexpansion of biomineralization and organic matrix genes for shell formation, and origination of lineage-specific orphan genes for settlement. Notably, the expanded biomineralization gene encoding alkaline phosphatase evolves a novel, highly conserved motif that may trigger the origin of barnacle shell formation. Unlike molluscs, barnacles adopt novel organic matrices and cement proteins for shell formation and settlement, respectively, and their calcareous shells have potentially originated from the cuticle system of crustaceans. Therefore, our study corroborates the idea that selection pressures driving convergent evolution may strongly act in organisms inhabiting similar environments regardless of phylogenetic distance. The convergence signatures shed light on the origin of the shell and sessile lifestyle of barnacles and molluscs. In addition, notable non-convergence signatures are also present and may contribute to morphological and functional specificities.

Phylogeny and shell form evolution of the hydrothermal vent asymmetrical barnacles (Cirripedia, Thoracicalcarea, Neoverrucidae).

Imbricaverruca and Neoverruca are two genera of hydrothermal vent asymmetrical barnacles in Neoverrucidae, but found in vents of the Southwest Pacific and Northwest Pacific Oceans, respectively. Imbricaverruca has a flattened operculum and the shell base with multiple whorls of imbricating plates, while Neoverruca has an inclined operculum and the shell base with fewer developed imbricating plates. It has been hypothesized that Imbricaverruca has apomorphic shell characters in Neoverrucidae. Although the monophyletic relationship of the vent barnacle members in the superfamily Neolepadoidea were confirmed based on molecular phylogeny, the relationships between Neobrachylepadidae and Neoverrucidae, and between Neoverruca and Imbricaverruca have not been determined because there are no molecular data on Imbricaverruca. In this study, we sequenced three nuclear (18S rDNA, 28S rDNA, histone 3) and one mitochondrial (CO1) genes of I. yamaguchii from the Southwest Pacific. Our phylogenetic results showed that Neobrahchylepadidae is the sister taxon to Neoverrucidae (Imbricaverruca + Neoverruca), and Imbricaverruca and Neoverruca are monophyletic sister taxa each other, which not supporting the previous hypothesis that Neoverruca is sister to the clade containing Neobrahchylepadidae and Neolepadidae. These implied that the differences in shell forms between Neoverruca and Imbricaverruca are a result of independent divergent evolution in different deep-sea basins.

Transcriptomic analysis of male three-spot swimming crab (Portunus sanguinolentus) infected with the parasitic barnacle Diplothylacus sinensis.

Diplothylacus sinensis is reported as an intriguing parasitic barnacle that can negatively affect the growth, molting, reproduction in several commercially important portunid crabs. To better understand the molecular mechanisms of host-parasite interactions, we characterized the gene expression profiles from the healthy and D. sinensis infected Portunus sanguinolentus by high-through sequence method. Totally, the transcriptomic analysis generated 52, 266, 600 and 51, 629, 604 high quality reads from the infected and control groups, respectively. The clean reads were assembled to 90,740 and 69,314 unigenes, with the average length of 760 bp and 709 bp, respectively. The expression analysis showed that 18,959 genes were significantly changed by the parasitism of D. sinensis, including 4769 activated genes and 14,190 suppressed genes. The differentially expressed genes were categorized into 258 KEGG pathways and 647 GO terms. The GO analysis mapped 13 DEGs related to immune system process and 32 DEGs related to immune response, respectively, suggesting a potential alteration of transcriptional expression patterns in complement cascades of P. sanguinolentus. Additionally, 4 representative molting-related genes were down-regulated in parasitized group, indicating D. sinensis infection appeared to suppress the producing of ecdysteroid hormones. In conclusion, the present study improves our understanding on parasite-host interaction mechanisms, which focuses the function of Ecdysone receptor, Toll-like receptor and cytokine receptor of crustacean crabs infestation with rhizocephalan parasites.

Histology and transcriptomic analyses of barnacles with different base materials and habitats shed lights on the duplication and chemical diversification of barnacle cement proteins.

BACKGROUND: Barnacles are sessile crustaceans that attach to underwater surfaces using barnacle cement proteins. Barnacles have a calcareous or chitinous membranous base, and their substratum varies from biotic (e.g. corals/sponges) to abiotic surfaces. In this study, we tested the hypothesis that the cement protein (CP) composition and chemical properties of different species vary according to the attachment substrate and/or the basal structure. We examined the histological structure of cement glands and explored the variations in cement protein homologs of 12 barnacle species with different attachment habitats and base materials. RESULTS: Cement gland cells in the rocky shore barnacles Tetraclita japonica formosana and Amphibalanus amphitrite are eosinophilic, while others are basophilic. Transcriptome analyses recovered CP homologs from all species except the scleractinian coral barnacle Galkinia sp. A phylogenomic analysis based on sequences of CP homologs did not reflect a clear phylogenetic pattern in attachment substrates. In some species, certain CPs have a remarkable number of paralogous sequences, suggesting that major duplication events occurred in CP genes. The examined CPs across taxa show consistent bias toward particular sets of amino acid. However, the predicted isoelectric point (pI) and hydropathy are highly divergent. In some species, conserved regions are highly repetitive. CONCLUSIONS: Instead of developing specific cement proteins for different attachment substrata, barnacles attached to different substrata rely on a highly duplicated cementation genetic toolkit to generate paralogous CP sequences with diverse chemical and biochemical properties. This general CP cocktail might be the key genetic feature enabling barnacles to adapt to a wide variety of substrata.

From tides to nucleotides: Genomic signatures of adaptation to environmental heterogeneity in barnacles.

The northern acorn barnacle (Semibalanus balanoides) is a robust system to study the genetic basis of adaptations to highly heterogeneous environments. Adult barnacles may be exposed to highly dissimilar levels of thermal stress depending on where they settle in the intertidal (i.e., closer to the upper or lower tidal boundary). For instance, barnacles near the upper tidal limit experience episodic summer temperatures above recorded heat coma levels. This differential stress at the microhabitat level is also dependent on the aspect of sun exposure. In the present study, we used pool-seq approaches to conduct a genome wide screen for loci responding to intertidal zonation across the North Atlantic basin (Maine, Rhode Island, and Norway). Our analysis discovered 382 genomic regions containing SNPs which are consistently zonated (i.e., SNPs whose frequencies vary depending on their position in the rocky intertidal) across all surveyed habitats. Notably, most zonated SNPs are young and private to the North Atlantic. These regions show high levels of genetic differentiation across ecologically extreme microhabitats concomitant with elevated levels of genetic variation and Tajima's D, suggesting the action of non-neutral processes. Overall, these findings support the hypothesis that spatially heterogeneous selection is a general and repeatable feature for this species, and that natural selection can maintain functional genetic variation in heterogeneous environments.

Engineered Escherichia coli Biofilms Produce Adhesive Nanomaterials Shaped by a Patterned 43 kDa Barnacle Cement Protein.

Barnacles integrate multiple protein components into distinct amyloid-like nanofibers arranged as a bulk material network for their permanent underwater attachment. The design principle for how chemistry is displayed using adhesive nanomaterials, and fragments of proteins that are responsible for their formation, remains a challenge to assess and is yet to be established. Here, we use engineered bacterial biofilms to display a library of amyloid materials outside of the cell using full-length and subdomain sequences from a major component of the barnacle adhesive. A staggered charged pattern is found throughout the full-length sequence of a 43 kDa cement protein (AACP43), establishing a conserved sequence design evolved by barnacles to make adhesive nanomaterials. AACP43 domain deletions vary in their propensity to aggregate and form fibers, as exported extracellular materials are characterized through staining, immunoblotting, scanning electron microscopy, and atomic force microscopy. Full-length AACP43 and its domains have a propensity to aggregate into nanofibers independent of all other barnacle glue components, shedding light on its function in the barnacle adhesive. Curliated Escherichia coli biofilms are a compatible system for heterologous expression and the study of foreign functional amyloid adhesive materials, used here to identify the c-terminal portion of AACP43 as critical in material formation. This approach allows us to establish a common sequence pattern between two otherwise dissimilar families of cement proteins, laying the foundation to elucidate adhesive chemistries by one of the most tenacious marine fouling organisms in the ocean.

Design of RGDS Peptide-Immobilized Self-Assembling ß-Strand Peptide from Barnacle Protein.

We designed three types of RGD-containing barnacle adhesive proteins using self-assembling peptides. In the present study, three types of RGD-containing peptides were synthesized by solid-phase peptide synthesis, and the secondary structures of these peptides were analyzed by CD and FT-IR spectroscopy. The mechanical properties of peptide hydrogels were characterized by a rheometer. We discuss the correlation between the peptide conformation, and cell attachment and cell spreading activity from the viewpoint of developing effective tissue engineering scaffolds. We created a peptide-coated cell culture substrate by coating peptides on a polystyrene plate. They significantly facilitated cell adhesion and spreading compared to a non-coated substrate. When the RGDS sequence was modified at N- or C-terminal of R-Y, it was found that the self-assembling ability was dependent on the strongly affects hydrogel formation and cell adhesion caused by its secondary structure.

Comparative transcriptomic analysis of deep- and shallow-water barnacle species (Cirripedia, Poecilasmatidae) provides insights into deep-sea adaptation of sessile crustaceans.

BACKGROUND: Barnacles are specialized marine organisms that differ from other crustaceans in possession of a calcareous shell, which is attached to submerged surfaces. Barnacles have a wide distribution, mostly in the intertidal zone and shallow waters, but a few species inhabit the deep-sea floor. It is of interest to investigate how such sessile crustaceans became adapted to extreme deep-sea environments. We sequenced the transcriptomes of a deep-sea barnacle, Glyptelasma gigas collected at a depth of 731 m from the northern area of the Zhongjiannan Basin, and a shallow-water coordinal relative, Octolasmis warwicki. The purpose of this study was to provide genetic resources for investigating adaptation mechanisms of deep-sea barnacles. RESULTS: Totals of 62,470 and 51,585 unigenes were assembled for G. gigas and O. warwicki, respectively, and functional annotation of these unigenes was made using public databases. Comparison of the protein-coding genes between the deep- and shallow-water barnacles, and with those of four other shallow-water crustaceans, revealed 26 gene families that had experienced significant expansion in G. gigas. Functional annotation showed that these expanded genes were predominately related to DNA repair, signal transduction and carbohydrate metabolism. Base substitution analysis on the 11,611 single-copy orthologs between G. gigas and O. warwicki indicated that 25 of them were distinctly positive selected in the deep-sea barnacle, including genes related to transcription, DNA repair, ligand binding, ion channels and energy metabolism, potentially indicating their importance for survival of G. gigas in the deep-sea environment. CONCLUSIONS: The barnacle G. gigas has adopted strategies of expansion of specific gene families and of positive selection of key genes to counteract the negative effects of high hydrostatic pressure, hypoxia, low temperature and food limitation on the deep-sea floor. These expanded gene families and genes under positive selection would tend to enhance the capacities of G. gigas for signal transduction, genetic information processing and energy metabolism, and facilitate networks for perceiving and responding physiologically to the environmental conditions in deep-sea habitats. In short, our results provide genomic evidence relating to deep-sea adaptation of G. gigas, which provide a basis for further biological studies of sessile crustaceans in the deep sea.

Transcriptome analyses suggest a molecular mechanism for the SIPC response of Amphibalanus amphitrite.

Barnacles are notorious marine fouling organisms. Their successful attachment to a substrate requires that they search for an appropriate habitat during their cyprid stage. A chemical cue called SIPC (Settlement-Inducing Protein Complex) has been shown to play a key role in the induction of cyprid gregarious settlement; however, the underlying biochemical mechanism remains unclear. Here, RNA-seq was used to examine the gene expression profiles of Amphibalanus amphitrite cyprids in response to SIPC and to identify SIPC-activated intracellular signaling pathways. A total of 389 unigenes were differentially expressed in response to SIPC, and cement protein genes were not among them. KEGG enrichment analysis suggested that SNARE interactions in the vesicular transport pathway were significantly influenced by SIPC treatment, indicating a possible role for SIPC in triggering protein transportation and secretion. Several genes with specific functions in metamorphosis were found among the differentially expressed genes (DEGs). GO (Gene Ontology) enrichment analysis revealed that the DEGs were significantly enriched in enamel mineralization pathways, suggesting that SIPC may also be involved in the activation of mineralization.

Microsatellite DNA markers applicable to paternity inference in the androdioecious gooseneck barnacle Octolasmis warwickii (Lepadiformes: Poecilasmatidae).

The gooseneck barnacle Octolasmis warwickii has a rare sexual system called androdioecy, in which hermaphrodites and dwarf males co-occur. It has been hypothesized that dwarf males can coexist with conspecific hermaphrodites when dwarf males are capable of leaving more offspring than hermaphrodites via male reproduction. This hypothesis of reproductive superiority of dwarf males can be validated by comparing the reproductive success between dwarf males and hermaphrodites through DNA marker-based parentage testing. In the present study, we developed microsatellite DNA markers for O. warwickii, and evaluated the power of these markers to infer parentage based on simulation analysis. Using next generation sequencing, we obtained 344 microsatellite sequences suitable for designing primer sets for amplification in polymerase chain reaction (PCR). Of these, we examined the PCR amplification efficiency of 54 primer sets, of which 11 passed our primer screening in a population sample (n = 35). The developed markers exhibited moderate to high levels of polymorphisms, and met Hardy-Weinberg equilibrium with little evidence of significant allelic association to each other. Our simulated paternity inference suggested that the combinational use of the markers allows a high resolution of parentage (success rate of > 99.9%) if all candidate fathers are available.

Insights into the Synthesis, Secretion and Curing of Barnacle Cyprid Adhesive via Transcriptomic and Proteomic Analyses of the Cement Gland.

Barnacles represent one of the model organisms used for antifouling research, however, knowledge regarding the molecular mechanisms underlying barnacle cyprid cementation is relatively scarce. Here, RNA-seq was used to obtain the transcriptomes of the cement glands where adhesive is generated and the remaining carcasses of Megabalanus volcano cyprids. Comparative transcriptomic analysis identified 9060 differentially expressed genes, with 4383 upregulated in the cement glands. Four cement proteins, named Mvcp113k, Mvcp130k, Mvcp52k and Mvlcp1-122k, were detected in the cement glands. The salivary secretion pathway was significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes, implying that the secretion of cyprid adhesive might be analogous to that of saliva. Lysyl oxidase had a higher expression level in the cement glands and was speculated to function in the curing of cyprid adhesive. Furthermore, the KEGG enrichment analysis of the 352 proteins identified in the cement gland proteome partially confirmed the comparative transcriptomic results. These results present insights into the molecular mechanisms underlying the synthesis, secretion and curing of barnacle cyprid adhesive and provide potential molecular targets for the development of environmentally friendly antifouling compounds.

The Quantitative Proteome of the Cement and Adhesive Gland of the Pedunculate Barnacle, Pollicipes pollicipes.

Adhesive secretion has a fundamental role in barnacles' survival, keeping them in an adequate position on the substrate under a variety of hydrologic regimes. It arouses special interest for industrial applications, such as antifouling strategies, underwater industrial and surgical glues, and dental composites. This study was focused on the goose barnacle Pollicipes pollicipes adhesion system, a species that lives in the Eastern Atlantic strongly exposed intertidal rocky shores and cliffs. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using a label-free LC-MS high-throughput proteomic analysis, searched against a custom transcriptome-derived database. Overall, 11,755 peptide sequences were identified in the gland while 2880 peptide sequences were detected in the cement, clustered in 1616 and 1568 protein groups, respectively. The gland proteome was dominated by proteins of the muscle, cytoskeleton, and some uncharacterized proteins, while the cement was, for the first time, reported to be composed by nearly 50% of proteins that are not canonical cement proteins, mainly unannotated proteins, chemical cues, and protease inhibitors, among others. Bulk adhesive proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Some unannotated proteins highly expressed in the proteomes, as well as at the transcriptomic level, showed similar physicochemical properties to the known surface-coupling barnacle adhesive proteins while the function of the others remains to be discovered. New quantitative and qualitative clues are provided to understand the diversity and function of proteins in the cement of stalked barnacles, contributing to the whole adhesion model in Cirripedia.

Small RNA sequencing of sessile serrated polyps identifies microRNA profile associated with colon cancer.

Sessile serrated adenoma/polyps (SSA/Ps) of the colon account for 20-30% of all colon cancers. Small non-coding RNAs, including microRNAs (miRNAs), may function as oncogenes or tumor suppressor genes involved in cancer development. Small RNA sequencing (RNA-seq) was used to characterize miRNA profiles in SSA/Ps, hyperplastic polyps (HPs), adenomatous polyps and paired uninvolved colon. Our 108 small RNA-seq samples' results were compared to small RNA-seq data from 212 colon cancers from the Cancer Genome Atlas. Twenty-three and six miRNAs were differentially expressed in SSA/Ps compared to paired uninvolved colon and HPs, respectively. Differential expression of MIR31-5p, MIR135B-5p and MIR378A-5p was confirmed by RT-qPCR. SSA/P-specific miRNAs are similarly expressed in colon cancers containing genomic aberrations described in serrated cancers. Correlation of miRNA expression with consensus molecular subtypes suggests more than one subtype is associated with the serrated neoplasia pathway. Canonical pathway analysis suggests many of these miRNAs target growth factor signaling pathways.

Comparison of seven methods for DNA extraction from prosomata of the acorn barnacle, Amphibalanus amphitrite.

Next generation sequencing (NGS) technologies can provide an understanding of the molecular processes involved in marine fouling by Amphibalanus spp. barnacles. Here, seven methods for extracting DNA from A. amphitrite prosomata were assessed with respect to recovery, purity and size distribution. Methods incorporating organic extractions generally resulted in low recovery of fragmented DNA. The most promising method was the commercial E.Z.N.A. Blood DNA Mini kit, which provided tens of micrograms of DNA of sufficient molecular weight for use in long-read NGS library preparation. Other kits resulted in DNA preps suitable for short read length NGS platforms.

Evaluating the occurrence of cryptic invasions of a rocky shore barnacle, Semibalanus cariosus, between the north-eastern Pacific and Japan.

Many coastal barnacles are introduced to non-native regions. However, data are lacking on cryptic invasion, which is defined as an invasion that remains unrecognised because the invader is mistaken for a native or previously introduced species or clade. In this work, cryptic invasions of an intertidal barnacle, Semibalanus cariosus, between Japan and the north-eastern Pacific were evaluated based on population genetic analyses. A significant genetic differentiation was found between the Japanese and north-eastern Pacific populations, suggesting a limited introduction of non-native genotypes between these regions. Haplotype frequencies did not differ significantly between the past (museum samples collected in 1971 from Hokkaido, Japan) and present Japanese populations, implying the rare occurrence of human-mediated migration from the north-eastern Pacific to Japan. Migrate-n analysis revealed a low level of directional gene flow in S. cariosus from the north-eastern Pacific to Japan, possibly by natural stepping-stone dispersal via directional water currents or human-mediated transport.

Comparative Exploration of the Structure-Activity Space of Cloned α-Like Octopamine Receptors from a Marine and a Terrestrial Arthropod.

The α-like octopamine receptors (OctR) are believed to be the evolutionary precursor to the vertebrate α2-adrenergic receptors (α2-ARs) based upon sequence similarity and the ability to interact with norepinephrine and a number of compounds that bind with high affinity to α2-ARs. Barnacles and fruit flies are two prominent model marine and terrestrial representatives of the Arthropoda phylum, and although α-like OctRs have been cloned from Balanus improvisus (BiOctR) and Drosophila melanogaster (DmOctR), little is known about the structure-activity space for these important species. A diverse panel of 22 probes spanning different structural classes were employed to interrogate the structure-activity of the BiOctR and DmOctR. While BiOctR and DmOctR exhibited similar functional profiles for mammalian biogenic amine G protein-coupled receptor agonists and antagonists, some ligands had dramatically different mechanisms of action. For instance, significant differences in the efficacy for some agonists were observed, including that vertebrate biogenic amines structurally related to octopamine acted as superagonists at the DmOctR but partial agonists at the BiOctR, and the two species diverged in their sensitivities to the α2-AR antagonist [3H]rauwolscine. Furthermore, sodium enhanced [3H]rauwolscine's interactions with the BiOctR, but not at a vertebrate α2-AR. Molecular mechanistic studies indicate that rauwolscine interacts with the BiOctR, DmOctR, and α2C-adrenergic receptor at an allosteric site. In addition, compounds that acted as agonists at a cloned α-like BiOctR also induced a hyperactivity response in Balanus cyprids mediated by the α-like OctR, suggesting that the receptor may serve as a higher throughput proxy for discovering compounds with potential cyprid deterrent properties.