Indian Journal of Geo-Marine Sciences Vol. 43(11), November 2014, pp._____

The Bacterial Basis of Biofouling: a Case Study

Michael G. Hadfield*, Audrey Asahina, Shaun Hennings and Brian Nedved

Kewalo Marine Laboratory, University of Hawaii at Manoa 41 Ahui St., Honolulu, Hawaii, 96813 USA *[E-mail: [email protected]]

Received 07 March 2014; revised 14 September 2014

For billions of years bacteria have profusely colonized all parts of the oceans and formed biofilms on the benthos. Thus, from their onset, evolving marine animals adapted to the microbial world throughout their life cycles. One of the major adaptations is the use of bacterial products as signals for recruitment by larvae of many species in the seven invertebrate phyla that make up most of the biofouling community. We describe here investigations on the recruitment biology of one such species, the circum-tropical serpulid polychaete Hydroides elegans. Insights gained from in-depth studies on adults and larvae of H. elegans include: apparently constant transport of these biofouling worms on the hulls of ships maintains a globally panmictic population; larvae complete metamorphosis with little or no de novo gene transcription or translation; larvae of this tube-worm settle selectively in response to specific biofilm-dwelling bacterial species; and biofilms provide not only a cue for settlement sites, but also increase the adhesion strength of the settling worms’ tubes on a substratum. Although biofilm-bacterial species are critical to larval-settlement induction, not all biofilm elements are inductive. Because studies on chemoreception systems in the larvae failed to find evidence for the presence of common chemoreceptors, we focused on the bacterial cues themselves. To do this, we studied a widely distributed and strongly inductive biofilm bacterium, Pseudoalteromonas luteoviolacea. Using molecular manipulations of its genome, we learned that it produces complex clusters of bacteriocins, multi-protein structures evolutionarily derived from phage-tail elements, which induce metamorphosis of H. elegans. But large questions remain: how do these complex structures bring about induction? Do larvae of other invertebrate species that settle in response to P. luteoviolacea also metamorphose in response to its bacteriocins? Do all inductive bacterial species produce bacteriocins? As a whole, the described studies on the recruitment of Hydroides elegans demonstrate the importance of in-depth investigations of model species for understanding the problem of biofouling as well as the ubiquity of essential animal-bacterial interactions in the sea.

[Keywords: larval settlement, biofilms, Hydroides elegans, metamorphosis, biofouling]

Introduction likely that these early larvae utilized Bacteria have proliferated in all of the chemical/biological cues from biofilm bacteria as world’s seas and oceans for most of Earth’s history, signals for the suitability of particular habitats, a i.e., for about 3.5 billion years (Noffke et al., 2013). practice still present among larvae of most extant For roughly 1.75 billion years, bacteria had the seas marine invertebrate species today (Hadfield, 2011). to themselves, evolving, diversifying intensively Dependence of contact with marine and filling every possible marine environment biofilms, generally, or specific biofilm-bacterial (Knoll, 2004). Not only were bacteria abundant, but species has been noted for members of every major they surely were the food of the first eukaryotes as phylum of marine invertebrate animals, including they evolved during the last 1.75 billion years. the seven phyla that characterize nearly all marine Evidence is now strong that the evolution of biofouling communities (Table 1) (Hadfield and animals began only about 700 – 800 million years Paul, 2001). Bacteria-associated attachment of algal ago (Knoll, 2004), and there can be little doubt that spores has also been recorded (Joint et al., 2007). the first benthic marine animals must have Our understanding of larval-bacterial interactions interacted intensively with the ubiquitous bacteria has increased many fold over the last 25 years, in the seas. How and when complex life histories especially through in-depth investigation of specific involving biphasic life cycles and pelagic larvae “model species,” reared in the laboratory, for which evolved aren’t precisely known, but when they did developmental, sensory and ecological information evolve, larvae could not have approached any has been gained. While these studies include many surface in the sea without encountering a dense species of sponges (Porifera), hydrozoans and bacterial film with which they had to interact in corals (Cnidaria), polychaetes (Annelida), order to settle and metamorphose. It appears highly bryozoans (Ectoprocta), mussels and oysters HADFIELD et al.: THE BACTERIAL BASIS OF BIOFOULING

(Mollusca), barnacles (Arthropoda) and tunicates Initial observations of larval settlement of (Urochordata) (Table 1), a few species stand out. H. elegans led us to suspect that the larvae were The barnacle Amphibalanus(Balanus) amphitrite responding to fouled surfaces; that is, surfaces that and the bryozoan Bugulaneritina have provided were obviously coated with a film of bacteria, numerous insights into the understanding of larval diatoms and other microorganisms. These surfaces responses to bacterial films, especially, for the were quickly colonized by settling larvae of H. barnacle, with regard to sensory perception of elegans while clean ones were not. Finding the settlement cues(Gohad et al., 2012, Maruzzo et al., species both easy to rear and induce to settle led to a 2012, Maruzzo et al., 2011). In this paper, we focus series of controlled laboratory experiments from on the tropical biofouling serpulid tubeworm which we learned the critical life history Hydroides elegans. Since we began studying the characteristics for the worm that were essential to recruitment processes of H. elegans in 1990, the utilizing it extensively in the laboratory. H. elegans, species has been mentioned in more than 1,500 as collected from the field, is dioecious and free scientific papers and is a major topic in at least 84, spawning; i.e., sexes are separate, fertilization is as indicated by inclusion of the species name in external and the larvae develop in the plankton. At paper titles. In this review, we focus principally on least some individuals are protandric, maturing first the contributions of our University of Hawaii as males and later transforming into females. The laboratory on the development of H. elegans as a species develops rapidly into planktotrophic- research model organism, while emphatically trochophore larvae within a day that develop into acknowledging that other laboratories have made metamorphically competent nectochaete larvae in 4 important contributions to understanding of the – 5 days at 25°C (Figure 2) (Hadfield et al., 1994, biology of the species, especially that of Dr. Pei- Carpizo-Ituarte and Hadfield, 1998). It is at this Yuan Qian at Hong Kong University of Science and point that settlement / metamorphic induction is Technology, whom we introduced to H. elegans in necessary. In a first set of experiments, we the mid-1990s. demonstrated that the percentage of a cohort of larvae of H. elegans that settled on a surface was Choosing the species strictly proportional to the time that the surface had In 1990, researchers in our laboratory been immersed in flowing seawater (Hadfield et al. initiated studies of the biofouling community in 1994). Furthermore, when we counted the Pearl Harbor, Hawaii, a port that has served as a abundance of various types of microorganisms that major United States naval base for more than 125 were on the surfaces that had been immersed for years. Similar to other such bays around the world, different periods, the tightest correlation with larval Pearl Harbor had long accumulated a large element settlement abundance was the density of short, rod- of the global, warm-water biofouling community, shaped bacteria. From this, we concluded that it was carried to it on the hulls of ships, barges and bacteria in this category to which the larvae structures like floating dry-docks (Carlton and responded by attaching and rapidly secreting a Eldredge, 2009). We learned early on that one of membranous primary tube within which they the major problems experienced by the U. S. Navy completed metamorphosis (Figure 3). Subsequent in Pearl Harbor was the accumulation of calcareous research confirmed this finding, adding that many tubes of the polychaete worm Hydroides elegans different bacterial species isolated from inductive Haswell (1883) on the hulls of ships and other biofilms induced larval settlement of H. structures (Figure 1). Because we quickly found the elegans(Unabia and Hadfield, 1999). Information species on test panels submerged in the harbor, we on the handling of H. elegans as an experimental immediately focused some of our efforts on organism in the laboratory was summarized by understanding the recruitment biology of H. Nedved and Hadfield (2009). elegans. It turned out to be an exceptionally facile species for studies of its life history in the Larval responses in Hydroides elegans laboratory. Our first attempts to observe larval While continuing to explore the variety of development and settlement of H. elegans were bacteria that induce settlement in the tubeworm, we successful, enabling us to begin many years of began to extensively investigate the mechanisms by experimentation that have led to a current which larvae recognize a bacterial settlement cue understanding of the recruitment processes of H. and translate that cue into a morphogenetic process. elegans that is arguably among the most extensive Holm et al. (1998) investigated the nature of the for marine invertebrate species. cellular receptor that larvae might use to detect specific bacterial substances, expecting to find INDIAN J. MAR. SCI., VOL 43, NO.11 NOVEMBER 2014

Fig. 1 – Adults of the serpulid tubeworm Hydroides elegans. A. Several adult worms on a submerged surface. B. Accumulation of tubes of Hydroides eleganson a surface after 1-month submersion in Pearl Harbor, HI. Scale bars = 1 cm

Fig. 2 - Larval development in Hydroides elegans at 25 °C. A. Lateral view of a trochophore larva (12 h post-fertilization). B. Dorsal view of a nectochaete larva (~120 h post-fertilization). Scale bars = 50 μm.

HADFIELD et al.: THE BACTERIAL BASIS OF BIOFOULING

Fig. 3 - Tube secretion in Hydroides elegans. A. Metamorphosing larva of H. elegans enclosed in the transparent primary tube rapidly that the larva rapidly secreted during settlement. B. Higher magnification image of the posterior portion of a metamorphosing juvenile and the outlines of the primary tube. C. Juvenile situated in the calcified secondary tube secreted at the anterior end of the primary tube (~ 5 h post settlement). Arrows indicate lateral margins of the secondary tube; arrowheads indicate lateral margins of the primary tube. Scale bars = 50 µm. evidence that a G-protein-coupled receptor would The extent to which the initial settlement be found, because this type of receptor has been and processes of metamorphosis are dependent on implicated in most instances of chemosensory de novo gene action or upon translation of reception in taste and smell (Buck and Axel, 1991, preexisting messenger RNAs in larvae of H. Ngai et al., 1993). However, the results were elegans was investigated by rearing larvae in the negative in experiments applying pharmacological presence of both transcriptional and translational substances that should either stimulate G-protein inhibitors and observing whether or not larvae coupled receptors, and therefore induce larval settled and completed metamorphosis(Carpizo- settlement in the absence of a bacterial film, or Ituarte and Hadfield, 2003). The surprising result inhibit settlement in the presence of a biofilm. Holm with either set of inhibitors was that larvae settled et al. (loc. cit.) concluded that bacterial-signal normally, secreted primary tubes and advanced reception and transduction in larvae of H. elegans through metamorphosis to the stage when the buds must rely on a different receptor type, with many of the branchialradioles typically begin to elongate, other known possibilities. Experiments were also and then arrested. From this we concluded that performed to provide greater understanding of the little, if any, de novo genetic transcription or way in which the bacterial cue stimulated translation was necessary for settlement and the metamorphosis. We had long known that 20 – 50 initiation of metamorphosis in H. elegans. mM potassium chloride added to natural seawater Based on experience in studies of a number would stimulate metamorphosis in other larval of different marine larval types, including H. types (e.g., Yoolet al., 1986), a finding that was elegans, we developed theoretical analyses of larval confirmed in H. elegans.It was additionally found development noting: (1) over the prior 20 – 30 years that exposing the larvae to a 3-hour pulse of 10 mM there had been increasing awareness that embryonic CsCl added to seawater was inductive (Carpizo- development for most larvae continues during the Ituarte and Hadfield, 1998). We concluded from planktonic phase to the establishment of this that the bacterial cue, in some way, activates “competence,” defined as the capacity to undergo the larval nervous system, which, in turn, stimulates metamorphic transformation while retaining the the morphogenetic responses specific to different larval morphology and biology until a specific tissues. stimulus is encountered; and (2) a large body of evidence revealed that larvae do not so much INDIAN J. MAR. SCI., VOL 43, NO.11 NOVEMBER 2014 Table 1 - Species whose larvae have been reported to settle in response to biofilm bacteria from seven phyla that include marine biofouling species. Phylum Organism Inducing Bacterial Strain Reference Porifera Coscinoderma matthewsi Natural Biofilm Abdul Wahab et al. (2011) Rhopaloeides odorabile Natural Biofilm Whalan and Webster (2014) Cnidaria Pocillopora damicornis Pseudoalteromonadaceae bacterium, Tran and Hadfield (2011) (coral) Pseudoalteromonas luteoviolacea, Thalassomonas agarivorans Acropora millepora (coral) Pseudoalteromonas citrea, Pseudoalteromonas Tebben et al. (2011) peptidolytica, Pseudoalteromonas sp. Montipora capitata (coral) Natural Biofilm Vermeij et al. (2008) Hydractinia echinata Pseudoalteromonas espejiana Seipp et al. (2007) Bryozoa Bugula neritina Vibrio ichthyoenteri, Pseudoalteromonas atlantica, Dobretsov and Qian (2006) Cytophaga sp., Tenacibaculum mesophilum, Exiguobacterium aurantiacum Annelida Hydroides elegans Pseudoalteromonas luteoviolacea Huang and Hadfield (2003) Pomatoceros lamarkii Natural Biofilm Hamer et al. (2001) Galeolaria caespitosa Natural Biofilms Shimeta et al. (2012) Mollusca Crepidula onyx (Slipper Natural Biofilms Chiu et al. (2007) limpet) Aulacomya maoriana Macrococcus sp., Bacillus sp., Pseudoalteromonas Alfaro et al. (2011) (mussel) sp. Mytilus galloprovincialis Alteromonas sp. Bao et al. (2007) (mussel) Teredo parksi (teredinid Pseudoalteromonas sp. and Vibrio sp. Pachu et al. (2012) wood borer) Crassostrea virginica Natural Biofilms Campbell et al. (2011) Arthropoda Balanus amphitrite Natural Biofilm Lau et al. (2005) (Barnacle) Balanus trigonus (Barnacle) Natural Biofilm Lau et al. (2005)

Balanus trigonus Natural Biofilm Thiyagarajan et al. (2006) Chordata Boltenia villosa Natural Biofilm Roberts et al. (2007)

“choose” the sites where they settle, but rather are Although the first papers from the our stimulated or triggered to settle by more-or-less laboratory had established the necessity for only a specific stimuli from specific environments. In the marine biofilm to stimulate settlement of H. latter case, the vocabulary became “settlement or elegans, other laboratories reported on a role for metamorphic induction,” rather than larval choice gregarious factors in stimulating settlement of the (Hadfield, 1998, 2000). We further commented on species (Bryan et al., 1997). To more deeply the broad phylogenetic occurrence of the competent investigate this question, we carried out laboratory state in invertebrate larvae, the condition wherein tests wherein we exposed competent larvae to most or all juvenile structures are already present in surfaces bearing either living H. elegans in their the larvae and that it had apparently evolved tubes, tubes of H. elegans from which the adults multiple times (Hadfield et al., 2001). The had been removed, or tube mimics consisting of 10- tubeworm Hydroides elegans exemplifies this well. mm long pieces of non-toxic, polyethylene surgical By the time the larva of this species is tubing 1.09 mm in diameter (Intramedic #7405, metamorphically competent, it has three well Becton Dickinson & Co.), i.e., about the same developed, setae-bearing segments and a complete diameter as the tubes of adult H. elegans, with or gut. The major stages of metamorphosis of H. without a biofilm (Walters et al., 1997). We found elegans are: (1) attachment to a firm, typically that larval settlement patterns were essentially the biofilmed surface; (2) secreting a membranous same on all three test surfaces. In addition, Walters primary tube from the larval epidermis; (3) et al. (1997) suspended plates with similar shedding the ciliarytrochal bands that provide the distributions of worms, empty worm tubes or tube mechanism for both larval swimming and filter mimics in Pearl Harbor for six days and afterwards feeding; and, (4) transforming the anterior, pre- noted both the numbers and locations to which trochal region of the body into the branchial crown larvae had recruited to the plates. Again, the result of tentacles (Carpizo-Ituarte, 1998, 1998, Nedved was that larvae of H. elegans settled in very similar and Hadfield, 2009). locations on all three, and more abundantly in the HADFIELD et al.: THE BACTERIAL BASIS OF BIOFOULING crevices formed between the tubes and the plate were then subjected to intense sequence analysis surfaces, most probably due to larval selection of using repeated PCR, often referred to as “genome biofilmed surfaces on the down-stream sides of the walking,” to characterize the genes whose tubes in flowing water. That is, the experiments disruption had led to the loss of inductive capacity demonstrated no gregarious preference by the (Huang et al., 2012). The two mutated genes were settling larvae; only the presence of a biofilm was found to lie close together in a single large operon of critical importance. containing at least seven genes and separated by only a single open-reading frame. Subsequent Bacterial induction of settlement and sequencing of the entire genome of P. luteoviolacea metamorphosis in Hydroides elegans allowed us to verify the gene sequences and their Although the nature of larval cell-surface positions relative to one another. In-frame deletion receptors for bacterial settlement cues continues to mutants for the seven genes lying sequentially in evade us, we have made significant progress in the operon revealed that each of the first four was analyzing the bacterial cues themselves. A first step individually required for the inductive activity, and was isolation of bacterial strains from marine the last three were not. Further analyses indicated biofilms that were strongly inductive for that differences in bacterial density or production of metamorphosis of larvae of H. elegans (Unabia and the extracellular polymeric substances (EPS) in Hadfield, 1999). We found that a wide variety of biofilms from the mutant strains were not bacteria from many bacterial groups had that responsible for the lack of inductive capacity. capacity. Next, a small set of strongly inductive and Comparisons of the sequenced genes in the operon non-inductive bacteria was selected for in-depth against known sequences in the NCBI protein data characterization, first by obtaining their identities bank failed to provide any exact identities, although from sequences of the 16S ribosomal RNA gene, the first one has similarity to a hypothetical phage- and then by examining their relative densities in tail protein and the second one has elements related mono-specific biofilms (Huang and Hadfield, to biofilm production in some bacteria. The third 2003). Of four bacterial species analyzed in this gene transcribes a protein that may function in cell study, two were found to be non-inductive and the adhesion. Most importantly, it was found that genes two others inductive, one producing greater whose products are essential to the settlement- settlement and metamorphosis than the other. The inducing capacity of P. luteoviolacea produce inductive activity of the bacteria was not found to apparently structural proteins rather than elements correlate with their taxonomic relationships. That is, that might simply be part of the mucus-rich EPS one species of the genus Cellulophaga, C. lytica, with which bacteria coat themselves in biofilms induced settlement of larvae of H. elegans and (Huang et al., 2012). another species in the same genus did not. The most The latest stage in our analysis of the inductive bacterial strain was identified as the inductive capacity of P. luteoviolacea included a species Pseudoalteromonas luteoviolacea. A more extended investigation of the genome of this bacterium species identified as Flexibactersp. was bacterium, especially a search for additional non-inductive. Further research revealed that a elements of phage-tail systems identified in other congener of the most inductive species, bacteria and called bacteriocins (Shikuma et al., Pseudoalteromonas atlantica, was non-inductive. 2014). Bacteriocins in several bacterial species are Furthermore, Huang and Hadfield (2003) found that known to be employed to kill other, presumably nearly equally inductive biofilms from P. competing, bacterial species (Michel-Briand and luteoviolacea were less dense than biofilms made Baysse, 2002, Riley and Wertz, 2002). They are from only C. lytica; clearly, the quality of specific complex organelles composed of the products of bacteria in a biofilm has greater importance in multiple genes that employ the injection apparatus larval recruitment than simply the density of any of bacteriophages to either perforate the cell given bacterial species. membranes of target bacteria or to inject substances Focusing on P. luteoviolacea, Huang et al. (toxins?) into other bacteria. Bacteriocins in a few (2012) employed transposon mutagenesis to obtain species have been found to kill insects (e.g., Yang two non-inductive strains of the bacterium; that is, et al., 2006). In P. luteoviolacea, we found biofilms made from either of two mutated bacterial additional genes for bacteriocin structures strains failed to induce settlement of H. elegans positioned upstream from the operon identified by under conditions identical with those in which the Huang et al. (2012). Again employing in-frame wild-type strain induced most larvae to settle and deletion mutation, we found three genes for phage- metamorphose. These two non-inductive strains tail like structures to be essential for the bacterium INDIAN J. MAR. SCI., VOL 43, NO.11 NOVEMBER 2014 to induce settlement of larvae of Hydroides elegans species originated before its journeys across the (Shikuma et al., 2014).A further analysis, seas on the hulls of ships began long ago. What we employing the methods of electron have been able to determine using genetic analyses cryotomography, revealed that single cells in a is that H. elegans continues to be distributed population of P. luteoviolacea synthesize great circum-globally, surely on ship hulls, to maintain numbers of bacteriocins that are linked into highly broadly interbreeding populations (Pettengill et al., organized arrays and released into the EPS by cell 2003, Pettengill et al., 2007). Because it is so easily rupture. The bacteriocin clusters are apparently reared in the laboratory, Miles et al (2007) carried retained in the EPS of the biofilm and interact with out extensive selection and inbreeding experiments larvae of Hydroides elegans to stimulate their for eleven generations to understand the capacity settlement and metamorphosis (Shikuma et al., for natural selection to impact egg size. They found 2014). significant heritability for the trait, indicating “… While we now have a significantly greater substantial potential for egg size to respond to knowledge of the characteristics of a bacterial varying selective pressures…,” with implications species that is important in initiating biofouling by for variations in larval size and longevity. the tubeworm Hydroides elegans, we have as yet no The physical relationships between settling understanding of how complex arrays of larvae of H. elegans and biofilms have been bacteriocins, derived from gene sets evolved to investigated in several ways. Zardus et al (2008) allow viruses to inject their DNA into living examined the surface-adhesion strength of primary bacterial cells, cause larvae to settle. Do the and secondary tubes on clean and biofilmed bacteriocins inject a substance into the polychaete surfaces. They did this by stimulating larvae to larvae that, in some manner, induces their settle on natural biofilms accumulated on glass settlement and metamorphosis? Or, do the slides or on clean slides where they had been bacteriocins simply interact with the membranes of induced to settle by exposure to 10 mM cesium sensitive cells on the larval surface, or perhaps cilia chloride in seawater. The slides bearing newly on such cells, puncturing them sufficiently to cause settled worms in primary tubes were then placed an ion flux that initiates an action potential that into a turbulent flow channel and exposed for four brings about metamorphosis? Data cited above on minutes to a wall-shear force of 50 pascals to ionic induction of metamorphosis in larvae of H. measure their resistance to dislodgement, after elegans and many other species suggest this might which the slides were removed from the flow be the case. Furthermore, we don’t yet know if channel and remaining juveniles counted. The result other inductive bacteria also produce bacteriocin was that significantly more juveniles remained on arrays that can induce settlement in H. elegans or if the biofilmed slides than on the clean ones. That is, the larvae respond to different cues from different the primary tubes of newly settled juveniles had bacterial species. And, finally, we don’t know stronger adhesion on biofilmed surfaces than on whether bacteriocin arrays in P. luteoviolacea are clean ones, which may shed some light on the the element that brings about metamorphosis in reason for selective settlement on biofilms by larvae coral larvae (Tran and Hadfield, 2011) and those of of H. elegans and other animals. Because larvae of an echinoid in Australia (Huggett et al., 2006), both some biofouling invertebrates have been reported to of which are known to settle in response to the same respond differentially to a range of surface-free bacterium. The genus Pseudoalteromonas has been energies, or wettabilities, we have examined the identified in many biofilms and implicated in the relationship between surface free energy, biofilm biology of other larval types (Hadfield, 2011, accumulation and settlement by H. elegans in the Tebben et al., 2011, Sneed et al., 2014). Studies on field and the laboratory (Huggett et al., 2009). The the interactions of larvae from other phyla with general result was that, after 10 days of submersion bacteria, in general, and with species of of a set of surfaces with a range of wettabilities, all Pseudoalteromonas, specifically, will be especially the surfaces were equally, and highly acceptable for enlightening. settlement by the larvae. That is, marine biofilm bacteria quickly form on surfaces with a broad Some other aspects of the recruitment biology of range of free energies and thus provide an equal Hydroides elegans stimulus for larval settlement, a finding with As noted above, Hydroides elegans has significance for those who may place hope in been recognized in biofouling communities in developing foul-resistant coatings based on initial tropical and warm-temperate seas around the world. wettability characteristics. We will likely never know exactly where the HADFIELD et al.: THE BACTERIAL BASIS OF BIOFOULING

Conclusions 5. Buck, L., and R. Axel. 1991. A novel multigene family Hydroides elegans has proven to be an may encode ordorant receptors: a molecular basis for odor recognition. Cell65:175-187. important model system for the study of larval 6. Campbell, A. H., D. W. Meritt, R. B. Franklin, E. L. development and metamorphosis of marine Boone, C. T. Nicely, and B. L. Brown. 2011. Effects of invertebrate animals, comparable to a few other age and composition of field-produced biofilms on oyster species such as the hydrozoan Hydrachtinia larval setting. Biofouling27:255-265. echinata, the bryozoan Bugula neretina, the oysters 7. Carlton, J. T., and L. G. Eldredge. 2009. Marine bioinvasions of Hawai‘i - the introduced and cryptogenic Ostrea edulis and Crassostrea gigas, the purple sea marine and estuarine animals and plants of the Hawaiian urchin Strongylocentrotus purpuratus, the barnacle archipelago. 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