New gregarine species (Apicomplexa) from tunicates show an evolutionary history of host switching and suggest a problem with the systematics of Lankesteria and Lecudina.

Apicomplexa (sensu stricto) are a diverse group of obligate parasites to a variety of animal species. Gregarines have been the subject of particular interest due to their diversity, phylogenetically basal position, and more recently, their symbiotic relationships with their hosts. In the present study, four new species of marine eugregarines infecting ascidian hosts (Lankesteria kaiteriteriensis sp. nov., L. dolabra sp. nov., L. savignyii sp. nov., and L. pollywoga sp. nov.) were described using a combination of morphological and molecular data. Phylogenetic analysis using small subunit rDNA sequences suggested that gregarines that parasitize ascidians and polychaetes share a common origin as traditionally hypothesized by predecessors in the discipline. However, Lankesteria and Lecudina species did not form clades as expected, but were instead intermixed amongst each other and their respective type species in the phylogeny. These two major genera are therefore taxonomically problematic. We hypothesize that the continued addition of new species from polychaete and tunicate hosts as well as the construction of multigene phylogenies that include type-material will further dissolve the currently accepted distinction between Lankesteria and Lecudina. The species discovered and described in the current study add new phylogenetic and taxonomic data to the knowledge of marine gregarine parasitism in ascidian hosts.

Tunic extract of the host ascidian attracts the causal agent of soft tunic syndrome, Azumiobodo hoyamushi (Kinetoplastea: Neobodonida).

Azumiobodo hoyamushi, a kinetoplastid flagellate, is the causative agent of soft tunic syndrome, an infectious disease of the edible ascidian Halocynthia roretzi. The flagellate is thought to invade the tunic matrix via a damaged area of the tunic on the siphon wall. We hypothesized that the flagellate locates the tunic entry site by a chemotactic response to soluble substances diffused from the host ascidians. To investigate this hypothesis, we examined whether the flagellate shows a chemotactic response to tissue extracts (tunic and other tissues) from the host ascidian H. roretzi. We tested extracts from 5 tissues as well as hemolymph. Only the tunic extract showed significant positive chemotactic activity, and the activity decreased with increasing dilution. Furthermore, autoclaved tunic extract, extracts from diseased individuals, and extract from the styelid ascidian Styela clava also had chemotactic activity, although the activities were lower than that of tunic extract from healthy H. roretzi. Ultrafiltration of the tunic extract through a 3 kDa cutoff membrane completely abrogated the activity; the ultrafiltration retentate still showed activity. Thus, the soluble factors that attract the flagellate are present exclusively in the tunic extract, and the chemotactic factors are larger than 3 kDa. Our experiments also suggested that the tunic extract contains both heat-stable and heat-labile factors. We conclude that the flagellate locates the tunic entry site by chemotaxis toward soluble factors that diffuse from a damaged area of the tunic on the siphon wall.

A new genus of Asterocheridae (Copepoda: Siphonostomatoida) ectoassociate of the ascidian Eudistoma vannamei Millar, 1977 (Polycitoridae) from Brazil.

Asterocheres Boeck, 1860 is the largest genus of the siphonostomatoid copepod family Asterocheridae, containing 63 valid species. The genus is known for its symbiotic relationships with many marine invertebrate taxa, especially sponges, cnidarians, bryozoans, and echinoderms. Recent studies have restricted the diagnosis of this genus. Consequently, many species are now considered as species inquirendae. The present paper describes a new species living externally on the tunic of Eudistoma vannamei Millar, 1977, an endemic ascidian from Brazil. As the new species does not fit Asterocheres in the strict sense, a new genus is erected to accommodate it. Setacheres gen. nov. is characterized by its possession of two distal setae on the third endopodal segment of P3, thus differing from the distal seta and spine pattern that is deemed as diagnostic of Asterocheres. A revision and comparison of Asterocheres´ species inquirendae revealed eight species sharing the same generic characteristics and were thus reallocated as members of the new genus.

Disinfection of fertilized eggs of the edible ascidian Halocynthia roretzi for prevention of soft tunic syndrome.

Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, was likely introduced to farming sites of the edible ascidian Halocynthia roretzi via ascidian spat. The source of infection is thought to be cysts of A. hoyamushi that reside in the substrates on which the ascidian spat are attached, but not the spat themselves. Thus, there is a need to develop methods to prevent contamination of the substrates with A. hoyamushi during seed production of the ascidian. We evaluated the protozoacidal effects of sodium hypochlorite and povidone-iodine against the flagellate and temporary cyst forms of A. hoyamushi. Additionally, we evaluated the effects of these disinfectants on the development of fertilized ascidian eggs. The flagellate form of A. hoyamushi was completely inactivated by povidone-iodine (5 ppm, 1 min) and sodium hypochlorite (1 ppm, 1 min). The temporary cysts of A. hoyamushi were completely inactivated by both disinfectants (5 ppm, 1 min). Disinfection with 50 ppm povidone-iodine for 15 min or 5 ppm sodium hypochlorite for 15 min had no effect on ascidian embryogenesis. Thus, horizontal transmission of A. hoyamushi via the substrates can be efficiently prevented by disinfecting ascidian eggs or tools used for spawning with povidone-iodine baths ranging from 5 ppm for 1 min to 50 ppm for 15 min without any side effects.

Cellulose is not degraded in the tunic of the edible ascidian Halocynthia roretzi contracting soft tunic syndrome.

Soft tunic syndrome is a fatal disease in the edible ascidian Halocynthia roretzi, causing serious damage to ascidian aquaculture in Korea and Japan. In diseased individuals, the tunic, an integumentary extracellular matrix of ascidians, softens and eventually tears. This is an infectious disease caused by the kinetoplastid flagellate Azumiobodo hoyamushi. However, the mechanism of tunic softening remains unknown. Because cellulose fibrils are the main component of the tunic, we compared the contents and structures of cellulose in healthy and diseased tunics by means of biochemical quantification and X-ray diffractometry. Unexpectedly, the cellulose contents and structures of cellulose microfibrils were almost the same regardless of the presence or absence of the disease. Therefore, it is unlikely that thinning of the microfibrils occurred in the softened tunic, because digestion should have resulted in decreases in crystallinity index and crystallite size. Moreover, cellulase was not detected in pure cultures of A. hoyamushi in biochemical and expressed sequence tag analyses. These results indicate that cellulose degradation does not occur in the softened tunic.

Encystment and excystment of kinetoplastid Azumiobodo hoyamushi, causal agent of soft tunic syndrome in ascidian aquaculture.

Soft tunic syndrome in the edible ascidian Halocynthia roretzi is caused by the kinetoplastid flagellate Azumiobodo hoyamushi, which was found to assume a fusiform cell form with 2 flagella in axenic, pure culture. When the flagellate form was incubated in sterilized artificial seawater (pH 8.4), some of the cells became cyst-like and adhered to the bottom of the culture plate. The cyst-like forms were spherical or cuboidal, and each had 2 flagella encapsulated in its cytoplasm. Encystment was also induced in culture medium alkalified to the pH of seawater (8.4) but not in unmodified (pH 7.2) or acidified media (pH 6.4). More than 95% of the cyst-like cells converted to the flagellate form within 1 d following transfer to seawater containing ascidian tunic extracts from host ascidians. The cyst-like cells were able to survive in seawater with no added nutrients for up to 2 wk at 20°C and for a few months at 5 to 15°C. The survival period in seawater depended on temperature: some cyst-like cells survived 3 mo at 10°C, and ca. 95% of these converted to flagellate forms in seawater containing tunic extracts. Thus, A. hoyamushi is able to persist under adverse conditions in a cyst-like form able to adhere to organic and inorganic substrata for protracted periods of time.

Molecular systematics of marine gregarine apicomplexans from Pacific tunicates, with descriptions of five novel species of Lankesteria.

The eugregarines are a group of apicomplexan parasites that mostly infect the intestines of invertebrates. The high level of morphological variation found within and among species of eugregarines makes it difficult to find consistent and reliable traits that unite even closely related lineages. Based mostly on traits observed with light microscopy, the majority of described eugregarines from marine invertebrates has been classified into a single group, the Lecudinidae. Our understanding of the overall diversity and phylogenetic relationships of lecudinids is very poor, mainly because only a modest amount of exploratory research has been done on the group and very few species of lecudinids have been characterized at the molecular phylogenetic level. In an attempt to understand the diversity of marine gregarines better, we surveyed lecudinids that infect the intestines of Pacific ascidians (i.e. sea squirts) using ultrastructural and molecular phylogenetic approaches; currently, these species fall within one genus, Lankesteria. We collected lecudinid gregarines from six ascidian host species, and our data demonstrated that each host was infected by a different species of Lankesteria: (i) Lankesteria hesperidiiformis sp. nov., isolated from Distaplia occidentalis, (ii) Lankesteria metandrocarpae sp. nov., isolated from Metandrocarpa taylori, (iii) Lankesteria halocynthiae sp. nov., isolated from Halocynthia aurantium, (iv) Lankesteria herdmaniae sp. nov., isolated from Herdmania momus, (v) Lankesteria cf. ritterellae, isolated from Ritterella rubra, and (vi) Lankesteria didemni sp. nov., isolated from Didemnum vexillum. Visualization of the trophozoites with scanning electron microscopy showed that four of these species were covered with epicytic folds, whereas two of the species were covered with a dense pattern of epicytic knobs. The molecular phylogenetic data suggested that species of Lankesteria with surface knobs form a clade that is nested within a paraphyletic assemblage species of Lankesteria with epicytic folds.

The sexual and mating system of the shrimp Odontonia katoi (Palaemonidae, Pontoniinae), a symbiotic guest of the ascidian Polycarpa aurata in the Coral Triangle.

Theory predicts that monogamy is adaptive in symbiotic crustaceans inhabiting relatively small and morphologically simple hosts in tropical environments where predation risk away from hosts is high. We tested this prediction in the shrimp Odontonia katoi, which inhabits the atrial chamber of the ascidian Polycarpa aurata in the Coral Triangle. Preliminary observations in O. katoi indicated that males were smaller than females, which is suggestive of sex change (protandry) in some symbiotic organisms. Thus, we first investigated the sexual system of O. katoi to determine if this shrimp was sequentially hermaphroditic. Morphological identification and size frequency distributions indicated that the population comprised males that, on average, were smaller than females. Gonad dissections demonstrated the absence of transitional individuals. Thus, O. katoi is a gonochoric species with reverse sexual dimorphism. The population distribution of O. katoi in its ascidian host did not differ significantly from a random distribution and shrimps inhabiting the same host individual as pairs were found with a frequency similar to that expected by chance alone. This is in contrast to that reported for other socially monogamous crustaceans in which pairs of heterosexual conspecifics are found in host individuals more frequently than expected by chance alone. Thus, the available information argues against monogamy in O. katoi. Furthermore, that a high frequency of solitary females were found brooding embryos and that the sex ratio was skewed toward females suggests that males might be roaming among hosts in search of receptive females in O. katoi. Symbiotic crustaceans can be used as a model system to understand the adaptive value of sexual and mating systems in marine invertebrates.

The molecular phylogeny of the type-species of Oodinium Chatton, 1912 (Dinoflagellata: Oodiniaceae), a highly divergent parasitic dinoflagellate with non-dinokaryotic characters.

Oodinium pouchetii (Lemmermann, 1899) Chatton, 1912, the first described parasitic dinoflagellate, is the type of the Oodiniaceae Chatton, 1920. In the taxonomical schemes, this family of metazoan parasites includes Amyloodinium Brown & Hovasse, 1946 and Piscinoodinium Lom, 1981 that are responsible of important damages in fish aquaculture. Species of Oodinium Chatton, 1912 have unique characteristics such as the possession of both non-dinokaryotic and dinokaryotic nuclei within the life-cycle, and the absence of the transversal (cingulum) and longitudinal (sulcus) surface grooves in the parasitic stage. We provide the first molecular data for the genus Oodinium from specimens of O. pouchetii infecting the chordate Oikopleura sp. (Tunicata: Appendicularia) off the coasts of Brazil. Although O. pouchetii lacks dinokaryotic characters in the parasitic stage, the SSU rDNA phylogeny revealed that it forms a distinct fast-evolved clade that branches among the dinokaryotic dinoflagellates. However, there is no clear relationship with other dinoflagellates. Hence, the taxonomic affinity of the family Oodiniaceae is unclear at the moment.

Quantitative assessment of Azumiobodo hoyamushi distribution in the tunic of soft tunic syndrome-affected ascidian Halocynthia roretzi using real-time polymerase chain reaction.

BACKGROUND: The kinetoplastid parasite, Azumiobodo hoyamushi, is the causative agent of soft tunic syndrome (STS) in ascidians and leads to their mass mortality in Korean waters. This study was conducted to quantify A. hoyamushi density during the development of STS in the tunics of ascidians (Halocynthia roretzi) using real-time polymerase chain reaction (qPCR). FINDINGS: The infection intensity of A. hoyamushi, as measured by qPCR, varied depending on the part of the tunic analyzed, as well as the stage of STS development. The highest infection intensity was recorded in the tunics of the siphons. The infection intensity of A. hoyamushi in the siphons was only 2.9 cell/tunic (area, 0.25 cm(2)) or 106.0 cell/gram tunic (GT) in the early phase of STS, but this value increased dramatically to 16,066 cells/tunic (0.25 cm(2)) or 617,004 cell/GT at the time of death. The number of A. hoyamushi parasites increased gradually and their distribution spread from the siphons to the other parts of the tunics. CONCLUSIONS: qPCR enabled the quantitation of A. hoyamushi and the results revealed that parasite density increased as STS progressed. In addition, our results suggested that the siphons might function as the portal of entry for A. hoyamushi during infection.

Azumiobodo hoyamushi, the kinetoplastid causing soft tunic syndrome in ascidians, may invade through the siphon wall.

The infectious kinetoplastid Azumiobodo hoyamushi causes 'soft tunic syndrome', a serious problem in aquaculture of the edible ascidian Halocynthia roretzi. Infection tests using diseased tunics demonstrated that juvenile (0.8 yr old) individuals never developed soft tunic syndrome, but all individuals in the other age groups (1.8, 2.8, and 3.8 yr old) showed the disease symptoms. In the infection tests, tunic softening was first observed at the tunic around siphons. Based on ultrastructural observation of the inner wall of the branchial siphon, the tunic lining the inner wall in juveniles (0.5 yr old) was completely covered with cuticle, which had a dense structure to prevent bacterial and protist invasion. In contrast, the tunic was often partly damaged and not covered with cuticle in healthy adults (≥2.5 yr old). The damaged tunic in the siphon wall could be an entrance for A. hoyamushi into the tunic of adult hosts.

The kinetoplastid parasite Azumiobodo hoyamushi, the causative agent of soft tunic syndrome of the sea squirt Halocynthia roretzi, resides in the East Sea of Korea.

Mass mortality of the edible sea squirt Halocynthia roretzi since the 1990s in the southern and eastern seas of Korea has caused large economic losses. The disease is characterized by symptoms of initially softened and thinned tunics that eventually rupture. Thus, the disease is called soft tunic syndrome (STS); however, the causative agent in these regions is unknown. In the present study, two kinetoplastid organisms were isolated from STS sea squirts collected from culture farms in Tongyeong located in the East Sea of Korea. Phylogenetic analysis of 18S rRNA sequences identified these organisms as Azumiobodo hoyamushi and Procryptobia sorokini. These kinetoplastids were injected into healthy sea squirts and cultured at 15°C for 13days. Sea squirts injected with A. hoyamushi showed 100% STS whereas, P. sorokini did not induce disease, thereby confirming A. hoyamushi as the causative agent of STS. A. hoyamushi flourishes in vitro at 10-15°C, and dies at temperatures below 5°C or above 20°C. The optimum salinity level for growth is 30-35psu, and death occurs below 25psu. These optima coincide with marine temperature and salinity levels between March and June on the southern coasts of Korea, the period when the syndrome occurs at the highest frequency. The identification here of A. hoyamushi as the causative agent of STS and our findings regarding its optimum growth conditions should lead to methods for reducing the incidence of STS.

In vitro and in vivo efficacy of drugs against the protozoan parasite Azumiobodo hoyamushi that causes soft tunic syndrome in the edible ascidian Halocynthia roretzi (Drasche).

It was discovered recently that infection by a protozoan parasite, Azumiobodo hoyamushi, is the most probable cause for soft tunic syndrome in an edible ascidian, Halocynthia roretzi (Drasche). In an attempt to develop measures to eradicate the causative parasite, various drugs were tested for efficacy in vitro and in vivo. Of the 20 antiprotozoal drugs having different action mechanisms, five were found potent (24-h EC50  < 10 mg L(-1) ) in their parasite-killing effects: formalin, H2 O2 , bithionol, ClO2 and bronopol. Moderately potent drugs (10 < 24-h EC50  < 100 mg L(-1) ) were quinine, fumagillin, amphotericin B, ketoconazole, povidone-iodine, chloramine-T and benzalkonium chloride. Seven compounds, metronidazole, albendazole, paromomycin, nalidixic acid, sulfamonomethoxine, KMnO4 , potassium monopersulphate and citric acid, exhibited EC50  > 100 mg L(-1) . When ascidians were artificially infected with A. hoyamushi, treated using 40 mg L(-1) formalin, bronopol, ClO2 , or H2 O2 for 1 h and then monitored for 24 h, very low mortality was observed. However, the number of surviving parasite cells in the ascidian tunic tissues was significantly reduced by treating with 40 mg L(-1) formalin or ClO2 for 1 h. The data suggest that we might be able to develop a disinfection measure using a treatment regimen involving commonly available drugs.

Detection of the kinetoplastid Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, in wild ascidians Halocynthia roretzi.

The occurrence of soft tunic syndrome in wild populations of the ascidian Halocynthia roretzi was monitored by diving at 5 to 6 sites in Miyagi Prefecture in Japan in summer 2010 and 2011. These sites were located at varying distances from farming sites at which the disease had previously been detected. All dead ascidians were collected, and their tunics were examined for Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, using 18S rRNA PCR. In both years, <1% of wild ascidians we observed (18 out of 2100 in 2010, and 30 out of 3100 in 2011) were dead. The flagellates were only detected in 8 out of 18 dead ascidians from 3 sites in 2010, and 4 out of 30 from 2 sites in 2011. Healthy ascidians were successfully experimentally infected with the disease by immersing tunic samples from diseased wild ascidians into the rearing water. When apparently healthy ascidians collected from the wild population were reared for 40 d using pathogen-free water, the tunics of some ascidians became softened. The flagellates were detected in these individuals, which were diagnosed with soft tunic syndrome. Our results suggest that soft tunic syndrome affects the wild population of ascidians in Japan.

The enigmatic life history of the symbiotic crab Tunicotheres moseri (Crustacea, Brachyura, Pinnotheridae): implications for its mating system and population structure.

Resource-monopolization theory predicts the adoption of a solitary habit in species using scarce, discrete, and small refuges. Life-history theory suggests that temporarily stable parental dwellings favor extended parental care in species that brood embryos. We tested these two predictions with the symbiotic crab Tunicotheres moseri. This species exhibits abbreviated development and inhabits the atrial chamber of the scarce, structurally simple, long-lived, and relatively small ascidian Phalusia nigra in the Caribbean. These host characteristics should favor a solitary habit and extended parental care (EPC) in T. moseri. As predicted, males and females of T. moseri inhabited ascidians solitarily with greater frequency than expected by chance alone. The male-female association pattern and reverse sexual dimorphism (males < females) additionally suggests a promiscuous "pure-search" mating system in T. moseri. Also in agreement with theoretical considerations, T. moseri displays EPC; in addition to embryos, females naturally retain larval stages, megalopae, and juveniles within their brooding pouches. This is the first record of EPC in a symbiotic crab and the second confirmed record of EPC in a marine brachyuran crab. This study supports predictions central to resource-monopolization and life-history theories.

Azumiobodo hoyamushi gen. nov. et sp. nov. (Euglenozoa, Kinetoplastea, Neobodonida): a pathogenic kinetoplastid causing the soft tunic syndrome in ascidian aquaculture.

We used morphological and genetic analyses to investigate a pathogenic kinetoplastid isolated from a diseased edible ascidian Halocynthia roretzi with soft tunic syndrome. The morphological characteristics of the kinetoplastid are similar to those in the order Neobodonida in the subclass Metakinetoplastida. However, the presence of unique globular bodies distinguishes this kinetoplastid from the other polykinetoplastic genera (i.e. Cruzella, Dimastigella and Rhynchobodo) in this order. These globular bodies are cytoplasmic inclusions without an outer delimiting membrane and are composed of a homologous granular matrix containing electron-dense bands. A phylogenetic tree based on 18S rRNA gene sequences also indicated that the kinetoplastid belongs to the order Neobodonida, although it forms an independent clade in this order. From these results, we propose a new genus in the order Neobodonida, i.e. Azumiobodo gen. nov., and Azumiobodo hoyamushi as the type species for the genus.

RNA-seq-based metatranscriptomic and microscopic investigation reveals novel metalloproteases of Neobodo sp. as potential virulence factors for soft tunic syndrome in Halocynthia roretzi.

Bodonids and trypanosomatids are derived from a common ancestor with the bodonids being a more primitive lineage. The Neobodonida, one of the three clades of bodonids, can be free-living, commensal or parasitic. Despite the ecological and evolutionary significance of these organisms, however, many of their biological and pathological features are currently unknown. Here, we employed metatranscriptomics using RNA-seq technology combined with field-emission microscopy to reveal the virulence factors of a recently described genus of Neobodonida that is considered to be responsible for ascidian soft tunic syndrome (AsSTS), but whose pathogenesis is unclear. Our microscopic observation of infected tunic tissues suggested putative virulence factors, enabling us to extract novel candidate transcripts; these included cysteine proteases of the families C1 and C2, serine proteases of S51 and S9 families, and metalloproteases grouped into families M1, M3, M8, M14, M16, M17, M24, M41, and M49. Protease activity/inhibition assays and the estimation of expression levels within gene clusters allowed us to identify metalloprotease-like enzymes as potential virulence attributes for AsSTS. Furthermore, a multimarker-based phylogenetic analysis using 1,184 concatenated amino acid sequences clarified the order Neobodo sp. In sum, we herein used metatranscriptomics to elucidate the in situ expression profiles of uncharacterized putative transcripts of Neobodo sp., combined these results with microscopic observation to select candidate genes relevant to pathogenesis, and used empirical screening to define important virulence factors.

Redescription of Cardiosporidium cionae (Van Gaver and Stephan, 1907) (Apicomplexa: Piroplasmida), a plasmodial parasite of ascidian haemocytes.

Cardiosporidium cionae (Apicomplexa), from the ascidian Ciona intestinalis L., is redescribed with novel ultrastructural, phylogenetic and prevalence data. Ultrastructural analysis of specimens of C. intestinalis collected from the Gulf of Naples showed sporonts and plasmodia of C. cionae within the host pericardial body. Several merogonic stages and free merozoites were found in the pericardial body, together with sexual stages. All stages showed typical apicomplexan cell organelles, i.e. apicoplasts, rhoptries and subpellicular microtubules. Merogonic stages of C. cionae were also produced inside haemocytes. A fragment of the rSSU gene of C. cionae was amplified by PCR using DNA extracted from the pericardial bodies. The amplified product showed closest affinity with other apicomplexan representatives and a 66bp unique insertion, specific for C. cionae, at position 1644. Neighbour-joining phylogenetic analysis placed C. cionae in a clade with other piroplasm genera, including Cytauxzoon, Babesia and Theileria spp. The parasite was found in different populations of C. intestinalis with highest prevalence in October-November. Ultrastructural and DNA data showed that the organism, described in 1907 from the same host but not illustrated in detail, is a member of a novel marine apicomplexan radiation of tunicate parasites.

First report on histology and ultrastructure of an intrahemocytic paramyxean parasite (IPP) from tunicate Halocynthia roretzi in Korea.

In 2004, epizootiological studies were conducted on mass mortalities of tunicates Halocynthia roretzi in Goje, Korea. The clinical characteristics of infected H. roretzi were weakness of the tunic, loss of elasticity, and finally death involving a rupture of the tunic. Histological studies revealed severe hemocyte infiltration in the connective tissue surrounding the intestine and mantle of infected H. roretzi. Hypertrophied eosinophilic hemocytes containing several cytoplasmic vacuoles were observed in the connective tissue surrounding the intestine, gill and mantle. Ultrastructural examination revealed the presence of a parasite in the cytoplasm of hemocytes. Secondary cells were observed in the primary cell of the parasite. Spore formation within primary cells suggests that the parasite may be an intrahemocytic paramyxean parasite (IPP) and may cause mass mortality of H. roretzi.

Defense of benthic invertebrates against surface colonization by larvae: a chemical arms race.

Sessile invertebrates evolved in a competitive milieu where space is a limiting resource, setting off an arms race between adults that must maintain clean surfaces and larvae that must locate and attach to a suitable substratum. I review the evidence that invertebrates chemically deter or kill the propagules of fouling animals and protists under natural conditions, and that chemosensory mechanisms may allow larvae to detect and avoid settling on chemically protected organisms. The fouling process is an ecologically complex web of interactions between basibionts, surface-colonizing microbes, and fouling larvae, all mediated by chemical signaling. Host-specific bacterial communities are maintained by many invertebrates, and may inhibit fouling by chemical deterrence of larvae, or by preventing biofilm formation by inductive strains. Larval settlement naturally occurs in a turbulent environment, yet the effects of waterborne versus surface-adsorbed chemical defenses have not been compared in flow, limiting our understanding of how larvae respond to toxic surfaces in the field. The importance of evaluating alternative hypotheses such as mechanical and physical defense is discussed, as is the need for ecologically relevant bioassays that quantify effects on larval behavior and identify compounds likely to play a defensive role in situ.