Solitary Tunicate Research Articles

Halocyntin and papillosin, two new antimicrobial peptides isolated from hemocytes of the solitary tunicate, Halocynthia papillosa

We report here the screening of five marine invertebrate species from two taxa (tunicates and echinoderms) for the presence of cationic antimicrobial peptides (AMP) in defence cells (hemocytes). Antimicrobial activities were detected only in the two tunicates Microcosmus sabatieri and Halocynthia papillosa. In addition, we report the isolation and characterization of two novel peptides from H. papillosa hemocytes. These molecules display antibacterial activity against Gram-positive and Gram-negative bacteria. Complete peptide characterization was obtained by a combination of Edman degradation and mass spectrometry. The mature molecules, named halocyntin and papillosin, comprise 26 and 34 amino acid residues, respectively. Their primary structure display no significant similarities with previously described AMP.

Size-Specific Predation on Marine Invertebrate Larvae

Predation on planktonic larval stages is frequently a major source of mortality for the offspring of benthic marine invertebrates. Mortality rate likely varies with larval size and developmental stage, but few experiments have measured how these factors affect predation rates. I used experimental reductions in egg size to test how variation in larval size affects the likelihood of predation during planktonic development. Blastomeres of the sand dollar Dendraster excentricus were separated at the two-cell stage to produce half-sized zygotes. Larvae resulting from this manipulation were tested for their susceptibility to predation relative to whole-sized siblings at four ages. Individuals from each size class were simultaneously presented as prey items to five predators (crab zoeae, crab megalopae, chaetognaths, solitary tunicates, and postlarval fish) in the laboratory. Four predators consumed significantly more half-sized larvae than whole-sized larvae, but one predator type (postlarval fish) consumed more whole-sized larvae. Predators that consumed more half-sized larvae also preferentially consumed younger larvae. In contrast, postlarval fish showed no significant prey preference based on larval age. These results suggest that assumptions of constant mortality rates during development should be modified to account for the effects of larval size and age.

The Role of Vanadium in the Chemical Defense of the Solitary Tunicate, Phallusia nigra

Ascidians (sea squirts) may defend themselves from predators, biofouling competitors, and bacterial infection by producing secondary metabolites or sequestering acid, but many species also accumulate heavy metals, most notably vanadium. The defensive functions of heavy metals in ascidians remain unclear, and to this end, the solitary Caribbean tunicate, Phallusia nigra, was studied to localize vanadium in its tissues and to assess the defensive properties of vanadium-containing compounds. As determined by flame atomic absorption spectroscopy, the internal tissues and blood contained the highest vanadium concentrations (mean values of 2280 and 1886 ppm dry mass, respectively), followed by the tunic surface (871 ppm dry mass). Results of laboratory feeding assays with the bluehead wrasse, Thalassoma bifasciatum, confirmed outcomes of past studies that demonstrated that vanadyl sulfate (VOSO4.6H20) and sodium vanadate (Na3VO4) were unpalatable to fish, although these salts do not accurately reflect the chelation environment or oxidation state of vanadium in living tunicates. Fresh preparations of whole tunic, internal tissues, and blood were unpalatable to fish, but freezing and thawing of internal tissues and blood rendered them palatable. Crude organic extracts of whole tunic and internal tissues contained vanadium metabolites (225 and 750 ppm dry mass, respectively) and were palatable to T. bifasciatum; crude extracts also exhibited no antimicrobial effects against a panel of four marine bacteria known to be pathogens of marine invertebrates (Vibrio parahaemolyticus, Vibrio harveyi, Leucothrix mucor, and Deleya marina). Nonacidic vanadium (+3) complexes neither deterred predation nor inhibited microbial growth, whereas acidic aqua vanadium (+3 and +4) complexes were unpalatable to 7 bifasciatum and exhibited antimicrobial activity. Difficulties in decoupling low pH from oxidation state and chelation environment of vanadium prevent definitive conclusions about the importance of some vanadium metabolites, but low pH appears to be the principal agent of chemical defense for P. nigra.

Production of transparent exopolymer particles (TEP) by benthic suspension feeders in coastal systems

In the marine environment, transparent exopolymer particles (TEP) are ubiquitous and abundant, playing a significant role in carbon cycling and the structuring of food webs. Previous studies have shown that phytoplankton, bacteria, and oysters contribute to the production of TEP through the release of exopolymers. However, little is known about other potential sources of TEP and TEP precursors, especially in coastal systems. It was hypothesized that suspension feeders contribute to the TEP pool in near-shore environments through the release of exopolymers in both the dissolved and particulate form, and tested these hypotheses in both laboratory and field experiments. In the laboratory, the production of TEP by several species of benthic suspension feeders (the blue mussel, Mytilus edulis; the bay scallop, Argopecten irradians; the slipper snail, Crepidula fornicata; and the solitary ascidians, Ciona intestinalis and Styela clava) was investigated from October to November 2002 and June 2003. Concentrations of TEP and DOC were determined by spectrophotometry after alcian blue staining and high-temperature, catalytic oxidation, respectively. Similar analyses were conducted on water samples from the field in July 2003, collected in close proximity to dense beds of mussels in the Poquonnock River, Connecticut, USA (41°19′ N, − 72°02′ W). Laboratory results indicated that actively-pumping blue mussels, bay scallops, slipper snails, and both species of solitary ascidians significantly enhanced TEP concentrations above background levels over a five-hour period. However, only the solitary tunicate S. clava significantly enhanced DOC concentrations above background levels over the same period of time. Field samples indicated that TEP and DOC concentrations were high in close proximity to dense beds of mussels. These results imply that a variety of benthic suspension feeders produce TEP during feeding activities which could lead to enhanced flocculation of organic matter and carbon deposition in near-shore waters.

Self-fertilization as an alternative mode of reproduction in the solitary tunicate Pyura chilensis

The hermaphroditic broadcasting tunicate Pyura chilensis Molina, 1782 is a sessile filter-feeder organism that occurs in intertidal and subtidal habitats along the Chilean and Peruvian coast. In natural populations, P. chilensis form dense aggregations, small patches or occur as isolated individuals. This suggests that self-fertilization could be a potential insurance against adverse conditions for cross-fertilization. In this study, P. chilensis were reared in the laboratory as isolated and paired individuals, to assess occurrence and success of fertilization, settlement and metamorphosis. Occurrence of self-fertilization was also compared between specimens forced to cross-fertilize and specimens maintained in reproductive isolation for different periods. We also manipulated cross- and self-fertilization using strip-spawned gametes. Our study shows that P. chilensis is a hermaphroditic species with adolescent gonochorism; that is, individuals first developed male function and then later male and female functions simultaneously as specimens increased in size (protandrous hermaphroditism). The results also show more frequent fertilization in paired specimens and in manipulated fertilization involving cross sperm. Moreover, no perceptible differences in fertilization, settlement, and metamorphosis success among self and outcross progeny were found. Prolonged periods of reproductive isolation resulted in more frequent self-fertilization. Occurrence of selfing and highly successful settlement and metamorphosis of progeny originating from reproductively-isolated specimens suggest that even though outcrossing predominates, selfing is an advantageous alternative when sources of allosperm are scarce.

Biological control of fouling in suspended scallop cultivation

Fouling of scallop shells and cultivation nets by living organisms is costly to remove and can reduce scallop growth. Here we investigate biological control of fouling in suspended scallop ( Pecten maximus) cultivation. In preliminary trials in the Irish Sea, off the Isle of Man, sea urchins ( Echinus esculentus and Psammechinus miliaris) and hermit crabs ( Pagurus spp.) removed fouling from nets more efficiently than did a range of other invertebrates. Sea urchins and hermit crabs were subsequently deployed from August 2000 to January 2001 at various densities (hermit crabs and E. esculentus at 1, 2, or 3 per net and P. miliaris at 3, 5 or 7 per net) in pearl nets containing scallops. After 6 months, the survival of biological control organisms and their effect on scallop growth and mortality, and fouling of nets and scallop shells, were assessed. Only P. miliaris was associated with increased scallop shell growth, but no biological control organism reduced scallop growth or survival. All three biological control organisms significantly reduced the weight of fouling on nets (often by as much as 50%) and fouling of scallop shells; results were largely independent of biological control organism density. Sea urchins were most effective, removing hydroids and solitary tunicates efficiently; they could thus be commercially exploited alongside scallops in a form of polyculture. These results suggest that biological control could be an efficient and environmentally sound method of addressing the problem of fouling in scallop cultivation.

Plicatamide, an Antimicrobial Octapeptide from Styela plicata Hemocytes

Plicatamide (Phe-Phe-His-Leu-His-Phe-His-dc Delta DOPA), where dc Delta DOPA represents decarboxy-(E)-alpha,beta-dehydro-3,4-dihydroxyphenylalanine, is a potently antimicrobial octapeptide from the blood cells of the solitary tunicate, Styela plicata. Wild type and methicillin-resistant Staphylococcus aureus (MRSA) responded to plicatamide exposure with a massive potassium efflux that began within seconds. Soon thereafter, treated bacteria largely ceased consuming oxygen, and most became nonviable. Native plicatamide also formed cation-selective channels in model lipid bilayers composed of bacterial lipids. Methicillin-resistant S. aureus treated with plicatamide for 5 min contained prominent mesosomes as well as multiple, small dome-shaped surface protrusions that suggested the involvement of osmotic forces in its antimicrobial effects. To ascertain the contribution of the C-terminal dc Delta DOPA residue to antimicrobial activity, we synthesized several analogues of plicatamide that lacked it. One of these peptides, PL-101 (Phe-Phe-His-Leu-His-Phe-His-Tyr-amide), closely resembled native plicatamide in its antimicrobial activity and its ability to induce potassium efflux. Plicatamide was potently hemolytic for human red blood cells but did not lyse ovine erythrocytes. The small size, rapid action, and potent anti-staphylococcal activity of plicatamide and PL-101 make them intriguing subjects for future antimicrobial peptide design.

Secretion of a collectin-like protein in tunicates is enhanced during inflammatory responses

The sub-cellular and humoral concentrations of a collectin-like protein from the solitary tunicate, Styela plicata, were measured after in vivo challenge with the inflammatory agent, zymosan. Tunicates were injected with zymosan before hemocytes and serum were harvested, subjected to western blotting and immunostained with an anti- S. plicata collectin antibody to determine the relative titers of collectin-like proteins. Concentrations of the predominant 43 kDa collectin polypeptide were found to decrease in hemocytes immediately after zymosan injection, before rising to levels that were six times higher than controls within 96 h. Similarly, immunohistochemistry showed that the frequency of collectin-positive hemocytes in the circulating hemolymph increased significantly within 96 h of injection. Levels of the 43 kDa polypeptide in serum mirrored those of hemocytes. Humoral collectin concentrations decreased immediately after zymosan injection before rising, within 96 h post-injection, to levels three times higher than controls. This response to an inflammatory stimulus resembles that of mammalian collectins like mannose-binding lectin. The data suggest that, like its mammalian counterparts, the tunicate collectin acts as an acute phase antigen recognition protein.

Halocidin: a new antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium

From hemocytes of the tunicate Halocynthia aurantium we purified a new antimicrobial peptide named halocidin. The native peptide had a mass of 3443 Da and comprised two different subunits containing 18 amino acid residues (WLNALLHHGLNCAKGVLA) and 15 residues (ALLHHGLNCAKGVLA), which were linked covalently by a single cystine disulfide bond. Two different monomers were separately synthesized and used to make three additional isoforms (15 residue homodimer, 18 residue homodimer, heterodimer). In antimicrobial assays performed with synthetic peptides of halocidin, it was confirmed that congeners of the 18 residue monomer were more active than those of the 15 residue monomer against methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa.

A complement component C3-like protein from the tunicate, Styela plicata

This study identifies a complement component C3-like protein in the solitary tunicate, Styela plicata. Three different polyclonal antibodies raised against C3 molecules from two species (humans and the tunicate, Halocynthia roretzi) were used to identify the C3-like protein in S. plicata hemolymph. The C3 cross-reactive protein is a 170 kDa heterodimer composed of polypeptides (116 and 80 kDa) that have molecular weights comparable to those of C3α and C3β from other species. Amino acid sequencing and amino acid composition analysis confirmed that the C3-like protein from S. plicata is closely related to C3 from H. roretzi.

Dicynthaurin: an antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium

We isolated a novel antimicrobial peptide, dicynthaurin, from hemocytes of a tunicate, Halocynthia aurantium. The native peptide had a mass of approximately 6.2 kDa and was composed of two 30-residue monomers without sequence homology to any previously identified peptides (ILQKAVLDCLKAAGSSLSKAAITAIYNKIT). Most cynthaurin molecules were C-terminally amidated and were linked covalently by a single cystine disulfide bond. When performed in membrane-mimetic environments, circular dichroism studies of dicynthaurin revealed largely α-helical conformations. Dicynthaurin’s broad-spectrum activity encompassed Gram-positive ( Micrococcus luteus, Staphylococcus aureus, Listeria monocytogenes) and Gram-negative bacteria ( Escherichia coli, Pseudomonas aeruginosa), but not Candida albicans, a fungus. Although dicynthaurin was purified from a marine invertebrate, its antimicrobial activity was optimal at NaCl concentrations below 100 mM. This suggests that the antimicrobial actions of this molecule may take place intracellularly (e.g., within a phagosome) rather than extracellularly.

Settlement of the gregarious tube worm Hydroides dianthus (Polychaeta: Serpulidae). I. Gregarious and nongregarious settlement

We conducted still-water time-course experiments on cultured larvae of the serpulid polychaete Hydroides dianthus (Verrill, 1873) to examine timing and patterns of gregarious and non- gregarious settlement to better understand the conditions under which larvae of a gregarious species colonize new habitats. We first confirmed that these worms are aggregated in the field, and quanti- fied patterns of association with a number of other common intertidal species. Patterns of negative association with spatial competitors is unlikely to have resulted from larval avoidance during settle- ment, because we found no difference in larval settlement in response to conspecifics with or without the solitary tunicate Ascidia interrupta present. We then examined the settlement of competent lar- vae in response to biofilm and conspecifics to determine the conditions under which the larvae of this gregarious species settle nongregariously. Larvae settled concurrently in response to both biofilm and conspecifics, starting approximately 4 d after fertilization, and response was of similar magnitude to both biofilm and to conspecifics for the first couple of days post-competency. While settlement in response to conspecifics continued in subsequent assays carried out over 70 d (at which time larvae began to die in culture), settlement in response to biofilm decreased abruptly after the peak, and ceased by Day 14. Settlement patterns were qualitatively similar regardless of previous exposure to substrata; similar results were obtained whether the whole population of larvae in culture was denied access to or daily provided access to 1 or both experimental substrata. Furthermore, these patterns of settlement did not change as larval planktonic period was prolonged. Of larvae settling during 24 h sample assays, the majority of larvae metamorphosed in response to biofilm during the first 6 h, whereas metamorphosis in the final 6 h (between 18 and 24 h) of the assays was primarily in response to conspecifics. These results are opposite to the pattern predicted by the desperate larva hypothesis, because some larvae settled in response to biofilm even after rejecting conspecifics the previous day. In fact, cultures of competent larvae appear to become more specific in terms of larval settlement in response to live adult conspecifics as the planktonic period is artificially prolonged.

Investigation of bioactivity of extracts from Moroccan solitary tunicate Cynthia savignyi

Extracts of the tunicate Cynthia savignyi from the Moroccan Atlantic sea have been investigated in five bioassays. The first is an antibacterial test against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Agrobacterium tumefaciens; the second is an antifungal test against three tomato pathogenic fungi, Botrytis cinerea, Fusarium oxysporum and Verticillium albo-atrum; the third is a test based to the ability to reduce DNA peak size in procedures using an HPLC system for detection of antitumour agents; the fourth is a toxicity test using larva of Artemia salina; and the last is an enzymatic inhibitory test against papain and trypsin. For all the bioassays, extracts of C. savignyi were found to be bioactive. This result suggests that this tunicate is able to produce biologically active agents required for an overall defence against their predators.

Styelins, Broad-Spectrum Antimicrobial Peptides from the Solitary Tunicate, Styela clava

Two novel phenylalanine-rich antimicrobial peptides, Styelin A and Styelin B, were purified from the hemocytes of Styela clava. The peptides had very similar masses (Styelin A, 3685.8; Styelin B, 3700.6) and amino acid compositions, and at least 17 of their first 20 N-terminal residues were identical. Both Styelins were effective against a panel of gram negative and gram positive bacterial pathogens of humans, usually acting with minimal inhibitory concentrations < 1.5 μg/ml (<0.5 μM), even in the presence of 100 mM NaCl. Styelins also killed marine bacteria, Psychrobacter immobilis and Planococcus citreus, in media containing 0.4 M NaCl. The presence of antimicrobial peptides (Styelins) in tunicate hemocytes is evidence that such molecules are ancient mediators of host defense within the vertebrate lineage. Peptide antibiotics from marine organisms could afford design templates for the development of topical microbicides that manifest broad-spectrum antibacterial activity in the presence of physiological or elevated NaCl concentrations.

Clavanins, α-helical antimicrobial peptides from tunicate hemocytes

Hemocytes from the invertebrate Styela clava, a solitary tunicate, contained a family of four α-helical antimicrobial peptides that were purified, sequenced and named clavanins A, B, C and D. Each clavanin contained 23 amino acid residues and was C-terminally amidated. The tunicate peptides resembled magainins in size, primary sequence and antibacterial activity. Synthetic clavanin A was prepared and displayed comparable antimicrobial activity to magainins and cecropins. The presence of α-helical antimicrobial peptides in the hemocytes of a urochordate suggests that such peptides are primeval effectors of innate immunity in the vertebrate lineage.

Adoptive transfer of alloimmune memory in the solitary tunicate,Styela plicata

The role of hemocytes in histocompatibility responses of the tunicate, Styela plicata, was tested by adoptive transfer. Hemocytes were transferred to naive hosts from compatible individuals that had been presensitized to allogeneic tissue. Forty-four percent of the hosts that received presensitized hemocytes were capable of eliminating subsequent allografts more rapidly than control tunicates injected with saline, plasma, or hemocytes that had not been sensitized to the original graft donor. This adoptive transfer of alloimmune memory was specific to the presensitizing allogeneic tissue type. The transfer of hemocyte populations enriched in lymphocyte-like cells by density gradient centrifugation also specifically enhanced alloreactivity. Hemocyte fractions depleted of lymphocyte-like cells were incapable of inducing alloimmune memory. These data confirm that allograft rejection in S. plicata is a cell-mediated response involving lymphocyte-like cells. © 1996 Wiley-Liss, Inc.

Cytotoxicity reactions in the solitary tunicate Styela plicata

Hemocytes from the tunicate, Styela plicata, undertake cytotoxic responses toward human KS62 tumor cells or rabbit red blood cells (RRBC). Reactions to both target cell types were found to require metabolic activity. However, it was not determined whether killing involves soluble factors or direct cell/cell contact. Differences in the response to varied effector: target ratios indicate that reactions against the K562 cells and RRBC are mediated by separate mechanisms. The existence of two discrete cytotoxic mechanisms was supported by experiments which demonstrated that pharmacological treatments have different effects on the cytotoxic destruction of K562 cells and RRBC. Moreover, cytotoxic reactions toward the different targets cells were shown to be mediated by different hemocyte sub-populations isolated from hemolymph by density gradient centrifugation.

Enzyme electrophoretic evidence for the prevalence of outcrossing in the hermaphroditic brooding ascidian Dendrodoa grossularia (Chordata, Urochordata)

The genetic structures of three relatively dense populations of the sessile solitary tunicate Dendrodoa grossularia (van Beneden) were examined using enzyme electrophoresis to study a single polymorphic locus ( Pgi). Little or no genetic differentiation was apparent on a scale of tens of metres, and only moderate differentiation was observed between localities 25–110 km apart. Levels of heterozygosity yielded estimates of the outcrossing rate ranging from 0.84 to 1.18 at the three localities; values of the fixation index F IS were not significantly different from zero. The minimum outcrossing rate giving no statistically significant deviation from observed genotype frequencies ( α = 0.05) ranged from 0.62 to 0.80 at the three localities. The genotypes of reared young of known brood parents confirmed the occurrence of extensive outcrossing during internal fertilization, and yielded an estimate of the outcrossing rate, based on 16 broods, in excess of 1.0. These results demonstrate unequivocally that exogenous sperm are able to reach the site of internal fertilization in a solitary brooding ascidian.

Cell Cooperation During Host Defense in the Solitary Tunicate Ciona intestinalis (L).

Phagocytosis in the solitary ascidian Ciona intestinalis was investigated using mixed and separated populations of blood cells in vitro. Only the vacuolar and granular amoebocytes were seen to ingest bacteria, and when the serine protease inhibitors, STI, or benzamidine were added to monolayers of mixed cell types, uptake was significantly reduced. Analyses carried out with isolated cells revealed that phagocytosis was enhanced by incubation of the bacteria in blood cell lysate supernatants (CLS) that had been pre-treated with LPS. By contrast, pre-incubation of the bacteria in CLS preparations that were inhibited by benzamidine produced lower levels of phagocytosis. Treatment of the bacteria with plasma also failed to promote uptake, and there was no detectable agglutination of the bacteria by CLS. As lysate supernatants made from morula cells, but not other cell types, were effective in promoting phagocytosis, we propose that opsonins are derived from the morula cells, and that phagocytosis involves cooperation between different cell populations. Moreover, as the morula cells are the principal repositories of prophenoloxidase and an associated serine protease (factors which are activated by LPS but blocked by STI or benzamidine), prophenoloxidase or the protease may be involved in the opsonic phenomenon in a similar way to that previously reported for arthropods.

Protamines, not histones, are the predominant basic proteins in sperm nuclei of solitary ascidian tunicates

AbstractIn this work we study the protein content in sperm nuclei from 10 species of ascidian tunicates. The 7 species that we have investigated cytochemically show type 3 intermediate sperm basic proteins, as observed previously for the solitary tunicate Styela montereyensis (Chiva, Rosenberg, and Kasinsky, J. Exp. Zool., 253:7–19, 1990). We have focused this study on two specific points: the presence of protamines and the presence of somatic histones. In 4 species belonging to the suborder Phlebobranchiata, sperm nuclei contain only a protein more basic than histones (protamine). This protamine has an electrophoretic mobility similar to core histone H4. The nuclei of sperm in the species belonging to the suborder Phlebobranchiata are also characterized by their complete lack of somatic histones. In 5 species from the suborder Stolidobranchiata, sperm nuclei contain a similar protamine. However, 2 species from the genus Styela contain additionally other protamines with different electrophoretic mobilities. For all species belonging to the suborder Stolidobranchiata, we also have found very small quantities of residual somatic histones. We conclude that during the process of spermiogenesis in ascidian tunicates protamines replace histones either completely (Phlebobranchiata) or almost completely (Stolidobranchiata). In this respect spermiogenesis in tunicates (class Ascidiacea) is similar to that in vertebrates which, in general, also display the substitution of somatic histones by protamines. On the other hand, this model is different from that in echinoderms, where the protein content of sperm nuclei consists mainly of somatic histones and sperm‐specific variants of histones. © 1992 Wiley‐Liss, Inc.