Stepwise metamorphosis of the tubeworm Hydroides elegans is mediated by a bacterial inducer and MAPK signaling.

Diverse animal taxa metamorphose between larval and juvenile phases in response to bacteria. Although bacteria-induced metamorphosis is widespread among metazoans, little is known about the molecular changes that occur in the animal upon stimulation by bacteria. Larvae of the tubeworm Hydroides elegans metamorphose in response to surface-bound Pseudoalteromonas luteoviolacea bacteria, producing ordered arrays of phage tail-like metamorphosis-associated contractile structures (MACs). Sequencing the Hydroides genome and transcripts during five developmental stages revealed that MACs induce the regulation of groups of genes important for tissue remodeling, innate immunity, and mitogen-activated protein kinase (MAPK) signaling. Using two MAC mutations that block P. luteoviolacea from inducing settlement or metamorphosis and three MAPK inhibitors, we established a sequence of bacteria-induced metamorphic events: MACs induce larval settlement; then, particular properties of MACs encoded by a specific locus in P. luteoviolacea initiate cilia loss and activate metamorphosis-associated transcription; finally, signaling through p38 and c-Jun N-terminal kinase (JNK) MAPK pathways alters gene expression and leads to morphological changes upon initiation of metamorphosis. Our results reveal that the intricate interaction between Hydroides and P. luteoviolacea can be dissected using genomic, genetic, and pharmacological tools. Hydroides' dependency on bacteria for metamorphosis highlights the importance of external stimuli to orchestrate animal development. The conservation of Hydroides genome content with distantly related deuterostomes (urchins, sea squirts, and humans) suggests that mechanisms of bacteria-induced metamorphosis in Hydroides may have conserved features in diverse animals. As a major biofouling agent, insight into the triggers of Hydroides metamorphosis might lead to practical strategies for fouling control.

First documentation and molecular confirmation of three trematode species (Platyhelminthes: Trematoda) infecting the polychaete Marenzelleria viridis (Annelida: Spionidae).

Polychaete worms are hosts to a wide range of marine parasites; yet, studies on trematodes using these ecologically important species as intermediate hosts are lacking. During examination of the spionid polychaete Marenzelleria viridis collected on the north shore of Long Island, New York, putative trematode cysts were discovered in the body cavity of these polychaetes. In order to verify these cysts as metacercariae of trematodes, specimens of the eastern mudsnail Ilyanassa obsoleta (a very common first intermediate host of trematodes in the region) were collected for molecular comparison. DNA barcoding using cytochrome C oxidase I regions confirmed the presence of three species of trematodes (Himasthla quissetensis, Lepocreadium setiferoides, and Zoogonus lasius) in both M. viridis and I. obsoleta hosts. Brown bodies were also recovered from polychaetes, and molecular testing confirmed the presence of L. setiferoides and Z. lasius, indicating an immune response of the polychaete leading to encapsulation of the cysts. From the 125 specimens of M. viridis collected in 2014, 95 (76.8 %) were infected with trematodes; of these 95 infected polychaetes, 86 (90.5 %) contained brown bodies. This is the first confirmation that trematodes use M. viridis as a second intermediate host and that this intermediate host demonstrates a clear immune response to metacercarial infection. Future research should explore the role of these polychaetes in trematode life cycles, the effectiveness of the immune response, and transmission pathways to vertebrate definitive hosts.

Role of proteasomes in non-specific immune response of marine annelids.

We investigated functioning of proteasomes and chaperones in Arenicola marina coelomocytes in conditions of lipopolysaccharide-induced inflammation. We observed the increase of chymotrypsin-like proteasome activity in coelomocytes 1 h after induction. Amount of proteasome subunits alpha- and beta-5 types increased as well. We also detected appearance of a new form of Hsp70 chaperone in infected coelomocytes. Our results allow us to consider the changes in proteasome structure and induction of chaperones as principle mechanisms in stress adaptation and defensive reactions development in annelids.

Toxicological evaluation of sediment samples spiked with human pharmaceutical products: Energy status and neuroendocrine effects in marine polychaetes Hediste diversicolor.

There is a lack of studies about the ecotoxicology of pharmaceutical products on marine environment. To predict possible adverse effects of pharmaceutical products on benthic biota, polychaetes Hediste diversicolor were exposed for 14-days to pharmaceutical-spiked sediments under laboratory conditions. Carbamazepine (CBZ), ibuprofen (IBP) and propranolol (PRO) at concentrations of 500ngg(-1), 50ngg(-1), 5ngg(-1), 0.5ngg(-1) and 0.05ngg(-1), fluoxetine (FX) and 17α-ethynylestradiol (EE2) at concentrations of 100ngg(-1), 10ngg(-1), 1ngg(-1), 0.1ngg(-1) and 0.01ngg(-1), including environmental concentrations (underlined), were spiked in marine sediment samples. After the exposure, cellular energy status (total lipids content - TLP; and mitochondrial electron transport activity - MET), metabolism of monoamines (monoamine oxidase activity - MAO) and inflammation properties (cyclooxygenase activity - COX) were observed in polychaetes. CBZ increased TLP content and MET activity, and decreased MAO activity in polychaetes. IBP did not interfere on the TLP level, but on the MET and MAO activities (environmental concentrations). FX did not cause changes in the energy status. Therefore, environmental concentration diminished MAO activity. EE2 did not affect the energy status, however, MAO activity was significantly lower in polychaetes exposed to environmental concentration. PRO increased TLP level in polychaetes, but not MET activity. MAO activity was significantly lower for polychaetes exposed to environmental concentration. Except FX, all pharmaceuticals showed anti-inflammatory properties confirmed by the decrease of COX activity. Pharmaceutical products affected H. diversicolor physiology and health. As a benthic top predator, adverse effects on sea-worms can potentially culminate in ecosystem perturbations.

Thymosin-ß12 characteristics and function in Urechis unicinctus.

ß-thymosin family comprise a series of heat-stable multifunctional polypeptides involved in actin regulation, anti-inflammation, wound healing, cell migration, angiogenesis, cardiac protection, antimicrobial processes and antiviral immunity. The roles of Tß12 (thymosin-ß12) in marine invertebrates is still largely unknown, especially in terms of antibacterial immunity. In this study, we cloned the Tß12 gene with an ORF of 126 bp coding 41 amino acids from Urechis unicinctus. Tissue distribution analysis by qRT-PCR used TBP as reference gene showed that Tß12 was widely expressed in all tissues, and the transcript levels were the highest in the body wall, followed by the coelomic fluid, and the lowest in the intestines and anal sacs. After LPS (lipopolysaccharides) injection, Tß12 expression in the body was first elevated significantly at 3 h (p < 0.05), indicating that the body wall was the first defense line of the innate immune system; in the coelomic fluid, the Tß12 mRNA levels increased after LPS injection, with a significant increase occurring at 6 h, showing that coelomic fluid functioned as the second defense line of the innate immune system. In the midgut and anal sacs, a significant increase in the Tß12 level occurred at 24 h, suggesting that the midgut and anal sacs may act as accessory organs for the innate immune system. Moreover, U. unicinctus Tß12 recombinants can effectively inhibit the growth of both gram-negative and gram-positive bacteria. These results indicate that U. unicinctus Tß12 plays important roles in innate antibacterial immune responses, which can deepen our understanding of Tß12 in marine invertebrates.

Genomic, transcriptomic, and proteomic insights into the symbiosis of deep-sea tubeworm holobionts.

Deep-sea hydrothermal vents and methane seeps are often densely populated by animals that host chemosynthetic symbiotic bacteria, but the molecular mechanisms of such host-symbiont relationship remain largely unclear. We characterized the symbiont genome of the seep-living siboglinid Paraescarpia echinospica and compared seven siboglinid-symbiont genomes. Our comparative analyses indicate that seep-living siboglinid endosymbionts have more virulence traits for establishing infections and modulating host-bacterium interaction than the vent-dwelling species, and have a high potential to resist environmental hazards. Metatranscriptome and metaproteome analyses of the Paraescarpia holobiont reveal that the symbiont is highly versatile in its energy use and efficient in carbon fixation. There is close cooperation within the holobiont in production and supply of nutrients, and the symbiont may be able to obtain nutrients from host cells using virulence factors. Moreover, the symbiont is speculated to have evolved strategies to mediate host protective immunity, resulting in weak expression of host innate immunity genes in the trophosome. Overall, our results reveal the interdependence of the tubeworm holobiont through mutual nutrient supply, a pathogen-type regulatory mechanism, and host-symbiont cooperation in energy utilization and nutrient production, which is a key adaptation allowing the tubeworm to thrive in deep-sea chemosynthetic environments.

Transgenerational Immune Priming in the Field: Maternal Environmental Experience Leads to Differential Immune Transfer to Oocytes in the Marine Annelid Hediste diversicolor.

Transgenerational immune priming (TGIP) is an intriguing form of parental care which leads to the plastic adjustment of the progeny's immunity according to parental immune experience. Such parental effect has been described in several vertebrate and invertebrate taxa. However, very few empirical studies have been conducted from the field, with natural host-parasite systems and real ecological settings, especially in invertebrates. We investigated TGIP in wild populations of the marine annelid Hediste diversicolor. Females laid eggs in a mud tube and thus shared the local microbial threats with the first developmental stages, thus meeting expectations for the evolution of TGIP. We evidenced that a maternal bacterial challenge led to the higher antibacterial defense of the produced oocytes, with higher efficiency in the case of Gram-positive bacterial challenge, pointing out a prevalent role of these bacteria in the evolutionary history of TGIP in this species. Underlying mechanisms might involve the antimicrobial peptide hedistin that was detected in the cytoplasm of oocytes and whose mRNAs were selectively stored in higher quantity in mature oocytes, after a maternal immune challenge. Finally, maternal immune transfer was significantly inhibited in females living in polluted areas, suggesting associated costs and the possible trade-off with female's protection.

Immune failure reveals vulnerability of populations exposed to pollution in the bioindicator species Hediste diversicolor.

Human activities on the shoreline generate a growing pollution, creating deleterious habitats in coastal zones. Some species nevertheless succeed in such harsh milieus, raising the question of their tolerance to environmental stress. The annelid Hediste diversicolor lives buried in the sediments, directly exposed to contaminants trapped in the mud. After verifying the similarity of their genetic contexts, we compared reproductive output and individual immune resistance measures of populations living in polluted vs. 'clean' sediments, and related these assessments with measures of phthalates and metal pollution, and associated toxicity indices. Chemical analyses predicted no toxicity to the local infauna, and phenological studies evidenced no direct cost of living in noxious habitats. However, populations exposed to pollutants showed a significantly reduced survival upon infection with a local pathogen. Surprisingly, physiological studies evidenced a basal overinflammatory state in the most exposed populations. This over-activated baseline immune phenotype likely generates self-damage leading to enhanced immune cell death rate and immune failure. Monitoring the immune status of individual worms living in anthropic areas could thus be used as a reliable source of information regarding the actual health of wild populations.

Transcriptional changes in the Japanese scallop (Mizuhopecten yessoensis) shellinfested by Polydora provide insights into the molecular mechanism of shell formation and immunomodulation.

The Japanese scallop (Mizuhopecten yessoensis) is one of the most important aquaculture species in Asian countries; however, it has suffered severe infection by Polydora in northern China in recent years, causing great economic losses. The Polydora parasitizes the shell of scallops, badly destroying the shell's structure. To investigate the molecular response mechanism of M. yessoensis to Polydora infestion, a comprehensive and niche-targeted cDNA sequence database for diseased scallops was constructed. Additionally, the transcriptional changes in the edge mantle, central mantle and hemocytes, tissues directly related to the disease, were first described in this study. The results showed that genes involved in shell formation and immunomodulation were significantly differentially expressed due to the infestation. Different transcriptional changes existed between the two mantle regions, indicating the different molecular functions likely responsible for the formation of different shell layers. The differential expression of genes for immune recognition, signal transduction and pathogen elimination presented an integrated immune response process in scallops. Moreover, neuromodulation and glycometabolism involved in the regulation process with relevant function significantly enriched. The study provides valuable information for mechanism study of shell formation and immunomodulation in scallops.

Hedistin: A novel antimicrobial peptide containing bromotryptophan constitutively expressed in the NK cells-like of the marine annelid, Nereis diversicolor.

A novel antimicrobial peptide, named hedistin was identified from the coelomocytes of Nereis diversicolor. Hedistin shows no obvious similarities with other known peptides and constitutes the first antimicrobial peptide containing bromotryptophans demonstrated in annelids. cDNA and mass spectrometry analysis revealed that, upon bacteria challenge, this peptide is secreted following processing of a precursor containing a signal peptide and prosequences. Hedistin was shown to possess an activity against a large spectrum of bacteria including the methicillin resistant Staphylococcus aureus and Vibrio alginolyticus. The gene was demonstrated to be constitutively and exclusively expressed in circulating NK cells like known to play an important role in the immunity of the sand worm. These data contrast with those observed in another annelid, the leech, in which genes coding for antimicrobial peptides are upregulated in a specific tissue and peptides are rapidly released into the hemolymph after septic injury.

IL-6 immunoreactivity changes during aging in the polychaete Ophryotrocha labronica (Polychaeta: Dorvilleidae).

IL-6 is a pleiotropic pro-inflammatory cytokine thought to play a role in age physiology, even if its possible modulation by aging mechanisms has not been fully defined. In this paper, the morpho-functional modifications and IL-6 immunoreactivity during aging in a simple invertebrate model, Ophryotrocha labronica, are reported. The comparison between newly-hatched, juveniles, young adult and 3-month-old females showed significant differences in the nervous and genital systems. There is evidence of nerve cell loss and a decline in oocyte growth and maturation at the gonad level. Immunohistochemistry reveals a different distribution of IL-6-like molecules, and a decreased number of reactive nerve cells in the central nervous system of aged O. labronica associated to the induced morphological modifications.

Antagonistic evolution of an antibiotic and its molecular chaperone: how to maintain a vital ectosymbiosis in a highly fluctuating habitat.

Evolution of antimicrobial peptides (AMPs) has been shown to be driven by recurrent duplications and balancing/positive selection in response to new or altered bacterial pathogens. We use Alvinella pompejana, the most eurythermal animal known on Earth, to decipher the selection patterns acting on AMP in an ecological rather than controlled infection approach. The preproalvinellacin multigenic family presents the uniqueness to encode a molecular chaperone (BRICHOS) together with an AMP (alvinellacin) that controls the vital ectosymbiosis of Alvinella. In stark contrast to what is observed in the context of the Red queen paradigm, we demonstrate that exhibiting a vital and highly conserved ecto-symbiosis in the face of thermal fluctuations has led to a peculiar selective trend promoting the adaptive diversification of the molecular chaperone of the AMP, but not of the AMP itself. Because BRICHOS stabilizes beta-stranded peptides, this polymorphism likely represents an eurythermal adaptation to stabilize the structure of alvinellacin, thus hinting at its efficiency to select and control the epibiosis across the range of temperatures experienced by the worm; Our results fill some knowledge gaps concerning the function of BRICHOS in invertebrates and offer perspectives for studying immune genes in an evolutionary ecological framework.

Allergy to sea fishing baits.

We report a new case of rhinitis and asthma caused by sea fishing baits. The results showed exposure to Sipunculus nudus (Phylum Sipuncula; order Sipunculida: Sipunculidae) to be the main cause of the allergic symptoms. The intervention of IgE was demonstrated, with the presence of cross-reactions with allergenic extracts from other worm species used as baits, belonging to different orders of Annelida.

Natural killer cells in a lower invertebrate, Nereis diversicolor.

Globular, non-adherent coelomocytes, called "G3 granulocytes", of the polychaetous annelid Nereis diversicolor display spontaneous cytotoxicity. These cells were found capable of killing invertebrate as well as vertebrate target cells by a contact-dependent cytolytic process. Cytotoxic activity of G3 granulocytes against foreign cells develops in three steps. At first, the cells become motile and form lamellipodia. In a second step, short, pointed pseudopodia arise from the edge of the lamellipodia and are making contact with the stimulating foreign object. In a third step, the G3 granulocytes release dense granules by exocytosis onto the foreign substrate or cell which finally will undergo lysis. Within few minutes after activation, the G3 granulocyte will alter its polarity, realigning both Golgi apparatus and centrosome towards the target cell. A pore-forming protein may be involved in the cytotoxic activity of the G3 granulocytes. These cells were observed to burst after contact with and release of granules onto an abiotic solid substrate, indicating that under certain circumstances the G3 granulocytes may be sensitive to their own cytotoxic activity. These data support the postulate of Franceschi et al. (Eur. J. Immunol. 21, 489-493 (1991) that a primitive natural killer cell-like activity had been developed early in phylogenesis. A simple method for preparing invertebrate coelomocytes for scanning electron microscopy is described.

Synthesis and characterization of a recombinant myohemerythrin protein encoded by a synthetic gene.

The antigenic epitopes of the myohemerythrin (MHr) molecule have been studied extensively. The critical amino acid residues responsible for its immune recognition have been identified by using synthetic peptides and the technique of epitope scanning. To assess the true relevance of these techniques for determining the molecular mechanism of antigenic recognition and immunogenicity, the results obtained with isolated peptides should be tested in the context of the folded protein. To this end, we have designed and constructed a synthetic MHr gene, in modular form, which will allow subsequent alterations of nucleotide sequence encoding epitopes of interest. We have produced the recombinant protein at high level, and have shown by several criteria that it possesses the chemical, physical and immunological properties of the native worm protein. Thus, we have developed a valuable system for detailed immunological studies of the structure and chemistry required for antibody binding to protein.

Evidence for the presence of subpopulations of Arenicola marina coelomocytes identified by their selective response towards Gram+ve and Gram-ve bacteria.

In Arenicola marina (L.), in vitro attachment and phagocytosis of bacteria appears to be serum-independent and thus, non-specific. Mixtures of Gram+ve and Gram-ve bacterial species (Gram+ve/-ve, of two Gram+ve (Gram+ve/+ve) and of two Gram-ve bacterial species (Gram-ve/-ve)were overlaid on coelomocyte monolayers, in order to introduce competition for cell surface 'receptors'. An enhanced total phagocytosis was only recorded in the Gram+ve/-ve mixtures in comparison with their controls. The results are interpreted in terms of coelomocyte subpopulations and indicate that A. marina coelomocytes may possess specific carbohydrate/glycoprotein determinants capable of recognizing basic carbohydrate differences between the cell walls of Gram+ve and Gram-ve bacteria.

Distribution and nature of membrane receptors for different plant lectins in the coelomocyte subpopulations of the Annelida Nereis diversicolor.

Studies on membrane receptors have been performed on the Nereis coelomocytes using various lectins. In the agglutination assay, only LCA and WGA appeared nonreactive. Fluorescent lectins showed the poor reactivity of the eleocytes and the diversity of the receptors according to the granulocyte types. Types I-granulocytes reacted only with Con A. Type II-granulocyte membrane contained mannose and galactose receptors (reactivity with Con A, PNA and SBA). The type III-granulocyte membrane revealed the presence of mannose and fucose receptors (UEA, AAA). Electron microscope investigations with HRP-DAB or mannosyl labelled Con A, RCAI and LTA have confirmed the distribution of the membrane receptors.

A novel GGNG-related neuropeptide from the polychaete Perinereis vancaurica.

The GGNG peptides are myoactive peptides so far identified from earthworms and leeches, which are the earthworm excitatory peptides (EEP) and the leech excitatory peptide (LEP), respectively. A novel GGNG peptide was isolated and structurally determined from a marine polychaete, Perinereis vancaurica, using a combination of immunological assay and high performance liquid chromatography (HPLC). The peptide was a pentadecapeptide whose amino acid sequence was similar to that of EEP and LEP, and showed myoactivity on isolated esophagus of P. vancaurica with a threshold concentration of 10(-10)M. The peptide was designated as polychaete excitatory peptide (PEP). Amidation of the alpha-carboxyl group of C-terminal residue occurred in PEP. This is the case for LEP, but not for EEP. The cDNA cloning revealed that the structure of the PEP precursor is more similar to the EEP precursor than to the LEP precursor. Immunohistochemical staining showed the presence of PEP in several neurons of central nervous system (CNS) as somata and neuropile structure, epithelial cells of the pharynx and epidermal cells throughout the body wall. Altogether these results support the physiological significance of PEP in regulation of the CNS neural activity and the peripheral myoactivity.

Finding of the same antigens in the polychaete, Pseudopotamilla occelata, as those in the vanadium-rich ascidian, Ascidia sydneiensis samea.

The polychaete Pseudopotamilla occelata is the first animal revealed to contain high levels of vanadium besides ascidians. The present experiment disclosed that P. occelata has the same antigens with those in the ascidian Ascidia syndneiensis samea, which were recognized by two types of antibodies, a polyclonal antibody against vanadium-associated proteins extracted from blood cells and a monoclonal antibody against vanadocytes in the vanadium-rich ascidian A. sydneiensis samea. There is, therefore, a possibility that similar mechanism works on the accumulation of vanadium between the Polychaeta and the Ascididae.

The role of the heart-body and of the extravasal tissue in disposal of foreign cells in two polychaete annelids.

Sheep red cells injected into the coelom of the polychaetes Neoamphitrite figulus and Arenicola marina are phagocytosed by coelomocytes and conveyed to the heart-body and extravasal ('chloragogen') tissue respectively. In the former case the phagocytes creep into the vessels, penetrate the heart-body tissue and bequeath the sheep red cells to the heart body cells. In Arenicola the phagocytes accumulate on the tips of the blind ending vessels and the red cells are similarly transferred to the extravasal tissues. These observations confirm that the heart-body and extravasal cells function not only in production of haemoglobin but in sequestering foreign material.