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The polychaete Nereis virens occurs commonly in marine sediments, is widely distributed, and is a popular bait species, as well as a potential replacement for wild-caught fish in commercial fish feed preparations. It is being considered as a potential co-extractive species for culture in integrated multi-trophic aquaculture operations. However, it is not known whether pesticides or drugs used to treat sea lice on farmed salmon, such as emamectin benzoate (EB), would adversely affect cultured or wild worms, because these compounds may persist in the environment. To determine the potential effects of EB to N. virens, bioassays were performed wherein worms were exposed in sand for 30 days to a concentration of 400 µg/kg dw (nominal). While no treatment-related mortality occurred, significant decreases in worm mass and marked behavioral changes (lack of burrowing) were observed in EB-treated sand compared with controls. These lab-based observations suggest a potential hazard to worms at sites where EB treatments have occurred.

Polychaetes are famous for their outstanding ability to regenerate lost body parts. Moreover, these worms possess a number of ancestral features in anatomy, development, and genetics, making them particularly suitable for comparative studies. Thus, fundamental as well as new undisclosed so far features of regenerative processes may be revealed, using polychaetes as a model. In the present work, we aimed to analyze the molecular basis of caudal regeneration in the nereid polychaete Alitta virens (formerly Nereis virens). We focused on homologues genes of RNA helicases Vasa and PL10 and ncRNA-binding proteins Piwi. These markers are suggested to play a significant role in maintenance of undifferentiated state of primordial germ cells and multipotent stem cells across invertebrates. In normal conditions, A. virens homologues of Vasa, PL10, and Piwi were differentially expressed in the subterminal growth zone and germline cells. Caudal amputation induced expression of studied genes de novo, which further accompanies all steps of regeneration. An early appearance of the transcripts in wound epithelium and internal blastemal cells suggests involvement of these genes in the well-known cell dedifferentiation events that assure polychaete regeneration. Provided interpretation of the gene expression dynamics implies the primary restoration of the pygidium and growth zone, which promotes following segment formation. Obtained results are valuable as a molecular fingerprint of the alterations occurring in regulatory state of locally regenerating tissues.

The capability of regenerating posterior segments and pygidial structures is ancestral for annelids and has been lost only a few times within this phylum. As one of the three major segmented taxa, annelids enable us to monitor reconstruction of lost tissues and organs. During regeneration, regional identities have to be imprinted onto the newly formed segments. In this study, we show spatial and temporal localization of expression of nine Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii. Hox genes are homeodomain genes encoding transcriptional regulators of axial patterning in bilaterian animals during development. We demonstrate that five Platynereis Hox genes belonging to paralog groups (PG) 1, 4, 5, 6, and 9-14 are expressed in domains of the regenerating nervous system consistent with providing positional information along the anteroposterior axis of the regenerate. We report that expression in regenerating neuromeres is limited to varying subsets of perikarya, called gangliosomes. Four of nine genes analyzed do not appear to be involved in axial patterning. Two genes, Pdu-Hox2 and Pdu-Hox3, are predominantly expressed in the growth zone region. For some Hox genes expression in newly formed coelomic epithelia can be observed. Platynereis Hox genes do not exhibit temporal or spatial colinearity. Although there are some similarities to previously reported expression patterns during larval and postlarval development in Nereididae (Kulakova et al. 2007), expression patterns observed during caudal regeneration also show unique patterns.

Standard microelectrode technique was used to study electrical activity of the isolated heart of the polychaete annelid, Arenicola marina. Typical pacemaker activity with slow diastolic depolarization was observed in all recordings. The average maximum diastolic potential (-58.4 +/- 3.2 mV), the average amplitude of the action potential (28.7 +/- 4.7 mV) and the average total duration of the action potential (2,434 +/- 430 ms) were determined. There has been no gradient of automaticity observed in our studies, which suggests that all regions of the Arenicola heart could possess pacemaker functions. Acetylcholine (ACh) produced a concentration dependent (5 x 10(-8)-5 x 10(-5) M) increase of the beating rate via increase in the rate of the diastolic depolarization. ACh (5 x 10(-5) M) increased beating rate by 2.5-fold compared to the control rate. A stronger action of ACh resulted in depolarization, block of action potential generation and contracture of the heart. The non-hydrolysable ACh analog carbacholine (10(-8)-10(-6) M) produced similar effects. All effects of ACh and carbacholine were abolished by 5 x 10(-6) M atropine. D-Tubocurarine (5 x 10(-5) M) did not significantly alter effects of ACh or carbacholine. Epinephrine (10(-8)-10(-6) M) caused the slowing of pacemaker activity and marked decrease of action potential duration. 10(-6) M epinephrine produced complete cardiac arrest. The effects of epinephrine were not significantly altered by the beta-blocker propranolol (5 x 10(-6) M). The beta-agonist isoproterenol (10(-7)-10(-5) M) and the alpha-agonist xylometazoline (10(-6)-10(-5) M) did not produce significant effects. Thus, cholinergic effects in the Arenicola heart are likely to be mediated via muscarinic receptors, while the nature of adrenergic effects needs further investigation.

Deep-sea vestimentiferan tubeworms, which live in symbiosis with bacteria, exhibit different life strategies according to their habitat. At unstable and relatively short-lived hydrothermal vents, they grow extremely fast, whereas their close relatives at stable and long-persisting cold seeps grow slowly and live up to 300 years. Growth and age differences are thought to occur because of ecological and physiological adaptations. However, the underlying mechanisms of cell proliferation and death, which are closely linked to homeostasis, growth, and longevity, are unknown. Here, we show by immunohistochemical and ultrastructural cell cycle analyses that cell proliferation activities of the two species studied are higher than in any other characterized invertebrate, being only comparable with tumor and wound-healing processes. The slow growth in Lamellibrachia luymesi from cold seeps results from balanced activities of proliferation and apoptosis in the epidermis. In contrast, Riftia pachyptila from hydrothermal vents grows fast because apoptosis is down-regulated in this tissue. The symbiont-housing organ, the trophosome, exhibits a complex cell cycle and terminal differentiation pattern in both species, and growth is regulated by proliferation. These mechanisms have similarities to the up- and down-regulation of proliferation or apoptosis in various types of tumor, although they occur in healthy animals in this study, thus providing significant insights into the underlying mechanisms of growth and longevity.

Quorum sensing (QS) signals have been considered to play important roles in biofilm development and in the attractiveness of biofilms to higher organisms in marine ecosystem. In this study, bacterial QS signalsacylated homoserine lactone derivatives (AHLs) were detected in 2-, 4-, and 6-day-old subtidal biofilms by using AHLs reporter strains. N-dodecanoyl-homoserine lactone (C12-HSL) was identified in 6-day-old biofilm at a concentration of 9.04 microg cm(-minus;2) (3.36 mmol l(-minus;1)). To investigate the possible role of AHLs in the consequent eventlarval settlement of the polychaete Hydroides elegans onto subtidal biofilmsseven biofilm-derived bacteria that effectively induced larval settlement of H. elegans, were screened for AHL production. One of them, the Vibrio sp. UST950701-007, produced N-hexanoyl-homoserine lactone (C6-HSL). Larval settlement bioassay showed that C6-HSL, C12-HSL, and 3-oxo-octanoyl-homoserine lactone (3-oxo-C8-HLS) at certain concentrations induced some initial larval settlement behaviors such as reducing swimming speed, crawling on the bottom. However, these AHLs did not effectively induce larval settlement in comparison to the effective settlement inducer 3-isobutyl-1-methylxanthine. The possible chemokinetic mechanism and indirect effects of AHLs on larval settlement are suggested.

Polychaetes, Eurythoe complanata, from the Gulf of Cariaco,Venezuela, were exposed to 0.3, 1.6, and 3.3% water-soluble fraction (WSF) of used crankcase oil during 15 and 21 days. The antioxidant enzymes glutathione peroxidase (GPX), glutathione reductase (GR), catalase (CAT), and glutathione-S-transferase (GST) were assayed in the body wall tissue. Furthermore, after chemical exposure, the polychaetes were cut into equal halves; then wound healing and the number of regenerated body segments were recorded periodically. GST activity was affected by all the experimental treatments, with activity increasing with WSF concentrations. GPx activity was altered for the contamination period. GR and CAT activities rose in response to increasing WSF concentrations, and were higher for long-term than for short-term exposures. The wound healing of the transected body regions was retarded by WSF exposure. WSF affected the tissue regeneration, which was almost abolished at 3.3% WSF. The exposure period did not affect the tissue-repairing responses. Alteration of GST in contaminated organisms suggested equivalent changes in detoxication of bioaccumulated organic contaminants. The variation of GR and CAT suggests induction of oxidative stress that could reduce the ability of WSF-exposed worms to repair damaged tissue.

The suitability of two polychaete worms, Australonereis ehlersi and Nephtys australiensis, and three bivalves, Mysella anomala, Tellina deltoidalis, and Soletellina alba, were assessed for their potential use in whole-sediment toxicity tests. All species except A. ehlersi, which could not be tested because of poor survival in water-only tests, survived in salinities ranging from 18 per thousand to 34 per thousand during the 96-hour exposure period. No mortality was observed in any of the species exposed to sediment compositions ranging from 100% silt to 100% sand for 10 days, thus demonstrating the high tolerance of the five species to a wide range of sediment types. All species showed decreased survival after exposure to highly sulfidic sediments in 10-day whole-sediment tests. In 96-hour water-only tests, survival decreased, and copper accumulation in body tissues increased with exposure to increasing copper concentration for all species except A. ehlersi, which again could not be tested because of its poor survival in the absence of sediment. S. alba and T. deltoidalis were the most sensitive species to aqueous copper (LC50s of 120 and 150 microg Cu/L, respectively). All species tested were relatively insensitive to dissolved zinc up to concentrations of approximately 1,000 microg/L. In addition and with the exception of N. australiensis, all species accumulated significant levels of zinc in their body tissues. Whole-sediment tests were conducted over a 10-day period with copper-spiked (1,300 microg/g) and zinc-spiked (4,000 microg/g) sediments equilibrated for sufficient time to ensure that pore water metal concentrations were well below concentrations shown to have any effect on organisms in water-only tests. Survival was decreased in the bivalves T. deltoidalis and S. alba after exposure to copper-spiked sediments, and all species-except T. deltoidalis, in which 100% mortality was observed-accumulated copper in their tissues. Exposure to zinc-spiked sediments significantly decreased the survival of only one species, T. deltoidalis. Both polychaetes appeared to regulate concentrations of zinc in their body tissues with no significant uptake of zinc occurring from the sediment phase. Of the five species assessed in this study, T. deltoidalis was found to be the most sensitive to copper- and zinc-contaminated sediments, and based on commonly used selection criteria (ASTM 2002a, ASTM 2002b, ASTM 2002c) is recommended for development as test species in whole-sediment toxicity tests., (Copyright 2004 Springer Science + Business Media, Inc.)

The polychaete Sabella spallanzanii (Gmelin) (Sabellidae) is a widely distributed species throughout the Mediterranean where it is commonly found in sheltered shallow areas and up to 30 m depth in more exposed waters. The sedentary habit, the filter-feeding behavior and its capability to colonize disturbed environments such as harbors, suggest the potential utility of this organism as a bioindicator in environmental monitoring programs. The aim of this work was a preliminary characterization of S. spallanzanii, integrating chemical data on trace metal concentrations with the biochemical analysis of antioxidant defenses which play an important role in mediating responses and adaptation to stressful environmental conditions. Organisms were collected in four locations of the Adriatic and Tyrrhenian Sea, characterized by a moderate impact or influenced by organic enrichment or sulphuric emissions. Trace metal concentrations (Cd, Cr, Cu, Fe, Mn, Ni, Pb, Zn) were comparable to those of other invertebrate species and quite typical for unpolluted environments. Analyses of individual antioxidants (superoxide dismutase, catalase, glutathione peroxidases, glutathione S-transferases, glutathione reductase) were integrated with measurement of the total oxyradical scavenging capacity toward specific forms of ROS; the TOSC assay, quantifying the overall capability of a tissue to neutralize specific reactive oxygen species provide a more integrated picture of the balance between prooxidant factors and antioxidant defenses, with important indications about the role of different ROS in appearance of oxidative disease. The antioxidant profile was influenced by the different physiological functions of the analyzed tissues, namely the branchial crown, involved in filtration of large volumes of seawater, and the thorax where digestive processes occur. The analysis of antioxidant defenses revealed significant variations in polychaetes from various sampling sites suggesting the influence of specific environmental conditions like those limiting oxygen availability. Our results confirm the role of antioxidants in adaptation to stressful environmental conditions and their importance in ecotoxicological studies for the sensitivity in revealing the occurrence of biological disturbance.

Reactive oxygen species (ROS) are subproducts of the oxidative metabolism known to initiate chain reactions with polyunsaturated fatty acids that generate lipid peroxides (LPO). The objective of this work was to adapt the ferrous oxidation/xylenol orange (FOX) assay to measure LPO in invertebrate tissues i.e.: from polychaeta (Laeonereis acuta) and crab (Chasmagnathus granulata) species. Whole polychaetes were homogenized in methanol 100%, being determined the optimal sample volume and the time required for color development. It was tested five sample volumes (8-30 microl), following color development up to 215 min. Absorbance stabilization was observed after 90 min, being linearly related with sample volume. A similar procedure was adopted for crab tissues (anterior gills, posterior gills, and hepatopancreas). Differences between species and between organs of the same species were observed when analyzed nonspecific absorbance increments after adding the standard cumene hydroperoxide (CHP). In polychaeta and crab anterior gills tissue, absorbance increments were lower (21-25%) than samples without tissue extracts (blanks) that received CHP. In crab posterior gills and hepatopancreas, the nonspecific increment was almost negligible. Correction formulae are given to account for these differences and simplified protocols for each tissue and species are also included. Great differences in the lipid peroxides content was detected between worms (127.05 +/- 19.32 nmoles CHP/g of wet tissue) respect to anterior gills, posterior gills, and hepatopancreas from the crab species (52.65 +/- 3.59, 30.54 +/- 4.73, and 48.51 +/- 8.78 nmoles CHP/g of wet tissue, respectively).

A novel member of the innexin family (cv-inx) has been isolated from the annelid polychaete worm Chaetopterus variopedatus using a PCR approach on genomic DNA and sequence analysis on genomic DNA clones. The gene is present in a HindIII-HindIII segment of 2250 bp containing an uninterrupted open reading frame of 1196 bp encoding a protein of 399 amino acids. The predicted protein shows the typical structural features of innexins and consensus sites for phosphorylation. Analyses on genomic DNA demonstrate that cv-inx is a single copy gene with no introns in the coding region, exactly corresponding to the cDNA sequence. The gene expression is regulated during development as shown by Northern blots analyses of the RNA and by immunoreaction with antibodies against the protein at several embryonic stages. The finding of an innexin in the phylum Annelida, outside of the Ecdysozoa clade, and its peculiar gene structure suggest the necessity to reconsider the current hypothesis on the origin and evolution of gap junctional proteins.

A toxicity database for ordnance compounds was generated using eight compounds of concern and marine toxicity tests with five species from different phyla. Toxicity tests and endpoints included fertilization success and embryological development with the sea urchin Arbacia punctulata; zoospore germination, germling length, and cell number with the green macroalga Ulva fasciata; survival and reproductive success of the polychaete Dinophilus gyrociliatus; larvae hatching and survival with the redfish Sciaenops ocellatus; and survival of juveniles of the opossum shrimp Americamysis bahia (formerly Mysidopsis bahia). The studied ordnance compounds were 2,4- and 2,6-dinitrotoluene, 2,4,6-trinitrotoluene, 1,3-dinitrobenzene, 1,3,5-trinitrobenzene, 2,4,6-trinitrophenylmethylnitramine (tetryl), 2,4,6-trinitrophenol (picric acid), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The most sensitive toxicity test endpoints overall were the macroalga zoospore germination and the polychaete reproduction tests. The most toxic ordnance compounds overall were tetryl and 1,3,5-trinitrobenzene. These were also the most degradable compounds, often being reduced to very low or below-detection levels at the end of the test exposure. Among the dinitro- and trinitrotoluenes and benzenes, toxicity tended to increase with the level of nitrogenation. Picric acid and RDX were the least toxic chemicals tested overall.

Oogenesis in Typosyllis pulchra begins with a short proliferative gonial phase. Gonial clusters, consisting of six to eight syncytial sibling cells, are surrounded by gonial peritoneum and are eventually liberated into the coelom. At about the same time as the onset of the coelomic phase, one gonial cell in each cluster differentiates, begins to accumulate yolk and increases in size, eventually to become a mature oocyte. Yolk is composed of lipid droplets and membrane-bound protein granules. The protein component appears to be at least partially synthesized by the oocyte. Yolk is composed of lipid droplets and membrane-bound protein granules. The protein component appears to be at least partially synthesized by the oocyte although there are some signs of endocytosis. Nurse cells and peritoneal cells are apparently able to manufacture some protein that may also be utilized by the oocyte. The nurse cells maintain cytoplasmic continuity with the oocyte, and mitochondria and ribosomes are often seen within the intercellular bridges between oocyte and nurse cell. Immediately prior to spawning, oocytes undergo prematuration and cytoplasmic reorganization. Ultrastructural changes which occur during oocyte growth and maturation are described.

Tryposyllis pulchra reproduces by the production of three to four gamete-bearing stolons (schizogamy) during consecutive 30--day periods. Although gonads are found in a large number of segments, only those in the posterior-most segments produce gametes and become incorporated into the developing stolon. The more anterior gonads remain undifferentiated and probably sexually undetermined until they are needed in future stolonizations. Gonial cells, which will eventually become either male or female, are ultrastructurally identical at the onset of each stolonization period. Spermatogenesis is marked by a short proliferative period followed by differentiation and spermiogenesis. The first ultrastructural signs of spermatogenesis were found in coelomic spermatogonia on day 10 of stolon formation. Spermatogonia are joined by intercellular bridges, which are maintained until the early spermatid stage. Synaptonemal complexes mark the onset of meiosis, which is apparently synchronized in the syncytial clusters of primary spermatocytes. Spermiogenesis occurs during the final 10 days of stonolization and a variety of stages is present within a single animal. All sperm mature by the time the stolon detaches. Acrosome formation and nuclear condensation are described in addition to the ultrastructure of mature sperm.

Light- and electron-microscopic autoradiographic studies were carried out on Owenia to detect selectively catecholaminergic and indolaminergic neurons at two appropriate body levels according to the regenerative properties of this annelid, i.e. in the 1st and the 4th abdominal segments. Autoradiographically, intense 3H-5-hydroxytryptamine accumulation is seen in the 1st abdominal segment, and a less intense autoradiographic reaction in the 4th segment. Roughly, a similar difference in the distribution of the label is seen at both levels following 3H-noradrenaline administration. These observations, confirmed by a quantitative uptake analysis, suggest the presence of catecholaminergic and indolaminergic neurons in Owenia.

Static acute toxicity bioassays were conducted at 20 degrees C and 20 o/33 salinity with CdCl2-2 1/2 H20, K2CrO4, HgCl2, NiCl2-6H2O, and ZnCl2 using adults of starfish, Asterias forbesi; sandworm, Nereis virens; hermit crab, Pagurus longicarpus; softshell clam, Mya arenaria; mudsnail, Nassarius obsoletus; and mummichog, Fundulus heteroclitus, a fish. Concentrations (mg/L metal) fatal to 50% of the organisms in 168 hr ranged from 0.004 (clam) to 0.8 (mummichog) for mercury; 0.7 (clam, worm, crab and starfish) to 40.0 (mummichog) for cadimium; 0.2 (crab) to 52.0 (mummichog) for zinc; 0.7 (sandworm) to 44.0 (mummichog) for hexavalent chromium; and 13.0 (starfish) to 150.0 (mummichog) for nickel. Biocidal action was restricted to a relatively narrow range for all species-metals combinations tested: i.e., mean LC-75/LC-25 rations for individual metals at 168 hr ranged between 2.82 (Zn) and 6.02 (Cd); for individual species this ratio extended from 2.76 (fish) to 4.46 (clam). It appears that acute toxicity evaluation of potentially hazardous metals in saline environments requires utilization of at least several representative species from divergent taxonomic and ecological niches.

The effects of light and dark on photoreceptors of the brackish-water polychaete annelid Nereis limnicola were studied by electron microscopy. Animals dark-adapted for one or two days exhibited well-formed straight microvilli (rhabdomeres) on the sensory cell processes. Continuous illumination of worms for one or two days caused extensive breakdown of the microvilli into vesicles and debris. Thirty minutes to three h of exposure of dark-adapted animals to light produced increasing severity of degradation of photoreceptoral microvilli. Light-adapted worms placed in darkness for one-half to three h showed progressive restoration of the microvilli to the dark-adapted condition. The products of degradation were internalized by both sensory and pigmented supportive cells by phagocytosis and pinocytosis.