Primary structure of myohemerythrin from the annelid Nereis diversicolor

The metal-free form of Nereis diversicolor myohemerythrin was purified from whole animal extracts by trichloroacetic acid precipitation and ion exchange chromatography. The amino acid sequence of myohemerythrin has been determined. The protein is composed of 120 residues, possesses an unblocked N-terminus and is devoid of cysteine residues. It bears 62% sequence identity with Themiste zostericola myohemerythrin, the only other member of this subfamily sequenced to date. Within the family of hemerythrins, homology is particularly high in the segments involved in the binding of the two iron atoms and in the beta-turn-rich N-terminal segment.     

Effects of light and dark on photoreceptors in the polychaete annelid Nereis limnicola

Summary The effects of light and dark on photoreceptors of the brackish-water polychaete annelid Nereis Hmnicola 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.     

Cellular reactions of the polychaete annelid Nereis diversicolor against coelomic parasites

We studied the cellular reactions of the polychaete Nereis diversicolor against different natural coelomic parasites: a yeast, a coccidian, and a supposed orthonectid. The coelomocytes engaged in these reactions are type 1 and type 2 granulocytes. We observed recognition processes, accumulation and flattening of coelomocytes into concentric sheets around the parasites, and then formation of thick capsules which become brown bodies. The parasites are first paralyzed (in case of motile stages), then show degeneration symptoms of the cell organelles, and are finally killed and turn into necrosed nodules. Encysted stages of coccidians may remain alive in the capsules. Life of small coccidian sporozoites is possible inside the coelomocytes.     

Identification of extracellular haemoglobin as the major high molecular weight cadmium-binding protein of the polychaete annelid Nereis diversicolor

1. A high molecular weight cadmium-binding protein called MP I was isolated from Nereis diversicolor exposed to 20 ppm of cadmium (CdCl₂) for 4 days via sea water.2. The protein had a molecular weight comprising between 20 × 10⁶ and 1.5 × 10⁶ Da. It shows high absorptions at 280 and 415 nm and contained iron in addition to cadmium.3. Two dimensional SDS-PAGE of unreduced and reduced MP I revealed a dissociation pattern similar to that of extracellular haemoglobins of polychaetes and oligochaetes.4. Furthermore, negatively stained MP I molecules examined by conventional transmission electron microscopy showed the common appearance of extracellular haemoglobins of annelids.5. Evidence of the extracellular haemoglobin nature for the MP I was found by the identical Chromatographie behaviour of MP I with the main Nereis blood component and, by the labelling of blood vessel content with ¹⁰⁹Cd visualized by autoradiography on paraffin-embedded sections of exposed Nereis.     

Expression and evolution studies of ets genes in a primitive coelomate,the polychaete annelid,Hediste (Nereis) diversicolor

The Ets family includes numerous proteins with a highly conserved DNA-binding domain of 85 amino acids named the ETS domain. Phylogenetic analyses from ETS domains revealed that this family could be divided into 13 groups, among them are ETS and ERG. The ets genes are present in the Metazoan kingdom and we have previously characterized the Nd ets and Nd erg genes in the polychaete annelid Hediste diversicolor. Here, we isolated a fragment encoding the ETS domain from Nd Ets, by genomic library screening. By Northern blot analysis, we showed that this gene was transcribed as one major mRNA of 2.6 kb and one minor mRNA of 3.2 kb. By in situ hybridization, we observed that Nd ets was expressed in the intestine and oocytes and that Nd erg was expressed in cellular clumps present in the coelomic cavity, in an area of proliferating cells situated between the last metamere and the pygidium. Finally, we showed that Nd erg shared the expression pattern of Nd ets in oocytes. Molecular modeling studies have revealed that the spatial structure of ETS domain of Nd Ets and Nd Erg was conserved, in comparison to the murine Ets-1 and human Fli-1 proteins, respectively.     

Cellular immunity in an annelid (Nereis diversicolor,Polychaeta): production of melanin by a subpopulation of granulocytes

Summary We attempted to identify the nature and origin of the pigment produced by the marine worm Nereis diversicolor in order to isolate, in inert brown capsules, foreign objects introduced into its body cavity. This brown pigment, characterized by cytochemical techniques, could be a melanin. The activity of the enzyme phenoloxidase responsible for melanin biosynthesis was detected by enzyme cytochemistry techniques in vacuoles and the Golgi apparatus of coelomocytes activated by the presence of foreign bodies. Morphological techniques combined with a monoclonal immunological probe enabled us to establish that the G₂ granulocytes contain both the precursor of the pigment in dense bodies and the capacity for phenoloxidase synthesis when activated to encapsulate foreign bodies. The G₂ granulocyte may therefore be compared to a melanocyte in which melanin is not stored as in mammals, but immediately extruded following synthesis in the form of a thick fluid.Abbreviations ACTH adrenocorticotropic hormone - Dopa L-3,4 dihydroxyphenylalanine - FITC fluorescein isothiocyanate - G ₁, G ₂, G ₃ granulocyte of types 1, 2, 3 - MSH melanocyte-stimulating hormone - proPo prophenoloxidase     

Antibacterial activity in the coelomic fluid of a marine annelid, Glycera dibranchiata

The coelomic fluid of the polychaete Glycera dibranchiata contained a naturally occurring antibacterial factor, probably serving as part of the organism's defense against bacterial infection. This factor was active against several Gram-negative bacteria, including Serratia marcescens, Pseudomonas aeruginosa, and certain Escherichia coli strains. Quantitative methods to measure this activity were developed. This permitted study of some of its fundamental properties such as dose response, kinetics, and temperature sensitivity. Preliminary data suggested that the antibacterial factor was a heat-labile protein, unrelated to lysozyme. This factor differed from previously described bacteriolytic substances of invertebrate origin and may represent a new type of antimicrobial protein.     

Nereis diversicolor effect on the stability of cohesive intertidal sediments

The effect of the polychaete Nereis diversicolor on the stability of natural cohesive sediments was investigated in the laboratory. Three densities (450, 600 and 1200 ind m⁻²) of N. diversicolor were used. Sediment shear strength was measured using a cone penetrometer. Sediment erodability was assessed using an annular flume (current velocities from 5 to 55 cm s⁻¹) in which flow velocity was increased incrementally, and water sampled to quantify suspended material in order to derive critical erosion velocity and erosion rates. At low current velocities ( <25 cm s⁻¹), we found N. diversicolor to have a stabilising effect, reflected by an increase of up to 20% in the critical erosion velocity. This is related to an enhancement of ~50% in shear strength, due probably to gallery building activities, responsible for the promotion of lateral compaction, an increase in the area of the sediment–water interface, and enhanced microphytobenthos production. Once the sediment began to erode, the stabilising effect of N. diversicolor reverses, leading to an increase of up to 40% in eroded matter due to compaction, which resulted in the erosion of larger aggregates. The balance between the effect of N. diversicolor on herbivory and microphytobenthos production due to the presence of galleries is discussed. Our results indicate that neither chlorophyll a, nor shear strength nor critical erosion velocity are good indicators of erodability. This underlines the need to include biogeochemical processes in any realistic sediment transport model.     

Ultrastructural study of oocyte maturation in the polychaete annelid Sabellaria alveolata

Summary

Maturation begins by a cortical reaction, which resembles that of the sea urchin egg, but can precede fertilization. Complete vitelline membrane elevation necessitates the dissolution of the cortical granule matrix (which can be prevented by concanavalin A) and the retraction of the microvilli at the egg surface (which is inhibited by acid pH). Later on, an aster, with centrioles, develops near the nuclear envelope, which becomes undulated before disruption. In contrast to all other species so far studied, nuclear pores do not disappear and can even be observed several minutes later, in remmants of the nuclear envelope. The meiotic spindle has typical centrioles and, at metaphase I, chromosomes are surrounded by endoplasmic reticulum.     

The fine structure of the compound sense organs on the cirri of Nereis diversicolor

Summary A study of the fine structure of the sense organs on the prostomial cirri and palps of Nereis diversicolor shows them to consist of two types of cell. There are between 7 and 15 sensory cells and a similar number of associated cells which contain many osmiophilic granules. The cell bodies of both are sub-epidermal, having a long distal process which reaches the surface in a raised sensory hillock. The sensory cells carry a cilium, which passes through the cuticle and emerges surrounded by a sheath formed from the outer layers of the epicuticle. Scanning electron micrographs show the surface of the cirrus to be covered by hair-like epicuticular microvilli, through which the sheathed cilia protrude. There is also a second type of sensory cell which occurs singly between the epithelial cells. The distal membrane of this cell is formed into a tuft of approximately 55 large microvilli which open through a pore in the epicuticle. It is suggested by their position and structure, that both these receptors resemble chemoreceptors.We should like to acknowledge the advice and technical help of Dr. J. A. Nott of the N.E.R.C. unit of Electron Microscopy, Menai Bridge, and Dr. P. E. Secker of the School of Electronic Engineering for use of the Cambridge Stereoscan. The work is supported by a grant from the Science Research Council to D.A.D.     

Specificity of d-glucose transport by the apical membrane of Nereis diversicolor epidermis

(1) The specificity of d-[6-³H]glucose influx by a Na⁺-dependent and phlorizin-sensitive transport system in the apical epidermal membrane of the polychaete worm, Nereis diversicolor, was investigated in vivo. (2) The inhibitory effect of eleven d-glucose analogues on d-[6-³H]glucose influx from a 5 μM external concentration was recorded. The inhibitors (each tested at 5, 50, 500 and 5000 μM) were selected to illuminate the configurational requirements for interaction with the d-glucose transport system. (3) The following compounds were found to be significant inhibitors: methyl α-d-glucoside, methyl β-d-glucoside, d-galactose, $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$, 2-deoxy-d-glucose, d-xylose, myo-inositol, β-d-fructose; the effect was graded according to inhibitor concentration. l-Glucose also inhibited d-glucose influx but to the same extent at all four concentrations tested, suggesting transport site heterogeneity. d-Mannose and l-arabinose did not inhibit influx. (4) The most potent inhibitor, methyl-α-d-glucoside, was itself a substrate, and its transport was inhibited by phlorizin and d-glucose, as well as by substitution of Na⁺ in the incubation medium with Li⁺ or choline⁺. (5) We conclude that the specificity of the Na⁺-dependent d-glucose transporter in the apical epidermal membrane of Nereis is similar to that in the apical membrane of vertebrate small intestinal and proximal tubular epithelium, and in the tapeworm integument.     

Autonomy for a specific gene product in oocytes: Experimental evidence in the polychaetous annelid,Platynereis dumerilii

Oocytes of Platynereis dumerilii in early vitellogenesis were injected into female worms with oocytes of similar diameter. The donor oocytes were labeled by the or gene controlling eye pigmentation and, after some weeks of growth, were spawned together with the host oocytes. In most cases, a few donor progeny could be found among the offspring produced by the hosts. Donor progeny were examined with respect to an or gene-dependent maternal effect which normally causes wild-type eye color in homozygous ($\text{or}\text{or}$) larvae originating from the crossings of heterozygous ($\text{or}{}^{\mathrm{+}}\text{or}$) females and homozygous ($\text{or}\text{or}$) males. This maternal effect was absent from homozygous ($\text{or}\text{or}$) larvae derived from homozygous ($\text{or}\text{or}$) donor oocytes which had developed in heterozygous females. Conversely, this maternal effect was observed in homozygous ($\text{or}\text{or}$) larvae derived from heterozygous ($\text{or}{}^{\mathrm{+}}\text{or}$) donor oocytes which had developed in homozygous ($\text{or}\text{or}$) host females. It is concluded that the oocyte genome is active at the or⁺ locus during oogenesis and that the oocyte is autonomous with respect to the product of synthesis of the or⁺ locus. In the present case, the “maternal effect” is therefore caused by synthetic activity in the growing oocyte. The results are discussed with respect to current information on gene products from animal genomes.     

Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis

Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4–5 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61–71% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis, and gametogenesis.