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Hemolymph clotting in crustaceans: Implications for neuropeptide extraction from invertebrate hemolymph.

The hemolymph of invertebrates contains factors that facilitate clotting as a defense mechanism for injury. However, the clotting process may impair the measurement of hormone titers by interfering with the extraction of peptides. Using hemolymph from freshwater crayfish, our results demonstrate that hemolymph clotting appears to reduce both the amount of an endogenous peptide(s) (Dippu-AST 11-like) extracted from hemolymph, as well as the amount of spiked peptide tracer ([125I]-Dippu-AST 11) recovered from hemolymph. The efficacy of peptide extraction from hemolymph was improved by collecting the hemolymph into a variety of different media prior to hemolymph coagulation. Hemolymph samples collected into media containing the anticoagulant, citrate, had the highest amount of endogenous Dippu-AST 11-like peptide extracted as well as the highest percent recovery of spiked tracer.

Animals↗

Signal transduction at fertilization: the Ca2+ release pathway in echinoderms and other invertebrate deuterostomes.

Gamete interaction and fusion triggers a number of events that lead to egg activation and development of a new organism. A key event at fertilization is the rise in intracellular calcium. In deuterostomes, this calcium is released from the egg's endoplasmic reticulum and is necessary for proper activation. This article reviews recent data regarding how gamete interaction triggers the initial calcium release, focusing on the echinoderms (invertebrate deuterostomes) as model systems. In eggs of these animals, Src-type kinases and phospholipase C-gamma are required components of the initial calcium trigger pathway in eggs.

Animals↗

Divergent IRES elements in invertebrates.

Viruses have evolved unique strategies and mechanisms to recruit ribosomes to ensure continued translation of their viral RNA during infection. The Dicistroviridae family of invertebrate viruses contains an unusual internal ribosome entry site (IRES), which can directly recruit ribosomes in the absence of initiation factors. Moreover, this IRES initiates translation at a non-AUG codon independent of an initiator Met-tRNA. Recent studies have shown that the IRES mimicks a tRNA to interact with and manipulate the ribosome. The presence of this divergent IRES likely allows translation of the dicistroviral RNA during infection when host translation is compromised. This review will explore the unique properties of this unprecedented mechanism of gene expression. Specific topics will examine structural components of the IRES, the mechanism of initiating translation at non-AUG codons and the regulation of this IRES in vivo. The existence of this mechanism suggests that the repertoire of open reading frames in our genome may be greater than anticipated.

Animals↗

The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins.

We have cloned the genes PANX1, PANX2 and PANX3, encoding putative gap junction proteins homologous to invertebrate innexins, which constitute a new family of mammalian proteins called pannexins. Phylogenetic analysis revealed that pannexins are highly conserved in worms, mollusks, insects and mammals, pointing to their important function. Both innexins and pannexins are predicted to have four transmembrane regions, two extracellular loops, one intracellular loop and intracellular N and C termini. Both the human and mouse genomes contain three pannexin-encoding genes. Mammalian pannexins PANX1 and PANX3 are closely related, with PANX2 more distant. The human and mouse pannexin-1 mRNAs are ubiquitously, although disproportionately, expressed in normal tissues. Human PANX2 is a brain-specific gene; its mouse orthologue, Panx2, is also expressed in certain cell types in developing brain. In silico evaluation of Panx3 expression predicts gene expression in osteoblasts and synovial fibroblasts. The apparent conservation of pannexins between species merits further investigation.

Amino Acid Sequence↗

Effects of m-CPP in altering neuronal function: blocking depolarization in invertebrate motor and sensory neurons but exciting rat dorsal horn neurons.

The compound m-chlorophenylpiperazine (m-CPP) is used clinically to manipulate serotonergic function, though its precise mechanisms of actions are not well understood. m-CPP alters synaptic transmission and neuronal function in vertebrates by non-selective agonistic actions on 5-HT(1) and 5-HT(2) receptors. In this study, we demonstrated that m-CPP did not appear to act through a 5-HT receptor in depressing neuronal function in the invertebrates (crayfish and Drosophila). Instead, m-CPP likely decreased sodium influx through voltage-gated sodium channels present in motor and primary sensory neurons. Intracellular axonal recordings showed that m-CPP reduced the amplitude of the action potentials in crayfish motor neurons. Quantal analysis of excitatory postsynaptic currents, recorded at neuromuscular junctions (NMJ) of crayfish and Drosophila, indicated a reduction in the number of presynaptic vesicular events, which produced a decrease in mean quantal content. m-CPP also decreased activity in primary sensory neurons in the crayfish. In contrast, serotonin produces an increase in synaptic strength at the crayfish NMJ and an increase in activity of sensory neurons; it produces no effect at the Drosophila NMJ. In the rat spinal cord, m-CPP enhances the occurrence of spontaneous excitatory postsynaptic potentials with no alteration in evoked currents.

Animals↗

The CRH-ACTH-biogenic amine axis in invertebrate immunocytes activated by PDGF and TGF-beta.

In immunocytes from the mollusc Mytilus galloprovincialis, the major pathway followed by platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 in provoking the release of norepinephrine, epinephrine and dopamine into cell-free hemolymph (serum) is mediated by a corticotropin-releasing hormone-adrenocorticotropin hormone (CRH-ACTH) biogenic amine axis. This axis not only annulled the inhibiting properties of PDGF-AB, it even reversed the latter's effect, while the inducing effect of TGF-beta1 was amplified. These findings show that non-classical immune-neuroendocrine molecules, such as PDGF-AB and TGF-beta1, are involved in building stress response, using the same conserved mechanisms present from invertebrates to vertebrates.

Adrenocorticotropic Hormone↗

The relative influence of temperature and food on the metabolism of a marine invertebrate.

Many benthic marine invertebrates exhibit a seasonal cycle in activities such as feeding, growth and reproduction. In temperate regions, this seasonality is typically correlated with coincident cycles in photoperiod, temperature and food availability and it can be difficult to determine which of these environmental factors is the key driver. Polar regions are characterised by greatly reduced seasonal variation in temperature, and an enhanced seasonality of food availability; they therefore form a natural laboratory for distinguishing the ecological effects of food from those of temperature. Here, we report a study of the common shallow water urchin Sterechinus neumayeri from Rothera Point, Antarctica. This species exhibits a marked seasonal variation in metabolic rate and feeding activity (which ceases completely in winter). In this study the metabolic rate of urchins collected in late winter and held in the laboratory without food was compared with that of wild urchins undertaking the transition to summer feeding and growth. Starved urchins showed a small rise in metabolic rate in summer which could be explained entirely by the small increase in temperature (Q(10)=2.5). At the same time, the wild population showed a much larger increase in metabolic rate related largely to the costs of feeding and growth. Rates of nitrogen excretion were also much larger in wild urchins, and the O:N atomic ratio indicated that starved urchins were depending to a greater extent on lipid and carbohydrate. Gut mass and test organic content showed no change in starved urchins, indicating that metabolic substrate was being provided by the gonad. The data suggest that in wild S. neumayeri only 15-20% of the summer increase in metabolism is caused directly by the temperature rise whereas 80-85% is caused by increased physiological activity associated with feeding, growth and spawning.

Journal Article↗

Detection of casein fragments in an invertebrate and in a vertebrate using in situ hybridization.

Using a non-radioactive in situ hybridization procedure it has been demonstrated that both invertebrates such as the mollusc Mytilus galloprovincialis and lower vertebrates such as the fish Cyprinus carpio express bovine alphaS1- and kappa-casein homologous mRNAs. In particular, positive results were found in molluscan immunocytes, and in cells located in different fish tissues: intestine, endocrine pancreas and kidney. These findings suggest that the casein genes are highly conserved throughout evolution.

Animals↗

Effects of chronic low-level PAH contamination of soil invertebrate communities.

Soils containing low levels of polycyclic aromatic hydrocarbons (PAHs) were collected from an abandoned industrial site. A study was conducted to evaluate the effects of these contaminants on soil invertebrates representing three levels of ecological hierarchy: the microfauna, mesofauna, and macrofauna. Nematodes were studied as representatives of the soil microfauna, microarthropods as representatives of the mesofauna, and earthworms as representatives of the macrofauna. Six sample plots representing a gradient of PAH contamination ranging from 5.28 to 80.46 mg/kg total PAHs were evaluated. Nematode community structure, including abundance and diversity of trophic and taxonomic groups; the total abundance of microarthropods (orders Collembola and Acarina); and earthworm (Eisenia andrei) growth were evaluated. Multiple regression analyses were used to evaluate trends in the responses of these target organisms to PAH concentrations and habitat variability. Abundance of omnivore/predator nematodes and microarthropod order Collembola; nematode taxonomic diversity; and the percent difference in earthworm weights exhibited positive associations with PAH concentrations. Total abundance of microarthropod order Acarina was negatively associated with PAH concentrations.

Animals↗

A synthesis of growth rates in marine epipelagic invertebrate zooplankton.

We present the most extensive study to date of globally compiled and analysed weight-specific growth rates in marine epi-pelagic invertebrate metazoan zooplankton. Using specified selection criteria, we analyse growth rates from a variety of zooplanktonic taxa, including both holo- and mero-planktonic forms, from over 110 published studies. Nine principal taxonomic groups are considered, the copepods (number of individual data points (n) = 2,528); crustaceans other than copepods (n = 253); cnidarians (n = 77); ctenophores (n = 27); chaetognaths (n = 87); pteropods (n = 8); polychaetes (n = 12); thaliaceans (n = 88); and larvaceans (n = 91). The copepods are further examined by subdividing them into broadcasters or sac-spawning species, and as nauplii (N1-N6), copepodites (C1-C5) and adults (C6). For each taxonomic group relationships between growth, temperature and body weight are examined using a variety of methods. Weight-specific growth tends to increase with increasing temperature and with decreasing body weight in the crustacean group. Growth does not relate to body weight in the case of chaetognaths and larvaceans, but does increase with temperature. In the cnidarian and ctenophore groups growth does not relate to temperature, but is negatively related to body size. For the thaliceans growth increases with both increasing body weight and temperature. In the entire broadcasting copepod data set, weight-specific growth increases with increasing temperature and decreasing body weight. In sac-spawners, growth increases with increasing temperature, and increases with decreasing body weight at temperatures below 20 degrees C, but decreases with body weight at temperatures above this. Comparison between the different taxa shows important differences and similarities. Our extensive synthesis of data generally confirms that larvaceans, pteropods, cnidarians and ctenophores have rates of weight-specific growth that are typically greater than the copepods, chaetognaths and other crustaceans of similar carbon weight. For the cnidarians, ctenophores and larvaceans growth rates are almost always greater than the general relationship describing copepod growth, and are also at the upper limits or beyond the maximum rates for copepods of a similar weight. For the pteropods, growth rates are generally greater than those of copepods, although the data set was limited to a single carnivorous species in a single study (i.e. Clione limacina). The thaliaceans have the highest growth rates for animals with body weights greater than around 1 mg C ind-1, with rates of up to 2.1 d-1 for Pegea bicaudata. Whilst the larvaceans can achieve rates of 2 d-1 in warm tropical waters (28 degrees C), and as high as > 3 d-1 for < 0.2 mg C individual-1 animals of Oikopleura diocia. These are possibly the highest rates every recorded in epi-pelagic metazoans. Reasons for the differences between taxonomic groups are discussed in relation to intrinsic and extrinsic factors and limitations. The importance of this investigation not only lies in it being the most comprehensive overview of patterns of growth to date, but because the data set highlight the gaps in measurements and current knowledge. We examine the inadequacies in the current data sets, and in the methods being used to measure growth and production. Most of the data are for animals collected from coastal and estuarine waters, and it is clear that for a fuller understanding there is an urgent need for work in the open ocean, and for investigations outside temperate regions. There is also a need to explore the role of food availability, and how food concentrations in incubations, and under food saturation, relate to those experienced in the natural environment.

Animals↗

An estimation of radiation doses to benthic invertebrates from sediments collected near a Canadian uranium mine.

A new method is described for calculating radiation doses to benthic invertebrates from radionuclide concentrations in freshwater sediment. Both internal and external radiation doses were estimated for all 14 principal radionuclides of the uranium-238 decay series. Sediments were collected from three sites downstream of a uranium mining operation in northern Saskatchewan, Canada. Sediments from two sites, located approximately 1.6 and 4.4 km downstream from mining operations, yielded absorbed doses to both larval midges, Chironomus tentans, and adult amphipods, Hyalella azteca, of 59-60 and 19 mGy/year, respectively, compared to 3.2 mGy/year for a nearby control site. External beta radiation from protactinium-234 (234Pa) and alpha radiation from uranium (U) contributed most of the dose at the impacted sites, whereas polonium-210 (210Po) was most important at the control site. If a weighting factor of 20 was employed for the greater biological effect of alpha vs. beta and gamma radiation, then total equivalent doses rose to 540-560 mGy/year at the site closest to uranium operations. Such equivalent doses are above the 360-mGy/year no-observed-effect level for reproductive effects in vertebrates from gamma radiation exposure. Data are not available to determine the effect of such doses on benthic organisms, but they are high enough to warrant concern. Detrimental effects have been observed in H. azteca at similar uranium concentration in laboratory toxicity tests, but it remains unclear whether the radiotoxicity or the chemotoxicity of uranium is responsible for these effects.

Animals↗

Complement systems in invertebrates. The ancient alternative and lectin pathways.

The complement system in higher vertebrates is composed of about thirty proteins that function in three activation cascades and converge in a single terminal pathway. It is believed that these cascades, as they function in the higher vertebrates, evolved from a few ancestral genes through a combination of gene duplications and divergences plus pathway duplication (perhaps as a result of genome duplication). Evidence of this evolutionary history is based on sequence analysis of complement components from animals in the vertebrate lineage. There are fewer components and reduced or absent pathways in lower vertebrates compared to mammals. Modern examples of the putatively ancestral complement system have been identified in sea urchins and tunicates, members of the echinoderm phylum and the protochordate subphylum, which are sister groups to the vertebrates. Thus far, this simpler system is composed of homologues of C3, factor B, and mannose binding protein associated serine protease suggesting the presence of simpler alternative and lectin pathways. Additional components are predicted to be present. A complete analysis of this invertebrate defense system, which evolved before the invention of rearranging genes, will provide keys to the primitive beginnings of innate immunity in the deuterostome lineage of animals.

Amino Acid Sequence↗

A comparison of techniques for detecting Invertebrate iridescent virus 6.

The aim of this study was to compare the sensitivity and precision of various methods for the detection and quantification of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae) isolated from a the stem-boring moth Chilo suppressalis, and to apply these techniques to the detection of covert infections in the wax moth, Galleria mellonella. The relationship between the virus concentration and absorbance at 260 nm was linear over the range of 1.6 x 10(9)-5.6 x 10(10) particles/ml. TCID(50) assays using 12 different cell lines indicated that two Drosophila lines, DL2 and DR1, had the highest susceptibility whereas cell lines from Aedes albopictus and Plutella xylostella were four orders of magnitude less sensitive. TCID(50) values for IIV-6 in Spodoptera frugiperda Sf9 cells gave the particle-infectivity ratios of 15-64 virus particles/IU. An insect bioassay involved injecting doses of 1-100 IIV-6 particles into the third instar G. mellonella larvae. The prevalence of patent infection was 20-26% at a dose of 1 particle per larva rising to 86-92% at 10 particles and 100% at doses of 50 or 100 particles. Of the insects that survived to adulthood, between 5.8 and 75% caused patent infections when injected into G. mellonella larvae, indicating that they were covertly infected. A PCR technique resulted in 95% detection at 1000 virus particles per insect. Of the insects that proved positive for covert infection by insect bioassay, 41% also proved positive by PCR analysis. It is concluded that the G. mellonella bioassay is highly reliable for detection of doses of 10 particles or more and for determining the relative activity of IIV-6 preparations at doses as low as 1 particle per insect. PCR had a slightly lower sensitivity followed by the insect cell culture assay.

Aedes↗

Octopus, which owns the most advanced brain in invertebrates, has two members of vasopressin/oxytocin superfamily as in vertebrates.

A novel member of the vasopressin/oxytocin superfamily, octopressin (OP), has been isolated from Octopus vulgaris. Since another peptide of this superfamily, cephalotocin (CT), was isolated from the same species [Neurosci. Lett. 134 (1992) 191], Octopus has two members of the superfamily as in vertebrates, an observation made for the first time in invertebrates. Octopressin caused contractions of the Octopus peripheral tissues such as oviduct, aorta, rectum, etc. Cephalotocin had no effects on tested tissues. The octopressin and cephalotocin precursors were composed of a signal peptide, a nonapeptide, and a neurophysin domain-the typical structural organizations of the superfamily precursors. Reverse transcription polymerase chain reaction (RT-PCR)/Southern blot analysis revealed that octopressin mRNA was expressed in the supraesophageal and subesophageal brains, and the buccal and gastric ganglia. Cephalotocin mRNA was expressed mostly in the subesophageal brain. In situ hybridization in the brain showed that octopressin mRNA was localized in many lobes. Expression of cephalotocin mRNA was almost limited in the ventral median vasomotor lobe. Some of the neurons in this lobe are the source of the neurosecretory system of the vena cava, where cephalotocin was originally isolated. These results suggest that octopressin may be a multifunctional neuropeptide contributing to reproduction, cardiac circulation, and feeding. Cephalotocin may play important roles in metabolism, homeostasis, etc., as a neurohormone.

Amino Acid Sequence↗

Primary structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata and its cDNA: structural similarity to the B-chain from plant lectin, ricin.

CEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc) specific lectin purified from a marine invertebrate Cucumaria echinata has a strong hemolytic activity especially toward human and rabbit erythrocytes. We determined the primary structure of the CEL-III by examining the amino acid sequences of the protein and the nucleotide sequence of the cDNA. The cDNA encoding CEL-III has 1823 nucleotides and an open reading frame of 1296 nucleotides. CEL-III is composed of 432 amino acid residues with a M(r) of 47¿ omitted¿457 and has six internal tandem repeats, each with of 40-50 amino acids, comprising the N-terminal two-thirds of the molecule. Similar repeats are found in the B-chains of cytotoxic plant lectins, such as ricin and abrin, where six repetitive sequences extend throughout the molecules. A hydropathy plot predicts hydrophobic segments in the C-terminal region of CEL-III. These findings suggest that the N-terminal region of CEL-III plays an important role in binding to carbohydrate receptors on the target cell membranes, an event which triggers an intermolecular hydrophobic interaction of the C-terminal region, the result being oligomerization of CEL-III to lead to pore-formation in erythrocyte membrane.

Abrin↗

2',5'-oligoadenylate synthetase from a lower invertebrate, the marine sponge Geodia cydonium, does not need dsRNA for its enzymatic activity.

Recently, the presence of 2',5'-linked oligoadenylates and a high 2',5'-oligoadenylate synthetase activity were discovered in a lower invertebrate, the marine sponge Geodia cydonium. It has been demonstrated that mammalian 2-5A synthetase isozymes require a dsRNA cofactor for their enzymatic activity. Our results show that, unlike mammalian 2-5A synthetases, the 2-5A synthetase from the sponge acts in a dsRNA-independent manner in vitro. A prolonged incubation of the G. cydonium extract with a high concentration of a micrococcal nuclease had no effect on the activity of the 2-5A synthetase. At the same time, the micrococcal nuclease was effective within 30 min in degrading dsRNA needed for the enzymatic activity in IFN-induced PC12 cells. These results indicate that the 2-5A synthetase from G. cydonium may be active per se or is activated by some other mechanism. The sponge enzyme is capable of synthesizing a series of 2-5A oligomers ranging from dimers to octamers. The accumulation of a dimer in the predominant proportion during the first stage of the reaction was observed, followed by a gradual increase in longer oligoadenylates. By its product profile and kinetics of formation, the sponge 2-5A synthetase behaves like a specific isoform of enzymes of the 2-5A synthetase family.

2',5'-Oligoadenylate Synthetase↗

Adhesive and growth properties of lectin from the ascidian Didemnum ternatanum on cultivated marine invertebrate cells.

The effects of N-Acetyl-D-glucosamine-specific lectin (M(r) 27 kDa) isolated from the ascidian Didemnum ternatanum on cultivated cells of molluscs and echinoderms were studied. This lectin was found to stimulate the growth or the differentiation of cultivated marine invertebrate cells depending on the stage of embryonic development at which primary cell cultures were obtained. In addition, it has been shown to increase the attachment of cells in primary cultures of these animals. The degree of attachment is considerably increased when collagen or polylysine substrates are used. Using scanning electron microscopy we have demonstrated the stage-specific effect of this lectin on embryonic sea urchin and molluscan cells. Intensive cell spreading and an alteration of cell shape were observed only at the gastrula stage, when the switching from maternal information to embryonic genes occurred. The ascidian lectin seems to have some characteristics of both an adhesive factor and a growth factor.

Animals↗

Partial purification and characterization of six transglutaminases from ordinary muscles of various fishes and marine invertebrates.

Six transglutaminases were prepared from ordinary muscles of scallop (Patinopecten yessoensis), botan shrimp (Pandalus nipponensis), squid (Todarodes pacificus), carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), and atka mackerel (Pleurogrammus azonus), and their physicochemical and enzymatic properties were compared with each other. The Km value of carp transglutaminase for monodansyl cadaverine, a kind of primary amines, was 0.33 mM, while the other enzymes had Km values of 0.01-0.03 mM. The Km, values for succinylated casein of scallop, botan shrimp, squid, carp, rainbow trout, and atka mackerel enzymes were 1.2, 0.3, 1.8, 0.3, 0.2, and 0.1 mg/ml, respectively. In the presence of 0.5 M NaCl, the activities of scallop, botan shrimp, and squid transglutaminases were further enhanced about 11-, 2-, and 6-fold, respectively. There was no effect of NaCl on the activities of fish enzymes. These increment in the activities were dependent of NaCl concentrations and could be exhibited by using KCl instead of NaCl. To date there are no reports on types of transglutaminases whose activities are stimulated by salts. These findings suggest there exist a novel type of TGase in marine invertebrate muscles in which the osmotic pressure is isotonic to sea water.

Amines↗