Strong natural selection causes microscale allozyme variation in a marine snail.

Natural selection is one of the most fundamental processes in biology. However, there is still a controversy over the importance of selection in microevolution of molecular traits. Despite the general lack of data most authors hold the view that selection on molecular characters may be important, but at lower rates than selection on most phenotypic traits. Here we present evidence that natural selection may contribute substantially to molecular variation on a scale of meters only. In populations of the marine snail Littorina saxatilis living on exposed rocky shores, steep microclines in allele frequencies between splash and surf zone groups are present in the enzyme aspartate aminotransferase (allozyme locus Aat; EC. 2.6.1.1). We followed one population over 7 years, including a period of strong natural perturbation. The surf zone part of the population dominated by the allele Aat100 was suddenly eliminated by a bloom of a toxin-producing microflagellate. Downshore migration of splash zone snails with predominantly Aat120 alleles resulted in a drastic increase in surf zone frequency of Aat120, from 0.4 to 0.8 over 2 years. Over the next four to six generations, however, the frequency of Aat120 returned to the original value. We estimated the coefficient of selection of Aat120 in the surf zone to be about 0.4. Earlier studies show similar or even sharper Aat clines in other countries. Thus, we conclude that microclinal selection is an important evolutionary force in this system.

Refolding of omega conotoxin peptide derived from marine snail, Conus magus, to evaluate its analgesic effects

Oxidative refolding is one of the key challenges hampering the development of peptide based compounds as therapeutics. The correct refolding for three disulfide peptide like w-Conotoxi n MVIIA is difficult and crucial for biological activity. This work advanced knowledge of chemical and biological for improve oxidative refolding of synthetic w-Conotoxi n MVIIA in base of Conus magus venom. The present study aimed to set up an appropriate and effective protocols for refolding of disulfide-rich w-Conotoxin MVIIA. In this study, the crude peptide was protected with Acm group, according to the right amino acid sequences (Synthesized by Australian Company). The crude peptide was purified by H PLC. To prepare the peptide to refolding, innovative deprotection applied molar ratio (AMR) method was performed based on mercury. Accuracy of deprotection was approved by reverse phase chromatography. The deprotected target peptide (omega-conotoxin) was determined by SDS-PAGE. Then the Oxidative refolding of target peptide was performed in six protocol based on Guanidinium chloride and oxidized and reduced Glutathione. Analgesic effect of refolded peptide was surveyed with formalin test in mice Balb/c. Non neurotoxic effects of target peptides were survey with ICV injection in mice model (C57/BL6). The innovative deprotection protocol performed based on the best ratio of mercury/2-mercaptoethanol adjusted to 1mg/10p1 in 90 minute. The results showed the yield and purity of omega-conotoxin MVIIA as 93 and 95%, respectively. Refolding of 40 mg omega Conotoxin with GSSG and GSH on ratio of 10:1 and 20 mM ammonium acetate showed the best analgesic effect compared with the other methods. The result showed 95.5% yield and 98% purity of omega-conotoxin MVIIA in this refolding method. Related refolding method reduced 85% pain in experimented mice using 7 ng of the peptide. That was 71.5 fold stronger than morphine and 2 times than standard Prialt®. And it was not neurotoxic in mice. In this study, refolding method for omega-conotoxin MVIIA was optimized in the fourth factor including: reducing the time, amount and number of reagent and increase the efficiency. We introduced new method for deprotection of omega-conotoxin MVIIA. Effective, economic and applied refolding and deprotecti on method was performed in this research may al so be applied to similar omega conotoxin peptides.

A sleep-inducing peptide from the venom of the Indian cone snail Conus araneosus

The marine snail Conus araneosus has unusual significance due to its confined distribution to coastal regions of southeast India and Sri Lanka. Due to its relative scarceness, this species has been poorly studied. In this work, we characterized the venom of C. araneosus to identify new venom peptides. We identified 14 novel compounds. We determined amino acid sequences from chemically-modified and unmodified crude venom using liquid chromatography-electrospray ionization mass spectrometry and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Ten sequences showed six Cys residues arranged in a pattern that is most commonly associated with the M-superfamily of conotoxins. Four other sequences had four Cys residues in a pattern that is most commonly associated with the T-superfamily of conotoxins. The post-translationally modified residue (pyroglutamate) was determined at the N-terminus of two sequences, ar3h and ar3i respectively. In addition, two sequences, ar3g and ar3h were C-terminally amidated. At a dose of 2 nmol, peptide ar3j elicited sleep when injected intraperitoneally into mice. To our knowledge, this is the first report of a peptide from a molluscivorous cone snail with sleep-inducing effects in mice. The novel peptides characterized herein extend the repertoire of unique peptides derived from cone snails and may add value to the therapeutic promise of conotoxins. (C) 2015 Elsevier Ltd. All rights reserved.

Mollusca Non-Grata: The influence of top-down control and residence time on the abundance, distribution, and behavior of non-native marine snails in Washington State

Thesis (Ph.D.)--University of Washington, 2016-06

The Role of Chemical Mechanisms in Neural Computation and Learning

Most computational models of neurons assume that their electrical characteristics are of paramount importance. However, all long-term changes in synaptic efficacy, as well as many short-term effects, are mediated by chemical mechanisms. This technical report explores the interaction between electrical and chemical mechanisms in neural learning and development. Two neural systems that exemplify this interaction are described and modelled. The first is the mechanisms underlying habituation, sensitization, and associative learning in the gill withdrawal reflex circuit in Aplysia, a marine snail. The second is the formation of retinotopic projections in the early visual pathway during embryonic development.

Differential antagonism by conotoxin p-TIA of contractions mediated by distinct alpha(1)-adrenoceptor subtypes in rat vas deferens, spleen and aorta

The ability of the conotoxin p-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa, to antagonize contractions induced by noradrenaline through activation of alpha(1A)-adrenoceptors in rat vas deferens, alpha(1B)-adrenoceptors in rat spleen and alpha(ID)-adrenoceptors in rat aorta, and to inhibit the binding of [I-125]HEAT (2-[[beta-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat alpha(1)-adrenoceptors was investigated. p-TIA (100 nM to 1 muM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (pA(2)similar to7.2, n=4). This suggests that p-TIA is a competitive antagonist of alpha(1A)- and alpha(1D)-adrenoceptors with no selectivity between these subtypes. Incubation of p-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that p-TIA is a non-competitive antagonist at alpha(1B)-adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of p-TIA in inhibiting contractions was examined with similar occupancies (similar to25%) at each subtype. Its potency (pIC(50)) was 12 times higher in spleen (8.3 +/- 0.1, n=4) than in vas deferens (7.2 +/- 0.1, n=4) or aorta (7.2 0.1, n=4). In radioligand binding assays, p-TIA decreased the number of binding sites (B,,,,,,) in membranes from HEK293 cells expressing the rat alpha(1B)-adrenoceptors without affecting affinity (K-D), In contrast, in HEK293 cells expressing rat alpha(1A)- or alpha(1D)-adrenoceptors, p-TTA decreased the KD without affecting the B-max. It is concluded that p-TIA will be useful for distinguishing the role of particular alpha(1)-adrenoceptor subtypes in native tissues. (C) 2004 Elsevier B.V. All rights reserved.

Long-lasting hyperexcitability induced by depolarization in the absence of detectable Ca2+ signals.

Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca(2+) signals produced by depolarization. Indeed, transient Ca(2+) signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca(2+)-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca(2+) transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca(2+) levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve-ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis-dependent hyperexcitability under conditions in which Ca(2+) entry is prevented (by bathing in nominally Ca(2+) -free solutions containing EGTA) and detectable Ca(2+) transients are eliminated (by adding BAPTA-AM). Disruption of Ca(2+) release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca(2+)-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.

A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated p114a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. p114a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an R-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of p114a revealed a novel signal sequence, indicating that p114a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of p114a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, p114a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50) 1.59 mu M) and neuronal (IC50 = 8.7 mu M for alpha 3 beta 4) and neuromuscular (IC50 = 0.54 mu M for alpha 1 beta 1 is an element of delta) subtypes of the nicotinic acetylcholine receptor ( nAChR). Similarities in sequence and structure are apparent between the middle loop of p114a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

Kinetics of tidal resuspension of microbiota: Testing the effects of sediment cohesiveness and bioturbation using flume experiments

Resuspension of the top few sediment layers of tidal mud flats is known to enhance planktonic biomass of microbiota (benthic diatoms and bacteria). This process is mainly controlled by tidal shear stress and cohesiveness of mud, and is also influenced by bioturbation activities. Laboratory experiments in a race track flume were performed to test the interactive effects of these factors on both the critical entrainment and resuspension kinetics of microbiota from silt-clay sediments from the Marennes-Oleron Bay, France. The marine snail Hydrobia ulvae was used to mimic surface bioturbation activities. As expected, the kinetics of microbial resuspension versus shear stress were largely controlled by the cohesiveness of silt-clay sediments. However, our results indicate that the effect of surface tracking by H. ulvae on microbial resuspension was clearly dependent on the interaction between sediment cohesiveness and shear velocity. Evidence was also found that microphytobenthos and bacteria are not simultaneously resuspended from silt-clay bioturbated sediments. This supports the theory that diatoms within the easily eroded mucus matrix behave actively and bacteria adhering to fine silt particles eroded at higher critical shear velocities behave passively.

Regulation of electrical transmission by protein kinase C in Aplysia bag cell neurons

Electrical synapses are composed of gap junctions, made from paired hemi-channels that allow for the transfer of current from one neuron to another. Gap junctions mediate electrical transmission in neurons, where they synchronize spiking and promote rapid transmission, thereby influencing the coordination, pattern, and frequency of firing. In the marine snail, Aplysia calfornica, two clusters of neuroendocrine bag cell neurons use electrical synapses to synchronize a 30-min burst of action potentials, known as the afterdischarge, which releases egg-laying hormone and induces reproduction. In culture, paired bag cell neurons present a junctional conductance that is non-rectifying and largely voltage-independent. During the afterdischarge, PKC is activated, which is known to increase voltage-gated Ca2+ current; yet, little is understood as to how this pathway impacts electrical transmission. The transfer of presynaptic spike-like waveforms (generated in voltage-clamp) to the postsynaptic cell (measured in current-clamp) was monitored with or without PKC activation. It was found that pretreatment with the PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced junctional conductance between bag cell neurons. Furthermore, in control, presynaptic action potential waveforms mainly evoked postsynaptic electrotonic potentials at both -60 and -40 mV. However, with PKC activation the presynaptic stimulus consistently elicited postsynaptic action potentials from resting potentials of -40 mV, and would occasionally result in firing from repetitive input at -60 mV. Moreover, to assess whether this enhanced electrical transmission genuinely reflects a greater junctional conductance or a change in postsynaptic responsiveness, a fast-phase junctional-like current was applied to single bag cell neurons. Neurons in PMA always fired action potentials in response to current injection as opposed to control, which were less likely to spike. This outcome did not change when the junctional-like current was artificially enhanced in control conditions. Also, in response to fast- and slow-phase electrotonic potential (ETP) waveforms, Ca2+ current was markedly larger in single PMA-treated neurons. These findings suggest that PKC activation may contribute to afterdischarge fidelity by recruiting postsynaptic Ca2+ current to promote synchronous network firing. Finally, Aplysia gap junction genes (innexins) were transfected into mouse N2A cells and characterized. This revealed a biophysical and pharmacological profile similar to native gap junctions.

Molluscicidal activity of some marine substances against the snail Biomphalaria glabrata (Mollusca, Planorbidae)

Freshwater snails of the genus Biomphalaria play a major role as intermediate hosts of Schistosoma mansoni, the etiologic agent of schistosomiasis. While Biomphalaria spp. control by molluscicides is one of the main strategies to reduce the snail population in infected areas, there are few effective molluscicides commercially available. Natural products may be considered as potentially useful and safe molluscicides. We have evaluated the molluscicidal activity of 12 extracts from ten marine organisms on adult and embryonic stages of Biomphalaria glabrata. Only extracts of the red algae Liagora farinosa and of the sponge Amphimedon viridis presented molluscicidal activity. Lethal concentration (LC)(50) values obtained were 120 mu g/mL for L. farinosa CH2Cl2 extract (apolar fraction) and 20 mu g/mL for A. viridis extract and halitoxin. The polar alga fraction and halitoxin had no effect on B. glabrata embryos. The algae apolar fraction was active on B. glabrata in all embryonic development stages, with LC50 values for blastulae at 42 mu g/mL, gastrulae at 124 mu g/mL, trochophore at 180 mu g/mL, and veliger at 222 mu g/mL. This is the first report of extracts from marine organisms which presented molluscicidal activity.

Quantifying ecosystem-level consequences of ocean acidification and warming by scaling up individual level responses of a predator snail and its trophic interactions

Understanding long‐term, ecosystem‐level impacts of climate change is challenging because experimental research frequently focuses on short‐term, individual‐level impacts in isolation. We address this shortcoming first through an interdisciplinary ensemble of novel experimental techniques to investigate the impacts of 14‐month exposure to ocean acidification and warming (OAW) on the physiology, activity, predatory behaviour and susceptibility to predation of an important marine gastropod (Nucella lapillus). We simultaneously estimated the potential impacts of these global drivers on N. lapillus population dynamics and dispersal parameters. We then used these data to parameterize a dynamic bioclimatic envelope model, to investigate the consequences of OAW on the distribution of the species in the wider NE Atlantic region by 2100. The model accounts also for changes in the distribution of resources, suitable habitat and environment simulated by finely resolved biogeochemical models, under three IPCC global emissions scenarios. The experiments showed that temperature had the greatest impact on individual‐level responses, while acidification had a similarly important role in the mediation of predatory behaviour and susceptibility to predators. Changes in Nucella predatory behaviour appeared to serve as a strategy to mitigate individual‐level impacts of acidification, but the development of this response may be limited in the presence of predators. The model projected significant large‐scale changes in the distribution of Nucella by the year 2100 that were exacerbated by rising greenhouse gas emissions. These changes were spatially heterogeneous, as the degree of impact of OAW on the combination of responses considered by the model varied depending on local‐environmental conditions and resource availability. Such changes in macro‐scale distributions cannot be predicted by investigating individual‐level impacts in isolation, or by considering climate stressors separately. Scaling up the results of experimental climate change research requires approaches that account for long‐term, multiscale responses to multiple stressors, in an ecosystem context.