The role of cyclic nucleotides in modulation of crayfish neuromuscular junctions by a neuropeptide /

DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and has been shown to increase the amount of transmitter released at neuromuscular junctions of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The involvement of these kinases led to the prediction that a G-protein-coupled receptor (GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR pathways seen in other organisms and in other cells. G-proteins can also act on an enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the question of whether or not DF2's effects on synaptic transmission in crayfish are mediated by the cyclic nucleotides cAMP and cGMP. The effects of DF2 on synaptic transmission were examined using deep abdominal extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal extensor muscle LI . A number of activators and inhibitors were used to determine whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide. Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP) mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in EPSP amplitude induced by the peptide. When both kinase inhibitors are applied simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in the data suggests that the PKA inhibitor blocks a portion of the response elicited by the PKG activator. Thus, the PKA inhibitor may be less specific for PKA. Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results support the hypothesis that cAMP and cGMP, acting through their respective protein kinase enzymes, mediate the ability of DFi to increase transmitter output.

Effects of reflection and social isolation on crayfish behaviour /

Visual stimuli and socialization influence exploratory behaviour in crayfish. The predominant components of spontaneous exploratory behaviour were determined by observing the activity of solitary adult crayfish (Procambarus clarkii) in a glass aquarium containing fresh water and no objects. Five distinct behaviours were observed: rearing up (climbing on the wall), turning around, cornering (facing the comer), backward walking, and crossing (crossing the midline of the aquarium). The frequency of rearing up, cornering and turning around decreased when reflection from the glass wall was blocked with black cardboard, black paint or non-reflective transparent plastic. In a tank containing mirrors on one side and non-reflective plastic on the other, crayfish cornered, reared up, and turned around more in front of the mirrors. Socialization was necessary for crayfish to respond to the reflection. Crayfish that were housed in pairs for two weeks exhibited more rearing up, turning around and cornering in front of the mirrors than in the non-reflective side. Crayfish isolated for two weeks did not show these differences. Socialized crayfish also exhibited more rearing up, turning around and cornering than did isolated crayfish. Thus, crayfish respond to visual stimuli provided by a glass tank, but the responds depends on socialization.

Responses to Reflection in Two Invertebrate Species

The present thesis investigates the responses to reflection in both the crayfish Procambarus clarkii and the fruit fly Drosophila melanogaster. Responses to reflection in crayfish depend on social status and the current work suggests that learning and memory consolidation are required for these responses to be altered. Crayfish were treated to either massed or spaced training fights prior to reflection testing. The results show that subordinate crayfish treated to spaced training display a response typical of subordinate crayfish but subordinate crayfish treated to massed training exhibit a response typical of dominant crayfish. Fruit flies are shown to be attracted to reflection and responses to reflection are described here for the first time. Responses in fruit flies are shown to be dependent on social status. The frequency of behaviours were altered in isolated flies but not socialized flies. The addition of pheromones cVA and 7,11-HD were used to investigate how the addition of chemical cues altered responses to reflection in fruit flies. Socialized fruit flies treated with cVA exhibited an increase in the frequency of behaviours on both mirrored and clear glass walls, while isolated flies exhibited a decrease. Socialized flies treated with 7,11-HD spent more time on mirrored walls compared to glass walls, whereas the frequency of all behaviours were decreased in isolated flies treated with 7,11-HD.

ATP pulse and calcium homeostasis in cells from hepatopancreas of Dilocarcinus pagei, a freshwater crab

Calcium (Ca) is critical for crustaceans due to their molting cycle and its presence in the carapace as calcium carbonate, apart from the usual functions of Ca, such as cell signalling. Ca transport in Dilocarcinus pagei, a freshwater crab, was studied in isolated cells from hepatopancreas to further characterize Ca transport mechanisms in these crabs. Cells were isolated and loaded with Fluo-3, a calcium fluorescent dye. Three different cell treatments were performed: Group 1 cells were Ca free during cell dissociation, and calcium was present (at 1mM) for fluorescence cell loading and transport experiments (FC); Group 2 cells were calcium free during cell dissociation and for transport experiments, but not during cell loading (LC); and Group 3 cells were Ca free during cell dissociation, cell loading and transport experiments (WC). Intracellular Ca was recorded through time after ATP was added to the cells and ATP caused an increase in Ca efflux within 30s in all cells. WC cells showed the smallest Ca efflux compared to the other cells, probably because it was intracellularly Ca ""depleted"". Vanadate and amiloride decreased the Ca efflux when ATP was added to the cells, while verapamil did not cause any effect in Ca efflux, confirming the presence of a Ca(2+)-ATPase sensitive to vanadate in hepatopancreas of D. pagei. In a different set of experiments, cells were also exposed to a Ca pulse of 1 and 10mM during 180s. 10mM Ca increased intracellular Ca compared to 1mM, and the increase was not recovered during the experimental time. Additionally, Ca influx was reduced by verapamil and amiloride, but not completely. The results suggest that Ca influx probably occurs through an undefined exchanger, apart from Ca channels (verapamil sensitive) and electrogenic 1Na(+)(1H(+))/1 Ca(2+) exchanger (amiloride-sensitive). Similarities between freshwater and seawater crabs, lobsters and crayfish in relation to plasma membrane Ca transporters, although the environment where they live is quite diverse, suggest that universal mechanisms for Ca homeostasis are widespread among crustaceans. (C) 2010 Elsevier Inc. All rights reserved.

Exotic species of freshwater decapod crustaceans in the state of São Paulo, Brazil: records and possible causes of their introduction

Based on recent surveys of the freshwater decapod fauna, distributional data of five exotic species of freshwater decapod crustaceans for the hydrographic basins of the state of São Paulo are presented, as part of a large initiative for a comprehensive survey of the state's biodiversity (BIOTA-FAPESP Program). These species are the North American crayfish Procambarus clarkii (Girard) (Cambaridae), the crab Dilocarcinus pagei Stimpson (Trichodactylidae) from the Amazon and Paraguay/lower Parana River Basins, and the palaemonid shrimps Macrobrachium rosenbergii (De Man), from the Indo-Pacific region, Macrobrachium amazonicum (Heller) and Macrobrachium jelskii (Miers), both from the Orinoco, Amazon and the Paraguay/lower Parana River Basins. Possible modes by which their introduction might have occurred are commented upon and potential consequences are discussed.

Microphallus fonti sp. n. (Digenea: Microphallidae) from the Red Swamp Crawfish in Southern United States

A new species of digenean, Microphallus fonti, is described from the red swamp crawfish in Louisiana, USA. It has a small pharynx and a rudimentary gut like M. opacus and possibly related species from crayfishes, but it differs from them by its relatively large male copulatory papilla and a conspicuous metraterm.

Stato ecologico di Punte Alberete e Valle Mandriole: popolamenti macrozoobentonici e chimica delle acque

Il presente studio si è proposto di fornire un contributo per la definizione dello stato ecologico delle zone umide d’acqua dolce ravennati (Punte Alberete, Valle Mandriole e uno stagno temporaneo creatosi nella limitrofa Pineta di San Vitale), attraverso lo studio della fauna macrobentonica presente nei sedimenti e nella vegetazione emergente. Sono stati prelevati campioni d’acqua con lo scopo di analizzare alcuni parametri chimico fisici, per relazionarli con i dati ecologici sulle comunità oggetto di studio. Inoltre è stato valutato il potenziale effetto sulle comunità macrozoobentoniche della attuale gestione di Valle Mandriole, che prevede il prosciugamento estivo della zona meridionale al fine di consentire operazioni di sflacio della vegetazione. Per valutare questo tipo di impatto è stato applicato un disegno di campionamento di tipo Beyond-BACI. Sono stati definiti quattro siti di controllo ed un sito potenzialmente impattato. Sono stati definiti due punti di campionamento su ciascun sito e sono stati campionati in totale quattro volte ciascuno, due volte prima del periodo estivo, corrispondente alla messa in secca del sito impattato, e due volte in autunno. I dati raccolti sul benthos e sulle acque sono poi stati analizzati attraverso l’analisi statistica multivariata PERMANOVA. I risultati dei test statistici non hanno rilevato nessun tipo di impatto associabile alla pratica di gestione attuata a Valle Mandriole, mentre l’analisi delle comunità macrozoobentoniche ha evidenziato, attraverso confronti temporali con dati storici e con altri studi, una situazione di degrado dal punto di vista delle stato ecologico di Punte Alberete e Valle Mandriole, molto probabilmente associabile all’abbondante presenza del gambero della Louisiana. La condizione di stato ecologico non soddisfacente può oltretutto essere imputabile anche alla qualità ecologica scarsa dei corsi d’acqua deputati al ricarico delle zone umide considerate. Lo studio ha inoltre sottolineato l’importanza dal punto di vista conservazionistico dello stagno in Pineta San Vitale.

C. Julii Caesaris Commentarii De bello Gallico et civili Pompejano, nec non A. Hirtii, aliorumque De bellis alexandrino, africano, et hispaniensi commentarii ... : cum integris notis Dionysii Vossii, Joannis Dauisii, et Samuelis Clarkii ; Cura et studio Francisci Oudendorpii ... ; [pars prima]

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C. Julii Caesaris Commentarii De bello civili Pompejano, nec non A. Hirtii, aliorumque De bellis alexandrino, africano, et hispaniensi commentarii ... : cum integris notis Dionysii Vossii, Joannis Dauisii, et Samuelis Clarkii ; Cura et studio Francisci Oudendorpii ... ; pars altera.

Mode of access: Internet.

C. Julii Caesaris De bellis Gallico et civili Pompejano, nec non A. Hirtii aliorumque De bellis Alexandrino, Africano, et Hispaniensi commentarii ... cum integris notis Dionysii Vossii, Joannis Davisii, et Samuelis Clarkii /

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Effects of a hydroxy-cinnamoyl conjugate of spermidine in the neuromuscular junction of crayfish /

N'-coumaroyl spermidine (NlCSpd) is a plant derived chemical which is proposed to belong to a class of low molecular weight neuroactive substances called phenolic polyamines. NlCSpd is stnicturally similar to glutamate receptor blocking toxins found in certain spiders and wasps, such as JSTX-3 and NSTX-3 found in Nephila spiders. The goal of the present study was to determine if plant-derived phenolic polyamines act like other structurally related chemicals found in Arthropod venoms, such as JSTX-3, and whether they can be classified in the same pharmacological group as the spider and wasp toxins. A comparison was made to determine the relative potencies of various phenolic polyamines fi-om plants and insect venoms. This comparison was done by measuring the effect of various concentrations ofNlCSpd on the amplitude of excitatory postsynaptic potentials (EPSPs) elicited in muscle of the crayfish Proccanbarus clarkii. NlCSpd was also tested on L-glutamate induced potentials to determine if a postsynaptic component to sj^naptic block occurs. NlCSpd and an analogue with an a longer polyamine chain, NlCSpm, blocked EPSPs in a dose dependent manner, NlCSpd having an IC50 of lOOnM. NlCSpd also blocked L-glutamate induced potentials. The two main components of the NlCSpd molecule alone are insufficient for activity. NlCSpd acts postsynaptically by interfering with crayfish glutamatergic synaptic transmission, likely blocking glutamate receptors by interacting with the same site(s) as other phenolic polyamines. Certain moieties on the polyamines molecule are necessary for activity while others are not.

Genetic variation and evolutionary divergence within and among populations, species, and genera of the Cambarinae

Seven crayfish species from three genera of the subfamily Cambarinae were electrophoretically examined for genetic variation at a total of twenty-six loci. Polymorphism was detected primarily at three loci: Ao-2, Lap, and Pgi. The average heterozygosities over-all loci for each species were found to be very low when compared to most other invertebrate species that have been examined electrophoretically. With the exception of Cambarus bartoni, the interpopulation genetic identities are high within any given species. The average interspecific identities are somewhat lower and the average intergeneric identities are lower still. Populations, species and genera conform to the expected taxonomic progression. The two samples of ~ bartoni show high genetic similarity at only 50 percent of the loci compared. Locus by locus identity comparisons among species yield U-shaped distributions of genetic identities. Construction of a phylogenetic dendrogram using species mean genetic distances values shows that species grouping is in agreement with morphological taxonomy with the exception of the high similarity between Orconectespropinquus and Procambarus pictus. This high similarity suggests the possibility of a regulatory change between the two species. It appears that the low heterozygosities, high interpopulation genetic identities, and taxonomic mispositioning can all be explained on the basis of low mutation rates.

A possible role for intrinsic calcium buffers in the long- term adaptation of crayfish neurons

Increasing the impulse activity of neurons in vivo over 3 or more days causes a reduction in transmitter release that persists for days or weeks (eg. Mercier and Atwood, 1989). This effect is usually accompanied by decreased synaptic fatigue. These two changes involve presynaptic mechanisms and indicate "long-term adaptation" (LTA) of nerve terminals. Previous experiments have shown that LTA requires extracellular calcium and protein synthesis (eg. Hong and Lnenicka, Soc. Neurosci. Abstr. 17:1322) and appears to involve communication between the cell body and the nerve terminals. The present study examines the possibility that the reduction in transmitter release is caused by an -increase in the calcium buffering ability within the nerve terminals. It examines the responses of adapted and control nerve terminals to exogenously applied calcium buffer, BAPTA-AM, which decreases transmitter release (Robitialle and Charlton, 1992). If LTA increases intrinsic Ca2+-buffering, the membrane permeant form of BAPTA should have less effect on adapted nerve terminals than on controls. Experiments are performed on the phasic abdominal extensor motor neurons of the crayfish, Procambarns clarkii. BAPTA-AM decreases excitatory postsynaptic potentials (EPSP's) of the phasic extensor muscles in a dosedependent manner between 5 and 50 JLM. LTA is elicited by in vivo stimulation at 2.5 Hz for 2-4 h per day over 3 days, which reduces EPSP's by over 50%. Experiments indicate that BAPTA-AM produces no significant change in EPSP reduction in adapted neurons when compared to controls. These results do not support the hypothesis that increased daily activity alters rapid intrinsic calcium buffers, that are able to reduce transmitter output in the same manner as BAPTA.

Modulation of muscle contraction by a FMRFamide-related peptide

A FMRFamide-like neuropeptide with the sequence "DRNFLRF-NH2" was recently isolated from pericardial organs of crayfish (Mercier et aI., Peptides, 14, 137-143, 1993). This neuropeptide, referred to as "DF2'" has already been shown to elicit cardioexcitation and to enhance synaptic transmission at neuromuscular junctions. Possible effects ofDF2 on muscle were investigated using superficial extensor muscles of the abdomen of the crayfish, Procambarus clar/ai. These muscles are of the tonic type and generate slow contractions that affect posture. DF2, at concentrations of 10-8 M or higher, increased muscle tonus and induced spontaneous, rhythmic contractions. These effects were antagonized by 5 rnM Mn2+ but not by lO-7M tetrodotoxin (TTX). Thus, they represent direct actions on muscle cells (rather than effects on motor neurons) and are likely to involve calcium influx. In contrast, deep abdominal extensor muscles, responsible for rapid swimming movements, and superficial flexor muscles do not generate contractions in response to the peptide. 2 Spontaneous contractions were also induced in the superficial extensor muscles by decreasing the temperature to II-13°C. Such contractions were also TTX-insensitive and they were antagonized by adding calcium channel blockers (Mn2+, Cd2+ or Ni2+) or by removing calcium from the bathing solution. This suggests that the spontaneous contractions depend on an influx of calcium from the extracellular solution. N-type and L-type voltage dependent calcium channel blockers did not reduce the effect of the peptide or the spontaneous contractions suggesting that calcium influx is not through N- or L-type calcium channels.

Locomotion patterns in two South American gymnophthalmid lizards: Vanzosaura rubricauda and Procellosaurinus tetradactylus

We quantified gait and stride characteristics (velocity, frequency, stride length, stance and swing duration, and duty factor) in the bursts of locomotion of two small, intermittently moving, closely related South American gymnophthalmid lizards: Vanzosaura rubricauda and Procellosaurinus tetradactylus. They occur in different environments: V rubricauda is widely distributed in open areas with various habitats and substrates, while P. tetradactylus is endemic to dunes in the semi-arid Brazilian Caatinga. Both use trot or walking trot characterised by a lateral sequence. For various substrates in a gradient of roughness (perspex, cardboard, sand, gravel), both species have low relative velocities in comparison with those reported for larger continuously moving lizards. To generate velocity, these animals increase stride frequency but decrease relative stride length. For these parameters, P. tetradactylus showed lower values than V rubricauda. In their relative range of velocities, no significant differences in stride length and frequency were recorded for gravel. However, the slopes of a correlation between velocity and its components were lower in P. tetradactylus on cardboard, whereas on sand this was only observed for velocity and stride length. The data showed that the difference in rhythmic parameters between both species increased with the smoothness of the substrates. Moreover, P. tetradactylus shows a highly specialised locomotor strategy involving lower stride length and frequency for generating lower velocities than in V. rubricauda. This suggests the evolution of a central motor pattern generator to control slower limb movements and to produce fewer and longer pauses in intermittent locomotion. (c) 2008 Elsevier GmbH. All rights reserved.