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.

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 of crayfish to a reflective environment /

This thesis examines how dominance status of crayfish alters responses to their own reflection. Crayfish are social animals that fight to develop a dominance hierarchy consisting of dominant and subordinate members. After socialization, crayfish were videotaped in an aquarium with mirrors on one half of the tank and a non-reflective plastic on the other half. Dominants paired for 14 days perform more cornering, turning, crossing and spent more time in a reflective environment versus a non-reflective environment. Subordinate crayfish exhibit more reverse walking in a mirrored environment while isolated crayfish show no preference for reflection. This change in behaviour occurs immediately for dominants paired for 30 min while subordinates require 3 days of pairing to exhibit the same behaviour as subordinate crayfish paired for 14 days. Thus, 30 min of pairing is required to enhance responses to a reflection observed in dominant crayfish while 3 days is required to decrease subordinate responses to a reflection. These findings propose that male socialized crayfish respond to their mirror image as they do a male conspecific. Their responses depend on both their dominance status and the length of socialization which suggests that crayfish are learning to behave in a characteristic manner as a result of their social experience.

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.

Isolation of DNA sequences with homology to a degenerate FaRP oligonucleotide from the crayfish Procambarus clarkii

The neuropeptide Th1RFamide with the sequence Phe-Met-Arg-Phe-amide was originally isolated in the clam Macrocallista nimbosa (price and Greenberg, 1977). Since its discovery, a large family ofFl\1RFamide-related peptides termed FaRPs have been found to be present in all major animal phyla with functions ranging from modulation of neuronal activity to alteration of muscular contractions. However, little is known about the genetics encoding these peptides, especially in invertebrates. As FaRP-encoding genes have yet to be investigated in the invertebrate Malacostracean subphylum, the isolation and characterization ofFaRP-encoding DNA and mRNA was pursued in this project. The immediate aims of this thesis were: (1) to amplify mRNA sequences of Procambarus clarkii using a degenerate oligonucleotide primer deduced from the common amino acid sequence ofisolated Procambarus FaRPS, (2) to determine if these amplification products encode FaRP gene sequences, and (3) to create a selective cDNA library of sequences recognized by the degenerate oligonucleotide primer. The polymerase chain reaction - rapid amplification of cDNA ends (PCR-RACE) is a procedure in which a single gene-specific primer is used in conjunction with a generalized 3' or 5' primer to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end of cDNA of interest (Frohman et aI., 1988). PCRRACE reactions were optimized with respect to primers used, buffer composition, cycle number, nature ofgenetic substrate to be amplified, annealing, extension and denaturation temperatures and times, and use of reamplification procedures. Amplification products were cloned into plasmid vectors and recombinant products were isolated, as were the recombinant plaques formed in the selective cDNA library. Labeled amplification products were hybridized to recombinant bacteriophage to determine ligated amplification product presence. When sequenced, the five isolated PCR-RACE amplification products were determined not to possess FaRP-encoding sequences. The 200bp, 450bp, and 1500bp sequences showed homology to the Caenorhabditis elegans cosmid K09A11, which encodes for cytochrome P450; transfer-RNA; transposase; and tRNA-Tyr, while the 500bp and 750bp sequences showed homology with the complete genome of the Vaccinia virus. Under the employed amplification conditions the degenerate oligonucleotide primer was observed to bind to and to amplify sequences with either 9 or 10bp of 17bp identity. The selective cDNA library was obselVed to be of extremely low titre. When library titre was increased, white. plaques were isolated. Amplification analysis of eight isolated Agt11 sequences from these plaques indicated an absence of an insertion sequence. The degenerate 17 base oligonucleotide primer synthesized from the common amino acid sequence ofisolated Procambarus FaRPs was thus determined to be non-specific in its binding under the conditions required for its use, and to be insufficient for the isolation and identification ofFaRP-encoding sequences. A more specific primer oflonger sequence, lower degeneracy, and higher melting temperature (TJ is recommended for further investigation into the FaRP-encoding genes of Procambarlls clarkii.

Reproduction in the temperate crayfish, Orconectes rusticus: intermale aggression, copulatary behaviour, and sperm competition

The chelipeds of Orconectes rusticus are sexually dimorphic; males possessing the larger. Males use their chelae in intermale aggressive interactions, both to threaten, and assault opponents. In dyadic interactions males with larger chelae were dominant over otherwise physically similar opponents. A high frequency of attack behaviour, coupled with a low frequency of threats during these interactions indicates that actual physical contact is required for opponent assessment. Large clawed males oriented females into the copulatory position faster than small clawed males. Females more frequently escaped the precopulatory-grasp attempts of small clawed males. Additionally, male-female pairs that included a large clawed male remained in copula longer than pairs that included a small clawed male. Sperm of the second male to mate took precedence over the sperm of the primary male. Sperm precedence was incomplete; about 900/0 paternity accrued to the second male.

Mechanisms of long-term modulation of synaptic transmission in crayfish

Please consult the paper edition of this thesis to read. It is available on the 5th Floor of the Library at Call Number: Z 9999 B56 M68 2007

Effect of intermittent feeding on growth in early juveniles of the crayfish Cherax quadricarinatus

Three experiments were conducted with juveniles of the crayfish Cherax quadricarinatus to investigate the effect of intermittent feeding regimes on growth and the ability to tolerate the shortage of food. In experiment 1, stage III juveniles were assigned to one of seven intermittent feeding groups (from FS1: 1 day fed/1 day non-fed to FS7: 7 days fed/7 days non-fed) and two control groups, continuously fed (CF) and continuously starved (CS) animals; this experiment comprised a short-term intermittent feeding period until the first molt, followed by a continuous feeding period. In the experiment 2, stage III juveniles were assigned to one of three intermittent feeding groups (FS2 to FS4) and one control group (CF); it consisted of a prolonged intermittent feeding period, until the end of the experiment In the experiment 3, stage VI and VII juveniles were assigned to one of three intermittent feeding groups (FS2 to FS4) and one control (CF); it also consisted of a prolonged intermittent feeding period. The red claw crayfish juveniles were able to tolerate periods of intermittent feeding and underwent compensatory growth after continuous feed was re-established. The ability of crayfish to tolerate intermittent feeding was influenced by developmental stage and duration of the intermittent feeding period. Stage III juveniles survived, but decreased growth, when subjected to prolonged intermittent feeding. However, they showed full compensatory growth when the intermittent feeding period was short and followed by continuous feeding. on the other hand, stage VI-VII tolerated 60 days of prolonged intermittent feeding without any change in growth and survival. The hepatosomatic index (based on wet weight) values of the treatments and the control were similar, suggesting that intermittent feeding may not be considered a nutritional stress condition. The relative pleon weight (based on wet weight) values of the treatments and control were similar suggesting low use of nutrients from the muscle to increase the chance for survival. The juveniles of C quadricarinatus can tolerate relatively long periods of low food availability and this is an important adaptation for their survival in changing/unpredictable environments and an attribute favorable for the production of the species. (C) 2011 Elsevier B.V. All rights reserved.

EFFECT OF FOOD SHORTAGE ON GROWTH, ENERGETIC RESERVES MOBILIZATION, AND WATER QUALITY IN JUVENILES OF THE REDCLAW CRAYFISH, CHERAX QUADRICARINATUS, REARED IN GROUPS

The aim of this study is to analyze the impact of food shortage on growth performance, by means of energetic reserves (proteins, glycogen and lipids) mobilization and hepatopancreas cells analysis in C. quadricarinatus juveniles maintained in groups, as well as the effect on culture water quality. Two experiments were performed, each of them with two feeding regimes during 45 days. The Control feeding regime, in which crayfish were fed daily (once a day) throughout the experimental period (DF), and the Cyclic feeding regime, in which juveniles were fed for 2 or 4 days (once a day) followed by 2 or 4 days of food deprivation (2F/2D and 4F/4D, respectively) in repeated cycles. Cyclic feeding influenced growth, biochemical composition from hepatopancreas and muscle, and water quality. Juveniles cyclically fed were unable to maintain a normal growth trajectory during 45 days. Apparent feed conversion ratio, apparent protein efficiency ratio, hepatosomatic index and relative pleon mass were similar in cyclic and daily fed animals and no structural damage was found in the hepatopancreas of juveniles subjected to cyclic feeding. The novelty of this study was the significant accumulation of proteins in pleonal muscle in both cyclic feeding regimes (approx. 18%) suggesting that the storage of this constitutive material during food shortage may be an adaptation for a compensatory growth when food becomes abundant again. The cyclic feeding regimes had a positive effect on water quality decreasing inorganic nitrogen concentration. This was due to the reduction in the amount of animal excretes and feces in the group that received approx. 50% less feed. Additionally, water pH was higher in cyclic feeding tanks, as a result of lower organic matter decomposition and consequent release of CO2. Accordingly, total ammonia in the water was significantly lower for the cyclic feeding regimes compared to their respective controls. This study suggests that the protocol of cyclic feeding could be applied at least 45 days in 1 g juveniles maintained in group conditions, without affecting the energetic reserves and hepatopancreas structure, emphasizing the high tolerance of this species to food restriction.

The crayfish plasma clotting protein: A vitellogenin-related protein responsible for clot formation in crustacean blood

Coagulation in crayfish blood is based on the transglutaminase-mediated crosslinking of a specific plasma clotting protein. Here we report the cloning of the subunit of this clotting protein from a crayfish hepatopancreas cDNA library. The ORF encodes a protein of 1,721 amino acids, including a signal peptide of 15 amino acids. Sequence analysis reveals that the clotting protein is homologous to vitellogenins, which are proteins found in vitellogenic females of egg-laying animals. The clotting protein and vitellogenins are all lipoproteins and share a limited sequence similarity to certain other lipoproteins (e.g., mammalian apolipoprotein B and microsomal triglyceride transfer protein) and contain a stretch with similarity to the D domain of mammalian von Willebrand factor. The crayfish clotting protein is present in both sexes, unlike the female-specific vitellogenins. Electron microscopy was used to visualize individual clotting protein molecules and to study the transglutaminase-mediated clotting reaction. In the presence of an endogenous transglutaminase, the purified clotting protein molecules rapidly assemble into long, flexible chains that occasionally branch.