Synapses vary widely in the probability of transmitter release. For instance, in response to an action potential the phasic synapses of the crayfish have a 100-1000-fold higher release probability than tonic synapses. The difference in release probability is attributed to differences in the exocytotic machinery such as the degree of “zippering” of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex. I used physiological and molecular approaches to determine if the zippered state of SNAREs associated with synaptic vesicles and the interaction between the SNARE complex and Complexin influence the probability of release at the synapse. I used three Botulinum neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by SNARE interaction (zippering) or open to proteolytic attack. Under low stimulation conditions, the light-chain fragment of botulinum B (BoNT/B-LC) but not BoNT/D-LC or tetanus neurotoxin (TeNT-LC) cleaved VAMP and inhibited evoked release at both phasic and tonic synapses. In addition, a peptide based on the C-terminal half of crayfish VAMP’s SNARE motif (Vc peptide) designed to interfere with SNARE complex zippering at the C-terminal end inhibited release at both synapses. The susceptibility of VAMP to only BoNT/B-LC and interference by the Vc peptide indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end with the C-terminal end exposed under resting conditions. I used a peptide containing part of the crayfish Complexin central α-helix domain to interfere with the interaction between Complexin and the SNARE complex. The peptide enhanced phasic evoked release and inhibited tonic evoked release under low stimulation but attenuated release at both synapses under intense stimulation. Therefore, Complexin appeared to exhibit a dual function under low synaptic activity but only promoted release under high synaptic activity. The results showed that the zippered state of the SNARE complex does not determine initial release probability as a similar zippered SNARE complex structure under resting conditions is common to both phasic and tonic synapses. However, Complexin may have a role in influencing the initial release probability of a synapse. Therefore, the interaction between the SNARE complex and Complexin is important for release but other factors contribute more significantly to synaptic strength.

Synapses vary widely in the probability of transmitter release. For instance, in response to an action potential the phasic synapses of the crayfish have a 100-1000-fold higher release probability than tonic synapses. The difference in release probability is attributed to differences in the exocytotic machinery such as the degree of “zippering” of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex. I used physiological and molecular approaches to determine if the zippered state of SNAREs associated with synaptic vesicles and the interaction between the SNARE complex and Complexin influence the probability of release at the synapse. I used three Botulinum neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by SNARE interaction (zippering) or open to proteolytic attack. Under low stimulation conditions, the light-chain fragment of botulinum B (BoNT/B-LC) but not BoNT/D-LC or tetanus neurotoxin (TeNT-LC) cleaved VAMP and inhibited evoked release at both phasic and tonic synapses. In addition, a peptide based on the C-terminal half of crayfish VAMP’s SNARE motif (Vc peptide) designed to interfere with SNARE complex zippering at the C-terminal end inhibited release at both synapses. The susceptibility of VAMP to only BoNT/B-LC and interference by the Vc peptide indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end with the C-terminal end exposed under resting conditions. I used a peptide containing part of the crayfish Complexin central α-helix domain to interfere with the interaction between Complexin and the SNARE complex. The peptide enhanced phasic evoked release and inhibited tonic evoked release under low stimulation but attenuated release at both synapses under intense stimulation. Therefore, Complexin appeared to exhibit a dual function under low synaptic activity but only promoted release under high synaptic activity. The results showed that the zippered state of the SNARE complex does not determine initial release probability as a similar zippered SNARE complex structure under resting conditions is common to both phasic and tonic synapses. However, Complexin may have a role in influencing the initial release probability of a synapse. Therefore, the interaction between the SNARE complex and Complexin is important for release but other factors contribute more significantly to synaptic strength.

Astacologic survey took place in ÚN Lipno and was conducted in time from 2012 till 2013. For the actual mapping of the occurrence of spiny-cheek crayfish, the locations of total 17 places were detected. These monitoring areas were carried out in two ways for collecting the samples. The trapping of all individuals was achieved with special instruments, like pots or traps by hands or with the help of nets. For the caught specimens of spiny-cheek crayfish, which were determined by sex, the length of carapace was measured and as well if possible, the postorbital carapace length was taken. Throughout the whole monitoring, 228 individuals of spiny-cheek crayfish were caught in the amount of 107 (47%) females (CL = 21.5 ? 7.3 mm) and 121 (53%) males (CL = 20.1 ? 5 mm). Another aim of this work was to demonstrate that crayfish figure in fish food menu. Catches of fish were carried out in two ways, using fishing rods or into the clutches. The stomachs and intestines contents were analyzed at caught fish samples. If the condition of the contents of stomachs or intestines were allowed, it was also identified, what kind of food it was. For our annual issues, were included mainly species of piscivor fish. Spiny-cheek crayfish were identified in the stomachs of two species of fish, namely perch and pike.

In the nature, crayfish are represented like predators or a part of food base of some fish. Also in free waters they acts as detrivors and are an important element in the nutrient and energy circle in aquatic ecosystem. The crayfish weren't able to avoid negative impacts of anthropogenic activities such as water pollution and devastation of habitats. For these reasons reproduction of the crayfish is necessary in hatcheries and farms. The main aim of my work was to study the reproductive biology of the crayfish focused on the process of mating, the interval between mating and laying eggs, fertilization and morphological structure of the male and female gametes. Among the secondary objectives there was included using of repeated electrostimulation of the crayfish and the possibility of hybrids of the noble crayfish (Astacus astacus) and the narrow-clawed crayfish (Astacus leptodactylus). The observation of the period of reproduction was realized on representativies of the noble crayfish and the narrow-clawed crayfish throughout natural mating in the tanks. The structure of gametes was observed on the samples taken from thenoble crayfish (Astacus astacus), the narrow-clawed crayfish (Astacus leptodactylus), the signal crayfish (Pacifastacus leniusculus), the red swamp crayfish (Procambarus clarkii) and spinycheek crayfish (Orconectes limosus). The experiments were carried out during the autumn season in 2012 and 2013. The results of observations of the reproduction of crayfish made us clear as for the period and intervals for all phases of the process of mating crayfish. For females the noble crayfish and the narrow-clawed crayfish was found oviposition without the presence of males. Their individual components have been described by using ultrastructure of males and female gametes. Repeated electrostimulation showed at the family Astacidae as a suitable tool for collecting large mass of sperm material. Hybridization experiment proved that it can occur to mate the two species of crayfish in areas where they occur simultaneously. These experiments helped to further knowledge of reproductive biology of crayfish.

Polychlorinated Biphenyls (PCB's) are organochlorine molecules which find various industrial and product applications. PCB's are of concern to biologists because they are toxic substances which have become global contaminants. They are also of concern to biologists and analytical chemists because they interfere with the determination of some organochlorine pesticide residues. PCB's were discovered to be environmental contaminants after they showed up as unidentified peaks in pesticide analysis using gas-liquid chromatography with an electron capture detector (GLC-EC). In the present experiment standard GLC-EC techniques were used to assay PCB's in Daphnia experimentally contaminated in the laboratory and in crayfish from the Willamette River. Daphnia were placed in water containing 0.1, 4, 50 or 100 parts per billion (ppb) PCB for 4 to 72 hours. There was no mortality in any of the experiments. The individual PCB compounds were apparently taken up equally, since relative peak heights were similar in the standard and the residues extracted from Daphnia. Final concentrations of PCB's in Daphnis ranged from 1200 times that of the water (at water concentrations of 100 ppb) to 104,000 times that of water (at water concentrations of 0.1 ppb). The lower concentrations used here approximate environmental PCB levels found in some areas of the U.S. and elsewhere. Assuming that chronic exposure to these lower concentrations would not strongly inhibit growth or reproduction of Daphnia,, the high biological magnification found here would suggest that Daphnia and related organisms may play an important role in the accumulation of PCB's in fresh water food webs. Digestive glands of crayfish taken from the Willamette River were analyzed for PCB's. Statistically significant regional differences were found in the concentrations of PCB's in these organisms in the lower Willamette Valley. Crayfish from the center of Portland have the highest residues (7 to 9 parts per million). A similar distribution has been previously reported for other urban areas. In the present investigation, however, the highest residues occurred in crayfish from the river at a point approximately in the center of the city itself, but two miles upstream from the major industrial areas. Thus, in this case, the major source of environmental contamination may have been released from manufactured goods (e.g., automobile tires, paints, etc.) rather than from industrial sewerage. I

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