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Od trusu k prostřenému ubrusu. Molekulární analýza potravy raka signálního
ŠKRABÁNEK, Jan
This thesis aimed to determine the diet of the signal crayfish in Křesánovský brook in 2020. In addition to that, we aimed to find and test suitable markers for the diet analysis of signal crayfish, which are described in the thesis. We analyzed the crayfish diet by examining DNA from faecal pellet samples collected monthly from April to September. By using group-specific DNA markers, we targeted the main dietary groups such as Heptagenidae (markers Hpt28Sf + Hpt28Sr), Ephemeroptera (Epa28Sf + Epa28Sr), Integripalpia and Spicipalpia (Inspi18Sf + Inspi18Sr), Plecoptera (Ple-gen A269 + Ple-gen S268), Chironomidae (Chiro18Sf + Chiro18Sr), and Gammaridae (Gamae28Sf + Gamae28Sr). We also focused on representatives of macrophytes Characeae (CHA677 + CHA1681R) and Eudicotyledons (MYR152F + MYR1696R). The results showed that the crayfish's diet varied during the sampling season. Gammaridae was the most common food item, found in 54.1% of the samples of faecal pellets. Heptagenidae, Integripalpia, and Spicipalpia were also significant, present in 31.3% of the samples. Plecoptera appeared in 20.8% of the samples, while Chironomidae and Characeae were found in 10.4%. Eudicotyledons were detected in 8.3% of the samples. Ephemeroptera were the least common food group, detected only in 2.1 %. Gammaridae were the most significant component of the crayfish diet in April (found in 7 samples), while Heptagenidae were the second most abundant component. The diet was more diverse in May, but Gammaridae remained the primary food source, followed by Integripalpia and Spicipalpia (in 5 samples). In June, Gammaridae was present in all samples, while Heptagenidae and Integripalpia/Spicipalpia were significant. In July, Heptagenidae was the most common food group (found in 5 samples), with Gammaridae found in 4 samples. No food items were detected in the samples from August and October, and only one sample contained Gammaridae in September. Overall, this study provides insights into both the seasonal variations in the diet of signal crayfish and markers suitable for its analysis.
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Aktivita raka mramorovaného v závislosti na reprodukčním cyklu
MARKOVÁ, Kateřina
The aim of this thesis was to elaborate a literature review on the topic of crayfish activity, in particular their feeding activity and the use of shelters, including an emphasis on the role of sex and the reproductive cycle. I also focused on the characteristics of the marbled crayfish, Procambarus virginalis Lyko, 2017 and its usability as a model organism. The experimental part of the thesis focused on comparing three groups of female marbled crayfish (with ovulum, with glair glands and without ovulum). Experiments have been carried out using a method for ecological assessment of feeding behavior and predator-prey relationship in various prey densities in the form of larvae of buzzer midge Chironomus plumosus (Linnaeus, 1758) by functional response analysis and subsequently tested for the effect of shelter presence at uniform prey densities levels. In our experiment, all three groups of tested females showed a type II functional response typical of predators. The results indicate that females without ovulum have been the most active in terms of food collection in all cases, with a generally positive effect of the presence of shelter. Females with employed ovulum are generally less active in terms of consumption, processing and overall aktivity due to hormonal, physiological and consequently behavioural changes. Females with glair glands behaved similarly to females with ovulum in our experiments, being more cautious and less active than females without ovulum. This was probably due to their increased gonadosomatic index (GSI) and, conversely, their decreased hepatopankreas to total body weight ratio, which renders these females unable to physiologically ingest large amounts of submitted food. Female GSI decreases again after ovulation, nevertheless, even females with already employed ovulum did not consume more food, as females prefer safety at this stage of the reproductive cycle. They are less active, move more slowly, spend more time in a shelter, tend to their eggs, and thus cannot devote themselves fully to hunting and prey processing. Regarding its life strategy, the marbled crayfish is a generally suitable model organism for use in various areas of research, however, it is important to take into account its rapid life and reproductive cycle and adapt the conditions of the given experiments to this fact, as the parallel use of female marbled crayfish at various stages of the reproductive cycle can modulate results in terms of their activity. Certain differences in the presented results may also be due to the origin of the experimental animals, as with very few exceptions all performed experiments with marbled crayfish have been carried out using laboratory individuals. A number of conditions that change seasonally in nature have been kept constant in the laboratory. Therefore, it is important to keep in mind that in wild, crayfish are also exposed to both biotic (predation pressure of various species of fish, birds or mammals) and abiotic (changes in temperature and water chemistry) factors that can influence food activity. Nevertheless, we assume that the patterns observed here will also be valid for female crayfish living in the wild nature. However, the lack of similar experiments leading to the elucidation of female activity depending on the stage of the reproductive cycle provides a room to investigate other, not just crayfish species.
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Regenerace smyslových orgánů u raků
KOR, Golara
The antennae are a crucial sensory organ in crayfish that plays vital roles in their survival, communication, and orientation within their environment. They are equipped with sensory hairs that enable crayfish to detect various stimuli. Crayfish possess the unique ability to regenerate their antennae. Hemocytes are specialized cells within crayfish and play a crucial role in their immune system and contribute to the process of regeneration. We applied cryo-scanning and Transmission electron microscopies to study sensory organ morphology and regeneration in crayfish. In Chapter 2, cryo-scanning electron microscopy was used to identify potential differences in antennal morphology in six different crayfish species including marbled crayfish Procambarus virginalis, Mexican dwarf crayfish Cambarellus patzcuarensis, red swamp crayfish Procambarus clarkii, signal crayfish Pacifastacus leniusculus, common yabby Cherax destructor, and spiny-cheek crayfish Faxonius limosus. We observed significant differences among the six crayfish species in the ratios of antenna length, segment length and width to carapace length, and the number of segments. Examination of ultrastructural features unveiled differences in the distribution patterns of sensory hairs along the antenna and the morphology of the antennal surface. The varying morphology of antennae among studied species possibly indicates an adaptation to the specific conditions of their respective habitats. Furthermore, the outcomes demonstrated that a combination of variations in both antennal morphological characteristics and biometric measurements could effectively help us to distinguish the different studied crayfish species. In Chapter 3, the ultrastructural behaviour of hemocytes during coagulation and phagocytosis in the initial phases of injury in marbled crayfish was explored using transmission electron microscopy. During the coagulation process, hemocytes experienced marked transformations in morphology. The cytoplasmic granules exhibited a change from electron-dense to electron-lucent forms with the progress of coagulation. The transformed granules containing amorphous, electron-lucent material were observed to combine and release their contents into the extracellular space as part of the coagulation process. Additionally, it was observed that the nucleus contents also play a role in the coagulation process. Furthermore, the amputation of the leg led to substantial muscle degeneration, and phagocytic hemocytes start to take up the necrotic tissues. Besides, we observed the digested remains from phagocytized necrotic tissues merged into granules and other cellular components, thereby enhancing the granularity of the hemocytes and altering their cellular morphology. However, it is essential to note that hemocyte degranulation during coagulation could potentially reduce their granularity. Since morphological features are critical for classifying hemocytes, these morphological changes during coagulation and phagocytosis must be considered significant factors. The degenerated material that stored inside phagocytic hemocytes may have application in immunological and regeneration processes. In Chapter 4, we employed transmission electron microscopy to explore the ultrastructural aspects of potential immune cell involvement in nerve regeneration within crayfish antennae post-amputation. The findings indicated that, during nerve regeneration, all three forms of hemocytes were present. However, the granules within semi-granulocytes and granulocytes mainly contributed to the generation of new organelles such as mitochondria, the Golgi apparatus, and nerve fibers in the regenerated nerves of crayfish antennae. We revealed the transformation of granules of hemocytes into diverse organelles during the process of nerve regeneration at the ultrastructural level. In conclusion, these granules function as compact repositories of adaptable materials carried by immune cells, capable of transformatio
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Problematika invazních druhů raků na území České republiky
Čuprová, Veronika
The diploma thesis following the bachelor thesis "Invasive species in the Czech Republic" is focused on the issue of invasive species of crayfish. The aim of the thesis is to describe the legislation, the current method of monitoring these species and the possibilities of their regulation. The thesis deals with the monitoring of invasive crayfish species at two selected sites in the Czech Republic and the application of appropriate regulatory interventions. Along with the monitoring, water samples were taken and analyses for the presence of crayfish plague were carried out. The results show that at the Prostřední rybník site, where the marbled crayfish occurs and where eradication measures have been underway since 2020, it has still not been completely successfully eradicated. From the interview conducted with local respondents, it was found that about half of the respondents are aware of this issue, thanks to the actions carried out to eliminate this species and the information boards posted in the area. In addition to the signal crayfish, a small population of the native endangered river crayfish is also present at Besének. Regulatory measures have also been implemented at this site in order to eliminate this invasive species as much as possible and to prevent potential threats to the native crayfish. Based on the results obtained, further possible control measures were proposed at both sites. In the future, more focus should be placed on education of this highly topical issue, as public awareness and understanding is considered one of the most effective preventive measures.
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Buněčné a molekulární charakteristiky hemolymfy u raků
KIFAYATULLAH, NA
The cellular and molecular components of the hemolymph are the major arm of the innate immune system in decapod crustaceans. In-depth knowledge of the hemolymph components, including hemocytes and hemolymph proteins, can enhance our understanding of innate immunity in crustaceans. We utilized transmission electron microscopy and quantitative proteomics to study the cellular and molecular aspects of coagulation and phagocytosis in the hemolymph. Chapter 2 reviews the cellular and molecular parameters of the innate immune system and the effects of environmental stressors and their abiotic and biotic stress mechanisms in decapod crustaceans. The innate immune system of decapod crustaceans heavily relies on hemocytes in the circulating hemolymph. Generally, three types of hemocytes are accepted based on their morphology, however, there is still a lack of consensus among researchers regarding the classification of hemocyte types. The key innate immune functions such as coagulation and phagocytosis are still poorly understood and require further investigation especially at a molecular level. Environmental stressors can adversely affect the immune responses of decapod crustaceans, increasing their susceptibility to diseases. However, the abiotic stress mechanism is poorly understood due to the lack of available literature and needs further investigation. In Chapter 3, transmission electron microscopy was used to investigate the ultrastructural behavior of hemocytes during coagulation and phagocytosis in the early stages of leg amputation injury in marbled crayfish Procambarus virginalis. The granular hemocytes were the first to be activated, and the morphology of cytoplasmic granules changed from electron-dense to electron-lucent forms in an expanding manner. The transformed granules containing amorphous electron-lucent materials merged and discharged their contents into the extracellular space for coagulation. We observed that the leftover nucleus from degranulated hemocytes participates in the process of coagulation, which could be confused with hyalinocytes in some previous studies. In addition, leg amputation caused massive muscle degeneration, followed by a significant influx of phagocytic hemocytes that removed a substantial amount of muscle fibers and organelles, such as mitochondria, generated from disintegrating and decaying muscle. Furthermore, we found that phagocytic hemocytes contained varying numbers of granules in their cytoplasm and, for the first time, discovered that these cells incorporate necrotic bodies resulting from degenerated muscles into their organelles, such as cytoplasmic granules and nucleus. The granular hemocytes were found to be the main cells that carry out phagocytic activity in the injury site. This study provides a comprehensive description of all the stages of morphological changes in hemocytes during coagulation and phagocytosis after injury in crayfish for the first time. In Chapter 4, proteomic analysis of non-clotted and clotted samples indicated that quantities of most proteins remained unchanged during the coagulation process, suggesting that necessary proteins for coagulation are pre-synthesized and stored before clot formation. Due to their open circulatory system, decapod crustaceans possess robust clotting mechanisms. Upon injury, pre-synthesized clotting factors are released, resulting in clot formation. Therefore, only a few proteins, such as C-type lectin domain-containing proteins, Laminin A chain, and Tropomyosin, were down-regulated during clotting, suggesting their possible roles in the structural integrity of cells. Their downregulation could facilitate degranulation, a crucial step for clot formation.
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Monitoring reakce raků na různé podněty: jsou raci dobrými bioindikátory?
MALINOVSKA, Viktoriia
Freshwater crayfish play a vital role in food webs and energy transfer between trophic levels, often acting as keystone species. Crayfish are prime species among invertebrates, considering their large size, abundance, and biodiversity. Crayfish belong to common model organisms in scientific research and have been used as bioindicators of water quality in laboratory and field studies. Freshwater crayfish are at direct risk while facing polluted aquatic environments, as many chemicals enter natural waters throughout the year. Studies have been shown that crayfish exhibit a variety of behavioral and physiological alterations in response to anthropogenic contaminants. In Chapter 2, we aimed to assess the cardiac and locomotor responses of crayfish Pacifastacus leniusculus to short-term pulse exposure to environmentally relevant concentrations of three pesticides (metazachlor, terbuthylazine, and thiacloprid). A significant increase in heart rate and distance moved was observed in metazachlor-exposed specimens compared to controls. We did not detect significant changes in crayfish cardiac and locomotor activity in terbuthylazine and thiacloprid groups. Most crayfish in these groups did not exhibit locomotor response, so they might not be stimulated to escape a contaminated area. This may lead to continuous exposure to environmental chemicals that are potentially harmful. In Chapter 3, we investigated the effects of UV filters Benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid (BP4) and 2-Phenylbenzimidazole-5-sulfonic acid (PBSA) on crayfish during 30 min exposure period. Specimens exposed to the tested compounds moved greater distances and spent more time in locomotion. Increased heart rate was also detected in treated groups. Such behavioral and physiological alterations of crayfish detected in both studies can potentially impair crayfish ability to respond to predators, and consequently affect abundance and the functional properties of macroinvertebrate communities. Along with use of crayfish in laboratory and field experiments, there is also an option to conduct case study with crayfish acting as bioindicators. We evaluated crayfish physiological responses to water disinfected with chlorine dioxide in long-term monitoring under operating conditions in local brewery (Chapter 4). The disruption of cardiac rhythmicity was detected with chaotic increases and decreases of heart rate regardless of the time of day. In natural environment, heart rate of crayfish is higher during night time considering nocturnal activity of these decapods. Nocturnalism is important adaptation for crayfish, and its impairment can lead to certain risks related to increased visibility to predators and alterations in foraging. Our results indicated that crayfish exhibit a cardiac and locomotor responses when shortly exposed to commonly used herbicide metazachlor and two widely applied UV filters BP4 and PBSA, suggesting that crayfish can potentially detect the appearance of these aquatic contaminants in water. The usefulness of crayfish in long-term monitoring of water quality have also been demonstrated. These findings expand our understanding of using crayfish as bioindicators in scientific research. In conclusion, this thesis provides the data from laboratory and case studies focusing on measurements of cardiac and locomotor endpoints in crayfish. Given that crayfish is at the base of the food chain in freshwater environments, it is of great importance to study the effects of different stimuli on such keystone species. Understanding further biological impacts of aquatic pollutants on freshwater crayfish and other non-target taxa will benefit our knowledge of threats to functional compositional changes in populations and communities.
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Aktivita raka signálního v podmínkách malého toku
DAVID, Jaroslav
The signal crayfish is one of the oldest invasive crayfish species in Europe, causing ecological and often economic damage in newly invaded locations. The invasion and associated problems can be monitored in real-time on the Křesanovský brook, where regular eradication measures have been carried out for six years. However, the detailed and comprehensive impact of these activities on the crayfish population as a whole and any changes in individual behaviour have not yet been studied in detail. To obtain relevant data for this purpose, this thesis video-recorded the situation in the stream in monthly cycles from May to September 2021, with recordings taken the night before and the night after thorough manual crayfish catching in the brook, evaluating the number and activity of observed crayfish. The results did not show that the trapping had changed the overall crayfish activity. Even with a high number of captured crayfish, the weakened crayfish population after trapping showed the same overall activity as the complete population before trapping (presumably due to an increase in individual activity in response to catching and accompanied habitat disturbance). Furthermore, the results underline the previously assumed low success rate of eradication efforts at the site and confirm that barriers in the stream are not sufficient to stop the signal crayfish spread. These findings will help to better understand the impact of eradication measures on signal crayfish populations. As this was the first research of its kind, this thesis opens up opportunities for methodological improvements and further development, as well as posing many new questions that need to be addressed. The ultimate goal of these efforts is to improve management interventions against invasive signal crayfish.
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Dynamika společenstva makrozoobentosu podhorského toku s početnou populací raka signálního.
ŠŤASTNÁ, Hana
Assessment of macroinvertebrates population at the Křesanov brook above the town of Vimperk. It contains a large population of the signal crayfish. It is non-native in Europe. Sampling was carried out in 2020. Macroinvertebrates sampling was done. Then, in the laboratory, its determination and detection of abundance and biomass was done. Statistical analyses were subsequently made using hydrobiological indices from the collected data. The analyses compared two stream segments that were different in some parameters.
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Effects of diphenhydramine on molecular defense mechanisms in signal crayfish within a bioaccumulation experiment
KAHUDOVÁ, Dominika
Diphenhydramine (DFH) is a medication widely used around the world to treat various types of allergies. Residues of DFH are often detected in surface waters, sediments, and fish tissues. The impact of xenobiotics, such as pharmaceutical residues, on aquatic biota can cause metabolic changes and toxicity in organisms. This study aimed to assess whether DFH affects the molecular defense mechanisms of signal crayfish (Pacifastacus leniusculus) and evaluate the absorption and elimination of the drug in the hemolymph of signal crayfish within a bioaccumulation experiment. Crayfish were exposed to three different concentrations of DFH (2, 20, and 200 ugl-1) for 96 hours. After the experiment, the concentration of DFH in the hemolymph was measured, and crayfish tissue was analyzed. In a second, ten-day experiment, only the concentration of DFH was monitored for absorption and elimination over time at pH values of 6.7 and 8.7. Crayfish were exposed to two concentrations of DFH (20 and 200 ugl-1) for 168 hours and then kept in clean water without DFH for 72 hours. Acute changes in oxidative stress biomarker activity and neurotoxicity biomarkers were observed in the hepatopancreas, gills, and muscles. Environmental and moderately increased concentrations of DFH caused an imbalance only in the activities of enzymes associated with glutathione metabolism. The most significant changes caused by oxidative stress were detected at sublethal concentrations in the muscles. The medication caused changes in the antioxidant defense mechanisms of signal crayfish, but acute neurotoxicity was not detected. The results of the ten-day experiment showed that a greater amount of the pharmaceutical was absorbed at pH close to the dissociation constant of DFH, but no linear dependence of the concentration of DFH in the hemolymph on the size of the crayfish was detected. The data obtained in this study complement the limited knowledge about the effect of DFH on aquatic invertebrates. The results will serve as a methodological basis for further research on the rates of absorption, elimination, and other parameters of bioaccumulation kinetics of pharmaceutically active substances in freshwater crustaceans.
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Sublethal effects of pesticide mixtures on crayfish
HOVORKOVÁ, Anna
Prometryn, a triazine herbicide, was banned in Europe in 2004. Metazachlor, a chloracetamide used to treat mainly oilseed crops, is one of the herbicides still in use. Individual pesticides or their metabolites can cause adverse effects in the aquatic environment, and the resulting mixtures of pesticides that can form in the environment are another issue in terms of adverse effects in the environment. The aim of this study was to assess the effect of mixtures of pesticides mixed from selected concentrations (0.01 mg.l-1, 0.1 mg.l-1, 1 mg.l-1). Pesticides were also tested individually at selected concentrations. The biochemical profile of hemolymph and antioxidant biomarkers and oxidative stress biomarker in hepatopancreatic, muscle and gill tissues of marbled crayfish were determined. Toxicity was assessed using a subchronic toxicity test performed over a period of 8 days. Metazachlor concentrations (0.1 and 1 mg.l-1) caused decreasing ALT, Cl-, Mg2+ and Ca2+ levels and increasing NH3 levels in hemolymph. The effect of all tested mixtures was observed on the increase of GLO, ALT and NH3 levels, here a synergistic effect of the mixtures was observed. The activity of antioxidant biomarkers was affected only in CAT for prometryn (0.01 mg.l-1) and metazachlor (0.01 and 1 mg.l-1). LPO levels were affected by metazachlor (0.01 mg.l-1). There was no effect of pesticide mixtures on LPO levels and antioxidant enzyme activities.
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