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CRISPR/Cas9 Genome editing in Pyrrhocoris apterus
BERTOLUTTI, Maly
The CRISPR/Cas9 technology is a cutting edge method to genetically modify specific targets in the genome, which allows to create mutants in non-model organisms. The aim of this project was to genetically modify the Pyrrhocoris apterus genomic sequence of the cryptochrome2 gene using the CRISPR/Cas9 technology. Specifically, this project aimed to create a mutant with removed or modified C-terminal part of the CRY2 protein, which should later allow for studying the role of this sequence in CRY2 function.
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Effect of the entomopathogenic fungus Isaria fumosorosea on physiological processes in insects
GAUTAM, Umesh Kumar
The project is focused on a study of the infection elicited by entomopathogenic fungus (EPF) Isaria fumosorosea in two model species - the firebug Pyrrhocoris apterus and cockroach Periplaneta americana - and a role of adipokinetic hormones (AKHs) during that. The AKHs are neuropeptides that belong to the AKH/RPCH family and are known mainly for the mobilisation of energy reserves. The AKH role during the fungal infection was monitored by determination of mortality, gene expression in CNS, level of anti-oxidative stress markers, general metabolism, level of nutrients and activity of digestive enzymes. The I. fumosorosea infection elicited severe stress in the infected insects as documented by increase of the AKH expression and AKH levels in CNS of both insect models. The mortality test showed that application of AKH increased the efficacy of EPF application. It is supposed that AKH stimulated higher turnover of fungal toxins by enhanced intensity of metabolism (documented by higher carbon dioxide production) and encouraged the fungus growth by enrichment of haemolymph (a medium for the fungus) by mobilised nutrients. Interestingly, I. fumosorosea affected also digestive processes with substantial inhibition in midgut and partial inhibition in the caeca of male cockroach's digestive enzymes. Another part of this work was to examine changes of the vitellogenin (Vg) level in P. apterus after the I. fumosorosea treatment. The infection significantly reduced the Vg transcript level (checked by quantitative real-time PCR) in the female fat body and protein level in haemolymph (checked polyacrylamide gel electrophoresis). Interestingly, in the male fat body, Vg transcript level significantly increased after the infection, but EPF did not change protein level of male hemolymph. The obtained results point to an interesting role of Vg in insect defence reactions against EPF and are a good example of a trade-off between the Vg function in reproduction and defence reaction. Results of the project might be useful for better understanding of the mechanism of action of entomopathogenic fungi versus AKH, and could be utilised for practical biocontrol of the insect pests in future.
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Precopulatory behavior in firebug Pyrrhocoris apterus
Křivánek, Miloš ; Jarošík, Vojtěch (advisor) ; Stejskal, Václav (referee)
The existing research works, dealing with pre-copulatory behaviour in Pyrrhocoris apterus (L.), show that this behaviour follows a simple pattern of mating. The aim of this work is to extend knowledge regarding this type of behaviour. Results were obtained by processing the video footage to monitor the behaviour of individuals of the Central European population (the Czech Republic) as occurred in the respective copulatory zone. Changes were observed in the occurrence and mating activity of each sex depending on climatic conditions and time. Furthermore, the course of individual phases of the mating behaviour, based on mutual interactions and under the influence of external factors, was also observed. These results were supplemented by adding data gained from laboratory experiments, taking place at a temperature of 22 ± 4 řC and involving a long- day type photoperiodic reaction. Based on the results gained, it had been revealed that the climatic conditions significantly influenced the course of behaviour. Individuals of both sexes are physically active during the photo-phase when the intensity of this activity changes in the course of the circadian rhythm. Females display a higher sensitivity to any variation in climate. Males who are influenced by the presence of receptive females show greater locomotive...
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Factors influencing effectiveness of aposematic signals against avian predators
Kuklová, Lucia ; Exnerová, Alice (advisor) ; Krištín, Anton (referee) ; Sedláček, František (referee)
This thesis focuses on various factors affecting effectiveness of aposematic signals against avian predators. Adult, wild-caught as well as hand-reared juvenile great tits (Parus major) were used as predators in the experiments. The thesis consists of the following four studies. In the first study, we compared the reactions of great tits from two geographically distant populations toward aposematic firebugs (Pyrrhocoris apterus) and their non-aposematic artificially made colour variant. The birds from the Bohemian population mostly avoided aposematic firebugs and attacked non-aposematic variant. Finnish birds, which lacked experience with firebugs from their natural environment, were less hesitant to attack both firebug colour forms. Although the Bohemian birds avoided the aposematic prey variant, they were not more neophobic than Finnish birds. We conclude that the geographic differences in reactions of the birds to aposematic prey can be explained by a different population-specific experience of the birds with local aposematic prey communities. In the second study, we compared effectiveness of two chemical defence strategies in leaf beetle larvae (Chrysomela lapponica) against great tits. The birds avoided larvae devoid of external secretions after the first attack, which indicates the presence...
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Využití bioluminiscenčních, histologických a imunohistochemických technik při studiu lokalizace produktů genové exprese v tkáních hmyzu
PAUCHOVÁ, Lucie
The genes of enzymes catalyzing the bioluminescence reactions are commonly used as reporter genes in molecular biology. These markers are applied especially, in the studies of daily oscillation of gene expression products involved in the circadian clock. Here is reported an improved technique for monitoring the clock gene expression on the single neuron level in the brain of Drosophila melanogaster by using the bioluminescence marker. A part from that another study is focused on localizing a newly identified neuropeptide TEFLa in different developmental stages of firebug Pyrrhocoris apterus. This neuropeptide was detected in the brain of larvae and adults, in the posterior region of the midgut of adults and in the gonades of larvae. Beside that, it was found in the region of amnion closure in embryos. Synthesis of TEFLa in the adults' gut is controlled by the circadian rhythms. Identification of TEFLa distribution helps to uncover the function of this neuropeptide.
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Study of adipokinetic hormone role in insects stressed by entomopathogenic nematodes
IBRAHIM, Emad Ahmed Sayed
In this thesis, the effect of infection elicited by entomopathogenic nematodes (EPNs) Steinernema carpocapsae on Pyrrhocoris apterus and Drosophila melanogaster models were evaluated, and a role of adipokinetic hormones (AKHs) during the infection was characterized. These were monitored by determination of mortality, and various biochemical and physiological characteristics such as AKH levels both in the central nervous system (CNS) and in hemolymph, AKH gene expression in CNS, level of anti-oxidative stress markers, general metabolism and level of nutrients in normal and genetically modified insects. At P. apterus the mortality tests revealed that application of AKH increases the efficacy of EPN treatment. This result was confirmed using the firebugs with AKH receptor deficiency. Further, the increase of AKH expression and AKH levels in CNS and hemolymph seemed to be coordinated after the nematode treatment. At the D. melanogaster model also, the effect of adenosine into the above-mentioned characteristics was included. For this, mutants in AKH (AHK1), adenosine receptor (AdoR1) genes, and in both these genes together (AHK1 AdoR1 double mutant) were employed. Altogether, the results confirmed the involvement of AKH, and partially also adenosine into the antistress defense reactions elicited by the nematobacterial infection. Finally, the last part of the study was focused on examination of the vitellogenin (Vg) role in the defense reaction in firebug body P. apterus affected by two entomopathogenic organisms, the nematode S. carpocapsae and the fungus Isaria fumosorosea. The results revealed that Vg proteins play an important role in the defense against both types of the infections and are also able to kill entomopathogenic bacteria Xenorhabdus nematophila, that are symbionts of S. carpocapsae and that increase toxicity of this nematode.
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Circadian clock genes in the circadian clock and photoperiodic timer in Pyrrhocoris apterus
CHODÁKOVÁ, Lenka
This thesis focuses on the circadian clock genes and their involvement in the photoperiodic time measurement in the linden bug, Pyrrhocoris apterus. Application of the molecular biology methods enabled us to propose the architecture of circadian clockwork. We also investigated the role of several previously undescribed players in the circadian clock. Furthermore, by using molecular biology methods we focused on the involvement of core circadian clock genes in the photoperiodism.
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Adipokinetic hormone activities in insect body infected by entomopathogenic nematode
IBRAHIM, Emad Ahmed Sayed
The role of adipokinetic hormone (AKH) in the firebug Pyrrhocoris apterus adults infected by the entomopathogenic nematode (EPN) Steinernema carpocapsae was examined in this study. It was found that co-application of EPN and AKH enhanced firebug mortality about 2.5 times within 24 h (from 20 to 51% in EPN vs. EPN + AKH treatments), and resulted in metabolism intensification, as carbon dioxide production in firebugs increased about 2.1 and 1.6 times compared to control- and EPN-treated insects, respectively. Accordingly, firebugs with reduced expression of AKH receptors showed a significantly lower mortality (by 1.6 to 2.9-folds), and lower general metabolism after EPN + AKH treatments. In addition, EPN application increased Akh gene expression in the corpora cardiaca (1.6 times), AKH level in the corpora cardiaca (1.3 times) and haemolymph (1.7 times), and lipid and carbohydrate amounts in the haemolymph. Thus, the outcomes of the present study demonstrate involvement of AKH into the antistress reaction elicited by the nematobacterial infection. The exact mechanism by which AKH acts is unknown, but results suggested that the increase of metabolism and nutrient amounts in haemolymph might play a role.
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Reactions of the jumping spider Evarcha arcuata to aposematic true bugs
Raška, Jan ; Exnerová, Alice (advisor) ; Šobotník, Jan (referee)
Jan Raška: Reactions of the jumping spider Evarcha arcuata to aposematic true bugs Abstract: This paper studies the effect of qualities of a prey on predatory behaviour of Evarcha arcuata (Araneae: Salticidae). Naturally coloured aposematic larveae of Pyrrhocoris apterus have been compared to two types of prey: 1) to identically coloured but in defensive secretion different larvae of Scantius aegyptius, and 2) differently coloured larvae of white mutants of P. apterus. The defensive secretion of S. aegyptius has been found less effective in the first interaction with predator than that of P. apterus, however, after several trials the difference of reactions of the spider has not been noticeable. When changed the species of prey, E. arcuata has symmetrically and considerably generalised between the two species. Significant difference has been established in memory test - while P. apterus has been avoided even after one day, S. aegyptius has been attacked as thought the spiders were naive. The learning sequence has been similar in both colour forms of P. apterus; the generalisation of the two colours has been symetrical as well. Both colour forms succeeded in memory test (e.g. spiders have proven difference in comparison with the naive ones), however, white mutants have been successfully attacked...
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Precopulatory behavior in firebug Pyrrhocoris apterus
Křivánek, Miloš ; Jarošík, Vojtěch (advisor) ; Stejskal, Václav (referee)
The existing research works, dealing with pre-copulatory behaviour in Pyrrhocoris apterus (L.), show that this behaviour follows a simple pattern of mating. The aim of this work is to extend knowledge regarding this type of behaviour. Results were obtained by processing the video footage to monitor the behaviour of individuals of the Central European population (the Czech Republic) as occurred in the respective copulatory zone. Changes were observed in the occurrence and mating activity of each sex depending on climatic conditions and time. Furthermore, the course of individual phases of the mating behaviour, based on mutual interactions and under the influence of external factors, was also observed. These results were supplemented by adding data gained from laboratory experiments, taking place at a temperature of 22 ± 4 řC and involving a long- day type photoperiodic reaction. Based on the results gained, it had been revealed that the climatic conditions significantly influenced the course of behaviour. Individuals of both sexes are physically active during the photo-phase when the intensity of this activity changes in the course of the circadian rhythm. Females display a higher sensitivity to any variation in climate. Males who are influenced by the presence of receptive females show greater locomotive...
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