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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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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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Polymorphism of microsatellite markers in selected \kur{Beauveria bassiana} strains/isolates
KRÁLOVÁ, Martina
\kur{Beauveria bassiana} is a entomopathogenic polyphagous fungus commonly found in soil and it is parasite of soil insects, mainly of the stages of insect that occur in soil. At the present time it is used in plant protection against more than 70 species of insects. In the Czech Republic \kur{Beauveria bassiana} has the greatest importance in the fight against bark beetle \kur{Ips typographus} in the NP Šumava in these days. This study was focused on the evaluation of genetic variability \kur{Beauveria bassiana} strains on the basis of microsatellite analysis and the comparison of four separation methods: electrophoresis in 2% agarose gel, electrophoresis in 3% synergel, chip electrophoresis and fluorescent capillary electrophoresis in term of the most precise separation of PCR products. We used 41 strains which were collected in the NP Šumava and 20 strains from long-term collection determined as an exotic in this study. This large geographical scale group contains the strains from whole world and in addition it was upgraded by the strains collected from the NP Krkonoše and South Moravia. For the microsatellite analysis there were used 11 pairs of primers but for inter-comparison of separative methods were chosen only 4 pairs of primers. The population of \kur{Beauveria bassiana} strains collected from the NP Šumava were evaluated by analysis of microsatellites as a conservative and fully closed regardless of the source and the location. The strains from the large geographical scale group showed the great genetic variability. In terms of separation, the best and most suitable separation method was proved, the fluorescent capillary electrophoresis. Despite of its difficult financial aspect, this method was evaluated as the most precise and the most sensitive. Its advantage is in possibility to detect the smallest differences in the length of single allele in the range 1-2 bp, which is for the gel electrophoresis impossible.
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