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Molecular Characterisation of Parvalbumin Gene: Evolutionary Insights and Forensic Applications for Fish Species Identification and Authentication
Mukherjee, Subham ; Horká, Petra (advisor) ; Kalous, Lukáš (referee) ; Flajšhans, Martin (referee)
Abstract Parvalbumin (pvalb), a low molecular weight calcium-binding protein, plays a crucial role in regulating Ca2+ switching in fast-twitch muscle fibres and has been identified as a major cause of fish-induced food allergies. The molecular evolution of pvalb genes in teleost fish and its cause, duplication of the whole genome, was investigated, revealing high diversity and complex gene repertoires, making detection and identification challenging. This study provides robust genomic evidence of the complex evolution of parvalbumin genes in teleost fish. In addition to its role as a potent allergen, the pvalb gene, a nuclear gene, can serve as a valuable molecular marker. Keeping this in mind, a real-time PCR assay is developed to detect and quantify two European anglerfish species simultaneously, Lophius piscatorius and Lophius budegassa, which are susceptible to illegal species substitutions in the global seafood trade. The assay targets the intronic region of the pvalb gene, demonstrating high specificity, efficiency, and robustness, making it a potential forensic tool to prevent food fraud and ensure the accurate identification of fish species. Furthermore, a standardised quantitative PCR-based method is presented for the β-pvalb gene in Lophius piscatorius, utilising a plasmid DNA calibrator...
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The study of structure and metabolism of mammalian and fish sperm in relation to the process of reproduction
Koubek, Pavel ; Pěknicová, Jana (advisor) ; Komrsková, Kateřina (referee) ; Flajšhans, Martin (referee)
4. CONCLUSION Duringthe studyof teleosteanand chondrosteanfish spermatozoa,a new complex protocol,optimizedforimmunofluorescencestainingofantigenson carpandsturgeon spermatozoa,was developed lt was derivedfrom the protocolused for the mammalianmodel;however,theprotocolrespectsthesimpleandbreakablestructure offishsperm.Inthisway,possibledamagetospermatozoaduringsamplepreparation is minimizedtogetherwithgenerationof nonspecificartefactswhichcouldinfluence the results.Therefore,it is crucialto createandfollowa suitableprotocolforsemen handlingand immunofluorescencestainingnotonlyforevaluationof semenquality butalsoforexperimentalstudreswhenthereproducibilityofthemethodis essential. In the study of energy metabolismon the model of demembranatedcarp spermatozoa,we provedtheexistenceof PCr shuttleand showedthe localizationof creatinekinaseisoenzymesalongthecarpspermflagellumbytheaidof monoclonal antibody.Moreover,withthe contributionof experimentsusinginhibitionof creatine kinase,we demonstratedthe possibilityof initiationand maintenanceof cAMP- independentflagellarmovementof demembranatedcarpspermatozoaonlyby the componentsofthePCr shuttle The studyof chondrosteanfishwas focusedon thefinestructureand morphologyof sterletspermatozoa,which was examinedby scanningand transmisionelectron...
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Molecular phylogeography of the tench Tinca tinca (Linnaeus, 1758)
Lajbner, Zdeněk ; Kotlík, Petr (advisor) ; Flajšhans, Martin (referee) ; Macholán, Miloš (referee)
The tench Tinca tinca (Linnaeus, 1758) is a valued table fish native to Europe and Asia, but which is now widely distributed in many temperate freshwater regions of the world as the result of human-mediated translocations. Spatial genetic analysis applied to sequence data from four unlinked loci (introns of three nuclear genes and mitochondrial DNA) defined two groups of populations that were little structured geographically but were significantly differentiated from each other, and it identified locations of major genetic breaks, which were concordant across genes and were driven by distributions of two major phylogroups. This pattern most reasonably reflects isolation in two principal glacial refugia and subsequent range expansions, with the Eastern and Western phylogroups remaining largely allopatric throughout the tench range. However, this phylogeographic variation was also present in European cultured breeds and some populations at the western edge of the native range contained the Eastern phylogroup. Thus, natural processes have played an important role in structuring tench populations, but human-aided dispersal have also contributed significantly, with the admixed genetic composition of cultured breeds most likely contributing to the introgression. I have then designed novel PCR-RFLP assays...
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Diploid, triploid and tetraploid silver crucian carp Carassius auratus from the lower reach of Dyje River: the first results of reproductive characteristics and experimental hybridization
Flajšhans, Martin ; Lusková, Věra ; Vetešník, Lukáš ; Halačka, Karel ; Rodina, Marek ; Lusk, Stanislav ; Gela, David
Diploid, triploid and tetraploid males of silver crucian carp, Carassius auratus, produced live motile spermatozoa, capable of fertilization. Flow cytometry for the relative DNA content in male gametes revealed spermatozoa of diploid males to be haploid, those of triploid males to be aneuploid and those of tetraploid male to be aneuploid and/or diploid. Concentration of spermatozoa and the percentage of live spermatozoa showed a tendency to drop with increasing ploidy level and this effect was significant for the tetraploid male compared to those of lower ploidy levels.
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The ploidy of Carassius auratus in the Czech Republic in light of the artificial reproduction
Halačka, Karel ; Vetešník, Lukáš ; Flajšhans, M.
State of population of Carassius "gibelio" (the sex ratio and ploidy level) was for a long time observed in the Dyje river floodplain, where it has expanded by natural migration from the Danube river through Morava river in 1975-1976. Results show the time dynamics for both parameters, with increasing proportion of males and increasing proportion of diploids of both sex and the formation diploid-polyploid complex. Diploid, triploid and tetraploid males and females of silver crucian carp, Carassius auratus, as well as males of silver Blicca bjoerkna and Rutilus rutilus, were used in mating. The results show different reaction of females on used sperm even in the same group of ploidy. The existence of some groups of the individuals with a different strategy of reproduction in frame single population could be the explanation.
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