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Phenotypic plasticity plays a critical role in the adaptation of organisms to new environments and can facilitate the emergence of new phenotypes and speciation. We study the transition of surface animals to a cave environment characterized by extreme conditions such as complete darkness and limited nutrients. We use the teleost fish Astyanax mexicanus and the isopod crustacean Asellus aquaticus, which exhibit both the ancestral surface form and the derived cave form. The cave morphs show numerous adaptations, most notably the loss of pigment and eyes. By exposing the surface forms of both species to complete darkness under laboratory conditions, we observed molecular and phenotypic changes. Most phenotypes of the surface forms kept in the dark changed toward the cave phenotype. We also found some changes going in the opposite direction, suggesting that both maladaptive and adaptive plasticity play a role in adaptation to cave environments. Whether this random adaptive outcome depends on a specific epigenetic process remains to be seen. In contrast, most phenotypes of cave forms remained unchanged, i.e., they are canalized. We hypothesize that epigenetic changes underlie the extensive phenotypic plasticity in the surface forms and expect that epigenetic changes will be less pronounced in the cave forms exposed to total darkness than in the control light/dark conditions. Our current experiments are focused on identifying the type of epigenetic molecules involved in plasticity and genetic assimilation in our model organisms.

Animals that are not fully troglomorphic are often neglected in cave biology studies. However, they can provide important insights into the early stages of cave colonization. This is especially true when a surface population is also present and available for comparative studies. Although fishes are not uncommon in caves of the Dinaric Karst, it is generally believed that there are no cave-adapted fishes in this region. Several genera of fishes, such as Delminichtys, Telestes, and Phoxinellus, are known to be associated with caves, as they have been reported to retreat to caves during dry periods and spend several months underground until favorable conditions return. Data on such populations and their occurrence in caves are sparse, but during recent intensive surveys of Sušik Cave, Velika Kapela Mt, Croatia, cavers have frequently encountered fish. Sušik cave is a sinkhole of the small stream inhabited by a single endemic fish species, Telestes karsticus. Therefore, we set out to investigate whether T. karsticus lives permanently in Sušik Cave, how large the cave population is, and whether it exhibits any cave-adaptive characteristics. Our results show that T. karsticus lives in Sušik Cave throughout the year and its population is estimated to be several hundred individuals. Moreover, we found morphological and physiological characteristics that clearly distinguish it from the neighboring surface population. Therefore, Telestes karsticus forms a permanent cave population and the presence of a conspecific surface population makes it a potentially important model system for understanding the initial adaptive processes that occur during cave colonization.

The teleost species Astyanax mexicanus consists of an ancestral river-dwelling form (surface fish, SF) and a derived cave form (cavefish). The surface fish colonized caves only 30000 years ago and evolved numerous morphological, physiological, and behavioral adaptations that characterize the modern cavefish. We have previously found that many cavefish traits can be triggered by exposure of SF to complete darkness (DD SF), which is a hallmark of all subterranean habitats. Many of these traits are adaptive, such as decreased metabolic rate and increased fat deposition, suggesting that they may have evolved through genetic assimilation of the original plastic response of SF to darkness. Others are not adaptive, such as retinal proliferation and increased melanophores, which were selected against during the evolution of eye degeneration and albinism in cavefish. Genes involved in different levels of molecular organization, including epigenetic ones, were differentially expressed in DD SF compared with controls maintained under 12/12h light/dark conditions (LD SF). DNA methylation is also known to be involved in eye degeneration in cavefish. Therefore, our next step is to investigate the epigenetic mechanisms involved in extensive phenotypic plasticity in SF and their possible role in transgenerational inheritance during the adaptive evolution of cavefish.

Phenotypic plasticity has the potential to be of paramount importance in evolution. We showed its significance in adaptation to cave environments in a teleost species Astyanax mexicanus. Having ancestral surface morphs (surface fish, SF) and derived cave morphs (cavefish, CF), we exposed the former to complete darkness, thus mimicking its' colonization of caves. The results showed changes at both the gene expression and phenotypic levels. Remarkably, most phenotypes of dark maintained SF (hormone levels, fat content, neurotransmitter levels, metabolic rate) changed in the direction of the CF phenotype. In addition, we found 356 differentially expressed genes (DEGs) and further analyzed their expression in SF and Pachon CF at four stages of development (10 hpf/24 hpf/1.5 dpf/3 dpf). A total of 67 genes in SF exposed to dark change in the same direction as in Pachon in either all four or in three out of four stages of development, whereas 79 genes showed partially or completely opposite direction of change in expression. Our results show that more than half of gene expression changes are not in the adaptive direction. The plasticity of many traits known to be involved in adaptation to caves appeared in a single generation, however, the response tends to be random in the adaptive outcome. Our next aim is to look for mutations in SF and CF genes, as well as changes in the possible regulatory elements associated with aforementioned DEGs, in order to identify the molecular basis of plasticity as well as adaptive evolution of Astyanax CF.

Aim: Efficient isolation of DNA from various biological samples is the basis for successful forensic DNA profiling. Chelating resins and silica-based methods are commonly employed affinity and non-affinity methods for DNA extraction using limited amounts of biologic material. We aimed to evaluate DNA isolation efficiency of the commercially available non-affinity (Chelex® InstaGene™ Matrix) and affinity (Qiagen® QIAamp Mini) isolation procedures by comparing DNA yield and quality metrics of STR profiling data obtained from blood and buccal samples. Materials and methods: Ten buccal swabs and 10 blood stains collected on FTA cards were used as a starting material. Both buccal and blood DNA samples were isolated with the use of InstaGene Matrix and QIAamp Mini kit. Qubit fluorometer was used for DNA quantification and 16 STR loci were amplified with the AmpFlSTR® Identifiler PlusTM kit and the ProFlex 3x32 well PCR system. Following capillary electrophoresis on an ABI310, STR profiles and relative fluorescence units (RFU) were collected and analyzed in relation to buccal (B) or blood (P) sample DNA yield obtained with either InstaGene (IB or IP) or QIAamp Mini (QB or QP) isolation method. Kruskal Wallis and Dunn's post hoc test, adjusted by the Benjamini-Hochberg FDR were used for statistical data analysis. Results: The highest RFU (mean±SD, 3800±1315) and DNA concentration values (1.22±1.45 ng/ul) were seen in buccal samples isolated with InstaGene Matrix, when compared to IP RFU (1153±489, P=1.6x10-3) or DNA (0.06±0.012, P=4.8x10-3), and QP RFU (910±614, P=2.2x10-3) or DNA (0.02±0.035, P=9x10-5) levels. Moreover, QB RFU (3592±831) and DNA values (0.83±0.4) were significantly higher than QP RFU (910±614, P=6.9x10-4) and DNA (0.02±0.035, P=8.8x10-5) measures, which contained the highest number of DNA measurements below the lower Qubit sensitivity limit. Conversely, no difference in RFU and DNA values were observed between IP and QP, or IB and QB sample groups. Conclusion: Compared to blood stains, buccal samples provide greater DNA yield and higher STR peak values, regardless of DNA isolation method used. Conversely, InstaGene Matrix might be more reliable for blood stain DNA extraction and subsequent STR processing.

Ciljevi istraživanja: Izolirati lipidne splavi i utvrditi njihov sastav koristeći biokemijsku izolaciju splavi na miševima prije procesa mijelinizacije, tijekom i nakon njega (pet, 15 i 30 dana starosti) te uporabom specifičnih protutijela potvrditi uspješnu izolaciju splavi i usporediti razlike u mijelinizaciji među prethodno navedenim starosnim skupinama u moždanom deblu miševa s isključenim genom St8Sia1 s nedostatkom u sintezi kompleksnih gangliozida ; odnosno utvrditi kako nedostatak istih utječe na organizaciju mijelinskih mikrodomena, a time i na održavanje njegova integriteta. Materijali i metode: U istraživanju su korišteni KO miševi St8Sia1 stari pet, 15 i 30 dana s nedostatkom u sintezi kompleksnih gangliozida b – serije, što je dokazano genotipizacijom pomoću specifičnih početnica. Po još neobjavljenom protokolu izolirati lipidne splavi i uzorke najzad frakcionirati. Odrediti koncentracije proteina prema Bradfordu potrebnih za Western blot analizu. Elektroforezom odvojiti proteine te ih prenijeti na membrane. Korištenjem primarnih i sekundarnih protutijela odrediti izražaj na markere flotilina i transferina, tj. markere splavi, odnosno ne-splavi te na mijelinu pridruženi glikoprotein (MAG) i mijelinski bazični protein (MBP), markere mijelinizacije. Membrane vizualizirati i slikati, a dobivene rezultate analizirati denzitometrijski pomoću računalnog programa Fiji. Rezultati: Statistički značajnu razliku usporedbom svih triju starosnih skupina miševa pokazuju flotilin i transferin, dok MBP razliku pokazuje usporedbom miševa 15 i 30 dana starosti. Međutim, prva se dva biljega, neočekivano, nalaze u svim frakcijama umjesto u specifičnim frakcijama za navedene biljege. Zaključak: S obzirom na rezultate flotilina i transferina izgledno je da izolacija splavi nije bila potpuna te da su izolirane stanične membrane.

Uvod: Neurogeneza, odnosno stvaranje novih živčanih stanica, osim u embrionalnom razvoju, odvija se i u odraslom mozgu i to uglavnom u područjima: u subventrikularnoj zoni (SVZ) i subgranularnoj zoni dentatnog girusa (DG) hipokampusa te u njušnoj lukovici. U tom procesu ulogu imaju i kompleksni gangliozidi koji su prisutni u svim tkivima, ali su najzastupljeniji u mozgu, a njihov nedostatak dovodi do neurodegeneracije i neuroloških poremećaja. Ciljevi: Odrediti povezanost nedostatka kompleksnih gangliozida i biljega upale i proliferacije stanica. Materijali i metode: U istraživanje su uključena dva tipa miša - genetički modificirani B4Galnt1 (KO) s nedostatkom u sintezi kompleksnih gangliozida i kontrolni miš divljeg tipa (WT). Imunohistokemijskom analizom pomoću protutijela Iba1, GFAP i PCNA detektirani su navedeni biljezi. Rezultati: Rezultati su analizirani računalnim programom Fiji te su statistički obrađeni. Dobiveni su rezultati pokazali statistički značajnu razliku kod sva tri biljega. U SVZ-u je uočeno da je Iba1 više izražen kod KO-a u odnosu na WT, dok su vrijednosti PCNA biljega bile veće u WT-u uspoređujući ga s KO mišem. S druge strane, u DG-u je pokazana veća ekspresija GFAP pozitivnih stanica u WT mišu nego u KO. Zaključak: Veće vrijednosti biljega Iba1 potvrđuju hipotezu o povećanom broju mikroglija stanica pri upalnom procesu, dok manje vrijednosti PCNA u KO mišu upućuju na moguće postojanje smanjenog ukupnog broja matičnih stanica u SVZ-u. Slično, manji broj GFAP pozitivnih stanica u hipokampusu KO miša moglo bi se povezati s njihovom slabijom mogućnošću diferencijacije za vrijeme upale.