Development of non-destructive approaches for sex identification in the proteus (Proteus anguinus, Urodela, Amphibia)

Prepoznava spola pri jamski repati dvoživki močeril (Proteus anguinus) je otežena zaradi odsotnosti spolnega dimorfizma in homomorfnih spolnih kromosomov. Namen naloge je bil razvoj nedestruktivnega in zanesljivega pristopa prepoznave spola na podlagi analize (i) spolno specifičnih genetskih markerjev z verižno reakcijo s polimerazo, (ii) proteina vitelogenina (Vtg) s poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecil sulfata ter (iii) hormonov testosterona (T) in 17-β-estradiola (E2) in njunih metabolitov z encimskoimunskimi testi. Rezultate omenjenih analiz smo delno ovrednotili s histološko analizo gonad. Ugotavljamo, da samice prepoznamo, ko se koncentracija E2 v krvi zviša nad 450 pg/ml, detekcija Vtg v krvni plazmi pa omogoča prepoznavo vitelogene stopnje oogeneze. Vtg zaznamo po postopnem večmesečnem zviševanju E2, čemur sledi tudi vidna zaznava oocitov skozi trebušno steno. Z večkratnimi meritvami E2 in Vtg v krvni plazmi samic smo potrdili zoritev oocitov, nepričakovano znižanje E2 in Vtg med oogenezo pa sovpada z degeneracijo oocitov, kar potrjuje tudi zmanjšanje velikosti in števila vidnih oocitov. Samce prepoznamo, ko se koncentracija T v krvi zviša nad 450 pg/ml, zvišanje T v krvi pa sovpada z zadebelitvijo kloake. Kljub temu izgled kloake ni zanesljiv znak za prepoznavo spola. Analize T in E2 v sluzi ter njunih metabolitov v iztrebkih z uporabljenimi postopki niso bile uspešne, prav tako Vtg nismo zasledili v sluzi in homogenatih jeter ter jajčnikov. Genetskih spolno specifičnih markerjev, ki so poznani pri nekaterih dvoživkah, pri močerilu nismo potrdili. Razviti pristopi sicer ne omogočajo prepoznave spola živali izven obdobja aktivne gametogeneze, vendar bodo pomembno orodje za namen razmnoževanja močerilov v ujetništvu.In the cave salamander, Proteus anguinus, sex identification is difficult due to the absence of sexual dimorphism and homomorphic sex chromosomes. The aim of this work was to develop a non-destructive and reliable approach for sex identification based on analysis of (i) sex-specific genetic markers with polymerase chain reaction, (ii) the protein vitellogenin (Vtg) by sodium dodecyl sulfate polyacrylamide gel electrophoresis and (iii) the testosterone (T) and 17-β-estradiol (E2) and their metabolites by enzyme-linked immunosorbent assays. The results were partially evaluated by histological analysis of the gonads. We found that females are identified when the concentration of E2 in the blood exceeds 450 pg/ml. In addition, Vtg allows the recognition of vitellogenic stage of oogenesis and is detected after a gradual increase in E2 over several months, followed by the visible oocytes. Multiple measurements of E2 and Vtg in female confirmed the maturation of the oocytes, while an unexpected decrease in E2 concentration and Vtg during oogenesis coicides with their degeneration, which was also confirmed by a decrease in the size and number of visible oocytes. Males are identified when the blood T concentration exceeds 450 pg/ml. The increase in T conc. coincides with a swollen cloaca, but sex identification by a swollen cloaca is not reliable. With the approaches used, the analysis of T and E2 in mucus and their metabolites in faeces was not successful. Vtg was not detected in mucus, liver and ovarian homogenates. Genetic sex-specific markers known in some amphibians could not be confirmed in P. anguinus. The developed approaches allow sex identification during the active gametogenesis and will be an important tool for the purpose of captive breeding of P. anguinus

A method for targeting a specified segment of DNA to a bacterial microorganelle

Encapsulation of a selected DNA molecule in a cell has important implications for bionanotechnology. Non-viral proteins that can be used as nucleic acid containers include proteinaceous subcellular bacterial microcompartments (MCPs) that self-assemble into a selectively permeable protein shell containing an enzymatic core. Here, we adapted a propanediol utilization (Pdu) MCP into a synthetic protein cage to package a specified DNA segment in vivo, thereby enabling subsequent affinity purification. To this end, we engineered the LacI transcription repressor to be routed, together with target DNA, into the lumen of a Strep-tagged Pdu shell. Sequencing of extracted DNA from the affinity-isolated MCPs shows that our strategy results in packaging of a DNA segment carrying multiple LacI binding sites, but not the flanking regions. Furthermore, we used LacI to drive the encapsulation of a DNA segment containing operators for LacI and for a second transcription factor

Environmental DNA in subterranean biology update: from “Where?” to “How many?”

Recent records of Proteus anguinus outside its historically known range (Gorički et al. 2017), discovered through detection of its DNA dissolved in groundwater (environmental DNA or eDNA), mark the beginning of a new era in the study and conservation of cryptic subterranean biodiversity. An upgraded technology, droplet digital PCR (ddPCR), initially developed for studies of gene expression, detection of genetically modified organisms and in medical diagnostics, is being tested for improved detection of the much smaller and rare stygobiont, the cave clam Congeria jalzici. In parallel to eDNA assay development for various stygobiotic species of the Dinaric Karst, a groundwater-sample library is being created. The samples will be available for future analysis of their species composition and will also serve as a source of information on any changes in species distribution over time. In another line of eDNA research, the utility of ddPCR for direct quantification of eDNA molecules in groundwater is being explored by using the large, accessible and well-characterized (Zakšek and Trontelj 2017) natural Proteus population in the Planina Cave (Slovenia) as a model. The eDNA methodology may in the future be applied in estimation and monitoring of Proteus population sizes without having to see, mark or otherwise disturb the animals themselves