11. Apr. 2022

It lives in complete darkness and will go hungry for several months. It is completely blind and moves through a vibration-sensitive auditory system. However, scientists still do not know much about the unique physiology of the cave salamander. Further research was facilitated by Markéta Tesařová from CEITEC BUT, who created a virtual 3D atlas of this protected animal using X-ray computer microtomography. 

The cave salamander (proteus anguinus) is the only European amphibian living underground. It belongs to the endangered species and most often occurs in the Dinaric Karst Caves in Slovenia, Croatia and Italy. It has developed a number of unique morphological, physiological and behavioural mechanisms for living in absolute darkness and cold. However, because it is protected, lives in inaccessible conditions and cannot be kept in a laboratory, it has remained a mystery to biologists. 

In the online database of Gigascience magazine, however, they now have a complete atlas of 3D models of the salamander, including its developmental stages, thanks to which they can study the exceptional creature through and through - – from the brain, cartilage to complex olfactory, visual or reproductive systems. The main author of the atlas is doctoral student Markéta Tesařová from the X-ray and computed tomography laboratory at CEITEC BUT. 

Originally, the side project took up 6 years of research, and another 10 specialist workplaces took part in it on an ongoing basis - mainly from Italy and Slovenia, but also from Sweden and the USA. "At that time, we collaborated with a Swedish laboratory on research into the regenerative abilities of salamanders. At that time, I was on a research stay in Trieste, Italy, and because the cave salamander is very popular in this area, they offered us to explore a rare archival collection of salamander preserved in alcohol, " Tesařová described. 

The individual specimens, some of which were as old as 30 years, then began researching at CEITEC BUT using X-ray computer microtomography. Although the salamander has been X-rayed in the past, the researchers have only described the skeletal system. 

"We were also the first to map cartilage and soft tissues, including the brain. We soaked the samples in heavy metal solutions to increase the contrast. Then we performed X-ray projections from different angles and mathematically modelled the individual parts in 3D, " added Tesařová, noting that the great advantage of the method was its non-destructiveness. In addition to 3D models of the cave salamander, Carpenter also created models of the Mexican axolotl, which is a salamander living in Mexican lakes. Thus, scientists can compare the physiology of amphibians. 

The X-ray of the samples itself took the doctoral student one year, but she spent the next five years processing the data. "It has not been easy to distinguish soft tissues from cartilaginous ones - for example, in the structures of the salamander's eye, which are stunted inside the head. I had to consult everything with biologists, speleologists and study older hand-drawn publications, " explained the doctoral student. 

During her studies, she was surprised that even in adults, the cartilaginous part, which is more typical of the younger stages of development, occurs. "We discussed that a lot, and one of the hypotheses is that the cartilage stayed with him because it helps to transmit sound to the auditory system. When hunting, salamander  tilts his chin to the ground and, by vibration, recognizes where the prey is. Both the lower jaw and the facial cartilage seem to help him with precise orientation in space, " she added. 

Although the research of the cave salamander at CEITEC BUT is now coming to an end, it is just the beginning for speleologists and biologists around the world. "We have been contacted by various research institutes which, based on 3D models, plan to study the physiology of the ear, the reproductive system or the predatory behaviour of the salamander, which can be reconstructed from the way muscles are clamped to cartilage and bones," Tesařová described. The just created 3D models could offer answers. 

The doctoral student will present her work at the 4th International SOS Proteus conference in Trieste in May, where she will also meet experts from other institutions with whom she has continuously collaborated in the creation of the 3D database. These include the Italian research institutes Elettra-Sincrotrone and the Speleovivarium Erwin Pichl, the Tular Cave Laboratory and the Slovenian Academy of Sciences and Arts. 

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