THE "PROTEUS PROJECT"
WEBPAGE No.4.
THE "PROTEUS PROJECT"
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"A JOINT STRATEGY FOR THE PROTECTION OF THE ENDANGERED UNDERGROUND ENDEM Proteus anguinus AND ITS NATURAL KARST HABITAT IN THE TREBIŠNJICA RIVER BASIN."
Eastern Hercegovina and the Hercegovinian Littoral, Bosnia & Hercegovina;  Dalmatian Littoral and Konavle, Croatia;  Old Hercegovina, Montenegro.
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"Zajednička Strategija za Zaštitu Ugroženog Podzemnog Endem Čovječje Ribice (Proteus anguinus) i Njezinog Prirodnog Krškog Staništa u Slivu Rijeke Trebišnjice."
Istočna Hercegovina, Bosna i Hercegovina.
"Заједничка Стратегијаза Заштите Угроженог Подземног Ендема Човјечије Рибице (Proteus anguinus) и Његовог Природног Кршког Станишта у Сливу Ријеке Требишњице."
Источна Херцеговина, Босна и Херцеговина.
"Együttműködési Stratégia a Veszélyeztetett Barlangi Vakgőte (Proteus anguinus) Természetes Karsztos Élőhelyének Védelmére a Trebišnjica Folyó Völgyében."
Kelet- Hercegovina, Bosznia - Hercegovina.
Webpage 4. for PROJECT INFORMATION SECTIONS 6. & 7.
Text Updated.02 May 2016.
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.....PROTEUS PROJECT WEBPAGE [4.] CONTENTS :-
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.....NOTE TO OUR READERS :
.....In this major revision of the "Proteus Project" Webpage [4.], we hope we have now sufficiently addressed all of the issues our readers have raised with us in regard to the threats associated with the protection
.....and conservation of Proteus anguinus.and its natural habitats.
.....In particular, a few of the issues that many hundreds of our readers have raised over the past few years in this regard have been concerned with the situation of Proteus in Slovenia, although we do have first-hand 
.....evidence that one particular issue that has been raised is also common to Bosnia and Hercegovina where it has overspilled from Slovenia and about which we hope we have dealt with in a suitably appropriate and 
.....proportionate manner below, by giving just a few examples of known instances.
.....However, we must advise that any concerns about Proteus in Slovenia must be taken directly to the Slovene Nature Protection Authorities in Ljubljana and cannot be addressed by our "Proteus Project"
.....operating mostly in Bosnia & Hercegovina.
.....This revision has also seen the entire removal of the former "Case Histories" section.
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.....Section 6...Synopsis of the Conservation Problems Associated with the Survival of Proteus anguinus and its Natural Karst Habitats in the Trebišnjica River Basin.
.....Section 7...Incidental Non-Project-Related Data Acquisition, Surface Flora and Fauna :
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.....Click on the underlined Links below to navigate to the other "Proteus Project" Webpages more quickly.
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.....Link to.."Proteus Project" Introductory Webpage :
.....Link to.."Proteus Project" Webpage [1.] :
.....Link to.."Proteus Project" Webpage [2.] :
.....Link to.."Proteus Project" Webpage [3.]:
.....Link to.."Trebinje Proteus Observatorium"
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Section 6...SYNOPSIS OF THE CONSERVATION PROBLEMS ASSOCIATED WITH THE SURVIVAL OF PROTEUS ANGUINUS AND ITS NATURAL KARST HABITATS 
IN THE TREBIŠNJICA RIVER BASIN  :
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The common universal problems affecting the survival of Proteus anguinus and associated hypogean fauna are:
habitat destruction by urbanization and civil engineering projects; agricultural activities; overgrazing; nutrient enrichment; chemical & biochemical pollution; hydrological & hydrographical changes;
over-exploitation of natural resources on the surface; introduction of predator species and / or competitor species; industrial, widespread small-scale commercial and domestic environmental degradation etc.
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The specific problems affecting Proteus and its natural habitats in the Trebišnjica River Basin in Bosnia & Hercegovina are  :-
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a)..Electroprivreda RS / HET Co. Trebinje Hydro-Electric Scheme, BiH :
b)..Industrial Air- and Water-borne Pollution from Gatačko Polje, RS-BiH :
c)..Development of HE "Ombla", Komolac, Dubrovnik, Croatia, (proposed) :
d)..Introduction of Foreign Fauna :
e)..Ubiquitous Dumping of General Domestic Waste, Commercial Waste and Farm Waste :
f)...Illicit Trading in Proteus Specimens and other hypogean fauna :
g)..The Absence or Inadequacy of Nature Protection & Conservation Laws at RS-BiH-Entity Level and at R.BiH State Level :
h)..Uncontrolled Publicity of Proteus Habitat Locations :
i)...Collecting of speleo-fauna specimens for private collections and / or for supposed scientific research :
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We now deal comparatively briefly with each of these problems below.
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ELEKTROPRIVREDA REPUBLIKE SRPSKE (ERS) / HET Co. TREBINJE HYDRO-ELECTRIC SCHEME, BiH :
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Prior to the gigantic civil engineering construction works associated with the building of infrastructure across Eastern Hercegovina to support a region-wide integrated hydro-electric generation scheme for the Hidroelektrane
na Trebišnjici Co. (HET), the prevailing natural environmental conditions in the karst of the area were very much different to those of today. 
The impressive hydro-electric scheme would line the entire length of the Trebišnjica Karst River with concrete and shotcrete downstream from Trebinje to its natural end at the Ponikva Ponor Zone near Hutovo, a distance
of 45 km. Two hydro-electric dams would be constructed upstream of Trebinje. In Popovo Polje, karst ponors would be concreted over, reservoirs would be built and many kilometres of tunnels would be constructed allowing the novelty of forward and reverse flows of water within the hydro-electric generating system, for maximum economic benefit.
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Theoretically at least, the hydromelioration of Popovo Polje would be at last achieved, relieving it of an average 253 days of flooding each year. Certainly no mean feat of engineering if it could be achieved, but at what
expense to the natural karst hydrographical regime and its rich karst biodiversity? As a rough general guide to the more instantly obvious effects we list the following:-
...(i)..The hitherto natural two-way water flow functionality of many estavelles has now been reduced to a managed permanent condition of one-way flow, whereby these karst features now only function as periodic karst springs.
..(ii)..Many karst ponors are no longer functional, having been permanently deprived of their water input, which in turn will have a direct effect on the hydrology of localised underground environments and habitats.
.(iii)..The entire karst hydrology and karst hydrography of the Popovo Polje area and the connected endokarst down the natural hydraulic gradient in the direction of the Croatian and Hercegovinian Adriatic seaboards and
the littoral karst has been massively affected, with many karst underground flowlines and flowpaths permanently altered in one way or another.
.(iv)..The dehydration of Popovo Polje would have an immeasureable and permanent detrimental effect on the karst underground in the whole region, altering the natural karst ecology and with it, the natural habitats of
Proteus and other species. Milanović (1987) claims that 37 known Proteus habitat sites were destroyed by the effects of the Hydroelectric-energy construction scheme.
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Elektroprivreda Republike Srpske (also known as Elektroprivreda RS) is a state-owned integrated power company in Republika Srpska, Bosnia and Herzegovina since 02 June 1992. It is the second-largest power utility in Bosnia and Hercegovina.
The active proposals by the Elektroprivreda Republike Srpske (ERS) since the end of the Bosnian War of the 1990's to complete the project "Multipurpose Use of the Waters of the Upper Horizons of the Trebišnjica". / "Višenamjensko korišćenje voda gornjih horizonata", will only add massive destruction to the region's important karst ecodiversity. A further step in realizing this Project was completed in June 2005., with the completion
of the excavation for the pilot tunnel from Fatničko Polje to Bilećko Jezero. This will lead to the complete destruction of the natural hydrological conditions and associated karst ecology of this Polje, yet again replicating the company's previous irresponsible behaviour in Popovo Polje. The further destruction of Proteus anguinus habitats is, therefore, inevitable.
The more recent activities to initiate the long-planned hydroelectric station in Dabarsko Polje, involving the resumption of the tunnelling scheme between Dabarsko Polje and Nevesinjsko Polje, will, of course, affect more
of the natural karst hydrography and impact the biodiversity even more than before. 
.INDUSTRIAL AIR- and WATER-BORNE POLLUTION FROM GATAČKO POLJE :
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Over the reporting period, we have now become aware of a problem associated with industrial pollution and which serves to illustrate how a local pollution problem can be felt on a region-wide basis in a karst area.
The incident concerns the production and bad storage practices of waste chemical ash from the furnaces of the Gacko Thermo-electric generating plant No. 1. in Gatačko Polje. This Polje is a major upstream catchment feeder to the Trebišnjica River both directly via the drowned Vrela Trebišnjica karst springs down valley of Bileća; at Vrela Čepelica in the Bileća Reservoir and indirectly via the "Obod", "Baba Jama" and other estavelles in Fatničko Polje.
We have been told by Stanko Dučić, a farmer in Fatnica, that this major pollution incident decimated the wildlife in the "Obod" Estavelle and even after four years, although the Gaovica Fish began returning, they were still poisonous and could not be eaten by epigean wildlife.
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Tons of ash from the power station had been purposely discharged into a ponor in Gatačko Polje and was washed into the undergound karst system. The water from Fatničko Polje feeds the submerged main spring sources
to the Trebišnjica River. The repeated incidence of this problem over many years may be a major contributor towards the increasing rarity of the once prolific Proteus. The problems associated with the TE Gacko I. Plant
are detailed in Section 13. of the Fatničko Polje Webpages (Anthropogenic Impact).
For all those concerned with the on-going problems of karst water quality in BiH Eastern Hercegovina and Croatian Dalmatia, we strongly advise that you consult the aforementioned Section 13.
INTRODUCTION OF FOREIGN FAUNA :
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There are various salmonoid and other fish which are native to the surface waters of the Trebišnjica River. Rainbow Trout and various carp such as Šaran to name but two. However, there is another fish that used to breed prolifically in the specialised hydrographical conditions of the Dinaric Karst in Eastern Hercegovina.
It was long ago in history that use was made of the natural food supply afforded by the annual fishing season in the inundated or flooded Popovo Polje.
Out of a total of 7 species of Gaovica salmonoid fish, all endemic to the former Yugoslavia, 3 species live in and are native to the region of Eastern Hercegovina, and are namely:
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.....Popovska Gaovica or "Popovo Gaovica"  (Paraphoxinus ghetaldi, Steindachner, 1882.)
.....[as of 2004, it has a revised Scientific Name:   Delminichthys ghetaldii, Steindachner, 1882.]
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.....Gatačka Gaovica or "Gacko Gaovica"  (Paraphoxinus metohiensis, Steindachner, 1901.)
.....Trebinjska Gaovica or "Trebinje Gaovica"  (Paraphoxinus pstrossi, Steindachner, 1882.)
.......Taxonomic Notes:  Following Eschmeyer (2004), Paraphoxinus pstrossi seems to have been regarded as the same species as Phoxinellus ghetaldii.
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Gaovica are the only salmonoid fish without scales on their skin. In English, they are often referred to as Minnows and they have a voracious appetite, instantly attacking anything that they can eat. 
Their range within the cave habitat is normally very limited to that area where the daylight penetrates. There are a few exceptions to this limitation that are known to us.
Proteus normally cannot be found where the Gaovica congregate or it would certainly be attacked as indeed are cave shrimps. One is certainly aware of the other.
The continued undisturbed existence of the endemic Paraphoxinus ghetaldi became relevant, in an unlikely way, to that of Proteus. This temporary dweller of the karst under-ground is about 10-17cm. long when fully grown. 
It spent the dry summer part of the year (ca 100 days) living in the waters of the karst underground in the syphonic pools and lakes. When the Periodic Lake appeared in Popovo Polje, Gaovica came to surface in great quantities through the numerous vrela and estavelles that opened in the polje floor. However, with the advent of the complete control of the Polje's hydrography by the engineering of the Trebišnjica Hydro-energy System 
and the susbsequent permanent non-reappearance of the periodic lake, the main source for this rich supply of food also dried up.
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With the demise of the Popovo Polje intermittent lake and the annual fish supply that went with it, the population of the Popovska Gaovica fish was confined to appearing in decreasing numbers at the mouths of various karst springs, estavelles and ponors. Fishing activities at the openings of estavelles, which for centuries had been an important source of nutrition for the inhabitants of Popovo Polje, have now completely vanished.
With the inability of this hitherto rich food supply to provide adequate sustenance for the growing human population in and around the area, a local person went to America to source a breed of fish that would thrive in the local rivers and provide a boost in the supply hitherto provided only by the Gaovica fish............
...........He duly returned with the species of fish known as the Californian Trout,.Salmo gairdneri irideus, locally known as "Kalifornijska pastrmka".
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The introduction of the Californian Trout certainly met the demands of the hungry human population. However, this even more voracious creature, which was considerably larger than the endemic fish species, became an instant threat to the Proteus population upon which it feasted. The harmony which had previously existed in the karst waters, virtually disappeared overnight!  To cap it all, this newcomer also delighted in eating the 
endemic species of Gaovica fish, a situation which is current.
However, we are very pleased to be able to report that, contrary to popular belief, the endemic Gaovica fish are certainly plentiful across its range in Eastern Hercegovina. We are presently unaware of any actual or intended conservation management scheme to breed or re-introduce the endemic Gaovica species on a large scale or of any plan to farm this species commercially or of any plan to curtail and limit the presence of the Californian 
Trout to that of fish farms.

Photo PP6d.1.  (Photo :  B. Lewarne, 07 August 2002.)
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Photo PP6d.2....(Photo :  Bogdan Prelević, 04 March 2002.)
UBIQUITOUS DUMPING OF GENERAL DOMESTIC WASTE, COMMERCIAL WASTE AND FARM WASTE :
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The urban area of Trebinje contains countless sites where domestic and other wastes are dumped randomly on the karst. Many of these dumping areas are on or in very hydrologically-active areas such as ponor zones, spring zones and estavelles, where water sinks into or rises out of the karst underground.
A general view of the waste materials thrown into Vrelo "Oko" at Zasad, in Trebinje. Clearly seen are various metal objects, lorry tyres and a lead-acid accumulator, building up on the steps leading down to the spring-cave entrance.  What hope is there for the future of the karst water environment and for Proteus when such irresponsible activities are allowed to happen?
A general view of the waste materials thrown into the Trebišnjica River at the Pridvorci Ponor Zone near Trebinje.  The area was used as an uncontrolled Municipal Refuse Dump by the whole village. 
Some of the older villagers did not want us to record this disgraceful scene and were clearly embarrassed at the situation they had caused.
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We should make it clear that both the locations cited and illustrated above are now totally clear of the rubbish; this was as a result of a the locally widespread publicity given by the Project to the unacceptable environmental situation at Pridvorci.
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In general terms, there are wider issues here which still have to be urgently addressed at household level, community level and at the political level, right across all of Bosnia & Hercegovina, if this problem is to be solved before a major national ill-health situation develops.
We are advised by employees in the Trebinje Hospital that the incidence of various cancers in the human population of Eastern Hercegovina, has increased by one order of magnitude over the past decade. It should also be borne in mind that the polluted waters of the Trebišnjica River go on to supply water resources in the neighbouring Croatia via a trifurcated, transboundary watershed.
 
 
.ILLICIT TRADING IN PROTEUS SPECIMENS AND OTHER CAVE FAUNA :
and the collecting of other speleo-fauna specimens for private collections, supposedly for scientific research :

There is the usual problem of illicit trading in Proteus specimens. Unscrupulous individuals have already approached Project Workers with offers of specimens for sale. We do not accept such offers and will use every means to deter or prevent the capturing and selling of this extremely rare species.

Historically, trading in Proteus specimens seems to have always taken place. Some of the extreme examples of this are detailed in an interesting article written by Shaw (1999)
Proteus was for a long time on the list of the Washington CITES convention, which prohibits trade in rare wild animals, but it has now been removed. Although all surrounding countries are Party to the CITES Convention, Bosnia and Hercegovina is not a signatory country to the CITES Convention. Even if it were, we suspect that unscrupulous dealers would not give a second thought to this. This animal appears regularly on price lists in Italian shops for wealthy aquarists and is also a much wanted item, dead or alive, for export to Asia. Even biologists and many other scientists from all over the world use this disreputable method to acquire specimens for their research. The live animal is also collected as a curiosity by aquarists in Italy and France and has been seen on sale in Paris.

It should be mentioned here that there is now an increasing number of "rogue cavers" visiting BiH for the purposes of specimen collection and uncontrolled recreational caving pursuits. Even reputable speleobiologists are directly or indirectly connected with unscrupulous specimen collectors. In this connection, there is one wealthy Slovak specimen collector who was, until recently, an annual visitor to the area of Eastern Hercegovina and Montenegrin Old Hercegovina. The Slovak collector (also a businessman) seems to specialise in collecting cave beetles such as Hadesia and Antroherpon. He fraternises with genuine speleologists and through this association is bringing everyone into disrepute. This individual is responsible for removing quantities of cave fauna from Hercegovina for no other reason than self-gratification and personal financial profit.
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Several amateur and professional so-called "speleobiologists" have also tried to persuade us that it is supposedly in the interest of scientific research that the endless repeated sampling of cave fauna species, some of whose survival status are already at the "endangered" status, together with their removal from Hercegovina year after year, is a necessary activity.
We ourselves are not persuaded of this. We reiterate that there can only be one responsible way forward in the strategy of researching the speleobiology of Hercegovina; that is to construct the appropriate facilities within Hercegovina and to undertake the work in Hercegovina.
Such work should be done in conjunction with a teaching facility such as a local or regional University, in order to ensure that local specialists are developed in preference to foreign ones. Some of these individuals have been witnessed by us as actually being responsible for removing quantities of cave fauna from Hercegovina for no other reason than academic self-gratification and / or personal financial profit.

"COLLECTING PROTEUS SPECIMENS FOR SCIENCE or FOR HELPING IT TOWARDS EXTINCTION?"
( *indicates the item is downloadable from Section 2. Bibliography on "Proteus Project" Webpage [2.].)
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In our deep-seated opinion, the amazing resources of modern science should be assisting with the prevention of harm to the critically endangered Proteus anguinus and should be focussed on efforts to protect the
species and its natural habitats. However, there is striking evidence that there are those in the scientific world who still employ the primitive approaches of the 18th and 19th centuries in their work to ingratiate themselves in what they regard as being justifiable research and claiming it to be of benefit to the conservation of the species.
However, not to be prudish about this, the rot began in the 18th century when Proteus was certainly far more plentiful in population numbers across its geographical range than it is now. So, we shall begin this tale of the unecessary death and slaughter of Proteus anguinus specimens by highlighting the published evidence, some of which can be downloaded from our extensive Bibliographical Section 2.

For each of the following pieces of referenced published work resulting from biological dissection or other biological investigation on living or dead specimens, there is a death toll of Proteus anguinus bodies.
Bearing in mind the changing attitudes in the intervening years from "human curiosity" and genuine "scientific interest" to that of the self-importance of "how many scientific papers can I publish", we leave you, our readers, to judge for yourselves as to whether any of the animal sacrifices was necessary and whether any of the results justified the respective deaths necessary to obtain those results.
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Extract from Published Reference 1. (2002)
BULOG, B., MIHAJL, K., JERAN, Z. and TOMAN, M.J. (2002).*.."Trace Element Concentrations in the Tissues of Proteus anguinus (Amphibia, Caudata) and the Surrounding Environment."
In:..Water, Air and Soil Pollution, 136., Extract from pp. 149-1??.
"MATERIALS AND METHODS : 

Extracts from Published Reference 2. (2005 relating to events on 17 July 1828.)
JUŽNIČ, S. (2005).*.."Babbage's Calculating Machines, The Proteus from Postojna Cave and The Carniolan Museum Society."
In:..Acta Carsologica, 34/1., Extracts from pp..213-214.
"BABBAGE IN POSTOJNA. During his travels, the famous Charles Babbage loved to collect things that were not directly connected with his own scientific pursuits, but might yet be highly interesting to other people he knew. If those findings were not too heavy, he was willing to purchase them because he was certainly rich enough. On one occasion Babbage visited Postojna (Adelsberg) in Carniola; he was not very fluent in the geography and in his letter sent back home put the town into neighboring Styria...... Babbage took a look at “human fishes” that he rightly recognized as the Proteus.
Babbage hired some local people to hunt as much samples of the Proteus as they could; eventually six was the highest number he could afford....... During the journey, Babbage placed the bottles in “large leathern bags lashed to the barouche seat of his calash”. During the nights, he usually placed the animals “in a large wash-hand basin of water, covered over with a napkin”. He frequently visited them with a candle during the nights. The animals behaved in an especially extraordinary manner during such night visits. One of them even jumped out from the vessel and received the real admiration of its new English owner. Babbage knew quite a deal about the famous Proteus anguinus and showed his samples to the people he met during his travels. The rare animals were matters of great interest to many naturalists Babbage visited in his rambles....... 
Babbage felt very sad when all of his Proteus anguinus died one after another during his voyage to Ljubljana, Vienna, Prague, and Berlin. The first of Babbage's new pets died at Vienna, and another in Prague. Just two were still alive in Berlin, among them probably his favourite one who used to jump out from the vessel......... So the Proteus anguinus “gloomy lives terminated”.....  Certainly, Babbage's butler was to blame for the tragedy of little animals; he changed their water from the ordinary kitchen pipe although he was supposed to use the river water (Shaw 1999, 240-244, 290-291)...... Babbage did not tell us the date of his visit in Postojna, but Shaw discovered his signature at the book of Postojna guests signed on July 17, 1828. Babbage's own story verified the date."
[Notes :  Clearly, Proteus was not considered as being "endangered" in the early 19th century, but was still regarded as being a rare species. A pointless destruction of another 6 protei by an Englishman!!]
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In:..Acta Carsologica, 34/1., Extracts from pp..216-217.
"On July 8, 1857, Dr. Karel Vesel  presented to the Museum two of Dr. Hyrtl's letters about the anatomy of Proteus anguinus from Črna jama in Postojna. Vesel received the letters a decade after Babbage's visit in 1842 and 1843, when the famous anatomist Hyrtl was still professor in Prague. The Italian Mauro Rusconi had previously discussed the same Proteus anatomy questions in his work that Vesel cited as Saggio sul proteo anguineo, probably the work published with a slightly different title in 1819 (Configliachi & Rusconi 1819). In the second letter, Hyrtl discussed his microscope observations.
He examined the organs of twelve of Vesel's excellent samples of the Proteus anguinus. Hyrtl later published the details of his discoveries in Müller's Archive. Hyrtl compared the skeleton of Proteus anguinus with Diluvium Ichthiosaurus (Ichtyosaurus) and another amphibian (Dežman 1858, 119-120)..... Back home again near the famous cave, [Vesel] grew interested in Proteus again and began his correspondence with somewhat younger Hyrtl. He sent several living Proteus of Črna jama to Hyrtl and therefore prepared his famous Bohemian friend for the anatomical discoveries."
[Notes :  So the Bohemian Czechs joined in the carnage at an early date!]
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Extract from Published Reference 3. (2010).
BIZJAK MALI, L., TALABER, I., ŽIBERT, U. & BULOG, B., (2010).*.."Oogenesis in Proteus: Stages of oocyte development."
In:..20th International Conference on Subterranean Biology, Postojna, Slovenia, 29 Aug - 03 Sept 2010. ICSB 2010 Abstract Book, Extract from pp.116-117, description of poster presentation.
"In comparison with the other amphibians Proteus has very extended reproductive period, continuing 30 years or more and it has an extremely long reproductive cycle, that is to say Proteus' female lays eggs at intervals of 6 years. Sexual maturity is also acquired very late, with males maturing earlier than females and it appears in 11 year-old males and 15 year-old females at 11-12°C. Opposite to the knowledge of reproduction, little is known about Proteus oogenesis; a process of differentiation and maturing of the oocyte, resulting in the mature ovum capable of ovulation. To gain a better understanding and elucidation of the Proteus reproductive cycles, we have begun with studies of its ovary and oogenesis. Ovaries of 30 females of Proteus anguinus anguinus, with snout-vent lengths of 225 - 270 mm were examined by light and transmission electron microscopy.
The gonad samples used in this research were taken from animals that had been collected in previous years (from 1972 to 2009) for other research purposes. ........"
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Published Reference 4. (2010).
15th – 18th September 2010: Scientific expedition to Bosnia and Herzegovina with  prof. dr. Boris Sket and doc. dr. Rok Kostanjšek; Dr Jure Jugovic
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Extract from Published Reference 5. (2013).
BIZJAK-MALI, L., SEPČIĆ, K. and BULOG, B. (2013).*.."Long-Term Starvation in Cave Salamander Effects on Liver Ultrastructure and Energy Reserve Mobilization."
In:..Journal of Morphology 00:00–00 (2013). Extract from pp. 
"MATERIALS AND METHODS : Proteus anguinus anguinus Laurenti, 1768 (Amphibia, Caudata) is a neotenic cave salamander living only in the underground waters of Dinaric Karst region from the Isonzo-Soča River in Italy in the northwest to the river Trebišnjica in Hercegovina in the southeast. Altogether almost 250 locations are known, most in Slovenia (Sket, 1997). It is the only vertebrate in Europe permanently living in the caves.
Adult specimens were collected in the Planina Cave (Planina, Slovenia) with the permission of the Ministry of the Environment and Spatial Planning of the Republic of Slovenia which administers rigorous national conservation laws aimed at protection of P. anguinus anguinus. The number of captured animals available for research is strictly limited and for this study, we used six animals with body lengths from 226 to 270 mm. All of them were sexually mature females with different oocytes stages in the ovarium. Vitellogenic stage IV oocytes were present in almost all females; only one female had previtellogenic stage II oocytes as the most mature oocyte stage. The animals were acclimated to laboratory conditions for three months before experimentation. Animals were kept in a speleological laboratory in darkness in experimental tanks containing aerated water at temperature of 10ºC and were fed with isopods once a week. After three months, two animals were sacrificed and served as controls. The other four animals were food deprived for four weeks (N52), and 18 months (N52).
SPECIMEN PREPARATION : All sampling was accomplished at midmorning. The animals were anaesthetized in 0.3 g/L tricaine methane sulphonate solution (Sandoz MS-222) before being sacrificed. Standard length and body mass were determi ned. The whole liver was removed and weighed for determination of the liver somatic index (liver mass 3 100/body mass). A part of the liver from three different regions (anterior, median, and posterior) from each animal was used for light and electron microscopic analysis, the remainder for determination of glycogen, lipid, and protein content."
[Notes :  Obviously, the Slovene Ministry responsible for giving permission is easily persuaded to do so!  Also, the claims that "....almost 250 locations are known, most in Slovenia..." are major inaccuracies.]