On the Theodoxus prevostianus (C. Pfeiffer, 1828) population of the Tükör Spring in Kács (Hungary)

: For the past nearly 15 years, we have been conducting freshwater malacological studies in the Kács spring area, a wetland of great importance in the South-Eastern Bükk (Hungary). The focus of our research is the species Theodoxus prevostianus (C. Pfeiffer, 1828). In the present study, population density records from the direct outlet of the tepidwater Tükör Spring are presented and compared. At the study site, the number of individuals of the accompanying malacofauna of the species under study was also recorded ( Microcolpia daudebartii (Prevost, 1821), Bythinella thermophila Glöer, Varga et Mrkvicka, 2015, Bythinella pannonica (Frauenfeld, 1865). The data were recorded in 2006 (general survey), 2010 (drastic reduction) and 2021 (relocation). On the stone slab squares used to record individual counts, we detected a high-density population in 2006, a drastically reduced population in 2010 and a high-density population again in 2021


Introduction
The Theodoxus prevostianus (C. Pfeiffer, 1828) (Gastropoda: Neritidae) is a rare, relict, endemic, highly protected freshwater snail species of the Pannonian biogeographical region (European Habitat Directive, Annex IV). Ovoid-hemispherical in shape, 6-9 mm wide by 2-6 mm high, with a shell of three convolutions, twisted to the right, bright black in colour (Fig. 1a), sometimes with dark purple hues providing a unique colouration (Fig. 1b). The last convolution becomes wider, and the aperture takes the shape of a crescent (Richnovszky & Pintér, 1979).
The few references in professional literature associate the species mainly with the direct outlet of tepid water (20-24ºC) karst springs. Earlier observations (Soós, 1943;Lukács, 1959) show that at lower water temperature ranges (min. 15.5ºC), the T. prevostianus is viable and able to reproduce.
They are typically found in the zones filled with solid bed material in the headwaters. They are present in high densities on the surface of the stones (Fig. 1c).
They are a dioecious species and they lay their eggs on the undersurface of stones or on the shells of other snails in the vicinity. Now only four natural populations are left in the world. In recent decades, its habitat has declined mainly due to human interventions (dredging, construction). On the IUCN Red List, the species is classified as "endangered".
In the Hungarian malacological bibliography from 1727 (Varga et al., 2005) onwards there have been several references to the population of T. pre- vostianus in Kács (Kormos, 1905a(Kormos, , 1905bSchréter, 1915;Wagner, 1927;Wagner, 1937;Vásárhelyi, 1956;Lukács, 1959;Varga, 1976). Apart from the data on presence, specific test results are reported in only a very limited number of publications. Such is the work of Lajos Soós, who introduced the species in the drainage ditch of the Roman Baths in Budapest in 1909, but unfortunately his initial success did not lead to lasting results (Soós, 1927). In 1955, Dezső Lukács and Imre Vajon moved some T. prevostianus individuals from Miskolctapolca to a "hot-water spring" in Eger. Unfortunately, the population did not survive (Lukács & Vajon, 1955). Zoltán Fehér et al. have enriched the understanding of the species with important phylogenetic research results (Fehér et al., 2007). A 2009 paper by Ioan Sîrbu and Anna Maria Benedek provided valuable information about the disappearance of the Răbăgani habitat (Sîrbu & Benedek, 2009).
The 10-12 springs of the spring group represent a range of water temperatures between 14 and 24°C, with significantly different water yields. Of these, the tepid water Tükör Spring (44 l/s) and the cold water spring of the Northern Regional Waterworks (80 l/s) have a more significant volume of water.
The Tükör Spring has its source in the side wall of a long tunnel under a Benedictine monastery built here hundreds of years ago (Fig. 1d, e). No longer in operation, the Benedictine monks established a significant spa culture in Kács.
In 1972, the cold spring was tapped. Unused water emerging from the spring house forms the cold water tributary After a distance of 100 m from their source, the two tributaries converge and continue their course under the name of Kács Stream (Fig. 1f).
The habitat of the T. prevostianus covers the tepid water tributary of the Tükör Spring and the first 800 m of the Kács Stream. Here, the spreading of the snails is physically hindered by a 3 m high waterfall. Note: recently the species has also been found in cold water springs, but the causal relationship is not addressed in this paper.
It is important to note that the tepid water spring is also home to the protected Microcolpia daudebartii (Prevost, 1821), and the recently described, new-toscience snail species Bythinella thermophila Glöer, Varga & Mrkvicka, 2015(Glöer et al., 2015. The Kács Spring waters are also inhabited by the protected Bythinella pannonica (Frauenfeld, 1865).
For the last 15 years we have been studying the distribution of the T. prevostianus population in the Kács springs. In 2010, we tried to establish a stable population of the species through habitat reconstruction.
In the study presented in this paper, we sought to answer the question of how many individuals of the T. prevostianus can populate (colonise) an area of a quadrate. This study is specifically designed to analyse the spring outlet of the Tükör Spring only. From the available results, it is not appropriate to draw general conclusions for the other sections of the tepid water tributary. Due to lack of space, it was not possible to set up more quadrates in the sample area, so no statistical analysis was performed. Our aim is to obtain informative data for habitat reconstruction studies of the tepid water spring.

Material and methods
At the exit of the tunnel, stone slab quadrates were placed perpendicular to the riverbed. For data collection, 2 observation sites were placed at the Tükör Spring on each test date (2006,2010,2021). Density counts were recorded by census from the surface of the 25×25 cm quadrates. The unevenness of the water surface and the sunlight that is refracted on it result in significant distortions and difficult calculability. To eliminate this problem, an empty aquarium was lowered 2 cm below the surface of the water above the snails to create a transparent mirror surface. In 2006, in the absence of suitable digital technology, the density counts were recorded on the spot. In 2010 and 2021, we were able to take underwater digital photos. The photos were overlaid with a spatial grid during computer processing to facilitate counting by individual censusing. Although the focus of our studies is on the T. prevostianus, we also recorded the density of the accompanying species at the sampling sites. Data were collected on a weekly to fortnightly basis for 3 months.

Results and discussion
By week 4 of our 2006 survey, approximately 300 T. prevostianus had colonised both quadrats (Table 1). In the 5-6th week of the tests, the snow started to melt in the mountains. Due to local orographic conditions, the meltwater is drained in a dry basin, which feeds into the bed of the Tükör Spring. The temperature of the 22°C spring water dropped by 6°C and the volume of water grew eightfold. The meltwater did not bring sediment with it. The snails partly drifted off the surface of the quadrates and partly sought shelter on the back walls of the quadrates, which were more protected from the current. From week 7, the bed was again filled only with tepid water from the Tükör Spring. From week 10 onwards, apart from fluctuations in density, no further increase was recorded. The surface of the quadrates provided a favourable habitat for 400-600 T. prevostianus.
In 2010, we were able to directly experience the habitat-destroying effects of the surplus water mentioned earlier, bringing with it a large mass of sediment. In this case, it was not meltwater, but a significant amount of precipitation that fell on the surrounding mountains and the spring area. The original volume of water in the Tükör Spring bed was increased by 15-20 times by the sudden downpour of rainwater. At the Tükör Spring and in the tepid water tributary draining its waters, 30-50 cm thick fine sediment was deposited. The deposition of sediment had an extremely negative impact on the entire habitat of the T. prevostianus. At the outlet of the Tükör Spring and its tepid water tributary, the solid, rocky substrate essential for the habitat needs of the T. prevostianus had completely disappeared. Even by our most conservative estimates, 95-99% of the snails had died.
At the Tükör Spring, the quadrate stones were placed in the muddy bed in early April, but no Neritidae appeared on the surface. On 25 April, the observation sites were moved closer to the shore of the riverbed, where we found T. prevostianus clinging to the overhanging riparian foliage and an artificial structure (a drinking water pipe in the riverbed) (Fig.  3a).
On average, only 15-30 individuals settled on the quadrates in 2010. From the second week onwards,   the number of individuals did not change, apart from minor fluctuations, and the steady upward trend of the previous period was not observed (Table 2).
By 2021, the fine sediment in the bed of the Tükör Spring had been completely washed away, and the bed is now covered by a stony substrate. Note: only  partial washout was observed in the tepid water tributary. The colonisation of the quadrates was continuous and uninterrupted. Maximum densities were recorded at the survey sites in week 12. With minor fluctuations, 300-400 T. prevostianus settled on the quadrate surfaces during this test cycle (Table 3).
The changes in the number of the individuals of T. prevostianus, based on the tables and explanations presented above, are also represented with a summary diagram (Fig. 4).
Although the analysis of the accompanying fauna is not the subject of this article, we cannot but note some findings.
Microcolpia daudebartii is found in abundance at the Tükör Spring and in the entire Kács habitat, mainly in the fine sediment zones. However, on all three study dates, active presence was observed for the first 4-5 weeks on all quadrates set out, followed by a steady decline in numbers and then total disappearance from the test sites. Particularly high densities of this species were recorded during the 2010 study. Whether this is due to the absence of the T. prevostianus, requires further analysis. In any case, we are experiencing a similar phenomenon in our current ongoing studies.
Bythinella thermophila and Bythinella pannonica as Bythinella sp. are listed in the recorded data. The differentiation between the two species is extremely difficult from a shell morphological point of view, and impossible without manual handling of the individuals.
The B. thermophila was described only in 2015, from this very branch of the tepid water spring of the Kács Tükör Spring (Glöer et al., 2015). No studies have been carried out so far and we have no factual data on the species. It is important to clarify whether the Bythinella population here is exclusively, or to a lesser or greater extent, thermophila or pannonica. One wonders, if it is a newly described species, how it could have spread in such a short time. It also makes one wonder if B. pannonica is also present in the snail population of the Tükör Spring, as it has only been known in the literature to occur in colder spring waters.
In 2006 and 2010, no Bythinella species were detected at the Tükör Spring during the data collection. In 2021, we recorded surprisingly large numbers, sometimes exceeding those of the T. prevostianus (Fig. 3b). Their study is extremely important because it represents a new element in the direct outlet of the Kács Tükör Spring. Note: Until 2010, we had only observed Bythinella in the immediate vicinity of the mouths of a few subaqueous creek springs in the tepid water tributaries, which we then assumed to be B. pannonica. Dezső Lukács had already issued a publication about it in the late 1950s under the species designation "Bythinella austriaca" (Lukács, 1959). The snail prefers the surface of stones as its habitat, so it may have a significant impact on the population of the T. prevostianus here. With regard to Bythinella, we will carry out further investigations in cooperation with a researcher specialised in Hydrobiidae.

Conclusion
The Theodoxus prevostianus (C. Pfeiffer, 1828) form a stable population at the outlet of the Tükör Spring under undisturbed hydroecological conditions. Meteorological and orographic factors in the Kács spring area can adversely affect this stability. After the solid surfaces important for the T. prevostianus were cleared, the relocation processes were activated. Since 2010, no episodes of rainwater runoff and sedimentation have been observed, but the risk remains. The results of the study will certainly provide useful guidance for our further explorations. The study of the rare snail fauna of the Kács spring area requires very complex planning and implementation, which we are striving to achieve.