ISSN 0392-6672

International Journal of Speleology

Official Journal of the Union Internationale de Spéléologie

Volume 42 (1) - January 2013 International Journal of Speleology Scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it Volume 42(1), January 2013

Original papers

Enrique P. Sánchez-Cañete, Penélope Serrano-Ortiz, Francisco Domingo, and Andrew S. Kowalski

Cave ventilation is influenced by variations in the CO2-dependent virtual temperature...... 1-8

Angela M. Garcia-Sanchez, Concepcion Ariza, Jose M. Ubeda, Pedro M. Martin-Sanchez, Valme Jurado, Fabiola Bastian, Claude Alabouvette, and Cesareo Saiz-Jimenez Free-living amoebae in sediments from the Lascaux Cave in France...... 9-13

Valquíria Aparecida Alves Bastos, Rodrigo Lopes Ferreira, Daniel Cardoso de Carvalho, Marina Lages Pugedo, and Luciana de Matos Alves Pinto The cave environment influencing the lipid profile and hepatic lipogenesis of the fish Ancistrus cryptophthalmus Reis, 1987 (Siluriformes: Loricariidae)...... 15-23

Emma Petrella and Fulvio Celico Mixing of water in a carbonate aquifer, southern Italy, analysed through stable isotope investigations...... 25-33

Naowarat Cheeptham, Tara Sadoway, Devon Rule, Kent Watson, Paul Moote, Laiel C. Soliman, Nicholas Azad, Kingsley K. Donkor, and Derrick Horne Cure from the cave: volcanic cave actinomycetes and their potential in drug discovery...... 35-47

Andrew C. Smith, Peter M. Wynn, and Philip A. Barker Natural and anthropogenic factors which influence aerosol distribution in Ingleborough Show Cave, UK...... 49-56

Bianca Rantin and Maria Elina Bichuette Phototactic behaviour of subterranean Copionodontinae Pinna, 1992 catfishes (Siluriformes, Trichomycteridae) from Chapada Diamantina, central Bahia, northeastern Brazil...... 57-63

Iva Tomova, Irina Lazarkevich, Anna Tomova, Margarita Kambourova, and Evgenia Vasileva-Tonkova Diversity and biosynthetic potential of culturable aerobic heterotrophic bacteria isolated from Magura Cave, Bulgaria...... 65-76

Karen J. Vanderwolf, David Malloch, Donald F. McAlpine, and Graham J.Forbes A world review on fungi, yeasts, and slime molds in caves...... 77-96 World Karst Science Reviews Cave and Karst Science of the British Research Association Volume 39(2), 2012...... I

Die Höhle des Verbands Österreichischer Höhlenforscher und des Verbands der Deutschen Höhlen- und Karstforscher e.V. Volume 63(1-4), 2012...... I

Journal of Cave and Karst Studies of the National Speleological Society Volume 74(1), April 2012...... II Volume 74(2), August 2012...... II Volume 74(3), December 2012...... III International Journal of Speleology 42 (1) 1-8 Tampa, FL (USA) January 2013

Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Official Journal of Union Internationale de Spéléologie

Cave ventilation is influenced by variations in the CO2-dependent virtual temperature Enrique P. Sánchez-Cañete1,2*, Penélope Serrano-Ortiz1,2, Francisco Domingo1,3, and Andrew S. Kowalski2,4 1 Departamento de Desertificación y Geo-ecologia, EEZA-CSIC, Ctra. Sacramento s/n, 04120, La cañada de San Urbano, Almería, Spain 2 Centro Andaluz de Medio Ambiente (CEAMA), 18006, Granada, Spain 3 Departamento de Física Aplicada, Universidad de Granada, Av. Fuentenueva s/n, 18071 Granada, Spain 4 Universidad Almería, Departamento Biología Vegetal & Ecología, E-04120 Carrera Sacramento s/n, 04120, Almería, Spain

Abstract: Dynamics and drivers of ventilation in caves are of growing interest for different fields of science.

Accumulated CO2 in caves can be exchanged with the atmosphere, modifying the internal CO2 content, affecting stalagmite growth rates, deteriorating rupestrian paintings, or creating new

minerals. Current estimates of cave ventilation neglect the role of high CO2 concentrations

in determining air density – approximated via the virtual temperature (Tv) –, affecting

buoyancy and therefore the release or storage of CO2. Here we try to improve knowledge and

understanding of cave ventilation through the use of Tv in CO2-rich air to explain buoyancy for

different values of temperature (T) and CO2 content. Also, we show differences between T

and Tv for 14 different experimental sites in the vadose zone, demonstrating the importance

of using the correct def inition of Tv to determine air buoyancy in caves. The calculation of Tv

(including CO2 effects) is currently available via internet using an Excel template, requiring the

input of CO2 (%), air temperature (ºC) and relative humidity (%).

Keywords: buoyancy; carbon dioxide; caves; soil ventilation; ventilation; virtual temperature Received 26 June 2012; Revised 4 September 2012; Accepted 11 September 2012

Citation: Sánchez-Cañete E.P., Serrano-Ortiz P., Domingo F. and Kowalski A.S. 2013. Cave

ventilation is inf luenced by variations in the CO2-dependent virtual temperature. International Journal of Speleology, 42 (1), 1-8. Tampa, FL (USA) ISSN 0392-6672 http://dx.doi.org/10.5038/1827-806X.42.1.1 INTRODUCTION al., 2009; Serrano-Ortiz et al., 2010; Sanchez-Cañete

et al., 2011), modifying the internal CO2 content and There is currently growing interest in characterizing affecting stalagmite growth rates (Banner et al., 2007; storage and ventilation of CO2 in caves, both from Baldini et al., 2008), deteriorating rupestrian paintings external (atmospheric) and internal (speleological) (Fernández et al., 1986) and creating new minerals perspectives. Measurements of rising atmospheric (Badino et al., 2011). However due to the complexity th CO2 by Keeling (1960) since the mid-20 century and peculiarity of caves, as well as the variety of reveal that anthropogenic activities are causing CO2 meteorological conditions that determine the degree accumulation in the atmosphere and forcing global and timing of ventilation (Fairchild & Baker, 2012), warming. Soils are a large pool of terrestrial carbon such exchanges are not well understood and their

(C), estimated to contain 2344 Pg C in solid form in contributions to regional atmospheric CO2 budgets the top 3 m (Jobbagy & Jackson, 2000) – three times remain unknown. the aboveground biomass C reservoir and double Estimation of cave ventilation can be realized by that of the atmosphere (Schlesinger, 1997) – and a number of means, the most common of which also have an enormous capacity to store gaseous has traditionally neglected the role of high CO2

CO2 in subsurface cracks, pores and cavities. The concentrations and requires ref inement. The drivers vadose zone is enriched in CO2 and some caves often implicated in the cave ventilation can be classif ied exceed 5% (volumetric CO2 fraction of 50,000 ppm; as either dynamic or static (Cigna, 1968). Dynamic Ek & Gewelt, 1985; Howarth & Stone, 1990; Denis drivers are defined by moving fluids such as water et al., 2005; Batiot-Guilhe et al., 2007; Benavente et or wind (Nachshon et al., 2012), while static drivers al., 2010) representing important air compositional include variations of pressure, temperature or air differences with respect to the external atmosphere, composition (water vapor, CO2, CH4, etc.). Ventilation currently near 395 ppm. Accumulated CO2 in caves rates can be measured directly using anemometers, can be exchanged with the atmosphere (Weisbrod et estimated indirectly through variations in Radon

*[email protected], [email protected] Cave ventilation is influenced by the virtual temperature 2 content (Hakl et al., 1997; Faimon et al., 2006), or and thus can determine exactly when ventilation other tracer gases (de Freitas et al., 1982) or variations is possible, and therefore when a cave can release in air density. Most commonly, air density variations or store CO2. Also we represent Tv -explaining the are approximated to evaluate buoyancy according to relative buoyancy relevant for cave ventilation- for temperature differences between the internal (Tint ) and different values of T and CO2 content. Then, we show exterior atmosphere (Text ), neglecting air composition differences between T and Tv , -calculated both with and

(Fernàndez-Cortes et al., 2006; Baldini et al., 2008; without accounting for CO2 content- for 14 different Liñan et al., 2008; Milanolo & Gabrovšek, 2009; experimental sites in the vadose zone, demonstrating

Faimon et al., 2012). Faimon et al. (2012) modelled the the importance of using the correct def inition of Tv to airflows into a cave, and found that the temperature determine air buoyancy in caves. explained more than 99% of variations in air density; therefore, temperature could be used as an alternative DERIVATIONS AND DEFINITIONS airflow predictor. However, de Freitas et al. (1982) concluded that reversal of airflow occurs when the For purposes of characterizing air buoyancy, densities in the cave and the exterior are equal, rather meteorologists define the virtual temperature (Tv ) as the than when thermal conditions of the cave and external temperature that dry air must have to equal the density of air are the same. For this reason, they suggest that the moist air at the same pressure. The virtual temperature gradient in virtual temperature (Tv ) between the cave for the atmosphere is approximated as (see appendix A): and outside air would be the appropriate indicator. In this sense, Kowalczk & Froelich (2010), improved (1) the determination of internal /external air densities where T and Tv are the absolute temperature (K) and by including the influence of water vapor, using virtual temperature (K) respectively and r is the mixing the traditional definition of the virtual temperature. ratio (dimensionless), defined as the ratio of the mass

Nevertheless, in cases where CO2 molar fractions of of water vapor to that of dry air. internal air exceed atmospheric values by an order Thus variations in the virtual temperature serve as a of magnitude or more, it is necessary to take into proxy for those in air density (Stull, 1988), which can account the heaviness of CO2 when calculating the be obtained through the equation of state for moist air: virtual temperature (Kowalski & Sanchez-Cañete, 2010). (2) -3 Whereas high CO2 values registered in cave air have where p, ρ and Rd are the pressure (Pa=J m ), air -3 been attributed most often to the seepage of CO2- density (kg m ) and particular gas constant for dry enriched water from the root zone, the possibility of air (286.97 J kg-1 K-1) respectively. Equation (2) makes sinking flows of dense, CO2-rich air should also be clear that, for a given altitude level (pressure), air considered. Biological CO2 is produced near the surface density is related directly to Tv , which serves therefore by respiration of plant roots and microorganisms as a surrogate variable for determining buoyancy. (Kuzyakov, 2006); in most caves, isotopic studies Equation (2) is only valid for the free atmosphere, confirm a clear biological origin of cave CO2 (Bourges while for caves or soils it should not be used due to et al., 2001, 2012). Soil CO2 generally increases with high concentrations of CO2 in the air. This equation, depth, from near-atmospheric concentrations at a normally used for assessing the buoyancy of an air mass few centimeters to an order of magnitude more a few by changes in its density, is valid in the atmosphere meters down (Amundson & Davidson, 1990, Atkinson, because the molar mass of dry air (md ) is very constant, -1 1977). High concentrations of CO2 at depth have 0.02897 kg·mol , since air composition is very constant been explained in terms of shallow CO2 dissolution, once water vapor has been excluded. However the air downward transport by seepage, and subsequent composition in soils or caves differs from that of the precipitation from water in deeper layers (Spötl et atmosphere due to higher amounts of CO2. al., 2005), whereas surface layers are depleted in CO2 The correct equations to calculate the virtual by exchange with the atmosphere. At depth and for temperature including CO2 effects were developed caves in particular, another input of CO2 could be due by Kowalski & Sanchez-Cañete (2010). Frequently to the injection of dense, CO2-rich air, f lowing down caves exhibit values exceeding 0.4% in volumetric through fissures due to differences in buoyancy, fraction of CO2, ten times the atmospheric whose characterization is poorly known and requires concentration (Howarth & Stone, 1990; Denis et information regarding Tv. This virtual temperature has al., 2005; Batiot-Guilhe et al., 2007; Benavente been little applied to soils and caves, but could explain et al., 2010). This CO2 increment with respect to why CO2 accumulates at depth yielding concentrations atmospheric concentrations provokes changes in much higher than those in the atmosphere. the composition of dry air and its molar mass (md ) Here we show the error produced in determining so that the definition of the virtual temperature the virtual temperature when not taking into account in eq. (1) is inappropriate. An approximation to

CO2 effects, and demonstrate its repercussions for calculate the virtual temperature (Tv ) including CO2 the determination of air buoyancy in caves. We try effects is via the following equation (see appendix): to improve knowledge and understanding of cave ventilation through the use of virtual temperature (3) in CO2-rich air. Accurate determinations of virtual where rv and rc are the water vapour and carbon dioxide temperature allow numerical evaluation of buoyancy, mixing ratios respectively (dimensionless).

International Journal of Speleology, 42 (1), 1-8. Tampa, FL (USA) January 2013 Sánchez-Cañete et al. 3 Therefore for determining air density in caves or temperatures is commonly used (Spötl et al., 2005; soils including CO2 effects, the virtual temperature Fernàndez-Cortes et al., 2006, 2009; Baldini et al., can be used in the ideal gas law with the particular 2008; Liñan et al., 2008; Milanolo & Gabrovšek, -1 -1 gas constant (Rnoa, 287.0 J K kg ) for the mixture 2009; Faimon et al., 2012). Thus, with similar of nitrogen (N2), oxygen (O2), and argon (Ar). values of Tex t and Tin t, the differences between virtual temperatures can be more than 10ºC (Fig. 2).

(4) Fixing the internal CO2 content (e.g., at 3% to This parameter can be computed exactly using an continue with the example presented above), we Excel template found at http://fisicaaplicada.ugr. can analyze differences between external and es/pages/tv/!/download, where it is only necessary internal virtual temperatures (Fig. 3) at different to enter values of CO2 (%), air temperature (ºC) and temperatures. Negative values indicate that relative humidity (%). the interior air is denser than the exterior and therefore stagnant. For example, a cave with 3%

RESULTS AND DISCUSSION CO2, 100% RH and 10ºC presents neutral buoyancy when the external temperature is approximately The difference between internal (cave) and 6ºC. Consistent with the results of Fig. 2, when external (atmosphere) virtual temperatures can both cave and external atmosphere are at 10ºC, be used to determine the potential for buoyancy Tv is lower than T by 4.3ºC. Higher values of the flows. The virtual temperature is a variable used external temperature imply stagnant air inside the traditionally by meteorologists to determine air cave. buoyancy. Knowing the internal and external virtual Quantifying T v –T for specific caves temperatures allows determination of air densities To determine when buoyancy flows are possible, (using equation 4) and therefore calculation of scientists must compare the differences between the possibility of buoyancy f lows. The following results are organized into two sections. First, general differences between T and Tv , including

CO2 effects and comparing the interior and exterior environments, are presented to highlight the importance of using the appropriate variable (Tv ) to characterize air density. Then, differences are shown for the conditions of specif ic caves selected from the literature.

Quantifying Tv –T for caves in general Differences between air temperature (T) and virtual temperature (Tv ) (eq. 3) at different volumetric fractions of CO2 are shown in Fig. 1, assuming 100% relative humidity (RH) as is typical for internal conditions. To give an example, a cave with 3% CO2, and 10ºC would have a Tv 3ºC lower than T (see dashed lines). Positive values (orange Fig. 1. Isopleths of the difference between the virtual temperature and color) indicate that the virtual temperature is temperature (Tv - T), as a function of volumetric fraction CO2 higher than the temperature due to the dominant (1% = 10.000 ppm) and temperature, for 100% of RH. influence of water vapor on compositionally determined air density (for low CO2 concentrations). This is greater for higher temperatures since warm air can store more water vapor than cold air, decreasing the molar mass below that of dry air (28.96 g mol-1) due to the increased importance of water vapor (18 g mol-1), thus reducing the density. Figure 2 shows that whenever the internal and external atmospheres have the same temperature and relative humidity, higher values of CO2 inside the cave explain stagnation of the cave environment.

For example, a cave with 3% CO2 and exterior and interior temperature of 10ºC would have a virtual temperature 4.3ºC colder than that of the external air; consequently the internal air is denser than the exterior and therefore stagnant. Whereas differences between external and Fig. 2. Isopleths of the difference between virtual temperatures of internal virtual temperatures are necessary for exterior and interior (Tvint - Tvext) both with identical temperature and the correct interpretation of cave ventilation, 100% of RH, as a function of temperature and volumetric fraction the difference between external and internal CO2. The exterior is considered to contain 0.0395% CO2.

International Journal of Speleology, 42 (1), 1-8. Tampa, FL (USA) January 2013 Cave ventilation is influenced by the virtual temperature 4 Virtual temperature differences between the exterior and interior are compared to distinguish between periods of stagnant versus buoyant cave air for each

experimental site, using their maximum values of CO2 and the mean T (Fig. 5). Differences between the virtual

temperatures (Tv_ext-Tv_int) increase with increasing CO2

molar fraction and therefore higher CO2 implies greater differences between internal and external densities, with the internal air denser than the external air and therefore causing stagnation (in the case of a cave lying below its entrance). For example, the Natural

Bridge Caverns (I) with 4% CO2 presents a difference of 6 ºC between the (mean annual) external and internal virtual temperatures. Therefore, the internal air is denser than that of the external atmosphere (on average), inducing its stagnation and explaining the Fig. 3. Isopleths of the difference between the virtual temperature of storage of CO2. In the Nerja cave borehole (M), with interior and exterior with 3% CO2 (Tvint – Tvext), each at 100% of RH, as a function of the internal and external temperatures. 6% and 0.0395% CO2 for the internal and external atmosphere, respectively, the virtual temperature in external and internal virtual temperatures. Thus, if the borehole is 8.9ºC lower than the outside (Fig. 5). virtual temperatures are equal (independent of the This difference in Tv implies that the internal air is amount of water vapor or CO2) both air masses will be denser, inhibiting convective ventilation. Therefore, in equilibrium. On the other hand, if air mass A has researchers who use differences between exterior/ a virtual temperature higher than air mass B, then interior air temperatures to determine ventilation, may air mass A will have the lower density. In this way, find differences between virtual temperatures close to comparing the virtual temperatures of air masses 9 °C, when the exterior/interior air temperature is the specifies their relative densities (from eq. 4) and thus same in both, and therefore over- or under-estimate the tendency to float or sink. the ventilation periods.

Maximum CO2 values and mean temperatures Differences between virtual temperatures in two air of 14 caves and boreholes of the world are shown masses indicate density differences between both, and in Table 1. These published data were taken as thus the potential for ventilation due to buoyancy. examples to calculate the differences between the However, two possible issues must be considered that virtual temperature and temperature. Although such hamper or facilitate ventilation of the cave. The first differences also depend on the temperature, caves in includes atmospheric conditions such as the wind excess of 1% CO2 generally present negative differences (Kowalczk & Froelich, 2010) and pressure changes between Tv and T, while lower values of CO2 present (Denis et al., 2005; Baldini et al., 2006) inside and positive differences (Fig. 4). However, for example, outside of the cave. The relevancy of buoyancy-induced the subtropical Hollow Ridge cave (D) presents more cave ventilation is greatest on days with atmospheric positive values of Tv-T than does the temperate Císařská stability, where there are little pressure changes and cave (C), despite similar volumetric fractions of CO2 low winds. During these days static processes (Cigna, (0.42 and 0.4%, respectively). Such differences are 1968) are dominant. The second issue is the number due to differences in water vapor content, according of entrances to the cave and their different altitudes to temperature (19.6 versus 9.6º C in Hollow Ridge and orientations (up or down). In caves with a single and Císařská cave, respectively). entrance the air will flow inward along the floor or roof, Table 1. Published carbon dioxide concentrations in cavity airspaces (*estimates).

Cave/ % vol. CO Mean temperature Country Name 2 ID Source Soil (Maximum) (ºC) Italy Cave Grotta di Ernesto 0.170* 8* A (Frisia et al., 2011) Bosnia and Herzegovina Cave Srednja Bijambarska Cave 0.220 6.2 B (Milanolo & Gabrovšek, 2009)

Czech Republic Cave Císařská Cave 0.4 9.6* C (Faimon & Licbinska, 2010)

USA (Florida) Cave Hollow Ridge Cave 0.422 19.6 D (Kowalczk & Froelich, 2010) Spain Cave Cave Castañar de Ibor 0.44 17 E (Fernández-Cortes et al., 2009) Ireland Cave Ballynamintra Cave 0.65 11.5 F (Baldini et al., 2008) Spain Borehole Sierra de Gádor 1.5 12 G (Sanchez-Cañete et al., 2011) France Cave Aven d’Orgnac 3.5 13 H (Bourges et al., 2001) USA(Texas) Cave Natural Bridge Caverns *4 20* I (Wong & Banner, 2010) Spain Cave Cova de les Rodes 4.9 17.2 (other sources) J (Ginés et al., 1987) Australia Cave Bayliss Cave 5.9 26 K (Howarth & Stone, 1990) France Skinhole Causse d’Aumelas 6 15.3 (other sources) L (Batiot-Guilhe et al., 2007) Spain Borehole Cave of Nerja 6 21 M (Benavente et al., 2010) France Cave Cave of Lascaux 6 17.6 (other sources) N (Denis et al., 2005)

International Journal of Speleology, 42 (1), 1-8. Tampa, FL (USA) January 2013 Sánchez-Cañete et al. 5

Fig. 5. Volumetric fraction of CO2 (%, dark gray) and differences between the virtual temperature of interior and an exterior (T -T Fig. 4. Volumetric fraction of CO (%, dark gray) and differences v_int v_ext 2 in ºC, light gray) in the different cavities. Both internal and external between virtual temperature (T ) and temperature (T), (ºC, light gray) v air are assumed to have the same temperature, which is the annual in the different cavities. mean for the cave. and return outward along the roof or floor, according REFERENCES to the sign of the density difference. However if the cave has many entrances at different levels, it may Amundson R. G. & Davidson E. A., 1990 - Carbon be necessary to monitor more than one entrance dioxide and nitrogenous gases in the soil atmosphere. (Cigna, 1968). Due to the strong spatial variability of Journal of Geochemical Exploration, 38: 13-41. the temperature, simply knowing T at a single point http://dx.doi.org/10.1016/0375-6742(90)90091-N v Atkinson T. C., 1977 - Carbon-dioxide in atmosphere of inside (and outside) the cave may not necessarily be unsaturated zone - important control of groundwater sufficient for determining the potential for ventilation. hardness in limestones. Journal of Hydrology, 35: 111- 123. http://dx.doi.org/10.1016/0022-1694(77)90080-4 CONCLUSIONS Badino G., Calaforra J. M., Forti P., Garofalo P. & Sanna L., 2011 - The present day genesis and evolution of cave We used the information of several caves together minerals inside the Ojo de la Reina Cave (Naica Mine, with gas law to demonstrate that the difference between Mexico). International Journal of Speleology, 40: 125- external and internal virtual temperatures including 131. http://dx.doi.org/10.5038/1827-806X.40.2.5 CO effects determines the buoyancy and should be Baldini J. U. L., Baldini L. M., McDermott F. & Clipson 2 N., 2006 - Carbon dioxide sources, sinks, and spatial used for the correct interpretation of cave ventilation. variability in shallow temperate zone caves: Evidence Often scientists estimate ventilation neglecting CO2 from Ballynamintra Cave, Ireland. Journal of Cave and effects, but this can cause errors close to 9 °C in Karst Studies, 68: 4-11. the difference between external and internal virtual Baldini J. U. L., McDermott F., Hoffmann D. L., temperatures when the air temperature is the same in Richards D. A. & Clipson N., 2008 - Very high- both. Thus, the common use of the difference between frequency and seasonal cave atmosphere P(CO2 ) external and internal temperatures could over- or variability: Implications for stalagmite growth and under-estimate the existence of ventilation processes, oxygen isotope-based paleoclimate records. Earth depending on CO content and relative humidity. and Planetary Science Letters, 272: 118-129. 2 http://dx.doi.org/10.1016/j.epsl.2008.04.031 Banner J. L., Guilfoyle A., James E. W., Stern L. A. & ACKNOWLEDGEMENTS Musgrove M., 2007 - Seasonal variations in modern speleothem calcite growth in Central Texas, USA. This research was funded by the Andalusian Journal of Sedimentary Research, 77: 615-622. regional government project GEOCARBO (P08- http://dx.doi.org/10.2110/jsr.2007.065 RNM-3721) and GLOCHARID, including European Batiot-Guilhe C., Seidel J. L., Jourde H., Hebrard O. & Union ERDF funds, with support from Spanish Bailly-Comte V., 2007 - Seasonal variations of CO2 and Ministry of Science and Innovation projects Carbored- Rn222 in a mediterranean sinkhole - spring (Causse d’Aumelas, SE France). II (CGL2010-22193-C04-02), SOILPROF (CGL2011- International Journal of Speleology, 36: 51-56. 15276-E) and CARBORAD (CGL2011-27493), as well Benavente J., Vadillo I., Carrasco F., Soler A., Liñan C. & as the European Community’s Seventh Framework Moral F., 2010 - Air Carbon Dioxide Contents in the Vadose Programme (FP7/2007-2013) under grant agreement Zone of a Mediterranean Karst. Vadose Zone Journal, 9: n° 244122. The authors thank three anonymous 126-136. http://dx.doi.org/10.2136/vzj2009.0027 reviewers for constructive comments that helped to Bourges F., Genthon P., Genty D., Mangin A. & d’Hulst D., substantially improve the manuscript. 2012 - Comment on “Carbon uptake by karsts in the Houzhai Basin, southwest China” by Junhua Yan et al. Journal of Geophysical Research-Biogeosciences, 117: G03006. Bourges F., Mangin A. & d’Hulst D., 2001 - Carbon dioxide in karst cavity atmosphere dynamics: the example of the Aven d’Orgnac (Ardeche). Comptes Rendus de l’ Académie des Sciences, Serie II Fasc. a, Sciences de la Terre et des Planètes, 333: 685-692.

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Villar E., 1986 - Natural ventilation of the Paintings - Hidden, abiotic CO2 flows and gaseous reservoirs in Room in the Altamira cave. Nature, 321: 586-588. the terrestrial carbon cycle: Review and perspectives. http://dx.doi.org/10.1038/321586a0 Agricultural and Forest Meteorology, 150: 321-329. Frisia S., Fairchild I. J., Fohlmeister J., Miorandi R., Spötl http://dx.doi.org/10.1016/j.agrformet.2010.01.002 C. & Borsato A., 2011 - Carbon mass-balance modelling Spötl C., Fairchild I. J. & Tooth A. F., 2005 - Cave air control on and carbon isotope exchange processes in dynamic dripwater geochemistry, Obir Caves (Austria): Implications for speleothem deposition in dynamically ventilated caves. caves. Geochimica Et Cosmochimica Acta, 75: 380-400. Geochimica Et Cosmochimica Acta, : 2451-2468. http://dx.doi.org/10.1016/j.gca.2010.10.021 69 http://dx.doi.org/10.1016/j.gca.2004.12.009 Ginés A., Hernández J., Ginés J. & Pol A., 1987 - Stull R. B., 1988. An introduction to Boundary Layer Observaciones sobre la concentración de dióxido de Meteorology. Kluwer Academic Publishers, Dordrecht, carbono en la Atmósfera de la Cova de les Rodes. The Netherlands, 667p. Endins, 13: 27-38. Weisbrod N., Dragila M. I., Nachshon U. & Pillersdorf Hakl J., Hunyadi I., Csige I., Geczy G., Lenart L. & M., 2009 - Falling through the cracks: The role Varhegyi A., 1997 - Radon transport phenomena studied of fractures in Earth-atmosphere gas exchange. in karst caves - International experiences on radon levels Geophysical Research Letters, 36: L02401. and exposures. Radiation Measurements, 28: 675-684. http://dx.doi.org/10.1029/2008GL036096 http://dx.doi.org/10.1016/S1350-4487(97)00163-7 Wong C. & Banner J. L., 2010 - Response of cave air

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International Journal of Speleology, 42 (1), 1-8. Tampa, FL (USA) January 2013 Sánchez-Cañete et al. 7 Appendix

The starting point for deriving the virtual temperature and eliminating volume, which is identical for both the is the ideal gas law: mixture and any individual component, it is found that:

(A1) (A11) where p, V, n, R* and T are the pressure (Pa), volume showing that the effective particular gas constant (m3), number of moles (moles), universal gas constant for a mixture (such as dry air) can be calculated by (8.314 m3 Pa K-1 mol-1) and absolute temperature (K) the (mass) weighted combination of the particular of the gas, respectively. Since the number of moles (n) constants for the individual components. To determine is equal to the mass (m, in kg) divided by the molar Rd it is necessary to know the composition of the dry mass (M, in kg mol-1), equation 1 can be written as: atmosphere, by mass (Table A1).

Table A1. Components of dry air with their particular gas constants and fractional contribution (A2) by mass (NOAA et al., 1976). Individual gas constant Gas % Mass Ri (J kg-1 K-1) By substituting the density, ρ = m V this can be N2 296.7 75.52 rewritten in the form: O 259.8 23.15 2 Ar 208.1 1.28 (A3) CO 188.9 0.05 When defining the particular gas constant (R) as 2 R=R*/M, the equation of state for the atmosphere can be written in the form convenient for meteorologists Substituting into equation A11: as: 75.52 ⋅ 296.7 + 23.15 ⋅ 259.8 +1.28 ⋅ 208.1+ 0.05 ⋅188.9 −1 −1 Rd = = 286.97 ≅ 287JKg K (A4) 100 where p, ρ, R and T are the pressure (Pa=J m-3), Moist air density (kg m-3), particular gas constant (J kg-1 K-1) and The equation of state for moist air is given in absolute temperature (K) of the gas, respectively. equation A5.d. The moist air density can be written The individual gas laws for any gas (suff ix “i”), using equation A6: including the mixture defined as dry air (suff ix “d”), (A12) water vapor (suffix “v”) and another mixture defined as moist air (suffix “m”) are described in the following where ρv and ρd are the densities of water vapor and equations dry air respectively. Using equations A11 and A12, (A5.a) and the principle that the effective particular gas constant for the mixture (now moist air) is the (mass) weighted combination of the particular constants for (A5.b) the individual components, yields the gas constant of

moist air (Rm), (A5.c) (A13)

Multiplying by  1  (A5.d)  m   d  Dry air 1  m  The equation of state for dry air is shown in equation  d  produces (A5.b), where water vapor is excluded from the air mass. Here, Rd is the gas constant for dry air, which can be determined by using the principle of mass conservation (A14) (A6) Considering that the mixing ratio (r) is defined as

and Dalton’s law of partial pressures: the ratio of the mass of water vapor (mv ) to that of dry mv air (md) r ≡ , and substituting this into equation A14 md (A7) gives By combination of equations A5.a, A6 and A7 we obtain (A15) (A8), To simplify, equation A15 is multiplied by 1− r    which compared with equation A5.b yields to give: 1− r 

(A9) (A16) The denominator of equation A16 can be approximated Replacing the density ( ρ = m V ) in equation A9 as unity when recognizing that every second-order term

is several orders of magnitude smaller. The numerator can be similarly simplified, leading to the following approximation: (A10) (A17).

International Journal of Speleology, 42 (1), 1-8. Tampa, FL (USA) January 2013 Cave ventilation is influenced by the virtual temperature 8

To simplify, the second term is The errors that arise when using equation multiplied by A25 as an approximation to T (including  R  v  d  CO effects) were evaluated explicitly for the R 2  d  range of gas concentrations typically found in terrestrial caves and found to be less than

(A18) 0.1% (0.3 K) for volumetric CO2 fractions of up

Substituting the gas constants of dry air (Rd=287 to 5%. -1 -1 J kg K ) and water vapor (Rv=461.51 J kg-1 K-1), For a calculation without error, the virtual equation A18 can be written and organized as: temperature should be defined using the particular gas constant for the mixture of moist

(A19) air including high concentrations of CO2, denoted

as Rmc and defined as: (A20)

(A21) (A26)

where Rnoa is the particular gas constant for the

(A22) mixture of nitrogen (N2), oxygen (O2), and argon (Ar) -1 -3 Substituting into equation A5.d we can write the (287.0 J K kg ), Rc is the particular gas constant -1 -3 equation of state for moist air for CO2 (188.9 J K kg ), Rv is the particular gas -1 -3 constant for water vapor (461.5 J K kg ), rv is the

(A23) water vapor mixing ratio (dimensionless), and rc is Rather than associating the varying water vapor effect the carbon dioxide mixing ratio (dimensionless).

(1+0.61r) with the gas constant (and thus producing a The exact expression for Tv is then variable constant), meteorologists traditionally associate this term with the temperature. Thus, through equation A23, the virtual temperature for the atmosphere is defined as: (A27) (A24)

CO2 rich-air which is programmed in an Excel file freely available at Equations to calculate the virtual temperature including http://fisicaaplicada.ugr.es/pages/tv/!/download Therefore for determining air density in CO2 effects were developed by Kowalski & Sanchez-Cañete (2010). An approximation to calculate the virtual temperature caves or soils including CO2 effects, the virtual temperature can be used in the ideal gas law (Tv) including CO2 effects is via the following equation: with the particular gas constant for the mixture

(A25) of nitrogen (N2), oxygen (O2), and argon (Ar)

Where Tv is the virtual temperature (K), and rv and rc are the water vapour and carbon dioxide mixing (A28). ratios respectively (dimensionless).

International Journal of Speleology, 42 (1), 1-8. Tampa, FL (USA). January 2013 International Journal of Speleology 42 (1) 9-13 Tampa, FL (USA) January 2013

Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Official Journal of Union Internationale de Spéléologie

Free-living amoebae in sediments from the Lascaux Cave in France Angela M. Garcia-Sanchez 1, Concepcion Ariza 2, Jose M. Ubeda 2, Pedro M. Martin-Sanchez 1, Valme Jurado 1, Fabiola Bastian 3, Claude Alabouvette 3, and Cesareo Saiz-Jimenez 1* 1 Instituto de Recursos Naturales y Agrobiología, IRNAS-CSIC, 41012 Sevilla, Spain 2 Universidad de Sevilla, Departamento de Microbiología y Parasitología, Facultad de Farmacia, 41012 Sevilla, Spain 3 UMR INRA-Université de Bourgogne, Microbiologie du Sol et de l’Environment, 21065 Dijon Cedex, France

Abstract: The Lascaux Cave in France is an old karstic channel where the running waters are collected in a pool and pumped to the exterior. It is well-known that water bodies in the vicinity of humans are suspected to be reservoirs of amoebae and associated bacteria. In fact, the free-living amoebae Acanthamoeba astronyxis, Acanthamoeba castellanii, Acanthamoeba sp. and Hartmannella vermiformis were identif ied in the sediments of the cave using phylogenetic analyses and morphological traits. Lascaux Cave sediments and rock walls are wet due to a relative humidity near saturation and water condensation, and this environment and the presence of abundant bacterial communities constitute an ideal habitat for amoebae. The data suggest the need to carry out a detailed survey on all the cave compartments in order to determine the relationship between amoebae and pathogenic bacteria.

Keywords: free living amoebae; Acanthamoeba; Hartmannella; Lascaux Cave; sediments Received 5 April 2012; Revised 19 September 2012; Accepted 20 September 2012

Citation: Garcia-Sanchez A.M., Ariza C., Ubeda J.M. , Martin-Sanchez P.M., Jurado V., Bastian F., Alabouvette C. and Saiz-Jimenez C. 2013. Free-living amoebae in sediments from the Lascaux Cave in France. International Journal of Speleology, 42 (1), 9-13. Tampa, FL (USA) ISSN 0392-6672 http://dx.doi.org/10.5038/1827-806X.42.1.2 artificial lighting system (Lefèvre, 1974). Further, INTRODUCTION fungal outbreaks of Fusarium solani and melanised fungi were evidenced in 2001 (Bastian et al., 2010; Free-living amoebae (henceforth, amoebae) are Martin-Sanchez et al., 2012). Lascaux Cave is different ubiquitous and found in a variety of habitats, largely from all other studied caves because was treated aquatic, but also in soils and sediments. In aquatic for years with benzalkonium chloride to control the environments, the ecological distribution of amoebae fungal outbreaks. encompasses natural and treated water, drinking A climate regulation system was installed inside water treatment plants, swimming pools, sewage, etc. the cave in 1957, with several replacements, the (Khan, 2006). However, amoebae have been scarcely last one in 2001. Therefore, it was not surprising investigated in caves (Coppellotti & Guidolin, 2003; that Bastian et al. (2009) found in many sediment Mulec, 2008; van Hengstum et al., 2009; Walochnik & samples the simultaneous presence of sequences Mulec, 2009; Mazei et al., 2012). Caves harbour pools, related to Legionella, Apifia, Aquicella, and bacterial lakes and subterranean rivers, and the sediments endosymbionts of amoebae, common in climate and rock walls are constantly wet due to a relative systems. The finding of these bacteria motivated us to humidity near saturation and water condensation. carry out a survey on the cave sediments in a search This environment and the presence of abundant for amoebae. bacterial communities (Bastian et al., 2009; Saiz- Jimenez et al., 2011) constitute an ideal habitat for MATERIAL AND METHODS amoebae. Lascaux is an uncommon cave which experienced Four samples were collected from Lascaux Cave a strong anthropogenic impact. In fact, after sediments (Fig. 1); on 17 February 2010 from a black opening in 1948, the Lascaux Cave was highly stain located in the Apse (M6), on 21 September impacted by massive tourism, and suffered several 2010 from the Painted Gallery (S1 and S2) and on microbial outbreaks, the f irst one of the Chlorophyta 29 September 2010 from the Chamber of Felines Bracteacoccus minor in 1963 due to the effect of an (DF). These sites corresponded to the areas in which

*[email protected] 10 Free-living amoebae in Lascaux Cave, France abundant bacterial endosymbionts were previously EukB (5’-TGATCCTTCTGCAGGTTCACCTAC-3’) as detected (Bastian et al., 2009). Cave environmental described by Díez et al. (2001). Clone library was data were reported elsewhere (Lastennet et al., 2011). constructed using this purified PCR product, using Samples were maintained at 5ºC for 48 hours, standard methods. during the transport to the laboratory. Upon arrival, the samples were incubated and subjected to cloning RESULTS AND DISCUSSION and axenisation following standard methods (Page, 1967a, b). Samples M6 and S2 showed an intense Direct microscopy observations allowed the fungal contamination, which required continual identification of trophozoites and cysts of the genera reseeding, in addition to antifungal treatments with Acanthamoeba and Hartmannella in sample S1 0.5% nystatin. (Table 1). In sample DF were also present species For taxonomical identification of amoebae standard of the genera Acanthamoeba and Hartmannella, methods described by Pussard & Pons (1977, 1979) but the species of Acanthamoeba was different to were used. Trophozoites (feeding stage of amoebae) and that found in sample S1 (Fig. 2). In sample S2 only cysts (surviving stage during adverse environmental a species of Acanthamoeba, similar to that found in periods) were observed in a Zeiss stereomicroscope sample DF, was observed. However, trophozoites or IVB, and the images captured with an Olympus cysts of Hartmannella, unlike the other two samples, camera SP-500 UZ. were not detected. In sample M6 only trophozoites of DNA extraction from the cultures was performed Acanthamoeba but no cysts were detected, therefore using the FastDNA SPIN Kit for Soil (MP Biomedicals, its morphology could not be def ined. OH, USA) according to the manufacturer’s The genus Acanthamoeba could be easily instructions. The specific detection of Acanthamoeba recognized in sample S1 because of the morphological spp. was performed by PCR using the primers set JDP1 characteristics of the trophozoites and cysts (Fig. (5’-GGCCCAGATCGTTTACCGTGAA-3’) and JDP2 2A, B). Based on the size and morphological features (5’-TCTCACAAGCTGCTAGGGGAGTCA-3’), which of cysts the Acanthamoeba was identified as A. specifically amplify a 450 bp fragment of the 18S astronyxis, characterized by large trophozoites and rDNA gene in the genus Acanthamoeba (Schroeder et cysts (≤18 μm), with smooth ectocyst and stellate al., 2001). The amplification products were sequenced endocyst (Page, 1967b; Pussard & Pons, 1977). The using the same primers and the obtained sequences 18S gene sequence showed a 99% similarity with were compared with those deposited in GenBank the type strain A. astronyxis (ATCC30137), isolated using the NCBI BLASTn algorithm. Phylogenetic from water, and genotype T7 according to Stothard relationships between the Acanthamoeba spp. isolated et al. (1998). In the phylogenetic analysis these two from Lascaux Cave and the closest related sequences strains also formed a homogenous cluster with 81% of from GenBank were conducted using MEGA 5.0 with bootstrap value confirming the species identification the Maximum-Likelihood method. Gaps were treated (Fig. 3). as missing data and bootstrap values were generated The Acanthamoeba sp. found in sample DF using 1000 replicates. The resulting topology was corresponded to a different species. The strain is compared with results from other treeing algorithms, close related to A. mauritaniensis because some including the Neighbor-Joining and Maximum- endocysts had fewer than 6-rays and the cysts showed Parsimony methods. reticulation even after fixation with formaldehyde in In order to identify other genera of amoebae, a the heat impregnation technique (Fig. 2C) (Pussard region of 18S rDNA was amplified using the primers & Pons, 1977). However, analysis of the 18S gene set EukA (5’-AACCTGGTTGATCCTGCCAGT-3’) and sequence from this strain showed a 99% similarity

Fig. 2. Morphology of amoebae. a) In vivo trophozoite with acanthopodia of Acanthamoeba astronyxis S1. b) Five ray-cyst of Acanthamoeba astronyxis S1. c) Cysts of Acanthamoeba sp. DF under heat impregnation. d) Cysts of Acanthamoeba castellanii S2 under PATAg-r staining. e) Cysts of Acanthamoeba castellanii S2 under heat impregnation. f) Trophozoite and cyst of Hartmannella vermiformis. Fig. 1. Map of Lascaux Cave and sampling location. The bar represents 10 μm.

International Journal of Speleology, 42 (1), 9-13. Tampa, FL (USA) January 2013 Garcia-Sanchez et al. 11 Table 1. Identification of free-living amoebae in Lascaux Cave. ATCC 50374 isolated from a yeast culture and the Genbank Morphological Samples Molecular identificationb Accession strain Ma ATCC 50370 from a keratitis patient. Also identif icationa No. showed 99% of similarity with strains of A. polyphaga, Acanthamoeba Acanthamoeba astronyxis (99%), S1 HE653911 and 96% similarity with A. mauritaniensis, both astronyxis genotype T7c species included in the genotype T4. This genotype Hartmannella Hartmannella vermiformis (100%) FR832469 vermiformis was reported as the one most commonly found in the Acanthamoeba sp., Acanthamoeba sp. (99%), DF HE653914 group IId genotype T14e environment (Booton et al., 2002) and was associated Hartmannella Not determined vermiformis to a high percentage of amoebic keratitis (Stothard et Acanthamoeba sp., Acanthamoeba castellanii (99%), al., 1998). The phylogenetic analysis (Fig. 3) and the S2 HE653912 group IId genotype T4c morphological traits (Pussard & Pons, 1977) suggested Acanthamoeba castellanii (99%), M6 Acanthamoeba sp. HE653913 that S2 and M6 strains would be A. castellanii. genotype T4c Strains of the genus Hartmannella was found in aMorphological identification based on microscopic studies of trophozoites and cysts. bMolecular identification based on phylogenetic study of 18S gene, and comparison with sample S1 and DF. The description of Page (1976) GenBank by BLAST algorithm (NCBI); similarity percentages are included. for the genus Hartmannella is coincident with our cClassification according to Stothard et al. (1998). dClassification according to Pussard and Pons (1977) strains, and they were identified as H. vermiformis e Genotype described by Horn et al. (1999) (Fig. 2F). Molecular data confirmed the identification with Acanthamoeba sp. strain UWET, used by of the strain isolated from sample S1 (Table 1). Horn et al. (1999) to describe the genotype T14. In Acanthamoeba castellanii genotype T4, and H. the phylogenetic analysis these two strains form a vermiformis were previously found in Slovenian caves homogenous cluster with 94% of bootstrap value, (Mulec, 2008; Walochnik & Mulec, 2009). clearly separated from A. mauritaniensis (Fig. 3). To understand the presence of amoebae it must be In the Acanthamoeba sp. S2 strain, the taken into account that the Lascaux Cave is an old morphological classification leads to a similar result karstic channel with many water percolation points to that obtained for the sample DF as denotes the and a main emergence point at the entrance air-lock. similarity in size of the cysts. However, periodic acid The running waters are collected in a pool under thiocarbohydrazide silver reduced staining (PATAg-r) the machinery room (a thermic and hygrometric (Pussard & Pons, 1979) shows a somewhat different regulation system is installed inside, near the cave feature. In this case, the opercular complexes are entrance) and pumped to the exterior (Lastennet et stained more markedly, showing very characteristic al., 2011). It is well-known that water bodies in the concentric circles (Fig. 2D). Heat impregnation shows vicinity of humans are a reservoir of amoebae and clearly the cysts reticulation, similar to that obtained associated bacteria (Atlas, 1999). in the sample DF (Fig. 2C, E). In sample M6 only In a previous paper (Bastian et al., 2009), 696 trophozoites of Acanthamoeba sp. were observed, but bacterial clones were retrieved from different Lascaux not the presence of cysts in the culture plates, thus Cave halls and galleries. Phylotypes related with preventing a morphological characterization. pathogenic bacteria amounted to 45%, from which The 18S gene sequences from S2 and M6 strains the presence of Afipia spp., Aquicella spp. and were identical, confirming that they are the same Legionella spp. were remarkable. In addition, other species. These Acanthamoeba showed a 99% of bacteria identified in the cave, such as Achromobacter similarity with different strains of A. castellanii xylosoxidans, Stenotrophomonas maltophilia and (genotype T4) including the type strain Castellani Pseudomonas fluorescens were reported to be responsible for complete lysis of amoebae or found as intracellular pathogens (Pagnier et al., 2008). Hence, the simultaneous presence of sequences of Afipia, Aquicella and Legionella in some cave halls, and the identification of Acanthamoeba spp. and H. vermiformis suggest a close relationship among intracellular pathogenic bacteria and amoebae in the cave. Many protozoan species have been found to harbour intracellular Legionella spp. These include H. vermiformis and among other Acanthamoeba, A. castellanii. For some authors it was clear that A. Fig. 3. Phylogenetic tree derived from 18S rDNA sequences castellanii provided an intracellular niche in which corresponding to the specific amplimer ASA.S1 showing the relationships between the Acanthamoeba strains isolated from Lascaux environmental strains of Legionella can proliferate. Cave (in bold) and their close relative sequences from GenBank. The In addition, growth of Legionella pneumophila in accession numbers are in parentheses. T1, T4, T7, T8, T9, T11 and T14 potable water occurs only in the presence of amoebae are recognised Acanthamoeba genotypes. The tree was constructed and L. pneumophila may remain culturable for up using the Maximum Likelihood method applying Hasegawa-Kishino- Yano model, based on a comparison of 317 nucleotides. The tree to 6 months in a medium containing A. castellanii, with the highest log likelihood (-739.4) is shown. It was rooted with whereas free-living Legionella within biofilms may be Protacanthamoeba bohemica (Acanthamoebida) as outgroup. inactivated within a few weeks (Lau & Ashbolt, 2009). Bootstrap values are expressed as percentages of 1,000 replications. Srikanth & Berk (1994) reported that amoebae from All nodes of the tree were also recovered using Neighbour Joining and Maximum Parsimony treeing algorithms. Bar, 0.01 substitutions per cooling tower, containing Legionella, may adapt to nucleotide position. biocides and may even be stimulated by them. The

International Journal of Speleology, 42 (1), 9-13. Tampa, FL (USA) January 2013 12 Free-living amoebae in Lascaux Cave, France biocides tested by these authors included quaternary Berk S.G., Ting R.S., Turner G.W. & Ashburn R.J., ammonium compounds (QAC) and isothiazoline 1998 - Production of respirable vesicles containing live derivatives. In Lascaux Cave benzalkonium chloride Legionella pneumophila cells by two Acanthamoeba spp. was in use from 2001 till 2004 and isothiazolines were Applied and Environmental Microbiology, 64: 279-286. Booton G.C., Kelly D.J., Chu Y.W., Seal D.V., Houang employed in 2008 (Bastian et al., 2010), therefore it is E., Lam D.S., Byers T.J. & Fuerst P.A., 2002 - 18S not surprising that amoebae living in the cave were not ribosomal DNA typing and tracking of Acanthamoeba killed, taking into account their adaptation, the formation species isolates from corneal scrape specimens, of resistant cysts and the relative rapid inactivation of contact lenses, lens cases, and home water supplies these biocides by biotic and abiotic processes. Berk et of Acanthamoeba keratitis patients in Hong Kong. al. (1998) reported that QAC and isothiazolines biocides Journal of Clinical Microbiology, 40:1621-1625. were found useless for eliminating Legionella because http://dx.doi.org/10.1128/JCM.40.5.1621-1625.2002 these bacteria within Acanthamoeba vesicles were viable Coppellotti O. & Guidolin L., 2003 – Taxonomy and ecology of ciliate fauna (Protozoa, Ciliophora) from karst caves in after biocide exposure. North-East Italy. Subterranean Biology, 1: 3-11. The known resistance of encysted amoebae and the Díez B., Pedrós-Alió C., Marsh T.L. & Massana endosymbionts to disinfection and biocides (Srikanth R., 2001 - Application of denaturing gradient gel & Berk, 1994; Berk et al., 1998), which were tested electrophoresis (DGGE) to study the diversity of and applied in Lascaux between 2001 and 2008, marine picoeukaryotic assemblages and comparison and the limited field of action in a cave with rock art of DGGE with other molecular techniques. Applied result in a pessimistic scenario regarding elimination and Environmental Microbiology, 67: 2942-2951. of these microorganisms. 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International Journal of Speleology, 42 (1), 9-13. Tampa, FL (USA) January 2013 Garcia-Sanchez et al. 13 Pussard M. & Pons R., 1977 - Morphologie de la Stothard D.R., Schroeder-Diedrich J.M., Awwad paroi kystique et taxonomie du genre Acanthamoeba M.H., Gast R.J., Ledee D.R., Rodriguez- (Protozoa, Amoebida). Protistologica, 13: 557-598. Zaragoza S., Dean C.L., Fuerst P.A. & Byers Pussard M. & Pons R., 1979 - Étude des pores kystiques de T.J. 1998 - The evolutionary history of the Naegleria (Vahlkampf iidae – Amoebida). Protistologica, genus Acanthamoeba and the identification 15: 163-175. of eight new 18S rRNA gene sequence types. Saiz-Jimenez C., Cuezva S., Jurado V., Fernandez-Cortes Journal of Eukaryotic Microbiology, 45: 45-54. A., Porca E., Benevante D., Cañaveras J.C. & Sanchez- http://dx.doi.org/10.1111/j.1550-7408.1998.tb05068.x Moral S., 2011 - Paleolithic art in peril: Policy and van Hengstum P.J., Reinhardt E.G., Beddows science collide at Altamira Cave. Science, 334: 42-43. P.A., Schwarcz H.P. & Gabriel J.J., 2009 - http://dx.doi.org/10.1126/science.1206788 Foraminifera and testate amoebae (thecamoebians) Schroeder J.M., Booton G.C., Hay J., Niszl I.A., Seal D.V., in an anchialine cave: Surface distributions Markus M.B., Fuerst P.A. & Byers T.J., 2001 - Use of from Aktun Ha (Carwash) cave system, Mexico. subgenic 18S ribosomal DNA PCR and sequencing for Limnology and Oceanography, 54: 391-396. genus and genotype identif ication of Acanthamoebae http://dx.doi.org/10.4319/lo.2009.54.1.0391 from humans with keratitis and from sewage sludge. Walochnik J. & Mulec J., 2009 - Free-living amoebae in Journal of Clinical Microbiology, 39:1903-1911. http://dx.doi.org/10.1128/JCM.39.5.1903-1911.2001 carbonate precipitating microhabitats of karst caves Srikanth S. & Berk S.G. 1994 - Adaptation of amoebae to and a new vahlkampfiid amoeba, Allovahlkampfia cooling tower biocides. Microbial Ecology, 27: 293-301. spelaea gen. nov., sp. nov. Acta Protozoologica 48: http://dx.doi.org/10.1007/BF00182412 25-33.

International Journal of Speleology, 42 (1), 9-13. Tampa, FL (USA) January 2013 14 International Journal of Speleology 42 (1) 15-23 Tampa, FL (USA) January 2013

Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Official Journal of Union Internationale de Spéléologie

The cave environment influencing the lipid profile and hepatic lipogenesis of the fish Ancistrus cryptophthalmus Reis, 1987 (Siluriformes: Loricariidae) Valquíria Aparecida Alves Bastos1, Rodrigo Lopes Ferreira2, Daniel Cardoso de Carvalho3, Marina Lages Pugedo3, and Luciana de Matos Alves Pinto1* 1 Universidade Federal de Lavras, Departamento de Química, Caixa Postal 3037, 37200-000, Lavras, MG, Brazil 2 Universidade Federal de Lavras, Departamento de Biologia, Caixa Postal 3037, 37200-000, Lavras, MG, Brazil 3 Pontifícia Universidade Católica de Minas Gerais, Programa de Mestrado em Zoologia de Vertebrados. Belo Horizonte, MG, Brazil

Abstract: The metabolism of hypogean organisms is frequently molded by the cave environment traits, especially food scarcity. The aim of the present work was to evaluate the influence of such environment on lipid composition and hepatic lipogenesis in the fish Ancistrus cryptophthalmus. For this, the species was compared to an epigean population of the species. A greater accumulation of total lipids was observed in the cave-dwelling fish (18.36 g/100 g tissue) compared to the surface fish (14.09 g/100 g tissue). The muscle fatty acid profile also varied between the populations. Arachidonic acid was only detected in the epigean fish, while docosahexaenoic acid was present in the cave fish. In the lipid profile of Ancistrus cryptophthalmus there was a higher proportion of saturated fatty acids, followed by monounsaturated and polyunsaturated fatty acids; Ancistrus sp. showed a predominance of monounsaturated fatty acids. Significant differences were also observed in the activities of the hepatic enzymes glucose-6-phosphate dehydrogenase and malic enzyme. The activities of these two enzymes were greater in the epigean animals. The differences could be related to different food availability observed in the two environments. An ecotone zone was observed, located next to the entrance of the Lapa do Angélica cave (Goiás State, Brazil), where the fishes showed characteristics that were intermediate between those of hypogean fishes from deeper within the cave, and the epigean population. It could be concluded that the characteristics of the cave environment significantly influenced the composition of muscle fatty acids and lipogenesis in the hypogean fish Ancistrus cryptophthalmus.

Keywords: fish; cave; fatty acids; malic enzyme; glucose-6-phosphate dehydrogenase; metabolism Received 31 May 2012; Revised 21 August 2012; Accepted 27 September 2012

Citation: Bastos V.A.A., Lopes Ferreira R., Carvalho D.C., Pugedo M.L. and de Matos Alves Pinto L., 2012. The cave environment inf luencing the lipid prof ile and hepatic lipogenesis of the fish Ancistrus cryptophthalmus Reis, 1987 (Siluriformes: Loricariidae). International Journal of Speleology, 42 (1), 15-23. Tampa, FL (USA) ISSN 0392-6672 http://dx.doi.org/10.5038/1827-806X.42.1.3

INTRODUCTION (Trajano & Bichuette, 2010). Globally, there are some troglobitic armored catfishes that belong to the Fish are important components of subterranean Loricariidae family (Ancistrus). There are two Brazilian aquatic communities, where they often are at the top fishes of this genus: Ancistrus cryptophthalmus, found of the food web. Globally, the number of known fish in the State of Goiás, central Brazil (Sabino & Trajano, species is currently around 28,000, of which 299 can 1997), and Ancistrus formoso, found in the State of be found in the hypogean environment, with at least Mato Grosso do Sul (Trajano & Bichuette, 2010). 164 showing some degree of troglomorphism (Romero Although the diversity of hypogean fishes is fairly & Paulson, 2001; Romero, 2009; Proudlove, 2010). well known, there exists little information concerning Brazil possesses a rich subterranean ichthyofauna, the metabolism of these organisms. Nevertheless, it is with more than 25 fish species that present believed that the animals are hypometabolic, and tend troglomorphism compared to the corresponding to develop elaborate mechanisms for both searching epigean species (Trajano & Bichuette, 2010). Most for food and improving the capacity for lipid storage of these troglobitic fishes belong to the Siluriformes, (Wilkens, 2005; Salin et al., 2010). especially the Trychomicteridae family, although there Lipids are important components of the diets of are representatives of other families and also of other many animals, and are an efficient source of both orders (such as the Characiformes and Gymnotiformes) energy and the fatty acids that are essential for

*[email protected] 16 Bastos et al. development. In ectothermic animals, such as fish, (the Angélica, Bezerra, São Vicente, and Passa Três lipids act as structural components responsible for the caves). They have poorly-developed ocular structures, maintenance of fluidity of the plasma membrane, as reduced body pigmentation, detritivore feeding habits, well as for the storage and supply of energy, and are and precocial lifestyles. These characteristics can be therefore essential for basic physiological functions interpreted as being specialized adaptations to the (Brown & Murphy, 1995). An ability to synthesize subterranean environment (Secutti & Trajano, 2009). a wide variety of fatty acids is essential for all living In addition to the hypogean populations, the same organisms. region harbors epigean populations of the genus In fish, biosynthesis of fatty acids occurs mainly in Ancistrus, which include an unidentified species the liver, and proceeds by the successive addition of (Fig. 1B). These animals, which are morphologically two carbon units, derived from the malonyl group, to distinct to the hypogean, have better developed ocular the growing lipid chain (Nelson & Cox, 2010). This structures, and intense pigmentation (Reis et al., 2006). process involves the action of specific enzymes such as The fish of the genus Ancistrus provide an excellent glucose-6-phosphate dehydrogenase, which provides model for studies concerning the adaptive evolution energy in the form of NADPH in the pentose phosphate of cave-dwelling groups, because there are relatively pathway, and the malic enzyme, which is involved in abundant populations of the animals inhabiting both the pyruvate/malate cycle (Wang et al., 2005). hypogean and epigean environments in the same region In most fish species, the fatty acid profile and the (Trajano & Bichuette, 2007). activity of the enzymes concerned with lipogenesis are affected by the composition of the diet and the METHODS frequency of feeding (Visentainer et al., 2005; Wang et al., 2005; Visentainer et al., 2007). For this reason, Capture and transport subterranean environments, which tend to be Ten individuals of Ancistrus cryptophtalmus strongly oligotrophic, can promote alterations such as (hypogean) and Ancistrus sp. (epigean) were collected diminution of metabolism in cave fish, and a tendency manually using a net, in May 2011. No consideration for greater storage of lipids (Wilkens, 2005). was given to differences between the animals in terms of The objective of this work was therefore to investigate physiological factors, age, size, weight, stage of maturity, the influence of the subterranean environment on or anatomical features, in order to obtain a sample that the lipid profile and hepatic lipogenesis of the fish was more representative of the overall population. In Ancistrus cryptophthalmus. This was achieved using addition, the fish were handled as little as possible to comparisons with epigean fish that inhabited the minimize situations of stress. external stretch of the same water channel present The hypogean fish were collected at two points in the Lapa do Angélica cave (Goiás State, Brazil). In within the Angélica cave. Five individuals were addition, the study sought to identify whether there were obtained near the entrance (site P, located around any differences between individuals of the hypogean 300 meter far from the entrance), and the remainder fishes that inhabited zones closer to the cave entrance deeper within the cave, approximately 1000 meters (where there was a greater supply of nutrients), and from the entrance (site P2) (Fig. 2). Since fish of fish that were found deeper within the cave. the species Ancistrus cryptophtalmus are highly sedentary and live their entire life in a stretch Study region of water less than 100 m long, there was a low The São Domingos karst region is located in the probability of movement of the animals between the Serra do Calcário, in the central-west Brazilian State two locations (Trajano & Bichuette, 2007). The non- of Goiás, where the predominant vegetation is that of troglobitic fish were collected in the epigean stretch the cerrado (Brazilian savannah). This area has a semi- humid tropical climate, with a dry period that extends from April to September (Klink & Machado, 2005). The region is known for its rich subterranean ichthyofauna, and 66 limestone caves have been recorded, amongst which is the Angélica cave in the Terra Ronca State Park (13º 31’ 22.2” S, 46º 22’ 55.5” W). The Angélica cave is the fourth largest Brazilian cave in terms of extent, with 14,100 meters of mapped galleries, 8,000 meters of which contain large, fast- flowing watercourses (Auler et al., 2001). The stream conduit has an average width of around 5 meters, and the water depth varies between 0.5 and 2 meters. In the dry season, the measured discharge of water at the mouth of the cave was 2.27 m3/s (Trajano & Bichuette, 2007).

Fish species studied The Ancistrus cryptophthalmus Reis, 1987 (Fig. 1A) is Fig. 1. Species of f ish studied: A) Ancistrus cryptophthalmus; B) found in four caves in the São Domingos karst region Ancistrus sp.

International Journal of Speleology, 42 (1), 15-23. Tampa, FL (USA) January 2013 The cave environment influencing on lipid metabolism of fish 17 of the Angélica River, at approximately 300 meters and 658 base pairs of the mitochondrial gene from the cave entrance. Cytochrome c Oxidase I (COI) amplified using the Measurements of water pH, temperature, and primers FishF1 and FishR1 (Ward et al., 2005) dissolved oxygen content were made in situ at the as described in Carvalho et al. (2011). Sequences time of collection, using a multiprobe sensor (YSI). were determined bi-directionally using the BigDye Samples of substrate were also collected from the Terminator v3.1Cycle Sequencing Kit (Applied riverbed at the three collection points, for later Biosystems, Inc.,Foster City, California, USA) analysis of organic matter using the procedure following the manufacture’s protocol on an Applied described by Davies (1974). Biosystems Inc. 3130 Genetic Analyzer. Each The animals were transported to Universidade recovered haplotype was deposited in GenBank Federal de Lavras, in groups of five, in plastic (Accession numbers: JX477622-JX477648). One containers (5 liter capacity) protected from light and previously described Ancistrus brevipinnis COI with constant aeration. The containers were filled to sequence (Genbank number: EU359402.1) was also the rim with water from the Angélica river in order to included in the analysis. avoid impacts that could cause a situation of stress DNA sequences were analyzed using SeqScape to the fish, according to the norms established by the v2.7® software (Applied Biosystems, Inc., Foster Commission for Ethics in the Use of Animals of the City, California, USA). Genetic distances were Federal University of Lavras (license number: 002/11). estimated using Kimura’s two-parameter (K2P) nucleotide substitution model (Kimura, 1980) using Genetic analysis MEGA version 5.10 (Tamura et al., 2011). Samples of fins and muscle were obtained from 27 fishes representing four populations (22 from Biochemical analysis Angélica Cave: 5 specimens from a hypogean After transport, the animals were desensitized in an population inside the cave, 5 from a hypogean ice bath, weighed, and eviscerated, with separation population near the surface and 11 from an epigean of the liver for use in the enymatic analyses. population outside the cave and described only Samples of muscle tissue were submitted to lipid as Ancistrus sp.). Five hypogean individuals from extraction and esterification using the method of Passa-Três Cave were also analyzed. All tissues Folch et al. (1957). The methyl esters of the fatty were stored in 95% ethanol. DNA was isolated acids were determined using a Shimadzu GC-2010

Fig. 2. Map of the Angélica Cave, indicating collection points P1 and P2.

International Journal of Speleology, 42 (1), 15-23. Tampa, FL (USA) January 2013 18 Bastos et al. gas chromatograph equipped with a flame ionization Cave, two polymorphic sites were recovered in two detector and a DB-WAX fused silica capillary column specimens, resulting in a divergence of 0.2% between (100 m × 0.25 mm). The operating conditions were haplotypes. When comparing Ancistrus sp and as follows: splitless injection mode; 1 µL injection Ancistrus cryptophthalmus with Ancistrus brevipinnis volume; 260 ºC detector temperature; 260 ºC injector a divergence of 6.9-7.2% was recovered. temperature; temperature program: 4 ºC min-1 to 140 The Ancistrus cryptophthalmus fish presented higher ºC, hold at 140 ºC for 5 minutes, ramp at 4 ºC min-1 to body weights, with values of between 2.09 and 2.17 g, 240 oC, hold at 240 oC for 30 minutes. and greater deposition of lipids in the muscle tissue The chromatographic peaks were identif ied by (17.6-18.36%), compared to the epigean population comparing retention times with those of a commercial whose average weight and muscle total lipid content standard (Supelco 37 Component FAME Mix, supplied were 1.21 g and 14.09%, respectively (Fig. 3). by Sigma-Aldrich). The fatty acids were quantified by The lipid profiles revealed the presence of 14 fatty normalization of the peak areas, and the values were acids in the Ancistrus cryptophthalmus individuals converted to percentages of the mass of muscle tissue, collected at site P1, and 10 in fish of the same species multiplying by the total lipid fraction content and by collected at site P2. The lipid profile of the epigean the conversion factor for fatty fish (0.9), as described Ancistrus sp. revealed the presence of 19 fatty acids by Holland et al. (1994). (Table 1). Amongst those fatty acids identified in both The samples of liver tissue were homogenized in species, the most prevalent were myristoleic (C14:1), 25 mM HEPES-KOH buffer at pH 7.4, centrifuged at palmitic (C16:0), palmitoleic (C16:1), oleic (C18:1ω9c), 10,000 rpm (4 °C for 30 minutes), and analyzed for the and linoleic (C18:2ω6c) acids. specific enzymatic activities of glucose-6-phosphate Significant differences between the hypogean and dehydrogenase and malic enzyme, according to the epigean fishes were observed in the proportions of methods of Graeve (1994) and Spina et al. (1966), saturated and monounsaturated fatty acids. While the respectively. The enzyme kinetics was monitored using cave fish from both sites within the cave showed high UV spectrophotometry (Shimadzu UV-1800). The total levels of saturated fatty acids (62.88% for P1, and 62.75% soluble protein contents of the livers were determined for P2), and smaller proportions of monounsaturated fatty using the method described by Bradford (1976). acids (24.54% for P1, and 24.76% for P2), the epigean fish All the results were tested statistically using analysis showed a higher content of monounsaturated (46.86%) of variance (ANOVA) and Tukey’s test to identify than saturated (39.22%) fatty acids. significant differences among the sample mean values. The total content of polyunsaturated fatty acids showed The software used was R (R Development Core Team, no significant difference between the populations, with 2011), and the significance level employed was 5%. the omega-6 series predominant over the omega-3 series in both cases. RESULTS In the analysis of the rates of lipogenesis, it was found that the specific activities of the hepatic enzymes At the time of sample collection, the river water at (glucose-6-phosphate dehydrogenase and malic enzyme) the epigean site presented a temperature of 24.8 ºC, differed significantly (P<0.05) between the hypogean and a dissolved oxygen content of 94.7%, and a pH of epigean fishes (Table 2). Higher specific enzyme activities 6.3. Inside the cave, where there was no significant in the epigean fish were indicative of greater lipogenesis difference between the conditions at the two collection in this population. points, the temperature was higher (25.3 ºC), and DISCUSSION the dissolved oxygen content was lower (91.5%). The water pH was the same at the hypogean and epigean Studies of metabolism are essential for sites. Physico-chemical information concerning the understanding the basic needs of animals, and collection points is important because the conditions were used here to elucidate the habits of the can significantly influence the metabolism of the fish. The organic matter content of the sedimented substrate differed substantially between the two environments, with values of 1.84 and 0.38 g/cm2 obtained at the epigean site and hypogean site P1 (closer to the cave entrance), respectively. The organic matter content was below the detection limit at the second hypogean site (deeper within the cave). After trimming unclear ends, a total of 556 base pairs (bp) were obtained from each specimen. Three haplotypes were recovered from all samples analyzed, with a K2P genetic divergence ranging from 0 to 0,2%. When comparing fishes collected only at Angélica Cave (5 individuals from a hypogean population inside the cave, 5 from a hypogean population near the surface and 11 from an epigian population) only one Fig. 3. Body weights and ether extract amounts of the epigean and troglobitic f ish. haplotype was recovered and no genetic divergence * Collected at site P1. was observed between populations. At Passa-Três ** Collected at site P2.

International Journal of Speleology, 42 (1), 15-23. Tampa, FL (USA) January 2013 The cave environment influencing on lipid metabolism of fish 19 Table 1. Fatty acid profiles of the species Ancistrus cryptophthalmus and Ancistrus sp. Values are shown as means ± standard deviation. Where the values expressed in % (on the same line) are followed by identical letters, differences between the values were not significant (p>0.05). Where the values expressed in g/100 g (on the same line) are followed by identical Greek letters, differences between the values were not significant (p>0.05). Differences were evaluated using analysis of variance (ANOVA) and Tukey’s test. nd = not determined.

Ancistrus cryp.** Ancistrus sp. Ancistrus cryp.* Fatty acid % g/100g % g/100g % g/100g

C14:0 0.81 ± 0.10b 0.10 ± 0.01β 5.01 ± 0.61a 0.85 ± 0.10α 5.93 ± 1.59a 0.96 ± 0.25α

C14:1 13.78 ±1.73a 1.79 ± 0.22α nd nd 14.22 ± 3.06a 2.27 ± 0.50α

C15:0 0.80 ± 0.08c 0.10 ± 0.01 ɣ 4.58 ± 0.70a 0.77 ± 0.11α 3.35 ± 0.25b 0.53 ± 0.04b

C 15:1 0.94 ± 0.15b 0.12 ± 0.01β 4.89 ± 0.56a 0.82 ± 0.09α nd nd

C16:0 25.67 ±1.66b 3.30 ± 0.21β 48.82 ±1.01a 8.18 ± 0.19α 49.54 ± 5.69a 7.78 ± 0.88α

C16:1 11.36 ±0.40a 1.45 ± 0.05α 1.88 ± 0.91b 0.31 ± 0.15β 10.54 ± 2.60a 1.64 ± 0.40α

C17:0 0.46 ± 0.10c 0.06 ± 0.01ɣ 3.50 ± 0.61a 0.58 ± 0.10α 1.55 ± 0.35b 0.24 ± 0.05β

C17:1 0.81 ± 0.15b 0.10 ± 0.01β 2.41 ± 0.45a 0.39 ± 0.07α nd nd

C18:0 8.06 ± 0.52 1.01 ± 0.06 nd nd nd nd

C18:1ω9c 19.40 ±0.56a 2.43 ± 0.07α 11.42 ±0.65b 1.86 ± 0.10β nd nd

C18:2ω6t 1.69 ± 0.34 0.21 ± 0.04 nd nd nd nd

C18:2ω6c 7.07 ± 0.60a 0.88 ± 0.07α 5.61 ± 1.155 a 0.91 ± 0.18α 7.01 ± 1.48a 1.06 ± 0.22α

C20:0 2.77 ± 0.21a 0.34 ± 0.02α 0.95 ± 0.24b 0.15 ± 0.03β 2.35 ± 0.31a 0.35 ± 0.04α

C18:3ω6 0.51 ± 0.36b 0.19 ± 0.04α 0.95 ± 0.41a 0.21 ± 0.06α 1.09 ± 0.34a 0.16 ± 0.05α

C20:1 0.51 ± 0.004 0.06 ± 0.004 nd nd nd nd

C22:0 0.63 ± 0.13 0.07 ± 0.01 nd nd nd nd

C20:3ω6 1.98 ± 0.32 0.24 ± 0.03 nd nd nd nd

C20:4ω6 0.38 ± 0.007 0.04 ± 0.008 nd nd nd nd

C22:2 nd nd 0.78 ± 0.11 0.13 ± 0.09 nd nd

C24:1 nd nd 3.92 ± 0.75 0.56 ± 0.10 nd nd

C22:6ω3 1.18 ± 0.20b 0.14 ±0.02β 4.85 ± 0.66a 0.74 ± 0.10α 4.36 ± 0.95a 0.63 ± 0.13α

∑ Saturates 39.22±1.98b 5.00 ± 0.25β 62.88 ±1.07a 10.55 ± 0.18α 62.75 ± 6.54a 9.86 ± 1.02α

∑Monounsaturates 46.83 ±1.47a 5.96 ±0.19α 24.54±1.24b 3.96 ± 0.20β 24.76 ± 5.06b 3.90 ± 0.81β

∑Polyunsaturates 13.94 ±1.10a 1.71 ± 0.13α 12.56 ±0.48a 2.00 ± 0.07α 12.47 ± 2.14a 1.86 ± 0.31α

∑ ω3 1.18 ± 0.20b 0.14 ± 0.02β 4.85 ± 0.66a 0.74 ± 0.10α 4.36 ± 0.95a 0.63 ± 0.13α

∑ ω6 12.75 ±1.14a 1.57 ± 0.14α 6.94 ± 1.12b 1.12 ± 0.18β 8.10 ± 1.80b 1.22 ± 0.26β

* Collected at site P1. ** Collected at site P2. cave fish. The hypogean environment is unusual al., 2010), provides a basis for an understanding when compared to epigean environments, since of the results obtained in the present work. The there is no photoperiod and food availability is hipogean fishes presented a higher weight and low. For many animals, these factors preclude greater lipid deposition in the muscle tissue than the establishment of viable populations in the epigean Ancistrus sp., which is provided with subterranean ecosystems (Culver et al., 2009). A a greater quantity of organic matter in its habitat. fish species would become actually established A large sinkhole at the entrance to the Angélica in the hypogean environment, if it develop cave enables transport of quantities of organic morphological, physiological, metabolic, and matter, mainly leaves and small invertebrates, into behavioral specializations (Mathieu & Hervant, the cave interior. However, fish of the Ancistrus 2006; Reis et al., 2006). genus are essentially grazers, so that even given In a study of the epigean fish species Micropterus the transfer of such organic matter, the amount of salmoides, it was shown that when subjected food available to these fish is considerably smaller to periods of fasting, the animals developed the in the cave habitat, and consists only of a film capacity to store proteins and lipids as a means of fine particulate organic matter (Power, 1990; of guaranteeing a supply of energy (Cyrino et al., Trajano & Bichuette, 2007). 2000). This ability to store nutrients, developed by It is recognized that a reduction in the availability various fish species as a result of fasting (Salin et of food is generally associated with a worsening of the

International Journal of Speleology, 42 (1), 15-23. Tampa, FL (USA) January 2013 20 Bastos et al. Table 2. Specif ic activities of glucose-6-phosphate dehydrogenase The elevated levels of palmitic (C16:0) and palmitoleic and the malic enzyme in epigean and hypogean fish. (C16:1) acids in both of the population studied, and Values are shown as means ± standard deviation. Where the values the lower levels of long chain fatty acids (C20 and C22), on the same line are followed by identical letters, differences between the values were not significant (p>0.05). Differences were evaluated are common characteristics of freshwater fish (Çelik et using analysis of variance (ANOVA) and Tukey’s test. al., 2005; Inhamuns et al., 2009). On the other hand,

Ancistrus sp. Ancistrus cryp.* Ancistrus cryp.** stearic acid (C18:0), which is also common in the Enzyme (U/mg of ptn) (U/mg of ptn) (U/mg of ptn) lipid profile of freshwater fish, was not found in the Glucose-6-phosphate 0.633 ± 0.116a 0.271 ± 0.097b 0.189 ± 0.098b dehydrogenase hypogean fish Ancistrus cryptophthalmus. Since the

Malic 0.048 ± 0.011a 0.034 ± 0.006a 0.013 ± 0.011b fatty acid composition reflects the diet of the animal, it can therefore be inferred that the diet of the cave- * Collected at site P1. ** Collected at site P2. dwelling fish was probably deficient in this fatty acid (Inhamuns et al., 2009). condition of fish (Oscoz et al., 2005). Nonetheless, Eicosenoic (C20:1), behenic (C22:0), eicosatrienoic metabolic changes, such as greater deposition of (C20:3ω3), and arachidonic (C20:4ω6) acids were body fat, occur in order to maintain a constant only detected in the epigean fish. Arachidonic acid nutritional status and bodily condition, even in (C20:4ω6), which is nutritionally and physiologically an environment with limited resources, such as a important for fish, is synthesized from the essential cave. linoleic acid (C18:2ω6) present in abundance in Although the total lipid contents of the muscle cave-dwelling fish (Martino, 2003). The absence tissues of the Ancistrus cryptophthalmus fish of arachidonic acid in individuals of the Ancistrus were higher, the variety of fatty acids in the lipid cryptophthalmus is suggestive of a deficiency in the profile was smaller than observed in the epigean specific desaturases and elongases responsible for population. Several studies have shown that the catalyzing the conversion of linoleic acid into longer profile of fatty acids found in the muscle tissues of chain derivatives (such as arachidonic acid). fish reflects the variability of these compounds in It should also be noted that conditions of stress, the diets of the animals (Visentainer et al., 2005; of any nature, predispose fish to various illnesses. Vieira et al., 2011). Valente et al. (2007) found Hence, in an environment where conditions are that in the fish species Dicentrarchus labrax, the adverse, such as that of a cave, compared to epigean provision of linoleic acid in the diet was directly environments a fraction of the polyunsaturated reflected in the muscle content of this fatty acid as fatty acids that are synthesized is directed to the well as its derivatives formed during the processes immunological system in order to maintain it of elongation and desaturation. The findings of the active, which could explain the low availability of earlier studies were corroborated by the results these compounds in the muscle tissues of Ancistrus obtained in the present work, where the hypogean cryptophthalmus (Sargent et al., 1999). fish that had a more restricted diet (in an habitat In fish, the balance between saturated and poor in organic matter) exhibited a reduced range of unsaturated fatty acids is intimately related to fatty acids in their muscle tissues, compared to the maintenance of the fluidity of the cell membranes, epigean organisms. and is greatly influenced by temperature (Ribeiro Regions close to the entrances of caves often et al., 2011). Generally, a decrease in temperature show structural, biological, and physical gradients, results in an increase in the degree of unsaturation, creating a zone of transition between the epigean and since unsaturates are required in the membrane hypogean systems. Such regions can be considered phospholipids to maintain flexibility and as ecotones, with distinct characteristics due to permeability (Lovell, 1991). Conversely, in higher the equilibrium established between the epigean temperature aquatic environments, it is often the zone, where resources are readily available, and saturated fatty acids that predominate in the lipid the hypogean zone, where the environment is more profiles (Visentainer et al., 2007). stable (Prous et al., 2004). This is supported by the present findings, where Due to the existence of an ecotone zone located Ancistrus cryptophthalmus showed higher levels close to site P1, the hypogean fishes collected at of saturated fatty acids, while monounsaturates this area presented a range of fatty acids that was predominated in the epigean fishes, since during the intermediate between those of the epigean fish and collection period, which took place on a sunny day in the animals collected at the site deeper within the the dry season, the hypogean site on the Angélica river cave. It is therefore possible that the diet of the was 0.5 ºC warmer than the epigean site. fish from site P1 was richer and more diversified Meanwhile, it is important to note that according than that of fish from deeper regions of the cave, to Secutti & Trajano (2009) there is a diurnal but less rich than that of their epigean population. temperature cycle in the Angélica cave, within a Even though the sample population was small range of around 22.5 ºC to 25 ºC, and that seasonal (5 fish at each collection point), the information changes in climate can affect the lipid profile of presented is representative of the total population fish (Inhamuns et al., 2009; Kalyoncu et al., 2009). of Ancistrus cryptophthalmus in the Angélica cave, Further work over a more extended period will be since it employed data that were biochemical in needed to fully investigate these effects. nature, rather than morphological measurements As observed for Ancistrus cryptophthalmus, other alone. studies of freshwater fish have also shown that when

International Journal of Speleology, 42 (1), 15-23. Tampa, FL (USA) January 2013 The cave environment influencing on lipid metabolism of fish 21 the proportion of saturated fatty acids in the muscle The specific activities of the two enzymes tissues increases, the level of polyunsaturated fatty investigated were lower in the Ancistrus acids decreases (Inhamuns et al., 2009). cryptophthalmus collected at site P2, compared to In addition to Ancistrus sp., other fish that are not the epigean population, which could have been cave-dwellers, such as cachara (Pseudoplatystoma due to a scarcity of food in the cave environment fasciatum), pintado (Pseudoplatystoma corruscans), (Catacutan & Coloso, 1997; Dias et al., 2004). pacu (Piaractus brachypomus), and dourado In the case of the malic enzyme, the (Salminus brasiliensis) present higher concentrations specific activities were the same for Ancistrus of monounsaturated fatty acids in their muscle cryptophthalmus collected at site P1 and the tissues (Ramos Filho et al., 2008; Sharma et al., Ancistrus sp.. Site P1 was located close to the 2010). entrance of the Angélica cave, in an area that Considering the polyunsaturated fatty acids, could be characterized as a transition zone, and Gutierrez & Silva (1993) reported that the great where the concentration of organic matter in the majority of freshwater fish caught in Brazil sedimented material was intermediate between are deficient in the omega-3 series fatty acids, the concentrations measured at site P2 and the such as eicosapentaenoic acid (C20:5ω3) and epigean site. Hence, the hypogean fish found in docosahexaenoic acid (C22:6ω3). These two this region of the cave retained the principal route compounds are produced endogenously from for production of NADPH (the pentose phosphate α-linolenic acid, found in large quantities in marine pathway), while at the same time the greater plants. availability of food induced higher activity of the Eicosapentaenoic acid (C20:5ω3) was not detected malic enzyme in the alternative route. in either Ancistrus cryptophthalmus or Ancistrus sp., The mtDNA COI region could correctly differentiate while docosahexaenoic acid (C22:6ω3) was found at Ancistrus brevipinnis from Ancistrus sp and A. higher concentrations in the troglobitic fish. cryptophthalmus, but could not differentiate the According to Sargent et al. (1999), docosahexaenoic epigean species Ancistrus sp from the hypogean acid (C22:6ω3) is extremely important for animal species A. cryptophthalmus. The lack of genetic development, and can be found at high levels in divergence observed between Ancistrus sp and neural tissues as well as the ocular apparatus A. cryptophthalmus is strong evidence that both of fish. Since the ocular structure of Ancistrus morphotypes correspond to the same species. cryptophthalmus is less developed than that of the Phenotypic plasticity and gene flow between corresponding epigean organisms, there is greater epigean and hypogean populations are possible availability of this fatty acid in the muscle tissue. hypotheses that should be tested in order to The greater part of lipid synthesis occurs in the explain the contradiction between morphological hepatic tissue of fish (Figueiredo-Silva et al., 2005; and molecular data. Valente et al., 2007). In most animals, the pentose The most remarkable ecological characteristics phosphate pathway, catalyzed by the glucose-6- presented by hypogean fish populations are their phosphate dehydrogenase enzyme, is the principle small size, restricted geographical distribution, source of NADPH (up to 60%) for the synthesis of vulnerability to environmental disturbances and fatty acids. The malic enzyme, important in the sensitivity to stress (Bichuette & Trajano, 2008). pyruvate/malate cycle, provides an alternative Due to the fragility of these populations, it is very route that can also contribute to the supply of important to elucidate the metabolism of cave NADPH. The contribution of each of these enzymes fishes in order to understand their habits and depends, amongst other factors, on the species create favorable conditions for their preservation. and the conditions to which the fish are subjected (Wang, 2005). CONCLUSIONS The specific enzyme activity values obtained here for both fish populations are in agreement It is concluded that the characteristics of the with data reported in the literature, according to cave environment, especially the quantity of which specific activity values for fish lie in the available food, had a significant influence on ranges 0.109-0.920 U/mg of protein for glucose-6- the composition of fatty acids in the muscles, phosphate dehydrogenase, and 0.013-0.082 U/mg as well as on lipogenesis, in the fish Ancistrus of protein for the malic enzyme (Borba et al., 2003; cryptophthalmus, which contributed to differences Valente et al., 2007). between the hypogean and epigean populations. It has been reported that the activities of the enzymes The existence of an ecotone zone was observed that participate in the processes of lipogenesis and close to the entrance of the Angélica cave, where lipid storage in fish (such as glucose-6-phosphate the characteristics of the fish were intermediate dehydrogenase and the malic enzyme) are affected by between those of hypogean and epigean populations. the composition of the diet of the animal (Valente et Since Ancistrus cryptophthalmus and Ancistrus al., 2007). Studies have shown that in many species sp. (undescribed and non-troglomorphic species) of fish lipogenesis is stimulated by diets rich in comprise the same species (as shown by the genetic carbohydrates (Dias et al., 2004), and is suppressed analysis), we cannot consider A. cryptophtalmus as when the diet contains a high lipid content (Catacutan a troglobitic species, but as a troglophilic species & Coloso, 1997; Wang et al., 2005). with troglomorphic populations.

International Journal of Speleology, 42 (1), 15-23. Tampa, FL (USA) January 2013 22 Bastos et al. ACKNOWLEDGEMENTS Dias J., Rueda-Jasso R., Panserat S., Conceição L. E. C., Gomes E. F. & Dinis M. T., 2004 - Effect The authors are grateful to CNPq (Conselho Nacional of dietary carbohydrate-to-lipid ratios on growth, de Desenvolvimento Científico e Tecnológico) for lipid deposition and metabolic hepatic enzymes in juvenile Senegalese sole (Solea senegalensis. financial support, to IBAMA (Instituto Brasileiro do Kaup). Aquaculture Research, 35: 1122-1130. Meio Ambiente e dos Recursos Naturais Renováveis) http://dx.doi.org/10.1111/j.1365-2109.2004.01135.x for licenses nº 23907-1 and nº 13295-2, and to Figueiredo-Silva A. C., Rema P., Bandarra N. M., SEMARH (Secretaria do Meio Ambiente e dos Recursos Nunes M. L. & Valente L. M. P., 2005 - Effects Hídricos de Goiás) for license no. 010/2011. Thanks of dietary conjugated linoleic acido in growth, are also due to Francisco Alexandre Costa Sampaio nutrient utilization, body composition, and and Matheus Henrique Simões for assistance in field hepatic lipogenesis in rainbow trout juveniles collections; to Regina Beatriz and Arnor for hospitality (Oncorhynchus mykiss). Aquaculture, 248: 163-172. http://dx.doi.org/10.1016/j.aquaculture.2005.04.022 in the Terra Ronca State Park; and to Grupo Bambuí Folch J., Lees M. & Stanley G. H. S., 1957 - Simple de Pesquisas Espeleológicas for providing the map of method for the isolation and purification of total lipids the Angélica cave. RLF also thank Conselho Nacional from animal tissues. Journal of Biological Chemistry, do Desenvolvimento Científico e Tecnológico (CNPq 226: 497-509. grant 301061/2011-4). Graeve K., 1994 – Purification, characterization and cDNA sequence of glucose -6-phosphate dehydrogenase from potato (Solanum tuberosum L.). Plant Journal, 5: 353-361. 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Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Off icial Journal of Union Internationale de Spéléologie

Mixing of water in a carbonate aquifer, southern Italy, analysed through stable isotope investigations Emma Petrella*1 and Fulvio Celico1,2 1 Department of Biosciences and Environment, University of Molise, Contrada Fonte Lappone, 86090, Pesche (IS), Italy 2 Department of Physics and Earth Sciences “Macedonio Melloni”, University of Parma, Parco Area delle Scienze 157A, 43100, Parma, Italy

Abstract: Mixing of water was analysed in a carbonate aquifer, southern Italy, through stable isotope investigations (18O,δ2H). The input signal (rainwater) was compared with the isotopic content of a 35-meter groundwater vertical prof ile, over a 1-year period. Within the studied aquifer, recharge and flow are diffuse in a well-connected fissure network. At the test site, the comparison between input and groundwater isotopic signals illustrates that no efficient mixing takes place in the whole unsaturated zone, between the fresh infiltration water and the stored water. When analysing the stable isotope composition of groundwater, significant variations were observed above the threshold elevation of 1062 m asl, while a nearly constant composition was observed below the same threshold. Thus, temporal variations in stable isotope composition of rainwater are completely attenuated just in the deeper phreatic zone. On the whole, taking into consideration also the results of previous studies in the same area, the investigations showed that physical characteristics of the carbonate bedrock, as well as aquifer heterogeneity, are factors of utmost importance in influencing the complete mixing of water. These findings suggest a more complex scenario at catchment scale.

Keywords: aquifer heterogeneity; carbonate aquifer; mixing processes; stable isotopes; southern Italy Received 21 November 2011; Revised 16 September 2012; Accepted 12 October 2012

Citation: Petrella E. and Celico F. 2013. Mixing of water in a carbonate aquifer, southern Italy, analysed through stable isotope investigations. International Journal of Speleology, 42 (1), 25-33. Tampa, FL (USA) ISSN 0392-6672 http://dx.doi.org/10.5038/1827-806X.42.1.4

INTRODUCTION in three cave sites in the eastern U.S.A. (Tumbling Creek Cave, Missouri; Shenandoah Caverns, Virginia; At catchment scale, hydrology in carbonate systems Indian Echo Caverns, Pennsylvania) a nearly complete can be characterised by (a) diffuse or concentrated mixing of water in the first 10 ÷ 15 meters of the recharge and (b) diffuse or concentrated flow vadose zone, even if the structural and lithological (Atkinson, 1977; Williams, 1983; White, 1988; Ford settings of the three cave systems were felt to be & Williams, 1989). In the present study the attention sufficiently different. Schwarz et al. (2009), in the is focused on a carbonate aquifer system where Blautopf Catchment, one of the largest karst areas in recharge and flow are diffuse in a well-connected Germany, found that the isotope signals in seepage fissure network (Celico et al., 2006; Petrella et al., water in the caves (analysed at a depth ranging from 8 2008), but little information is available concerning to 45 meters from the surface) were almost completely mixing of water within the same system. Thus, the buffered and ranged around an average δ18O value. main purpose of this research was the identification Conversely, Cruz Jr. et al. (2005), in the Santana cave of the compartments of the aquifer system where systems in subtropical Brazil, found that the isotopic these processes take place. signal of water collected at drip sites, from 100 to 300 Several studies have been carried out in carbonate meters below ground, are directly related in a non- aquifers using stable isotopes (δ18O and δ2H) as linear fashion with rainwater input into the karst. conservative tracers (e.g. Maloszewski et al., 2002) to Within the present study, mixing processes were investigate the role of the unsaturated zone only or of analysed at site scale through isotopic investigations both the saturated and the unsaturated ones in mixing (δ18O, δ2H) over a 1-year period, by comparing the processes. However, different findings were obtained input signal (rainwater) with the isotopic content of in different test sites. Yonge et al. (1985) found a 35-meter groundwater vertical profile. Due to the

*[email protected] 26 Petrella and Celico absence of accessible caves and conduits, cave drip- cataclasite is observed, allow significant groundwater water studies were not possible. Thus, the types of flowthrough, and the interdependence of hydraulic vadose flow (preferential or fracture fed; matrix heads up- and down-gradient of these faults has been or seepage fed; Baker & Brundson, 2003) were not observed (Celico et al., 2006). The aquifer behaves as directly investigated, contrary to other studies (eg. a basin-in-series system, where the hydraulic head Baker et al., 1997; Genty & Deflandre, 1998; Baker shows a step-like shape and seasonal and temporary et al., 2000; Kaufman et al., 2003; Tooth & Fairchild, springs occur along some fault zones (Petrella et al., 2003; Spötl et al., 2005; Karmann et al., 2007; 2009a). At the basin scale, the groundwater flows McDonald & Drysdale, 2007; Lange et al., 2010). westwards towards the perennial spring (Fig. 1). The role of the unsaturated zone (thickness ranging The carbonate medium is laterally and vertically from 27 to 62 meters during the observation period) well connected in the subsurface, and the fracture was analysed based on observations made within the spacing is sufficiently dense to apply the continuum groundwater profile in a purpose-drilled well. approach to describe groundwater flow at the metric scale (Petrella et al., 2007). A significant vertical DESCRIPTION OF THE FIELD SITE heterogeneity of the carbonate bedrock has been found, due to differences in fissuring and karstification with The focus of the work lies on a carbonate aquifer depth (Petrella et al., 2007). Darcy’s law can be applied (Acqua dei Faggi, Longano, southern Italy; Fig. 1) in epikarstic horizons with some karstification, and formed predominantly of limestone (Monte Calvello groundwater flow is also expected to be laminar in and Monaci Formations; Cretaceous-Oligocene) diffusely karstified epikarst and in the underlying and subordinately of lower permeability rocks fissured bedrock (Petrella et al., 2008). (Macchiagodena Formation; Oligocene-Burdigalian) The groundwater responds rapidly to recharge (De Corso et al., 1998). The Macchiagodena Formation events, due to fast and diffuse rainwater infiltration. consists mainly of marls and marly limestone. Monte The funnelling effect into larger shafts does not play Calvello and Monaci Formations are mainly made up an important role in the hydrogeological behaviour of of calcarenites and calcirudites with intergranular the aquifer (Petrella et al., 2007). At the aquifer scale, material composed predominantly of spatic cement, the thickness of the unsaturated medium ranges biocalcarenites and biocalcirudites. Carbonates have from a few centimetres (Celico et al., 2006) to some very low primary porosity, but are extensively fissured. hundreds of meters (Petrella et al., 2009a), depending Natural gradient tracer tests yielded an opening on the area and the groundwater level fluctuations. porosity given by fissures of 2.3 x 10-4 (Petrella et al., 2008). Lugeon tests showed hydraulic conductivity MATERIALS AND METHODS of limestone in the order of 10-6 m s-1 (Petrella et al., 2007). Carbonate rocks are exposed or lie below a Hydrogeological investigations cover of pyroclastic origin (Naclerio et al., 2008). The hydraulic head was measured on an hourly The aquifer is bounded by fault zones that act as basis with a pressure transducer, from May 2008 barriers to groundwater flow and compartmentalise to August 2009, at well P1 (Fig. 1). The well P1 was the aquifer system (Celico et al., 2006; Petrella et al., drilled in order to analyse mixing processes in one 2009a). However, some fault zones, where calcite-filled of the sites where the unsaturated zone is relatively

Fig. 1. Hydrogeological map (1 Quaternary deposits; 2 marls and clays; 3 marls and marly limestone; 4 limestone; 5 dolostone; 6 aquifer boundary; 7 fault; 8 perennial spring; 9 seasonal spring; 10 well; 11 main groundwater flow direction; 12 Rain Water Sampler [RWS]; 13 elevation contour line; the numbers are expressed in m asl; 14 location of the well shown in Fig. 8).

International Journal of Speleology, 42 (1), 25-33. Tampa, FL (USA) January 2013 Mixing of water in a carbonate aquifer 27 thin, if compared with the rest of the studied Isotopic analyses aquifer system. The well (130 m deep) is drilled Isotopic analyses (δ18O, δ2H) were carried out at the through a few meters of cover of pyroclastic origin Laboratorio di Geochimica Isotopica of the University and then through limestone. It is full-screened, of Parma (Italy), and at the Istituto di Geoscienze e except for the first 5 meters that are sealed in order Georisorse of the CNR, Pisa (Italy), using Isotope Ratio to prevent the local infiltration of surface water. Mass Spectrometry (IRMS). The analytical precision This well is the same borehole that was used in was ±0.1‰ for δ18O, ±1.0‰ for δ2H. a previous study (Petrella et al., 2009b), during The compositions of δ18O and δ2H are reported in which experimental data demonstrated the local δ‰ vs. V-SMOW (Vienna Standard Mean Ocean diffuse infiltration of rainwater through topsoil and Water) standard. limestone bedrock, and showed the effect of this infiltration on the shallower groundwater in terms Mixing analysis of salinity modification. Other investigations, in The two-component isotopic separation technique Petrella et al. (2009b), were carried out only to (Pinder & Jones, 1969; Sklash & Farvolden, 1979) was determine the origin of the lower- and the higher- used to calculate the proportion of freshwater mixed salinity groundwater which were detected during with pre-event groundwater along the investigated this survey. groundwater profile, during the main recharge events. This well is therefore useful to analyse other The mixing was calculated using eq. (1): processes, such as mixing, related to the interaction between fresh infiltration water and groundwater. XT CT = XA CA + XB CB

Water sampling where XT is groundwater at any depth, XA is pre- Rainwater event groundwater (corresponding to the deepest 18 2 Rainwater samples for isotopic (δ O, δ H) analyses sampled one along the investigated profile), XB is the were collected at a rain sampler (RWS1), located event water (corresponding to rainwater collected at 18 within the aquifer system, close to P1, at 1150 RWS1), CT, CA, and CB are δ O composition (‰) of meters above sea level (m asl; Fig. 1). The sampling groundwater at any depth, of pre-event groundwater, was carried out on a monthly basis from May 2008 and of event water, respectively. to August 2009, to compare the input isotopic signal with the isotopic content of groundwater, during the RESULTS same observation period. A more detailed analysis of rainwater isotopic Input isotopic signal content was obtained collecting samples on a With regard to stable isotopes composition of weekly basis, from April 2006 to January 2008, rainfall (Tab. 1) it is possible to calculate the following at another rain sampler (RWS2) located within the equation that describes the Local Meteoric Water Line same aquifer system (1014 m asl). Polyethylene (LMWL): bottles (10 L) containing about 300 ml of vaseline oil to prevent evaporation processes even under δ2H = 7.7 δ18O + 12.7 (R2=0.96; n of samples=15) very hot summer conditions were used to collect the samples. Oil contamination was carefully This equation is very close (Fig. 2) to the Western avoided while syringing the water samples out of Mediterranean Meteoric Water Line (WMMWL; δ2H the bottle. = 8 δ 18O + 13.7) found by Celle-Jeanton et al. (2001) in several stations located in the Mediterranean Groundwater seaside countries. All rainwater samples fall Groundwater samples for stable isotope (δ18O, between the Global Meteoric Water Line (GMWL; δ2H) analyses were collected at P1 at different δ2H = 8 δ18O + 10; Craig, 1961) and the Easter depths, using a 5-m-depth interval. Preliminary Mediterranean Water Line (EMWL; δ2H = 8 δ18O investigations suggested that a maximum + 22; Gat & Carmi, 1970) underlining that the investigated depth of 35 m below the groundwater variability is influenced by both the Atlantic and table was necessary to thoroughly analyse the Mediterranean air masses. This is also evident variations of the groundwater isotopic signal at from the deuterium excess values (d in Table 1). the study site. Collection was carried out 10 times, From May 2008 to August 2009 the weighted mean from June 2008 to August 2009. isotopic composition for the precipitation was A stainless steel bailer was used (250 cm3 volume) -7.88‰ for δ18O and -47.38‰ for δ2H. However, fitted with a one-way valve at the lower end and a significant temporal variability of the signal attached to 6 mm plastic tubing at the upper end. was observed in monthly cumulated rainfall, with The upper end of the tubing was attached at the values ranging from -10.48‰ and -5.11‰ for surface to a pump. The bailer was pressurised δ18O and from -70.80‰ to -24.30‰ for δ2H. The with air before lowering it to the chosen sampling significant temporal variability of the input signal is depth. Once at the chosen depth, the pressure was more thoroughly observed in the samples collected released, allowing water to enter the bailer. After weekly from April 2006 to January 2008 in RWS2, the bailer was withdrawn, a sample of water was with values ranging from -15.70‰ and -2.61‰ for transferred to a polyethylene bottle. δ18O and from -117.46‰ to -9.58‰ for δ2H.

International Journal of Speleology, 42 (1), 25-33. Tampa, FL (USA) January 2013 28 Petrella and Celico Table 1. Isotopic and d-excess values in RWS1 rainwater samples.

δ18O ‰ δ2H ‰ d-excess (vs. V-SMOW) (vs. V-SMOW)

04/06/2008 -8.01 -51.40 12.68

07/07/2008 -5.31 -31.00 11.48

31/07/2008 -5.78 -31.70 14.54

03/09/2008 -5.84 -31.90 14.82

06/10/2008 -8.39 -49.30 17.82

03/11/2008 -5.11 -24.30 16.58

04/12/2008 -7.93 -42.40 21.04

09/01/2009 -9.50 -59.50 16.50

03/02/2009 -10.48 -70.80 13.04 Fig. 3. Variations of δ18O below the hydraulic head. Full lines are 06/03/2009 -8.25 -49.90 16.10 referred to “type A” prof iles, while dashed lines are referred to “type B” prof iles. 09/04/2009 -5.38 -27.90 15.14

07/05/2009 -7.75 -46.50 15.50 isotopic composition varied significantly (variations were greater than the 2σ error of the analyses) within 10/06/2009 -5.77 -34.30 11.86 the whole investigated profile.

06/07/2009 -7.59 -47.90 12.82 Comparison between rain- and groundwater signals 06/08/2009 - - - The temporal variations of the input function were strongly, but not completely reduced at the groundwater table. Variations of groundwater Groundwater isotopic signal isotopic content were observed at shallower After plotting the isotopic data of groundwater depths below the hydraulic head, and were clearly collected at different depths at P1 in the δ18O vs. δ2H due to mixing between groundwater and fresh scattergram (Fig. 2) it is observed that all samples are infiltration water. In fact, taking into account only well situated along the LMWL, therefore suggesting the recharge period (from November 2008 to May a complete meteoric origin of groundwater within 2009) when the rainfall produces huge effective the whole investigated profile. However, interesting infiltration, the mixing analysis (Tab.2) shows that differences in groundwater isotopic content were fresh infiltration water is up to 40% of the whole observed with depth. Two main types of profiles (A groundwater within the upper 10 meters below and B) were detected within the 35-m-deep interval. the hydraulic head in early November 2008 and in Type A (full lines in Fig. 3) was observed during February 2009. Below this depth, the percentage late recession, when the isotopic composition was of fresh infiltration water was negligible (less than homogeneous (variations were lower than the 2σ error 5%). The negative values of infiltrated water are in of the analyses) down to the maximum investigated Table 2. Percentage of fresh water in the whole groundwater, at depth. Type B (dashed lines in Fig. 3) was observed different depths, during huge effective infiltration. during recharge and early recession, when the

Depth below 03/11/2008 03/02/2009 groundwater level (m)

0 30.1 34.6

-5 31.3 39.4

-10 28.8 31.1

-15 0.3 5.1

-20 -0.9 -1.6

Fig. 2. δ18O vs. δ2H relationship in rainfall (triangles; Local Meteoric -25 0.6 -6.3 Water Line [LMWL]) and groundwater samples (full dots). The Global Meteoric Water Line (GMWL; Craig, 1961), the Eastern -30 1.3 -3.9 Mediterranean Meteoric Water Line (EMMWL; Gat & Carmi, 1970) and the Western Mediterranean Meteoric Water Line (WMMWL; -35 0.0 0.0 Celle-Jeanton et al., 2001) are given for reference.

International Journal of Speleology, 42 (1), 25-33. Tampa, FL (USA) January 2013 Mixing of water in a carbonate aquifer 29 the range of the error related to the two-component and groundwater isotopic signals illustrates that no isotopic separation technique. This phenomenon complete mixing takes place in the whole unsaturated has been thoroughly investigated and quantified zone, between the fresh infiltration water and the during this recharge period, because the strong stored water. This observation, together with the fast difference between the isotopic content of rainwater response of the hydraulic head to recharge events, and that of pre-event groundwater, and the huge suggests the widespread existence of an important effective infiltration both allowed to distinguish vadose flow in larger fissures at the study site, even in a detailed manner the influence of event water though the mobilisation of pre-event water from on pre-event groundwater (Fig. 4). This time span smaller fissures and rock matrix cannot be excluded. is enough to experimentally verify how mixing of Existence and importance of diffuse and fast vadose water takes place within the unsaturated zone flow in larger fissures is also in agreement with and the shallower saturated zone. In early April the short transit time observed when studying 2009, isotopically heavier rainwater caused a new contaminant transport within the same system, slight increase in isotopic content of the same induced by diffuse migration of microorganisms groundwater layer, but the difference in isotopic through the soil and the limestone bedrock (Celico et composition between end-members is not useful al., 2004a and b; Naclerio et al., 2008, 2009). to thoroughly quantify the mixing, as carried out during the previous events. The isotopic content DISCUSSION AND CONCLUSIONS remained nearly constant in early May 2009, due to precipitation that was characterised by When analysing the stable isotope composition a composition very close to that of groundwater of groundwater as a function of the depth below (Fig. 4). In such a scenario, the slight increase ground surface, significant variations (greater than in isotopic content in the deeper groundwater, the 2σ error of the analyses) were observed above that was observed from February 2009 onwards, the threshold elevation of 1062 m asl (~78 meters indicates a mixing between the upper and below the ground surface), while a nearly constant isotopically heavier, and the lower and isotopically composition was observed below the same threshold lighter groundwater layer detected in November (Fig. 5). These differences do not correspond to 2008 (Fig. 4). The absence of precipitation in significant differences in terms of residence time, as June and July 2009 emphasised the results of demonstrated in a former study (3H content in both this mixing in terms of isotopic composition of the the upper and the lower groundwater layers is 4.5 different groundwater layers (Fig. 4). Tritium Units; Petrella et al., 2009b). The slight difference between the weighted mean These findings suggest that temporal variations isotopic composition of rainwater (-7.88‰ for δ18O in stable isotope composition of rainwater are not and -47.38‰ for δ2H) and the nearly-constant completely damped in the whole unsaturated medium, isotopic content of the deeper groundwater layer as well as in the upper phreatic zone, during recharge. (-8.22‰ ± 0.15 for δ18O, and -49.30‰ ±1.88 for Since the dampening of the signal is well observed δ2H) is explained taking into consideration that the in the deeper phreatic zone, close to the depth rainwater sampler (RWS1) is at an elevation lower than at which geophysical investigations detected the the mean altitude of the catchment area (δ18O vertical transition between limestone with a well-connected gradient -0.16‰/100m, Petrella et al., 2009a). fissure network (ρ 2500 Ω m) and limestone with As a matter of fact, the comparison between input a poorly-developed fissure network (ρ 10000 Ω ≌ ≌

Fig. 5. Schematic of the attenuation of temporal isotope (δ18O) variations in recharge waters during infiltration through the Fig. 4. Temporal variations of δ18O of groundwater at different depths unsaturated zone (UZ) and flow within the shallower (SG) and the below the hydraulic head (coloured lines with dots; the dots show the deeper (DG) groundwater, and the critical depth below which isotopic date of water collection), variations of monthly weighted mean δ18O of variability is less than the analytical precision at the test site. The dots rainwater (blue dots; numbers are the amount of rainfall in mm), and show the isotopic composition of precipitation at the test site during variation of the hydraulic head (dashed line). the study.

International Journal of Speleology, 42 (1), 25-33. Tampa, FL (USA) January 2013 30 Petrella and Celico m)(Petrella et al., 2009b), physical characteristics Conversely, where the hydraulic head fluctuates of the carbonate bedrock and aquifer heterogeneity in the deeper limestone bedrock, a more complex are factors of utmost importance in influencing the scenario is expected. From the top to the bottom, complete mixing of water. the sequence is made of (a) epikarst, (b) limestone In a wider context, taking into consideration with well-developed fissure network mainly due physical and hydraulic properties of the aquifer to stress release fracturing, (c) limestone with at catchment scale, as well as its heterogeneities, poorly-developed fissure network, (d) limestone different scenarios are expected at the same scale, with well-developed fissure network and relatively regarding mixing of water. The whole setting can be high effective porosity, due to mixing of water depicted as follows. with different chemical composition, (e) deeper Coexistence of an upper several-meters-thick limestone bedrock with poorly developed fissure limestone bedrock with a well-developed fracture network (box B in Fig. 6). In this second scenario, network (thicker than the epikarst only) and a the complete mixing of water might occur within lower limestone bedrock with a poorly-developed the unsaturated zone. fracture network are principally due to stress release At catchment scale, mixing processes are further fracturing, which occurs because of unloading stress influenced by discontinuous heterogeneities, due where rock has been removed. This phenomenon to fault zones, where physical characteristics of causes an increased density of joints and bedding the carbonate bedrock can be locally influenced plane partings in the near-surface bedrock also by tectonics, with coexistence (at metric, (Ferguson, 1967; Sasowsky & White, 1994). However, decametric or hectometric scale) of fault core vertical heterogeneity can be further emphasised by and damage zone (eg. Mollema & Antonellini, mixing processes between groundwater and fresh 1999; Salvini et al., 1999; Billi et al., 2003; Billi, infiltration water just below the water table of an 2005; Agosta & Aydin, 2006; Billi et al., 2007; unconfined aquifer. When saturated waters with Balsamo et al., 2008; Storti & Balsamo, 2010). different chemical composition mix, new aggressivity Fault cores can include single slip surfaces arises and widening of fissures can be caused, due to (Caine et al., 1991), highly indurated, cataclastic carbonate dissolution (e.g., Gabrovšek & Dreybrodt, zones (Chester & Logan, 1986), brecciated and 2000; Romanov et al., 2003; Gabrovšek & Dreybrodt, geochemically altered zones (Sibson, 1977), or 2010). In the studied aquifer, great hydraulic head unconsolidated clay-rich gouge zones (Anderson fluctuations were observed during each hydrologic et al., 1983). The damage zone includes fault- year in several wells (30 to 100 meters; Petrella et al., 2007 and 2009a), therefore suggesting that this process can influence the widening of the fissures at really different depths and within relatively thick horizons, depending on the zone of the aquifer system, and the phase of the hydrologic year. For example, in the test site where the well P1 has been drilled, the depth at which the contrast between the more and the less fissured bedrock has been detected (~70 meters below the ground, Petrella et al., 2009b) is close to the maximum depth at which the hydraulic head has been recorded from 2005 on (~73 meters below the ground). Moreover, taking into consideration that in high flow the hydraulic head at this site rises close to the ground surface, the mixing between waters with different chemical composition occurs within a several tens of meters- thick limestone bedrock. In a wider context, these statements suggest the existence of two different scenarios at least, at catchment scale. Where the uppermost limestone bedrock is nearly completely involved in head fluctuations during the hydrologic year, the following sequence is expected, from the top to the bottom: (a) epikarst, (b) limestone bedrock with well-developed fissure network and relatively high effective porosity, due to stress release Fig. 6. Conceptual model (not to scale) showing both layered and fracturing and mixing of water with different chemical discontinuous heterogeneities influencing mixing processes within the composition, (c) limestone bedrock with poorly- aquifer system (1 topsoil; 2 epikarst; 3 limestone bedrock with well- developed opening network (box A in Fig. 6). In this developed fissure network due to (a) stress release fracturing and/or scenario, the complete mixing of water is expected (b) carbonate dissolution due to mixing between water with different chemical composition; 4 limestone bedrock with poorly-developed to occur within the phreatic zone, as observed in the fissure network; 5 fault zone; 6 spring; 7 hydraulic head in low- [solid studied test site. line], and in high-flow period [dashed line]; 8 groundwater flow).

International Journal of Speleology, 42 (1), 25-33. Tampa, FL (USA) January 2013 Mixing of water in a carbonate aquifer 31

Fig. 7. Non-permanent haloclines observed in P1, within the protolith. No nearby faults are observed upgradient of P1 (modif ied from Petrella et al., 2009b). related subsidiary structures and can enhance fault zone permeability relative to the core and the undeformed protolith (e.g. Chester & Logan, 1986; Andersson et al., 1991; Goddard & Evans, 1995). Thus, mixing of water is expected to occur within these zones, due to coexistence of thinner and thicker openings in a complex architecture (box C in Fig. 6). The hypothesis that mixing processes can occur through fault zones is supported by comparison of electrical conductivity (EC) vertical profiles in groundwater within the protolith and close to fault zones, downgradient of the faults. In former studies (Petrella et al., 2009a, 2009b), non-permanent haloclines were observed within the protolith of the studied aquifer system, due to non-complete mixing between fresh-infiltration water and pre- event water in the shallower groundwater (in Fig. 7, examples of haloclines observed in the same well P1 used within this study are shown; no nearby faults are observed upgradient of P1; Fig. 1). On the contrary, a few tens of meters downgradient of a normal fault, the EC vertical profile is homogeneous down to the maximum investigated depth throughout the hydrologic year, despite recharge and layered heterogeneity of the carbonate bedrock (Fig. 8). We interpret this phenomenon as the result of mixing, through the fault zone, between lower and higher salinity groundwater layers flowing upgradient of the fault. Summarising, mixing processes in the studied aquifer system are influenced by both the layered and the discontinuous heterogeneity. Thus, different scenarios coexist at catchment scale, and this complex setting must be taken into consideration when interpreting chemographs Fig. 8. Homogeneous EC vertical prof iles observed in a well located a few tens of meters downgradient of a normal fault, in high (Scenario and isotopic signals of perennial, seasonal and “A”) and in low (Scenario “B”) flow periods (modified from Petrella et temporary spring-waters. al., 2008).

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International Journal of Speleology, 42 (1), 25-33. Tampa, FL (USA) January 2013 Mixing of water in a carbonate aquifer 33 Kaufman A., Bar-Matthews M., Ayalon A. & Carmi I., Pinder G.F. & Jones J.F., 1969 - Determination of the 2003 - The vadose flow above Soreq Cave, Israel: groundwater component of peak discharge from the A tritium study of the cave waters. Journal of chemistry of total runoff water. Water Resources Hydrology, 273: 155-163. Research, 5: 438-445. http://dx.doi.org/10.1016/S0022-1694(02)00394-3 http://dx.doi.org/10.1029/WR005i002p00438 Lange J., Arbel Y., Grodek T. & Greenbaum N., Romanov D., Gabrovšek F. & Dreybrodt W., 2003 - 2010 - Water percolation process studies in a The impact of hydrochemical boundary conditions on Mediterranean karst area. Hydrological Processes, the evolution of limestone karst aquifers. Journal of 24: 1866-1879. http://dx.doi.org/10.1002/hyp.7624 Hydrology, 276: 240-253. 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Terra Nova, 19: 82-88. Storti F. & Balsamo F., 2010 - Impact of ephemeral http://dx.doi.org/10.1111/j.1365-3121.2006.00720.x cataclastic fabrics on laser diffraction particle Petrella E., Falasca A. & Celico F., 2008 - Natural- size distribution analysis in loose carbonate fault gradient tracer experiments in epikarst: A test study breccia. Journal of Structural Geology, 32: 507- in the Acqua dei Faggi experimental site, southern 522. http://dx.doi.org/10.1016/j.jsg.2010.02.006 Italy. Geofluids, 8: 159-166. Tooth A.F. & Fairchild I.J., 2003 - Soil and karst http://dx.doi.org/10.1111/j.1468-8123.2008.00214.x hydrological controls on the geochemical evolution Petrella E., Capuano P., Carcione M. & Celico F., of speleothems-forming drip waters, Crag Cave, 2009a - A high-altitude temporary spring in a southwest Ireland. 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Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Official Journal of Union Internationale de Spéléologie

Cure from the cave: volcanic cave actinomycetes and their potential in drug discovery Naowarat Cheeptham1*, Tara Sadoway1, Devon Rule1, Kent Watson3, Paul Moote1, Laiel C. Soliman2, Nicholas Azad2, Kingsley K. Donkor2, and Derrick Horne4 1 Department of Biological Sciences, Thompson Rivers University, Kamloops, British Columbia, Canada 2 Department of Physical Sciences, Thompson Rivers University, Kamloops, British Columbia, Canada 3 Department of Natural Resource Sciences, Faculty of Science, Thompson Rivers University, Kamloops, British Columbia, Canada 4 BioImaging Facility, University of British Columbia, Vancouver, British Columbia, Canada Abstract: Volcanic caves have been little studied for their potential as sources of novel microbial species and bioactive compounds with new scaffolds. We present the first study of volcanic cave microbiology from Canada and suggest that this habitat has great potential for the isolation of novel bioactive substances. Sample locations were plotted on a contour map that was compiled in ArcView 3.2. Over 400 bacterial isolates were obtained from the Helmcken Falls cave in Wells Gray Provincial Park, British Columbia. From our preliminary screen, of 400 isolates tested, 1% showed activity against extended spectrum ß-lactamase E. coli, 1.75% against Escherichia coli, 2.25% against Acinetobacter baumannii, and 26.50% against Klebsiella pneumoniae. In addition, 10.25% showed activity against Micrococcus luteus, 2% against methicillin resistant Staphylococcus aureus, 9.25% against Mycobacterium smegmatis, 6.25% Pseudomonas aeruginosa and 7.5% against Candida albicans. Chemical and physical characteristics of three rock wall samples were studied using scanning electron microscopy and flame atomic absorption spectrometry. Calcium (Ca), iron (Fe), and aluminum (Al) were the most abundant components while magnesium (Mg), sodium (Na), arsenic (As), lead (Pb), chromium (Cr), and barium (Ba) were second most abundant with cadmium (Cd) and potassium (K) were the least abundant in our samples. Scanning electron microscopy (SEM) showed the presence of microscopic life forms in all three rock wall samples. 16S rRNA gene sequencing of 82 isolates revealed that 65 (79.3%) of the strains belong to the Streptomyces genus and 5 (6.1%) were members of Bacillus, Pseudomonas, Nocardia and Erwinia genera. Interestingly, twelve (14.6%) of the 16S rRNA sequences showed similarity to unidentif ied ribosomal RNA sequences in the library databases, the sequences of these isolates need to be further investigated using the EzTaxon-e database (http://eztaxon-e. ezbiocloud.net/) to determine whether or not these are novel species. Nevertheless, this suggests the possibility that they could be unstudied or rare bacteria. The Helmcken Falls cave microbiome possesses a great diversity of microbes with the potential for studies of novel microbial interactions and the isolation of new types of antimicrobial agents. Keywords: volcanic cave; actinomycetes; bioprospecting; antimicrobial activity; multidrug resistant bacteria Received 23 July 2012; Revised 23 October 2012; Accepted 25 October 2012 Citation: Cheeptham N., Sadoway T., Rule D., Watson K., Moote P., Soliman L.C., Azad N., Donkor K.K. and Horne D. 2013. Cure from the cave: volcanic cave actinomycetes and their potential in drug discovery. International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) ISSN 0392-6672 http://dx.doi.org/10.5038/1827-806X.42.1.5

INTRODUCTION well understood, hence, there is a need to investigate further (Northup & Welbourn, 1997; Barton et al., Volcanic cave actinomycetes, a group of Gram- 2001; Northup & Lavoie, 2001; Barton & Luiszer, positive filamentous bacteria, are relatively less 2005; Barton, 2006; Barton & Jurado, 2007; Barton studied for their capacity as potential sources of novel & Northup, 2007; Snider et al., 2009; Engel, 2010; species and new antimicrobials with different mode Lavoie et al., 2010; Bhullar et al., 2012). Antibiotic of actions. In such nutrient-deprived environment, resistance in pathogens is surfacing at an increasingly their original relationships in their own communities alarming rate in hospitals and communities around and with other organisms in the same habitats, their the world. When dealing with multidrug resistant contributions to cave formation and degradation, as pathogens, currently available antibiotics have well as their evolutionary patterns and their potential often fallen short for their lack of specific inhibitory to be used to produce bioactive compounds are not activities or with negative side effects. Therefore, there

*[email protected] 36 Cheeptham et al. is a greater need than ever to discover and identify been few published reports on analyses of microbes novel microorganisms that can produce new scaffolds found in volcanic caves in North America and of bioactive compounds to fight existing and emerging modest number of reports on the potential use of resistant infectious agents. In a recent study, Bhullar microbes from volcanic caves worldwide regarding and colleagues discovered that antibiotic resistance drug discovery (Northup et al., 2004, 2011). Cave is innate in bacteria and it is not linked with habitats are of interest as the main focus for a new antibiotic uses as it was generally thought of when pool of microbial diversity and as a major source of isolated cave bacteria were subjects of such study microorganisms that produce antimicrobial agents. (Bhullar et al., 2012). The implication of this study Cave-adapted organisms are classic examples of suggests that there are potentially far more unfound regressive evolution: over time, unused, unusual antibiotics in the environments than was previously and obvious functions become reduced, while other estimated. Since streptomycin was discovered in functions become more pronounced. A good example 1943, it has been shown that actinomycetes, Gram- of this type of evolution is seen in cavefish: these positive bacteria with a high G+C content, are fish lose their eyes and pigmentation over time, but still a reliable and prolific source of new bioactive gain in other sensory systems to compensate for compounds (Lazzarini et al., 2009; Nakaew et al., the absence of vision (Yoshizawa et al., 2010). The 2009a,b). It is known that approximately two-thirds bases for these developmental changes are genetic. of naturally occurring antimicrobial products are Theoretically, the same process could occur in cave produced from actinomycetes (Okami & Hotta, 1988; adapted microorganisms, which need to adapt to Baltz, 2007; Atta et al., 2012). Large numbers of the extreme starvation environment in the cave novel actinomycetes were reported to be present system. Recent work done by Northup and colleagues in unexplored habitats worldwide including cave demonstrated that cave bacteria are more sensitive environments (Groth et al., 1999; Jones, 2001; Groth to ultraviolet radiation (UVR) than those of surface et al., 2001; Nakaew et al., 2009a, b). Jurado et al. bacteria (Snider et al., 2009). Since these bacteria (2010) reported that 34 novel species of actinomycetes live in the dark hence losing an unneeded trait such were isolated from subterranean environments in the as UVR resistance makes sense for their survival decade 2000-2009, as gathered in the International when present in low-nutrient habitats (Snider et al., Journal of Systematic and Evolutionary Microbiology. 2009). Such changes and adaptations might lead These novel actinomycetes were exemplified by the the microorganisms to acquire different pathways description of Beutenbergia cavernae, a new genus of for their physiology and metabolism which may be L-lysine-containing actinomycete (Groth et al., 1999) beneficial for drug discovery. For these reasons, cave and Agromyces subbeticus isolated from a cave in microorganisms are viewed as a potential resource for southern Spain (Jurado et al., 2005). studies on genetics and on the mechanisms by which The significance of microbial diversity in search and they produce bioactive compounds. These studies discovery of novel metabolites has been underlined may also provide solutions to current problems in in more recent articles (Bull et al., 2000; Langer et medicine, biology, agriculture, environment and other al., 2006; Tripathi et al., 2007; Beloqui et al., 2008; disciplines. Singh & Pelaez, 2008). Consequently, our study was This initial work is undoubtedly the first on the to search in previously uncharacterized habitats isolation of Canadian volcanic cave actinomycetes to uncover novel actinomycetes that may produce and their screening for antimicrobial agent(s) that is antimicrobial agents against a number of antibiotic a part of a larger study on microbial diversity in caves resistant microorganisms such as Klebsiella of Canada. pneumoniae KPC-1 and Acinetobacter baumannii. A volcanic cave habitat was chosen as a potential MATERIALS AND METHODS source of antimicrobial producing actinomycete strains because of the caves’ unique characteristics Cave Description and the little amount of study of similar habitats. In The Helmcken Falls cave entrance is situated general, caves’ unique characteristics include limited approximately 550 metres at a bearing of 2980 from amount of light, low bioavailable organic nutrients, the top of the Helmcken Falls (51º57’14.47” N, high humidity, high concentration of minerals with 120º10’36.48” W) (Google Earth, 2012). The falls is a constant temperature all year round. In this study, about 141 metres high (Fig. 1) and is located near the Helmcken Falls basaltic volcanic cave, in the interior mouth of the Murtle River where it joins the Clearwater of British Columbia of Canada was the source of River, British Columbia, Canada. This cave has limited microorganisms. Knowledge of cave microbial diversity visits from tourists and human activities (less than 10 is very limited, despite the fact that caves are found people a year) since the unmarked trail leading to the abundantly throughout the world. Microbial diversity entrance of the cave is very rough and it takes about has been studied in Karstic caves (caves in limestone two and a half hours to hike from the park’s parking formations) in order to better understand the impacts lot, (elevation 753 metres), to the entrance of the cave of microbes on cave formation/degradation and to (Fig. 2). The opening (51º57’22.45” N, 120º11’02.11” determine how this knowledge can be used for cave W) is located at the top of a colluvial talus slope at conservation (Laiz et al., 1999; Engel et al., 2001; an elevation of 652 metres. The Murtle River, directly Barton et al., 2004, 2006, 2007; Barton & Luiszer, below the cave entrance, is at 603 metres (Google 2005; Gonzalez et al., 2006). However, there have Earth, 2012). The interior of the cave, as shown in the

International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 Volcanic cave actinomycetes and their potential in drug discovery 37

Fig. 3. Panoramic images from cave interior. a) (top) shows the entrance view taken from the back of the cave. The entrance has almost been completely closed by angular colluvial blocks that have fallen from the outside cliff face. Talus debris slopes from the back and sides of the cave to the lowest point in the center of the image. This rubble suggests that there has been a collapse of material in conjunction with water erosion. Fluvial sands were deposited along the upper slopes in contact with the cave roof. b) (bottom) shows the back view at the end of the cave taken Fig. 1. Helmcken Falls, Wells Gray Provincial Park, British Columbia, from the lowest point of the cave entrance. The fluvial sand (yellowish Canada. The cave is located 100 metres almost vertically directly brown) is easily seen along the upper parts of the talus slope. It does not below from where this photograph was taken. The bowl at the base of occur within the angular blocks. Soil samples were taken from the sand the fall has been eroded by water action. which suggests that the cave was scoured by water action.

Fig. 3a and 3b, indicates that the formation of this cave the Clearwater River, it cut through an existing lava may have originated as a lava tube that was modified tube and water eroded the walls of the circular tube. by river action. A typical lava tube is formed by a lava The roof of the tube collapsed forming a flat ceiling flow developing a solidified crust over hot lava that while the thicker material around the outside of the continues to flow beneath leaving the shell behind as tube collapsed and formed the sloping talus that rises an open tube with a circular or oval cross sectional from the center of the cave floor upwards to meet the shape. However, this cave has a flat basalt roof and cave roof. Sediment loaded water entered the collapsed upward sloping sides composed of angular colluvial cavern, deposited sands and fine gravels, laminated in blocks. Fluvial sands, stratified in some places, are some places, along the upper colluvial slopes between found along the edges of the cave where the upward the floor and ceiling. The other possibility is that the sloping floor meets the ceiling. The cave entrance is cave was created totally by water erosion similar to the almost completely closed by angular colluvial rubble large bowl that can be seen at the base of Helmcken that has fallen from the 100 metre high cliff that rises Falls today (Fig. 1). Similar flat lenticular cave above the entrance. It is theorized that, as the Murtle structures are visible on the opposite canyon walls. River eroded back through the basaltic flows from The cave is 5 m high, 72 m long and 20 m wide. The cave has only one big room, the main room is where all of our sediments and rock samples were collected. Near the entrance of the cave, the only place of light exposure, biofilms of phototrophic microorganisms were observed. There were a number of areas with incoming drips of water and where we found a type of decoration, cave popcorns. The cave floor, walls, and ceiling are composed of basalt. A preliminary map showing the cave area and contours was produced in ArcView 3.2 (Fig. 4 and 5).

Cave mapping The cave mapping has been conducted since 2005 as an on-going project. A Trimble Global Positioning (GPS) unit was used to locate an approximate latitude Fig. 2. The entrance to the cave is almost completely closed by large and longitude position outside the Helmcken Falls angular colluvial blocks that have fallen from the 100 metre high cliff cave entrance. The coordinates were 51º57’26.25664” face which rises vertically to the parking lot above. Before the cave N and 120º11’08.79861” W. The GPS elevation entrance would have been 5 or more metres high. The curved nature was 674.278 m above sea level (ASL). This position of the opening and the curvature of the lava flow suggest this cave may have been a lava tube that was affected by water and freeze translated into UTM coordinates of 693363.005 m.E. thaw action as the Murtle River cut through it. and 5760029.507 m.N. for Zone 10U. This position

International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 38 Cheeptham et al. was not differentially corrected and the position was cave entrance direction changed to 28º for a distance approximate at best. Obtaining an accurate GPS of 20 m at a slope of -29º. From Station 3 the direction position within the narrow conf ines of the Helmcken changed to 66º for a distance of 9 m and a slope of Falls Canyon was not possible and was known to be -10.5º which placed Station 4 at, what was initially an estimate only. In 2006 this position was used as the thought as, the lowest point in the cave. From Station 4 starting point to produce the contour map of the cave a line 26 m long at a bearing of 66º with a positive slope interior (Fig. 4 and 5). of 9º ended at Station 5 at the back of the cave. With the In 2009, using BC Online Cadastre (Government base line established, 11 line lengths and slopes were of BC 2012) and Google Earth (2012) imagery of measured at 90º on either side of the base line to the Helmcken Falls, the following position was recorded for cave edges (Fig. 4). the cave entrance: 51º57’22.45” N and 120º11’02.11” The GPS point, compass bearing, slope and distance W. This position translated into UTM coordinates of measurements were taken in order to map cave area 693495.892 m.E. and 5759916.901 m.N. for Zone and to produce a contour map of the cave interior (Fig. 10U. The BC Online Cadastre elevation is closer to 650 5). The field data was entered into an Excel spreadsheet m ASL. The 2004 imagery used by Google Earth and (data not shown). The GPS point was converted to UTM BC Online Cadastre is the same and the resolution Zone 10U coordinates. Each line of data was entered into within the Helmcken Canyon itself is not high quality, a separate spreadsheet which calculated the location is distorted and the cave entrance location may still and elevation of each station. Points were created at one have a positional error associated with it. However, the metre intervals along each surveyed line. The elevations new position is more accurate than the original GPS were then labeled for each point on the map. The cave position but it does not affect the contour map results area polygon was created by connecting the outermost with respect to shape and relative elevations within the dot of each line. The half metre contour lines were cave. mapped as a line theme by drawing arcs using the point From the initial GPS position (Station 0) (Fig. 4) a spot elevations as guides to line placement (Fig. 5). distance of 5.16 m at a compass bearing of 25º was A panorama of thirty-five mm digital photographs, measured to the cave entrance (Station 1). From Station taken with an 18 mm wide angle lens, was taken of 1 a distance of 17 m was measured on a bearing of 38º the Helmcken Falls cave interior. These images were and a slope of -21.5º to Station 2. From Station 2 the stitched together using Photoshop CS3 (Fig. 3a and 3b).

Fig. 4. Base map points of the Helmcken Falls cave in Wells Gray Provincial Park were established to produce the contour map.

International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 Volcanic cave actinomycetes and their potential in drug discovery 39

Fig. 5. Cave contour and sample site locations map. Sample collection and bacterial isolation Hickey Tresner (HT), Bennett’s Agar, Starch Casein Nitrate In total, 15 samples were collected from the Helmcken Agar; Actinomycetes Isolation Agar and Nutrient Agar (Table Falls cave, in accordance with Park Use Permit#102172 2). Isolation plates were then cultured by incubating at 25oC (Fig. 1, 2, 5; Table 1). Sample materials collected included for 4 weeks. During incubation, colonies of bacteria, fungi, rocks from inside the cave and cave wall, sediment, and and actinomycetes present were enumerated on a weekly speleothems (cave popcorns). All samples were collected basis. Only actinomycete cultures were then sub-cultured using aseptic techniques and placed in Zip-Lock bags. until purified on HT agar medium for antimicrobial These samples were transported to the Thompson Rivers production studies. University (TRU) microbiology laboratory in a cooler packed with ice and kept cold until isolation could occur. Within 24 Antimicrobial Activity Screening h of sampling, the samples were diluted in sterile saline and The ability of isolated actinomycetes to produce plated onto six different isolation media: Modified Soil Agar, bioactive compounds was first determined using plug

Table 1. Description of sample sites and material gathered from the cave. Site ID Material Description T (oC) pH Near cave entrance 1 Soil Coarse Sand / Gravel 7.6 6.42

Near cave entrance 2 Soil Coarse Sand / Gravel 7.8 6.81

South wall of cave 3 Soil Coarse Sand / Gravel 7.7 6.81

South wall of cave 4 Soil Coarse Sand / Gravel 7.5 6.98

East corner of cave 5 Soil Coarse Sand / Gravel 7.5 7.03

East corner of cave 5.1 Wall rocks Rock chips with some white deposits N/A N/A

East corner of cave 5.2 Floor rocks Rock chips with off white deposits N/A N/A

East corner of cave 6 Soil Coarse Sand / Gravel 7.6 7.19

Middle of cave 7 Soil Rich organic soil 8 6.31

Adjacent room on cave’s Northwestern side 8 Soil Coarse Sand / Gravel 7.7 7.97

South wall of cave 9 Soil Coarse Sand / Gravel 7.6 8.38

South wall of cave 9.1 Soil Coarse Sand / Gravel 7.6 8.38

South wall of cave (closer to entrance) 11 Soil Coarse Sand / Gravel 7.5 8.22

The deepest part of cave 12 Soil Coarse Sand / Gravel 7.5 8.22

East wall of cave 13 Cave Popcorn White calcium deposits from ceiling 12.2 N/A

International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 40 Cheeptham et al. Table 2. Six isolation media used in this study. were weighed out. Samples were then carefully Hickey Tresner Modified Soil Agar Bennett’s Agar transferred into 100-mL volumetric flasks. Using a (HT) (M. Soil) (BA) squirt bottle, sides of the flasks were washed down 10.0g Dextrin 4.0g cave soil 1.0g Beef Extract with approximately 10 mL of 18 MΩwater. A 7-mL 1.0g Yeast Extract 1.0g Yeast Extract 10g Glucose aliquot of concentrated HCl was added to each flask,

1.0g Beef Extract 0.5g NaH2PO4 2.0g N-Z amine which was placed on a hotplate and allowed to boil

0.02g CaCl2 1.7g KCl 1.0g Yeast Extract for 10 min. Samples were then cooled for 5 min. A

2.0g N-Z Amine 0.5g MgSO4 15.0g Bacto Agar 5-mL aliquot of concentrated nitric acid was added to

15.0g Bacto Agar 0.02g CaCO3 pH 8.0 each flask and boiled for an additional 10 min. When pH 9.0 15.0g Bacto Agar 1L De-ionized Water the samples turned clear and yellowish in colour,

1L De-ionized Water pH 7.0 20µg/mL Nalidixic Acid they were taken off the hotplate. Approximately 5

20µg/mL Nalidixic Acid 1L De-ionized Water mL of 20 % lanthanum chloride (LaCl3) and 15 mL of

20µg/mL Nalidixic Acid concentrated HCl were added into each flask. Samples were then placed in a cool water bath before diluting to mark with 18 MΩ water. Flasks were shaken 20 Humic Acid Actinomycete Starch Casein Nitrate times to ensure homogeneity. The atomic absorption Vitamin Agar Agar (Act) Agar (SCNA) (HVA) analysis was done with a Varian Spectra AA 55B. A 2.0g Sodium Caseinate 10.0g Soluble Starch 1g Humic Acid lean (oxidizing) air-acetylene flame was used for both

0.1g L-Asgargine 0.3g Vitamin Free Casein 1.0g Yeast Extract calcium and magnesium analyses. The wavelengths used for calcium and magnesium were 422.7 nm and 4.0g Sodium Propionate 2.0g Sodium Nitrate 0.5g NaH PO 2 4 285.2 nm respectively. Samples were analyzed in 0.5g K HPO 2.0g Sodium Chloride 1.7g KCl 2 4 triplicates. 0.1g Magnesium Sulfalte 2.0g K2HPO4 0.5g MgSO4 0.001g Ferrous Sulfate 0.05g Magnesium Sulfate 0.02g CaCO 3 Scanning Electron Microscopy 15.0g Bacto Agar 0.02g Calcium Carbonate 15.0g Bacto Agar Three cave rock, cave wall, and popcorn samples 5mL Glycerol 0.01g Ferrous Sulfate pH 7.0 (Sample number 13 cave popcorn, Sample 5.1 rock pH 8.0 15.0g Bacto Agar 1L De-ionized Water from cave wall and Sample 5.2 rocks from cave f loor) 1L De-ionized Water pH 8.0 20µg/mL Nalidixic Acid (Fig. 5) were also sent to the BioImaging Facility at the 20µg/mL Nalidixic Acid 1L De-ionized Water University of British Columbia, Vancouver, Canada 20µg/mL Nalidixic Acid for Scanning Electron Microscopy (SEM). Samples needed to be freeze-dried and freeze drying was done assays conducted by placing solid fermentation material in a Flexi-DryTM MP (FTS Systems, USA). After freeze- from each culture plate, as agar plugs, on to pathogen- drying at -20ºC, frozen samples were osmium fumed in seeded agar plates. The microorganisms used as the the tubes for approximately one hour, then transferred targets in this screen are Candida albicans, Micrococcus to polypropylene petri dishes with a saturated filter luteus, Mycobacterium smegmatis, Pseudomonas paper (4% OSO4 aqueous) and 16% formaldehyde aeruginosa, Methicillin Resistant Staphylococcus aureus (~ 0.5 mL not touching the sample) for another two (MRSA), multidrug resistant (MDR) Acinetobacter hours. The samples were inverted after one hour baumannii, Klebsiella pneumoniae, and Extended and further osmium fumed over one weekend. Then, Spectrum ß-lactamase (ESBL) Escherichia coli. Each samples were mounted using a bulk sample holder, isolated actinomycete was cultured on HT agar medium coated with 12 nm of gold using a Cressington 208 HR for 10 days at 25ºC and then was used as the plug assay sputter coater (Cressington, Watford, England, UK). material. To prepare pathogen-seeded assay plate, the Samples were imaged at various accelerating voltages, respective target organism was grown in an appropriate modes, and working distances on a Hitachi S4700 liquid medium (HT or NB) and once the growth reached FESEM (Hitachi, Tokyo, Japan). log phase around OD 0.5 when read at 600 nm, 1% (v/v) of the culture was transferred to 250 ml molten 16S rRNA analysis nutrient agar, mixed well, and solidified. Each plug Of 99 isolates showing inhibitory activities against from the isolated actinomycetes was then placed on pathogenic microorganisms used in the plug assay, the prepared seeded assay plates and was incubated only 82 isolates were studied for their genotypic and at 37ºC overnight. The zone of inhibition was observed phenotypic characterizations due to limitation in and the diameter of each was measured and recorded. funding. Microscopic and macroscopic morphologies Positive controls used in this study were tetracycline, were observed under a light microscope and a nystatin and ampicillin depending on the pathogens dissecting microscope (Motic). Eighty-two isolates used. Negative controls were sterile water impregnated were subjected to 16S rRNA gene sequencing to disks. clarify taxonomic status using polymerase chain reaction (PCR) conditions carried out at Macrogen, Metal analysis of cave environmental samples Inc., Seoul, Korea as described herein. Template All environmental samples collected at the DNA of each isolate was prepared. Colonies of each Helmcken Falls cave were analyzed to determine the isolate were picked up with a sterilized toothpick and mineral components using flame atomic absorption suspended in 0.5 mL of sterile saline in a 1.5-mL spectrometry (FAAS). These samples were pulverized centrifuge tube. The culture was then centrifuged at with a mortar and pestle and 0.5000 ± 0.0010 g 10,000 rpm for 10 min. After removal of supernatant,

International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 Volcanic cave actinomycetes and their potential in drug discovery 41 the pellet was suspended in 0.5 mL of InstaGene Hickey Tresner, Bennett’s Agar, Starch Casein Nitrate Matrix (Bio-Rad, USA) and was incubated 56ºC for Agar; Actinomycetes Isolation Agar, and Nutrient 30 min. and then heated to 100ºC for 10 min. After Agar. Of the isolation media used, Modified Soil agar heating, the supernatant was used for PCR. For the yielded the greatest number of actinomycetes (data PCR process, 1 μL of each template DNA sample not shown here). The macroscopic morphological was added to 20 μL of PCR reaction solution. The diversity noticed on this medium was substantially 27F/1492R primers were used to amplify the 1400bp less than the others - the medium showcasing the bacterial 16S rRNA gene (Table 3). The amplification best morphological diversity were SCNA, Bennett’s cycles were performed 35 times at 94ºC for 2 min, Agar, and Actinomycetes Isolation Agar respectively. 94ºC for 45 s, 55ºC for 60 s, 72ºC for 60 s, 72ºC In this study, we selectively used only culturing for 5 min. Escherichia coli genomic DNA was used techniques favoring actinomycetes to isolate this as a positive control in the PCR. Then, the PCR prolific antibiotic-producing group of bacteria, our products of each isolate were removed from the focus is not to determine the whole constitution of unincorporated PCR primers and dNTPs by using cave microbiome at this point in time. Because of Montage PCR Clean up kit (Millipore). For sequencing, their often unique biochemistry, these cave organisms 518F/800R or 27F/1492R primers were used as have been the target to identify new drugs including seen in the Table 3. Sequencing was performed by antibiotics, and are used as models to evaluate the using Big Dye terminator cycle sequencing kit v.3.1 possibility of life forms on other planets (Boston et al., (Applied BioSystems, USA). Sequencing products 2001; Nakaew, Pathom-aree, & Lumyong 2009a,b; were resolved on an Applied Biosystems model Yücel & Yamaç, 2010; Northup et al., 2011). Though, 3730XL automated DNA sequencing system (Applied a major drawback of isolation through culturing BioSystems, USA) at Macrogen, Inc., Seoul, Korea. method is that it is generally now understood that less than 0.1% of soil microbial community were Phylogenetic analysis likely culturable in a lab setting (Amann et al., 1995; Sequences generated in this study (1,000 to 1,400 Hill et al., 2000). bp long) were submitted to BLAST and RDB to identify In this study, over 400 bacterial isolates were isolated the closest relatives based on 16S rRNA sequences. from the environmental samples collected from All sequences were manually aligned and analyzed the cave. With the preliminary screen, 400 isolates using SeqPup version 0.6f. The evolutionary history were screened and most of the cave actinomycetes was inferred using the Maximum Parsimony method. tested demonstrated antimicrobial activities against The bootstrap consensus tree inferred from 5000 the target bacteria with a unique inhibitory pattern. replicates is taken to represent the evolutionary history Overall, 10.25% showed inhibitory activity against of the taxa analyzed (Felsenstein, 1985). Branches M. luteus, 2% against S. aureus, 9.25% against M. corresponding to partitions reproduced in less than smegmatis, 1.75% against E. coli, 1% against ESBL 50% bootstrap replicates are collapsed. The MP tree E. coli, 2.25% against A. baumannii, 26.50% against was obtained using the Close-Neighbor-Interchange K. pneumoniae, 6.255 P. aeruginosa and 7.5% against algorithm (Nei & Kumar, 2000) with search level 1 in C. albicans (Table 4). These findings demonstrate which the initial trees were obtained with the random a promisingly high probability of discovering a addition of sequences (10 replicates). The analysis novel compound with different modes of action. In involved 68 nucleotide sequences. Codon positions particular, with such a high proportion of hits against included were 1st+2nd+3rd+Noncoding. All positions these important nosocomial pathogens, this gives containing gaps and missing data were eliminated. us confidence that cave bacteria can be the source There were a total of 1309 positions in the final of novel compounds that provide precursors of new dataset. Evolutionary analyses were conducted in drugs to combat Gram-negative antibiotic resistant MEGA5 (Tamura et al., 2011). bacteria. Additionally, chemical and physical characteristics Nucleotide sequence accession numbers of the cave environmental samples were studied The nucleotide sequences of the 82 isolates using FAAS and SEM. A chemotaxonomic study obtained from this study have been deposited in (unpublished results) and phylogenetic analysis of GenBank database under the accession numbers bacterial isolates was conducted for preliminary JQ422118-JQ422184 identification. With the FAAS, aluminum (Al), arsenic (As), barium (Ba), calcium (Ca), cadmium (Cd), RESULTS AND DISCUSSION chromium (Cr), iron (Fe), lead (Pb), magnesium (Mg), Six different media were used for the isolation of potassium (K), sodium (Na) were measured in all actinomycetes from cave soils - Modified Soil Agar, samples. The results demonstrated that each of the samples is unique and composed of different amounts Table 3. Primers used in amplification and 16S rRNA sequencing and types of minerals (Fig. 6 to 8). The resulting values Primers Sequences Amplification Sequencing of each sample are categorized in three groups of most abundant (0-40% wt/wt), second abundant (0-10% 27F AgAgTTTgA TCM TGG CTC Ag used wt/wt), and least abundant ((0-0.5% wt/wt) of the 1492R TAC ggY TAC CTT gTTACg ACT T used compositions observed and measured. Ca, Fe, and Al 518F CCA gCAgCCgCggTA ATA Cg used were found to be the most abundant components (Fig. 800R TAC CAgggT ATC TAA TCC used 6) while Mg, Na, As, Pb, Cr, and Ba were the second

International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 42 Cheeptham et al. Table 4. Antimicrobial activities of cave actinomycetes screened New Mexico, USA, and Portugal (Fig. 10A and 10B) against variety of pathogens (Northup et al., 2011). The evidence of microbial Size of inhibitory zone (diameter, mm)* Target life in these three samples observed in our study microorganisms more may not have fully addressed their identity and 7 to 12 13 to 17 18 to 22 than 22 Total their roles in cave secondary mineral formation. M. luteus 26 (6.5) 12 (3) 2 (0.5) 1 (0.25) 41 (10.25) However, it definitely leads to certain questions in MRSA 5 (1.25) 3 (0.75) 0 (0) 0 (0) 8 (2) microbes-mineral interaction in cave environments.

M. smegmatis 30 (7.5) 5 (1.25) 2 (0.5) 0 (0) 37 (9.25) It has long been known that calcification (CaCO3 deposition) is a general occurrence in soil bacteria E. coli 7 (1) 0 (0) 0 (0) 0 (0) 7 (1) (Boquet et al., 1973). In particular, for bacteria that ESBLE. coli 4 (1) 0 (0) 0 (0) 0 (0) 4 (1) live in caves which can be rich in calcium, it may be A. baumannii 3 (0.75) 2 (0.25) 4 (1) 0 (0.75) 9 (2.25) beneficial to have a vital physiological adaptation of 2+ P. aeruginosa 15 (3.75) 5 (1.25) 2 (0.5) 3 (0.75) 25 (6.25) removing toxic Ca ions by calcification to survive and thrive in such microenvironment (Banks et K. pneumoniae 74 (18.50) 22 (5.50) 3 (0.75) 7 (1.75) 106 (26.50) al., 2010). Biomineralization and bioprecipitation C. albicans 22 (5.5) 3 (0.75) 3 (0.75) 2 (0.5) 30 (7.5) in particular calcium carbonate precipitation in Note: Calculated values in bold represent number in total of cave the isolated bacteria is under investigation in our isolates that showed inhibitory activity in each category. Values in the laboratory. Not only can we understand roles of brackets are shown as percentage calculated from 400 cave isolates microorganisms in cave formation and degradation screened. *Diameter of the paper disks used in this study was 6 mm (Toyo Advantec, Japan) 40.00 most abundant (Fig. 7) with Cd and K as the least 35.00 abundant in our samples (Fig. 8). In Northup and 30.00

Lavoie 2001, similar observations were found in 25.00 % Fe limestone and basalts, in which they are often rich in reduced sulfur, iron, and manganese (Northup 20.00 % Ca Wt/Wt (%) Wt/Wt & Lavoie, 2001). In our study, it is worth noting 15.00 % Al that there are two samples (samples 9.1 and 11) 10.00 with high percentage of arsenic. In the course of 5.00 determining mineral composition in our samples, it 0.00 is found that interference is minimized by cesium 1 2 3 4 5 5.1 5.2 6 7 8 9 9.1 11 12 13 chloride and lanthanum (III) chloride. Expanding Sample ID instrumental methods for the analysis of more Fig. 6. Most abundant components found in the cave samples. cationic species and incorporating protocols for 10.00 the analysis of several anionic species (non-metals) 9.00 are advised for further investigation. Nevertheless, 8.00 this set of baseline data will lend itself to further 7.00

% Mg mineral-microbe’s interaction study. Furthermore, 6.00 % Na analyses of mineral compositions and identification 5.00 % As of bacterial communities will enhance an (%) Wt/Wt 4.00 % Pb understanding of cave microorganisms’ roles in 3.00 % Cr both cave formation and degradation. Analyses of 2.00 % Ba mineral compositions of each sample will also lead 1.00 to identification of optimal growth conditions and 0.00 metabolite production in cave bacteria. 1 2 3 4 5 5.1 5.2 6 7 8 9 9.1 11 12 13 Scanning electron micrographs showed the Sample ID Fig. 7. Second most abundant components found in the cave presence of microscopic life forms in all three samples. samples; popcorn (sample#13), rock from cave wall 0.35 (sample#5.1) and rock from cave floor (sample#5.2) (Fig. 9). The SEM images presented diverse biological 0.30 morphologies observed in the samples. Fig. 9A to 9C demonstrated a variety of microbial populations in 0.25 the popcorn sample; a filamentous chain, a biofilm of short thin rods and some filamentous growth and 0.20 fossilized spheroids. In Fig. 9D to 9E, only bacilli % Cd Wt/Wt (%) Wt/Wt 0.15 on and around mineral deposits were observed in % K the rock from cave wall sample. Additionally, a 0.10 population of mixed microorganisms in biofilm- formed fashion was apparent in the rock from cave 0.05 floor sample (Fig. 9F to 9H). Our SEM images in Fig. 9B and 9F are very similar to those found in 0.00 1 2 3 4 5 5.1 5.2 6 7 8 9 9.1 11 12 13 a recent study done in three basaltic lava caves Sample ID from three different climate conditions in Hawaii, Fig. 8. Least abundant components found in cave samples.

International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 Volcanic cave actinomycetes and their potential in drug discovery 43 a) e)

b) f)

c) g)

d) h)

Fig. 9. SEM images of not-yet-identified microorganisms observed in some white speleothem (popcorn) found in Helmcken Falls volcanic cave in British Columbia, Canada; a) network of filamentous chain; b) a biofilm of short thin rods and some filamentous growth on top of crystal; c) fossilized spheroids (connected as a ring); d) bacilli on the surface; e) bacilli on and around mineral deposits; f) a biofilm of a mixed population; g) a connecting chain of rods with spike; h) long chain of rods on mineral deposits. Fig. 9a-c are from Sample#13 cave popcorn, Fig. 9d-e are from Sample#5.1 rock from cave wall, Fig. 9f-h from Sample# 5.2 rock from cave floor.

International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 44 Cheeptham et al.

N C 015151.1 V

ul

c PseudomonasPseudomonas peli P sp. Pa ni

s a e t a m 2 2 out 1 Erwinia sp. S 132 123

M 72 novs M Nocardia sp. P M P P 86 P M P M 185A M M ki P M 58B 107A 470 a P M 87B Streptomyces coelescens N K P 119 M PM P M P 240A 350 D M M 351 126 P 100 110A M R016 P 258 M 100 P 58C C18 M Streptomyces phaeochromogenes 100 P E25 100 PM 72 B-2156 E9 R009 RRL 100 D NC39 R 043854.1 N 89 N PM276A PM87A PM 69 246 PM108 100 PM306 99 PM124

PM182B 55 99 PM185B

PM198 PM106 83 58 PM106 D PM198C R031 P R014 83 M184 338 D PM 99 N 309 63 R 043354.1 N PM P 53 M67 RRL M 51 P B-12114 PM 277B 230 P PM 93 M 233 277A 97 P M M P 43 59 M P P M 99 P 73B R006 M P 59A M 262 D 261A P M 260 M 266 D M P P P M M 59B P P R011 P M

P P 341 D

P P M R012

M M M 60 261C M

234 Legend

9 0

425C 4 73A

3 5

M 2 259C 267 D P 425A M R045 ◊ Streptomyces microflavus M P P M P ▼ Streptomyces finlayi □ Streptomyces candidus ■ Streptomyces hypolithicus ○ Streptomyces cirratus ♦ Streptomyces drozdowiczii Streptomyces durmitorensis ● Streptomyces sampsonii Fig. 10. Maximum Parsimony analyses of taxa (continued on p. 45) The evolutionary history was inferred using the Maximum Parsimony method. The bootstrap consensus tree inferred from 5000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The MP tree was obtained using the Close-Neighbor-Interchange algorithm (Nei & Kumar, 2000) with search level 1 in which the initial trees were obtained with the random addition of sequences (10 replicates). The analysis involved 68 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 1309 positions in the f inal dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). through such study, but we also can understand for survival of the constitutant species is vital. what factors influence microbial-induced calcite 16S rRNA gene sequences revealed that 79.3% of precipitation, such information can shed light to the 82 isolates sequenced belong to the Streptomyces potential application in soil improvement (Ng et al., genus and 6.1% were in Bacillus, Pseudomonas, 2012). With a better understanding of the microflora Nocardia, and Erwinia genera (Fig. 10). This was in cave environments, a shift from basic questions in line with our focus for this study; isolation identifying the cavern microflora to more complex media used are favorable to actinomycetes. With ecological, community and application questions dominance of Streptomyces genus identified from that evaluate their contributions in biological and the sequencing results, S. microflavus, S. finlayii, S. geological chemistry of the cave, the ecological role candidus, S. hypolithicus, S. cirratus, S. drozdowiczii, in the cave biomes, special adaptations necessary S. durmitorensis, and S. sampsonii were species

International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 Volcanic cave actinomycetes and their potential in drug discovery 45

N C 015151.1 V

Pseudomonas sp. JQ422175 ul Pseudomonas peli JQ422184 c a ni

s a e t Erwinia sp. JQ422180 a m Nocardia sp. JQ422130 out

novs JQ422127 JQ422131 JQ422128 JQ422153 JQ422121 ki Streptomyces coelescens JQ422176 a JQ422157 JQ422119 JQ422173 JQ422123 JQ422161 JQ422172 100 JQ422126

100 JQ422163 Streptomyces phaeochromogenes JQ422179 100 JQ422120 JQ422178 JQ422182 72 B-2156 RRL 100 JQ422181 JQ422177 NR 043854.1 N JQ422169 89 JQ422122 JQ422137 69 JQ422125 100 JQ422142 99 JQ422129

JQ422132 55 99 JQ422134

JQ422136 JQ422124 83 58 JQ422156 JQ422135 83 JQ422133 JQ422170 99 N 63 R 043354.1 N JQ422143 JQ422152 RRL 51 JQ422148 JQ422158 B-1211 4 JQ422159 JQ422141 93

97 JQ422149 JQ422140 JQ422155

JQ422118 99 JQ422150 JQ422167 JQ422166 JQ422151 JQ422171 JQ422138 JQ422183 JQ422160

JQ422174 JQ422154 JQ422165 JQ422162 JQ422164 Legend JQ422168

JQ422139 ◊ Streptomyces microflavus

JQ422145 JQ422144 ▼ Streptomyces finlayi □ Streptomyces candidus ■ Streptomyces hypolithicus ○ Streptomyces cirratus ♦ Streptomyces drozdowiczii Streptomyces durmitorensis ● Streptomyces sampsonii Fig. 10. Maximum Parsimony analyses of taxa (continued) preference. Some isolates (14.6% of the 82 isolated better understand bacterial compositions in caves, sequenced) showed a similarity to unidentified a combination of techniques (culture-dependent ribosomal RNA sequences in databases (data not and molecular based approaches) needs to be shown here). Further investigation is needed for considered for an inclusive interpretation. such sequences. In other studies, diverse species In conclusion, there are more questions needed of bacteria including actinomycetes in the genus of to be answered arising from this preliminary study. Streptomyces were found in lava tube caves (Moya Nevertheless, the Helmcken Falls cave microbiome et al., 2009; Northup et al., 2011). With a number possesses a great diversity of microbes with the of cave-microbial diversity studies being reported, potential for studies of novel microbial interactions it is worth noting that each cave is unique and and the isolation of new types of antimicrobial diverse in its microbial compositions, however, agents. This study suggests a high possibility of there is a trend of finding dominating communities finding new antimicrobial agent(s) from previously shared in many caves. In general, Actinobacteria, unknown actinomycetes in volcanic cave habitats. Acidobacteria, Proteobacteria and others have been This is the first work of antimicrobial activities repeatedly found to show up in different caves, this against a variety of multidrug resistant pathogens may also suggest that caves may consist of a core from volcanic cave actinomycetes isolated in set of microbial phyla (Northup et al., 2011). To Canada.

International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 46 Cheeptham et al. ACKNOWLEDGEMENTS Barton H.A., Taylor N.M., Kreate M.P., Springer A.C., Oehrle S.A. & Bertog J.L, 2007 - The impact Our sincere thanks go to TRU internal grants (SAC of host rock geochemistry on bacterial community structure in oligotrophic cave environments. grants, CUEF and Research Innovation Funds) for its International Journal of Speleology, 36: 93-104. financial support and to the Ministry of Forests, Lands http://dx.doi.org/10.5038/1827-806X.36.2.5 and Natural Resource Operations for Park Use Permit Barton H.A., Taylor N.M., Lubbers B.R. & Pemberton #102172. This work could have not been completed A.C., 2006 - DNA extraction from low-biomass carbonate without the initial work of my former undergraduate rock: An improved method with reduced contamination research students in particular Eric Bottos, Stephanie and the low-biomass contaminant database. Richards, Sylvia Kay, and Alexa Daneluk. Thanks Journal of Microbiological Methods, 66: 21-31. http://dx.doi.org/10.1016/j.mimet.2005.10.005 also go to colleagues at the Department of Biological Beloqui A., de María P.D., Golyshin P.N. & Ferrer Sciences in particular Drs. Tom Dickinson, Ken M., 2008 - Recent trends in industrial microbiology. Wagner, and Ms. Carolynne Fardy for their assistance, Current Opinions in Microbiology, 11 (3): 240-248. continued support and expertise. Also, our thanks http://dx.doi.org/10.1016/j.mib.2008.04.005 go to Dr. Nancy Van Wagoner, TRU former Associate Bhullar K., Waglechner N., Pawlowski A., Koteva K., VP Research, Innovation and Graduate Studies for Banks E.D., Johnston M.D., Barton H.A. & Wright her true belief in my research and insights to cave G.D., 2012 - Antibiotic resistance is prevalent in an isolated cave microbiome. PLoS ONE : e34953. formation. Our sincere gratitude goes to Dr. Julian 7 (4) http://dx.doi.org/10.1371/journal.pone.0034953 Davies for donating the bacterial pathogens used in Boquet E., Boronate A. & Ramos-Cormenzana A., 1973 this project. Last but not least, our thanks go to Dr. - Production of calcite (calcium carbonate) crystals by Cesareo Saiz-Jimenez of the Instituto de Recursos soil bacteria is a general phenomenon. Nature, 246: Naturales y Agrobiologia de Sevilla Spain for insights 527-529. http://dx.doi.org/10.1038/246527a0 in regards to manuscript preparation. Boston P.J., Spilde M.N., Northup D.E. , Melim L.A., Soroka D.S., Kleina L.G., Lavoie K.H., Hose L.D., REFERENCES Mallory L.M., Dahm C.N., Crossey L.J. & Schelble R.T., 2001 - Cave biosignature suites: Microbes, minerals and Mars. Astrobiology Journal, 1 (1): 25-55. 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International Journal of Speleology, 42 (1), 35-47. Tampa, FL (USA) January 2013 48 International Journal of Speleology 42 (1) 49-56 Tampa, FL (USA) January 2013

Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Official Journal of Union Internationale de Spéléologie

Natural and anthropogenic factors which influence aerosol distribution in Ingleborough Show Cave, UK Andrew C. Smith1*, Peter M. Wynn1, and Philip A. Barker1 1 Lancaster Environment Centre, University of Lancaster, LA14YQ, UK

Abstract: Monitoring in Ingleborough Show Cave (N. Yorkshire, UK) reveals the influence of tourism and cave management techniques on different parameters of the cave atmosphere. Exploratory aerosol monitoring identified a 0.015 ± 0.03 mg/m³ (≈70%) reduction in airborne particulates within the f irst 75 meters of cave passage and two major aerosol sources within this artificially ventilated show cave. Autogenic aerosol production was identified close to active stream ways (increases of <0.012 mg/m³), suggesting the expulsion of water-borne aerosols during turbulent water flow. The presence of tourist groups also increased aerosol concentrations within the cave (increases of <0.021 mg/m³), either by transporting them from an allogenic source or through the disturbance of particles which had previously been deposited within the cave environment. Exploratory aerosol data is presented alongside more routine analytical monitoring, helping to contextualise the impact of cave management strategies on natural cave atmospherics.

Keywords: aerosol; cave; tourism; temperature; carbon dioxide; ventilation

Received 31 August 2012; Revised 19 November 2012; Accepted 19 November 2012

Citation: Smith A.C., Wynn P.M. and Barker P.A. 2013. Natural and anthropogenic factors which influence aerosol distribution in Ingleborough Show Cave, UK. International Journal of Speleology, 42 (1), 49-56. Tampa, FL (USA) ISSN 0392-6672 http://dx.doi.org/10.5038/1827-806X.42.1.6

INTRODUCTION composition and temperature of cave air over short temporal scales (Baker & Genty, 1998; Faimon et Understanding cave atmospherics is becoming al., 2006, 2012b; Liñan et al., 2008; Dominguez- increasingly important for the interpretation of Villar et al., 2010; Benavente et al., 2011). A less isotopic and trace element signatures encapsulated well defined impact of cave tourism is the possible in karstic speleothem deposits. The cave atmosphere introduction of foreign atmospheric particulates offers an important control over speleothem into the cave environment (Kertész et al., 1999). For chemistry, acting to modify the composite signal example some studies have attributed speleothem derived from the external atmosphere, vegetation discolouration to an increase in fossil fuel derived and epikarst (McDermott 2004; Fairchild et al., particulates, transported into cave systems by 2006). Cave ventilation, carbon dioxide degassing visitors (Jeong et al., 2003; Chang et al., 2008). and temperature fluctuations actively control Other studies have investigated natural and speleothem growth dynamics and therefore dictate anthropogenic transport mechanisms which lead how and when speleothems incorporate chemical to the dispersal of biological aerosols, especially components from the hydrological system (Spötl et important for cave managers when combating al., 2005; Faimon et al., 2006, 2012a; Baldini 2010). unwanted plant growth (Mulec et al., 2012). Despite Cave atmospherics are controlled by soil these studies, comparatively little attention has productivity, cave geometry and pressure been focused upon either natural or anthropogenic differences between the external and cave introduction of atmospheric particulates in the atmospheres (Cigna 1967; Spötl et al., 2005; size fraction 0.1 -10 µm (referred to in this study Baldini et al., 2006). However, caves that are open as aerosols). As scientific interest in speleothem to the public may be subject to additional controls, chemistry increases, understanding the role of the which influence their atmospheric composition and cave atmosphere in transporting and depositing ventilation regimes (Lario & Soler 2010). Human aerosols is becoming more important (Fairchild et visitors are known to change the carbon dioxide al., 2010; Dredge et al., 2012 in review).

*[email protected] 50 Smith, Wynn and Barker Here, we undertake an exploratory assessment show cave, reducing radon build up and ensuring a of aerosol presence within a cave environment by safe working environment for cave guides, who may measuring aerosol concentrations, and logging their enter the cave hundreds of times every year (Hayland, distribution throughout Ingleborough Show Cave. 1995). Studies have indicated that Ingleborough Cave This study aims to categorise sources of aerosols has relatively low radon levels (annual average 610 within the cave environment and identify zones of Bq/m³; summer average 1140 Bq/m³) in comparison maximum and minimum aerosol concentration. with adjacent caving locations (Hayland, 1995). The Aerosol monitoring is incorporated into a larger scale introduction of this fan however, alters natural cave characterisation of show cave atmospherics during atmospherics. Here we assume that three discrete the peak tourist season (June to October 2009). atmospheric scenarios occur within the cave. The term background is used to distinguish relatively stable SITE DESCRIPTION atmospheric conditions (either naturally occurring or as a result of fan operation) from more dynamic Ingleborough Cave (54°8’N, 2°22’W and 270 atmospheric conditions driven by tourism. m.a.s.l.) (Fig. 1) is situated 2 km outside the village 1) Natural atmospherics, occurring whilst the cave of Clapham, Yorkshire and functions as a resurgence is not open for tourism and the fan is not operating. for the Gaping Gill system (Gascoyne et al., 1983). 2) Fan induced background atmospherics, occurring The cave is developed in 100-200 m thick, well jointed whilst the fan is on but visitors and staff are not Carboniferous Limestones with a North-West dip of present within the system. 1-4° (Sweeting, 1950; Waltham, 1970). Whilst little 3) A combination of fan activity and tourists within is known about the chemical records encapsulated the cave system. within the speleothems from this system, the first This study measures changes in cave air

500 m of passages (total length 17 km) is open to temperature, pCO2 and aerosol concentration at public access as a show cave, providing an excellent different spatial and temporal scales to assess these opportunity to assess the impact of cave tourism on three atmospheric regimes. natural underground atmospheric conditions. Cave modifications include a concrete path from 0 – 500 METHODS m depth into the cave for ease of access, whilst timer operated lighting is used to minimise unwanted Monitoring was undertaken from June - October vegetation growth within the cave. 2009 with repeat surveys of aerosol concentration To reduce the concentrations of potentially harmful in February 2011. Cave monitoring characterised radon gas within the cave system a fan has been atmospheric properties before, during and after tourist installed at the cave entrance, pumping external visits, with the aim of identifying the magnitude and air into the cave throughout the tourist day (09:00- duration of atmospheric changes within the show 17:00). Inward air movement of up to 1.8 m/s was cave. Tourist group numbers fluctuated between 4 recorded next to the fan using a Kestrel 4500 airflow and 40 individuals. monitor, (range 0.6 - 40 m/s, error 0.1 m/s and Large spatial scale atmospheric monitoring was an accuracy of ±3 ‰), in contrast to the natural undertaken either by continuous in-situ logging or by “summer season” exhalation of cave air recorded at the formation of cave transects. In-situ logging was speeds of 1.4 m/s at ground level when the fan is undertaken if several logging devices were available switched off. Fan operation occurs whenever people for monitoring (limited to temperature data). Transects are in the cave system, predominately the height of are comprised of data obtained at a number of pre- the summer tourist season but also during weekend identified locations from the entrance to end of the winter tourist visits and staff activities. This fan is show cave (identified in Table 1 and Fig. 1) when only designed to effectively ventilate the entirety of the one logging device was available.

Temperature Cave air temperature was continuously logged at

different cave locations throughout June, July and

August, using 4 inter-calibrated Tiny-Tag Plus2

100m loggers. These loggers have an operational range

between -40 and +85 °C, limit of detection of 0.01

°C, measurement uncertainty of ±0.02 °C and are

Bedrock waterproof to 15 m making them ideal for operating in Forest Plantation depth high humidity cave environments. Sampling intervals were set between 1 second and 3 minutes. (5) Stal Gallery (8) Waterfall (10) 1st bells (3) Old Cave (12) End cave 0m (1) External Carbon Dioxide

(2) Cave entrance (4) Eldon Hall (6) Pillar (11) Long Gallery Horizontal Scale (9) Oxbow Cave atmospheric CO2 concentration was measured door and fan Hall

(7) Abyss 0m 100m in parts per million (ppm) using a Vaisala GM70

Fig. 1. Location of Ingleborough Cave and cave survey. Survey monitor and GMP221 probe with measurement adapted from the original created by Taylor & Hodgson, Land uncertainty of ±2 % and operational range of 0 – Surveyors of Lancaster (1838). 10,000 ppm. The wide operational range and high

International Journal of Speleology, 42 (1), 49-56. Tampa, FL (USA) January 2013 Aerosol distribution and cave atmospherics within a British show cave 51

Table 1: Site number and names correspond to Fig. 1. Cave air temperature data was collected by continuous logging, whilst CO2 and aerosol data was obtained during discrete cave transects, all data was collected between June and August 2009, unless stated. Where appropriate, measurement ranges are presented and data is related to fan operation. The range refers to the amplitude of data fluctuation around the presented average value.

-3 Location Depth into Average Temperature (°C) CO2 (ppm) Average Aerosol concentration (mg/m ) cave system (m)

Fan on (range) 09:00- Fan off (range) 17:00- Fan On Fan Off July 09 (range) Feb 11 (range) 17:00 09:00

(1) External 0 13.13(1.50) 12.33 (1.68) Atmospheric ≈ 380 0.018 (0.010) 0.022 (0.043)

2) Cave Entrance -5 10.20 (0.40) 10.06 (0.35) 380 380 0.009 (0.004) 0.012 (0.073)

(3) Old Cave -25 - - 400 380 0.004 (0.007) 0.013 (0.042)

(4) Eldon Hall -60 10.23 9.55 410 980 0.002 (0.004) 0.007 (0.019)

5) Stal Gallery -100 - - 410 1280 0.001 (0.002) 0.005 (0.028)

(6) Pillar Hall -180 0.003 (0.004) 0.006 (0.015)

7) Abyss -200 9.31 (0.08) 9.30 (0.049) 420 1300 0.015 (0.026) 0.013 (0.015)

(8) Waterfall -250 0.010 (0.065)

(9) Oxbow -320 0 0.004 (0.036)

10) 1st bells -350 0.001 (0.059)

(11) Long Gallery -400 9.27 9.20 490 1240 0 0.003 (0.037)

(12) End Cave -500 9.20 (0.16) 9.18 (0.065) 520 1190 0 0.003 (0.019) stability (±5 % over 2 years) of this handheld device not in operation. Two continuous logging overnight makes it an ideal piece of equipment to operate both studies, taken exactly 1 month apart (17-18th June during transects and in situ logging scenarios. and 17-18th July) in Eldon Hall (100 m into the cave

system) exhibit CO2 increases occurring immediately Aerosols after the fan is switched off (Fig. 2). The July study

Aerosol quantities were measured using a Sidepak- represents the most extreme CO2 concentrations AM510 aerosol pump and extension tube. The measured in Ingleborough with overnight values in AM510 has an operating range of 0.001 - 20 mg/ excess of 3500 ppm (2200 ppm higher than transect m³ and a limit of detection of 0.001 mg/m³, ideal for maximums) (Fig. 2). Overnight CO2 values during both logging very low aerosol concentrations found within studies show a distinctive CO2 ‘pulse’, which gradually cave environments. The AM510 was used with returns to what may be described as a ‘natural an impactor head of 10 microns sampling aerosol background minimum’. fractions between 0.1 and 10 µm, pump rate of 1.2 l/ The high natural background level in July is min and measurement uncertainty of 0.001 mg/m3. immediately disrupted once the fan is switched

Monitoring was restricted to short intervals due to the on, with an extremely rapid reduction in CO2 high relative humidity nature of cave environments concentration. This rapid readjustment due to fan and the unknown impact this may have on the operation would not have been expected in June as previously untested AM510 pump. natural background levels already approximate those Aerosol monitoring focused upon establishing 1) the induced by the fan. decay of aerosol quantities with increasing distance from the cave entrance, 2) the transport of aerosols Fan induced background atmospherics by tourists and 3) sites of aerosol production within The largest control that the fan exerted over the cave. the monitored cave atmospherics is its control of carbon dioxide concentration, demonstrated in a

RESULTS Overnight CO2 maximums 4000 June July 800 Natural background atmospherics 3500 700 Natural cave atmospherics were continuously 3000 600 (ppm)

(ppm) logged during overnight periods when monitoring 2500 500 June July was unaffected by human activity or fan operation. 2000 400

Temperatures reduce with distance into the system 1500 Radon fan off 300 at ≈17:00 Natural background minimum Concentration Concentration 2 from 10.06 °C inside the entrance to 9.18 °C at the 2 1000 200 CO CO end of the show cave (Table 1, data recorded between 500 Radon fan induced 100 background level June and August 2009). The amplitude of recorded 0 0 temperatures also decreases with distance into the 13:00 18:00 23:00 04:00 09:00 cave, from 0.35 °C in the cave entrance to 0.07 °C Time at the end of the show cave. The observed reduction Fig. 2. Continuous logging of carbon dioxide concentrations (5 in temperature range with distance demonstrates the min log) from Eldon Hall, between the 17th and 18th of June (Blue Diamonds) and 17th and 18th of July (Gray Diamonds). CO increases diminishing influence of the external environment on 2 are observed as the tourist day finishes (≈17:00), values reach a the cave microclimate. natural peak, eventually recovering to natural background minimum Carbon dioxide concentrations are highest during values. The influence of the fan can be observed at 09:00 in July, periods when the show cave is closed and the fan is forcing a rapid reduction in CO2 concentrations.

International Journal of Speleology, 42 (1), 49-56. Tampa, FL (USA) January 2013 52 Smith, Wynn and Barker transect undertaken on the 4th of June (10:00) with to average values similar to those recorded outside the a three minuet logging period (Table 1). Fan induced cave entrance (0.015 mg/m3) with maximum values of background carbon dioxide levels remain stable 0.056 mg/m3. Aerosol concentrations were measured and low during cave opening times, especially close at 5 m intervals to establish zones of maximum to the cave entrance (380-410 ppm). When the fan production and settling gradients surrounding this is operating, maximum carbon dioxide levels rise to autogenic aerosol source (Fig. 4). 520 ppm at 500 m depth into the cave, exhibiting a Maximum aerosol values within this subsection gradual, but relatively minor increase of approximately of the cave occurred at zones of maximum water 120 ppm from the entrance to the end of the show turbulence, focussed at the Abyss. Aerosol values cave (Table 1). remained higher than expected until the second Data presented in Table 1 shows average cave air waterfall, resulting from the presence of an active temperatures recorded at hourly intervals during stream running alongside the path. Lower aerosol two monitoring periods. The first between 4th-17th of values were recorded before and after this section of June (loggers sited in Eldon Hall and Long Gallery), turbulent stream flow, as can be seen in Fig. 3 and the second from 15th July-18th August (loggers sited 4. Aerosol levels then decline to almost zero at the at the cave entrance, Abyss, cave end and external to termination of the show cave where no active stream the entrance). A rapid stabilisation of temperatures ways are present (Fig. 3). is observed between 60 m (Eldon Hall) and 200 m (Abyss) into the cave. Short term perturbations due to tourism Aerosol concentration transects were taken The influence of tourist groups within the exclusively during cave opening hours on the 14th cave system is superimposed upon fan induced of July 2009 and 28th of February 2011, with the background atmospheric conditions. Groups often fan in operation. Aerosol measurements therefore overprint the cave microclimate on small spatial and incorporate not only naturally produced cave temporal scales. aerosols but also any aerosols derived from artificial High resolution temperature studies were ventilation, reflecting a maximum value that may be undertaken, focused upon capturing the impact of expected within the cave system. individual tourist groups in the Old Cave (22nd-23rd External atmospheric aerosol concentrations had an June and 29th June) and at the end of the show cave average value of 0.018 – 0.022 mg/m3. With increasing (18th-22nd June). The end of the show cave offers a distance into the cave, aerosol concentrations stable atmospheric environment to test temperature decreased to average readings of ≈0.003 - 0 mg/m3 variations associated with tourist groups. If tourists at 500 m (Fig. 3). Aerosol concentrations decrease are absent, the coolest sections of the air column with distance from the entrance, such that by 75 m are the floor (due to the density of cold air) and the into the cave, aerosol levels reach values similar to ceiling. Under monitored summer conditions, warm those measured at 500 m depth (Table 1 and Fig. 3). air rises and is rapidly cooled by overlying bedrock, The reduction in aerosol concentration was observed creating atmospheric convection (Fig. 5a). Cooling by during 2 separate transects, both incorporating the the bedrock exceeds warm air replenishment, creating same 12 monitoring sites but occurring 1.5 years a zone of cold air close to the cave ceiling (Figs. 5a apart and under different seasonal conditions. and 6). Bedrock and cave air temperature variations The measured reduction of total aerosol concentration during the winter season mean that this process may reverses at the Abyss; this region of the cave has the not be dominant year round. only active waterfalls close to the tourist path. The During tourist visits, anthropogenic warming of the active stream continues close to the path between the air column causes temperatures to increase; the most Abyss and the second waterfall, both marked in Fig. 3 dramatic of which occur at the roof of the chamber and 4. Aerosol concentrations in this section increased (2.5 m) (Fig. 6). One tourist group of 35 people is seen

Location within cave system 0.025 Door Eldon hall Abyss Long gallery End show cave

100m 0.020 ) 3 28th Feb 2011 (mg/m

14th July 2009 0.015

0.010

concentration

Aerosol Concentration (mg/m‐3) Aerosol Concentration (mg/m3) 0.005 Aerosol 0.0115 – 0.014 High 0.0090 – 0.0115 0.0065 – 0.009 0.0040 – 0.0065 0.000 0m 5m Low ‐20 30 80 130 180 230 280 330 380 430 480 Horizontal distance into cave system (m)

Fig. 3. Average aerosol concentrations throughout Ingleborough Show Fig. 4. Aerosol concentrations taken during a high resolution (5 m Cave taken during two cave transects. The ‘bracketed’ section around intervals) transect from Pillar Hall to the main Oxbow, taken on 28th the Abyss represents a 100 m zone of aerosol production by turbulent Feb 2011. Values are average aerosol concentration from 3 minutes water flow. The fan is operational but tourists are not present within of logging at each location. The fan is operational but tourists are not the cave system. present within the cave system.

International Journal of Speleology, 42 (1), 1-8. Tampa, FL (USA) January 2013 Aerosol distribution and cave atmospherics within a British show cave 53

Floor level to increase end chamber cave air temperature by Tourist Group 9.48 0.75 meters 0.12°C during a 5 minute period, between 12:25 and th 1.5 meters 12:30 on 18 June (Fig. 6). This temperature increase 9.44 2.5 meters was then replicated by three subsequent groups 9.4 (numbers unknown) throughout the day. Sections (°C) of the air column which show the most dramatic 9.36

Natural air increases in temperature also represent the regions of 9.32 column most dramatic cooling after tourist visits. Temperature stratification 9.28

Accumulations of carbon dioxide are recorded Atmospheric recovery period during periods of high tourist activity (Fig. 7). Using 9.24 07:00 09:00 11:00 13:00 15:00 17:00 19:00 21:00 23:00 01:00 03:00 05:00 the suggestion of Faimon et al., (2012a) that an Time average human exhales 15 l of air per minute with Fig. 6. Continuously logged, height based temperature profile during th 3% CO2 volume, large tourist groups (40 people) in five tourist visits to the End Chamber, 18 June 2009. Overnight Ingleborough could be expected to exhale ≈600 l of air temperature stratification (23.00-05.00) represents natural cave air per minute, reflecting a considerable anthropogenic temperatures and structure, which both differ during periods of tourist activity. Tourist group numbers 4, 35, 35, unknown and unknown. CO2 input into the cave atmosphere. Insufficient removal of CO2 even during fan operation can therefore studies suggest that under natural conditions, lead to persistently high daytime atmospheric values temperature stratification is controlled by heat loss (950 ppm) during periods of sustained tourist activity to the surrounding bedrock. During the summer (Fig. 7). Full atmospheric recovery from regular group season cooler air dominates the upper and lowermost visits (≈20 persons every 30 mins) is almost achieved reaches of the passage, creating two zones of colder two hours after the last visitors leave the cave (Fig. 7). air, close to the cave bedrock. This stratification Human activity within the cave also acts to originates from a natural convection system. increase the base level of aerosol concentration. An Natural carbon dioxide values are influenced by the in-situ tourist group study (1 second log interval) operation of the fan; when the fan is turned off at the was undertaken on the 2nd of October 2009 in the end of the day, a pulse of air flows through the cave Old Cave between 11:00 and 13:00 encompassing 4 system towards the entrance, as is expected under a small tourist groups (≈30 people total). Monitoring summer ventilation regime (Spötl et al., 2005). This shows an increase in aerosol base level by 0.021 pulse represents a fourth more complex atmospheric mg/m3, taking an hour to return to fan induced scenario, in relation to the three scenarios (Fan off, background levels after tourist activity (0.004 mg/ fan on and fan on with tourists) interpreted earlier. m3) (Fig. 8). This would be an atmospheric transition between fan induced artificial and natural background

DISCUSSION conditions, where CO2 rich cave air trapped deep within the system is flushed through the cave after Natural background atmospheric conditions the fan is turned off. Spatial investigations undertaken in the period These results reflect a summer atmospheric summer – autumn 2009 in Ingleborough Show system; strong ventilation during the winter season Cave reveal a decline in average air temperatures may alter the observed atmospheric regime. In this and a dampened variability with depth into the cave way CO2 concentration is used as a proxy to assess system. Temperature stabilisation indicates the air movement within the system after the fan is external atmosphere has a diminishing influence turned off and the cave begins to return to natural on cave temperature with depth, possibly due to microclimatic conditions. the cooling effects of cave bedrock (Spötl et al., 1100 2005; Russell & MacLean, 2008). High-resolution Tourist group 1000

a. Natural atmospheric conditions b. Atmospheric conditions during tourism 900 (ppm)

Heat exchange 800 Atmospheric re‐ Heat produced by stratification: warmest tourist activity Concentration Zones of cold air temperatures 700 Atmospheric air build up occur in the convection uppermost sections of

the cave chamber Dioxide 600 Carbon

500 Atmospheric recovery period

400 Fig. 5. Diagrammatic representation of cave air temperature 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 stratification. a) Natural conditions with cool air pockets in regions Time close to surrounding bedrock, natural convention is insufficient to cause heating of the uppermost air pocket. b) Tourist input of warm Fig. 7 Cumulative increases in atmospheric CO2 concentrations air causes a re-stratification of the air column, dramatically warming measured within the Old Cave during four tourist group visits (20, the uppermost sections of the passage and creating a top down, 18, 20 and 19 persons) on July 12th. Cave atmospheric recovery is thermally stratified air column. observed by approximately 15.00hrs.

International Journal of Speleology, 42 (1), 49-56. Tampa, FL (USA) January 2013 54 Smith, Wynn and Barker

increases become cumulative during frequent tourist 0.14 visits in a similar fashion to CO2 concentrations. Tourist

activity also acts to alter the natural stratification of the 0.12 air column by: 1) Disturbing atmospheric convection

0.1 Tourists exit cave during tourist movement. 2) The addition of body (mg/m³) heat, which rises to the uppermost levels of the cave 0.08 Aerosol base level chamber, preferentially heating this zone and causing increase 0.06 the re-stratification of the air column (Fig. 5b). concentration Heat given off from visitors warms air close to

Aerosol 0.04 the cave ceiling, creating a thermally stratified atmospheric environment. After visitors leave, cave 0.02 air temperatures return to natural stratification Aerosol recovery period 0 within approximately 12 hours. This process of re- 11:30 11:40 11:50 12:00 12:10 12:20 12:30 12:40 12:50 13:00 Time stratification is driven primarily by bedrock cooling and possibly by vapour condensation, especially Fig. 8. Aerosol concentration increase during 4 tourist group visits (numbers unknown) to the Old Cave on the 2nd of October 2009, within the deepest sections of the cave system where demonstrating aerosol base level increase and decay over a 1 hour external air temperatures have little influence over period. cave air temperature. Middle sections of the cave atmosphere therefore exhibit smaller temperature Impact of fan operation and tourist groups on cave atmospherics fluctuations than either the floor or ceiling zones, a result of both reduced heating during tourist Ingleborough cave exhibits two distinct tourist interaction (Fig. 6) and increased distance from the related atmospheric regimes; one driven solely cooling influence of surrounding bedrock (Fig. 5b). by the operation of the fan, the other derived from a combination of fan operation and tourist group Cave aerosols activity. Aerosol monitoring in Ingleborough was only undertaken during periods of fan operation, so Carbon dioxide and temperature truly natural conditions cannot be discussed. The Carbon dioxide dynamics are artificially modified influence of the fan is to move external air into the by the fan throughout the show cave; driving CO2 cave system. Further discussion of cave aerosols in concentrations below levels which are naturally Ingleborough is undertaken in relation to the location observed. The fan forces cave air deeper into the of aerosol production. Ingleborough system, increasing the proportion of Allogenic Aerosols: Aerosol concentrations decay air derived from the external atmosphere. Individual rapidly within the first 75 m of the cave system. tourist groups have a secondary influence on the Primary findings suggest that this distance decay is artificially stable, low pCO2 atmosphere created by invariant across different seasons. Previous studies the fan. Increases in CO2 concentration are associated (Kertész et al., 1999) have suggested that aerosol with the presence of tourist groups (Fig. 7). Although dynamics are driven by natural cave ventilation cave atmospheric recovery does occur, CO2 build up regimes. However, the seasonal invariance observed due to tourism can be identified. During consecutive at Ingleborough suggests the fan promotes the group visits, cave atmospheric recovery cannot inward movement of aerosols in the entrance zone, be completed, producing cumulatively high CO2 independent of the natural ventilation regime of the concentrations over busy tourist periods. Only after cave system. The rapid decay of aerosols in the first the fan is turned off at the end of the tourist day can 75 m of cave passage may be caused by the deposition atmospheric recovery begin to occur. of larger aerosol fractions from the air column and A diurnal state of airflow switching exists in the limited ability of allogenically sourced aerosols Ingleborough, at the end of the show cave day a peak to penetrate deep into the cave system (Mulec et in CO2 concentration is measured in passages close to al., 2012). Although aerosol size fractions were not the cave entrance (0-75 m depth) (Fig. 2). This CO2 peak measured during this study it is assumed that larger is possibly derived from the natural exhalation of cave aerosol particulates would be deposited first, nano air, observed in Ingleborough and other cave systems sized particulates may therefore penetrate deeper into during the summer season due to differences in air the system. pressure (Spotl et al., 2005). During fan operation this Autogenic Aerosols: Aerosol distance decay is natural ventilation regime cannot dominate airflow reversed around the major stream passage of the due to the inward flux of air. However, once the fan Abyss, supporting a previous hypothesis from Gadoros is switched off natural ventilation dynamics resumes & Cser (1986), who suggested active streams are an and cool, CO2 rich cave air flows out of the main autogenic source of cave aerosols. Concentrations entrance. The observed CO2 peak is therefore forced reach highest in-cave levels at locations of most by daytime fan operation, representing a previously turbulent water flows. Bubble-bursting or water unidentified atmospheric control the fan has over aerosol dispersal mechanisms may therefore control cave atmospherics. aerosol release (Mulec et al., 2012). The decay of Cave tourism offers a strong control over temperature aerosol concentration towards the cave interior variation within Ingleborough (Fig. 6). Temperature reveals few other substantial autogenic sources of

International Journal of Speleology, 42 (1), 49-56. Tampa, FL (USA) January 2013 Aerosol distribution and cave atmospherics within a British show cave 55 aerosol production, although in caves with numerous highlighting the contrast between natural and active speleothems water splashing is thought to be a anthropogenic systems. Cave tourism overprints the possible aerosol source (Mulec et al., 2012). Further low base levels derived from artificial air pumping, monitoring of aerosol concentrations during variable causing increases in temperature, carbon dioxide stream flows would offer a secondary test to this and aerosol concentration. These increases are most assertion and a method of quantifying natural aerosol dramatic during busy periods where atmospheric production by underground streams. recovery is limited. Anthropogenic Aerosols: Aerosol increases observed Aerosols within the cave environment are shown during tourist group visits may be a result of two to decrease with distance from the cave entrance, mechanisms. Firstly, tourists may act as a vector suggesting a settling of larger particulates from the for aerosol transport; particulates attach to clothing increasingly stagnant air column. Passing tourist and are transported from outside the cave. Once groups cause short term increases in aerosol detached they become an aerosol source within the concentration, possibly through the re-suspension cave. Human activity therefore enables particulates of previously settled particulates or by transporting to penetrate deeper into cave systems than would allogenic aerosols into the cave system. Natural be observed naturally. This transport of particulates increases in aerosol concentrations were also may offer a possible explanation for the extremely observed surrounding regions of turbulent water flow, high levels of pollutant aerosols found within South identifying a significant autogenic source of aerosols Korean caves (Chang et al., 2008). This assertion may within cave environments. It is therefore suggested be tested by identifying aerosols sourced solely from that speleothems growing on tourist paths, close the external environment, such as anthropogenically to cave entrances or near stream passages, may be derived pollutants, and monitoring the extent to prone to incorporating higher levels of atmospheric which they infiltrate into the cave atmosphere under aerosols. These findings should be considered natural conditions and again during tourist activity. when choosing speleothems for scientific analysis, The identification of aerosol chemistry is however, especially those sourced from within active show more complex than the aerosol concentration logging caves. Investigation of aerosol uptake into actively that was undertaken in this study; a comprehensive depositing speleothems is required, with the aim review of aerosol identification strategy is given by of quantifying aerosol contribution to the chemical Dredge et al., 2012 (in review). record encapsulated in speleothems. Secondly, human visitors may re-entrain aerosols into the cave atmosphere by kicking dust from the ACKNOWLEDGMENTS path and touching cave walls. The re-suspension of settled dust particulates from the floor and The authors would like to thank Bob and Sue Jarman walls of the cave may be the cause of the small for information about and access to Ingleborough but stable increase in aerosol base levels observed show cave and cave guide John Cordingley for his during tourist activity in Ingleborough. This second information and assistance, all of which helped process is more likely to consistently add higher facilitate this work. Thanks also to Dr. Duncan Whyatt concentrations of particulates into the atmosphere, who provided insightful comments and suggestions, as it does not require the transportation of helping to improve this paper. Finally we would like particulates into the cave system. Chemical analysis to thank three anonymous reviewers for their useful of cave aerosols collected during tourist activity comments and suggestions. may help to identify if the aerosols are sourced allogenically, or if aerosol increases primarily result REFERENCES from the re-suspension of autogenic particulates derived from cave walls and floors. Baldini J.U.L., Baldini L.M., McDermott F. & Clipson. The incorporation of autogenic or allogenic aerosols N., 2006 - Carbon Dioxide sources, sinks and spatial into speleothem deposits may possibly influence variability in shallow temperate zone caves: Evidence the chemical record (Dredge et al., 2012 in review). from Ballynamintra Cave, Ireland. Journal of Cave and It is therefore essential for further work to establish Karst Studies, 68 (1): 4-11. 1) the extent of aerosol transport and entrainment Baldini J.U.L., 2010 - Cave atmosphere controls on stalagmite growth rate and palaeoclimate records. by humans and 2) the extent to which aerosols are In: Pedley H. M. & Rogerson M. (Eds.), Tufas and naturally incorporated into speleothems. This is speleothems: Unravelling the microbial and physical especially important for caves where tourist activities controls. Geological Society Special Publication, 336: and scientific analysis of speleothems operate in close 283-294. proximity to one another. Baker A. & Genty D., 1998 - Environmental pressures on conserving cave speleothems: Effects of changing surface CONCLUSION land use and increased cave tourism. Environmental Management, 53: 165-175. 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International Journal of Speleology, 42 (1), 49-56. Tampa, FL (USA) January 2013 56 Smith, Wynn and Barker Chang S.J., Jeong G.Y. & Kim S.J., 2008 - The origin Hayland R. Q., 1995 - Spatial and temporal variations of black carbon on speleothems in tourist caves of radon and radon daughter concentrations within in South Korea: Chemical characterization and limestone caves. Doctoral thesis, University of source discrimination by radiocarbon measurement. Huddersf ield. Atmospheric Environment, 42: 1790-1800. Jeong G. Y., Kim S. J., & Change S. J., 2003 - Black http://dx.doi.org/10.1016/j.atmosenv.2007.11.042 carbon pollution of speleothems by fine urban aerosols Cigna A. A., 1967 – An analytical study of air circulation in tourist caves. American Mineralogist, 88: 1872-1878. in caves. International Journal of Speleology, 3: 41-54. Kertesz Zs., Borbely-Kiss I., & Hunyadi I., 1999 - Study Dominguez-Villar D., Fairchild I.J., Carrasco R.M., of aerosols collected in a speleotherapeutic cave situated Pedraza J., & Baker A., 2010 - The effect of visitors in a touristic cave and the resulting constraints on natural below Budapest, Hungary. Nuclear Instruments thermal conditions for palaeoclimate studies (Eagle Cave, and Methods in Physics Research B, 150: 384-391. Central Spain). Acta Carsologica, 39 (3): 491-502. http://dx.doi.org/10.1016/S0168-583X(98)00899-4 Dredge J., Fairchild I.J., Harrison R.M., Fernàndez-Cortés A., Lario J., & Soler V., 2010 - Microclimate monitoring Sánchez-Moral S., Gunn J., Jurado V., Smith A., Spötl C., of Pozalagua Cave (Vizcaya, Spain): Application Mattey D., Wynn P.M. & Grassineau N., 2012 - Cave aerosols: to management and protection of show caves. Distribution and contribution to speleothem geochemistry. Journal of Cave and Karst Studies, 72 (3): 169-180. Quaternary Science Reviews, under review. http://dx.doi.org/10.4311/jcks2009lsc0093

Faimon J., Stelcl J. & Sas D., 2006 - Anthropogenic CO2- Liñan C., Vadillo I., & Carrasco F., 2008 - Carbon dioxide flux into cave atmosphere and its environmental impact: concentration in air within the Nerja Cave (Malaga, Andalusia, A case study in the Cisařská Cave (Moravian Karst, Czech Spain). International Journal of Speleology, 37 (2): 99-106. Republic). Science of the Total Environment, 369: 231-245. http://dx.doi.org/10.5038/1827-806X.37.2.2 http://dx.doi.org/10.1016/j.scitotenv.2006.04.006 McDermott F., 2004 - Palaeo-climate reconstruction Faimon J., Troppová D., Baldik V., & Novotny R., 2012a from stable isotope variations in speleothems: A - Air circulation and its impact on microclimatic variables review. Quaternary Science Reviews, 23: 901-918. in the Cisařská Cave (Moravian Karst, Czech Republic). http://dx.doi.org/10.1016/j.quascirev.2003.06.021 International Journal of Climatology, 32: 599-623. http://dx.doi.org/10.1002/joc.2298 Mulec J., Vaupotič J., Walochnik J., 2012 - Prokaryotic Faimon J., Ličbinska M., & Zajiček P., 2012b - Relationship and eukaryotic airborne microorganisums as tracers between carbon dioxide in Balcarka Cave and adjacent of microclimatic changes in the underground (Postojna soils in the Moravian Karst region of the Czech Republic. Cave, Slovenia). Microbial Ecology, 64: 654-667. International Journal of Speleology, 41 (1): 17-28. http://dx.doi.org/10.1007/s00248-012-0059-1 http://dx.doi.org/10.5038/1827-806X.41.1.3 Russell M. J., & MacLean V. L., 2008 - Management Fairchild I.J., Smith C.L., Baker A., Fuller L., Spötl C., Mattey issues in a Tasmanian tourist cave: Potential D., McDermott F., & E.I.M.F., 2006 - Modification and microclimatic impacts of cave modifications. Journal preservation of environmental signals in speleothems. Earth- of Environmental Managment, 87: 474-483. Science Reviews, 75: 105-153. http://dx.doi.org/10.1016/j.jenvman.2007.01.012 http://dx.doi.org/10.1016/j.earscirev.2005.08.003 Spötl C., Fairchild I.J., & Tooth A.F., 2005 - Cave air control on Fairchild I.J., Spötl C., Frisia S., Borsato A., Susini J., Wynn drip-water geochemistry, Obir Caves (Austria): Implications P.M., Cauzid J. & EIMF., 2010 - Petrology and geochemistry for speleothems deposition in dynamically ventilated caves. of annually laminated stalagmites from an Alpine cave Geochimica et Cosmochimica Acta, 69 (10): 2451-2468. (Obir, Austria): Seasonal cave physiology. Geological http://dx.doi.org/10.1016/j.gca.2004.12.009 Society, London, Special Publications, 336: 295-321. http://dx.doi.org/10.1144/SP336.16 Sweeting M.M., 1950 – Erosion cycles and limestone Gadoros M., & Cser F., 1986 - Aerosols in caves - theoretical caverns in the Ingleborough district. The Geographical considerations. 9th International Congress of Speleology Journal, 115 (1/3): 63-78. (Barcelona), 2: 90-92. http://dx.doi.org/10.2307/1789020 Gascoyne M., Ford D.C., & Schwarcz P., 1983 - Taylor & Hodgson Land Surveyors of Lancaster., 1838 - Rates of cave and landform development in the Cave survey. Yorkshire Dales from speleothem age data. Earth Waltham A.C., 1970 - Cave development in the limestone of Surface Processes and Landforms, 8 (6): 557-568. the Ingleborough district. The Geographical Journal, 136 http://dx.doi.org/10.1002/esp.3290080607 (4): 574-585. http://dx.doi.org/10.2307/1796185

International Journal of Speleology, 42 (1), 49-56. Tampa, FL (USA) January 2013 International Journal of Speleology 42 (1) 57-63 Tampa, FL (USA) January 2013

Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Official Journal of Union Internationale de Spéléologie

Phototactic behaviour of subterranean Copionodontinae Pinna, 1992 catfishes (Siluriformes, Trichomycteridae) from Chapada Diamantina, central Bahia, northeastern Brazil Bianca Rantin1* and Maria Elina Bichuette1 1Laboratório de Estudos Subterrâneos, Universidade Federal de São Carlos, São Carlos, São Paulo, Brasil. Rod. Washington Luís, km 235. CP. 676. CEP: 13565-905. Web site: http://www.biosub.ufscar.br

Abstract: The phototactic behaviour of three Copionodontinae (Trichomycteridae) catfish species (two troglobites and one epigean) from Chapada Diamantina was studied in order to detect modifications related to isolation in the subterranean environment. Differences in response under different luminosities were detected and, unlike other cavefish, Copionodontinae cave species have shown to be more photophobic than the epigean syntopic to them. The troglobitic Glaphyropoma spinosum is the most photophobic, presenting this behaviour under all light intensities, and more homogeneous regarding morphological characters. It suggests that this population is probably isolated for a longer time in the subterranean environment compared to Copionodon sp. n., the other cave species, which is only photophobic under low light intensities. The indifference to light exhibited by the epigean species C. pecten could be an answer to a recent predation pressure, an ecological aspect, and perhaps this character-state is under fixation in this population. There are also evidences that the skin has a relevant role in the perception of light for the Copionodontinae species.

Keywords: reaction to light; photophobia; cave catfish; Glaphyropoma; Copionodon

Received 10 April 2012; Revised 7 August 2012; Accepted 26 November 2012

Citation: Rantin B. and Bichuette M.E. 2013. Phototactic behaviour of subterranean Copionodontinae Pinna, 1992 catfishes (Siluriformes, Trichomycteridae) from Chapada Diamantina, central Bahia, northeastern Brazil. International Journal of Speleology, 42 (1), 57-63. Tampa, FL (USA) ISSN 0392-6672 http://dx.doi.org/10.5038/1827-806X.42.1.7

INTRODUCTION 1998; Trajano & Borowsky, 2003; Parzefall & Trajano, 2010). According to Trajano & Gerhard (1997) the Most troglobite specializations involve character phototactic behaviour displayed by some troglobite regression, frequently assigned to the loss of function catfishes may vary in the same species according to or to the relaxing of stabilizing selective pressure the stimulus context, i.e., between controlled tests (Wilkens, 1992). Besides the classic troglomorphisms with choice chamber in the laboratory and sudden (changes in morphological characters), like eye and illumination in the natural environment. Therefore, melanic pigmentation regressions until total loss of the responses are part of different behavioural traits, these characters, there are also regressions of some displayed in different functional contexts and possibly behaviour patterns (Parzefall & Trajano, 2010). following independent evolutionary paths (Trajano & Therefore, subterranean organisms are good models Gerhard, 1997). for studies of ecology/behaviour and evolution There are around 170 species of subterranean fishes (Trajano & Bockmann, 1999). in the world and 25 of them occur in Brazil (15% of Reaction to light represents the best studied the total richness) (Proudlove, 2010; G. Proudlove, behaviour on subterranean organisms and, pers. comm.). Most of Brazilian subterranean considering the fishes, at least ten families were fishes belong to the Order Siluriformes: catfishes studied regarding this aspect (Characidae - genus (Trichomycteridae, Heptapteridae and Callichthyiidae Astyanax Baird & Girard 1854, Cyprinidae, families) and armoured catfishes (Family Loricariidae) Balitoridae, Heptapteridae, Trichomycteridae, (Proudlove, 2010). Considering the Trichomycteridae Loricariidae, Clariidae, Amblyopsidae, Bythitidae, and catfishes, the subfamily Copionodontinae Pinna, Poecilidae), with no taxonomic evidence in the obtained 1992 is endemic of Chapada Diamantina, central results (e.g., Trajano & Gerhard, 1997; Parzefall, Bahia, and nowadays there are records of two genera

*[email protected] 58 Rantin & Bichuette and five species in the region, two of them occurring character-state. However, to polarize this character, and coexisting (syntopic) in the sandstone/quartzitic comparing the troglobitic species with at least two caves of Chapada Diamantina, showing variability other epigean taxa is necessary (Maddison et al., 1984). in the troglomorphic characters (regression of eyes Following this thought, Poulson (1964) and Romero and melanic pigmentation): Glaphyropoma spinosum (1985) consider that the maintenance of photophobia Bichuette, Pinna & Trajano, 2008 and Copionodon sp., in troglobitic fishes is a relict of the epigean existence this latter represents a new species, under description and not an adaptation to the cave life. (M. E. Bichuette, in prep.). In this study the reaction to light was analyzed Compared to other trichomicterids, copionodontines in laboratory, and eventual observations were show plesiomorphic condition of many morphological made in the natural environment, in order to character-states, shared with its possible sister group detect modifications related to the isolation in Trichogenes Britski & Ortega 1983 (for instance, the the subterranean environment. Copionodontinae complete infraorbital latero-sensory canal), and a unique catfishes probably represent a basal group in the primitive condition that suggests its basal position phylogeny of trichomycterid (Pinna, 1992) and within Trichomycteridae (the more anteriorly located the study of behaviour traits can provide valuable dorsal fin) (Pinna, 1992). Many of these conditions are information to corroborate the relationships within relevant to understand the relationships among other Trichomycteridae. Thus, we compare the response to families of Siluriformes and also within the Loricarioidea light in three copionodontine species from the same group (Pinna, 1997). region: Glaphyropoma spinosum and Copionodon new Behaviour studies of Brazilian subterranean fishes species (both troglobites) and the epigean species included Heptapteridae catfishes - Pimelodella kronei Copionodon pecten. Miranda-Ribeiro 1907 (e.g. Trajano, 1989; Hoenen, 1996), Taunayia sp. (= Rhamdiopsis sp. 2) (Trajano MATERIALS AND METHODS & Gerhard, 1997; Trajano & Bockmann, 1999) and Rhamdiopsis krugi (Mendes, 1995; Bichuette, in Study area prep.); Trichomycteridae catfishes - Trichomycterus Chapada Diamantina region (Fig. 1) is an extension itacarambiensis Trajano & Pinna 1996 (Trajano & of Serra do Espinhaço and its relief is composed of Gerhard, 1997), Ituglanis passensis Fernández & hills, mountains and subterranean galleries formed Bichuette 2002, I. bambui Bichuette & Trajano 2004, and I. ramiroi Bichuette & Trajano 2004 (Bichuette, 2003) and Loricariidae armoured catfishes - Ancistrus cryptophtalmus Reis 1987 and A. formoso Sabino & Trajano 1992 (Trajano & Souza, 1994; Bessa & Trajano, 2001). A huge variation is observed in the phototactic behaviour of Brazilian subterranean fishes: many species exhibit from a photophobia to a tendency to photofilia, with cases of indifference to light [e.g., Taunayia sp. (= Rhamdiopsis sp. 2 - Trajano & Gerhard, 1997)]. Moreover, a variation in the response to light is observed for intraspecific and interspecific contexts (populational and individual) and the variability of this behaviour is related to variation of morphological characters, for example, eyes and melanic pigmentation (e.g., for the armoured catfishes Ancistrus spp. and Ituglanis catfishes - Bessa & Trajano, 2001; Bichuette, 2003); and to the developing life cycle (Secutti & Trajano, 2009). When compared to the epigean relatives (in the case of known and/or existing species), some tendencies are observed, for example, the epigean fish show marked photophobic habits compared to the troglobitic ones (Langecker, 1992; Trajano, 2003). Langecker (1992) proposed that the maintenance of photophobia in the Fig. 1 Map of Chapada Dimantina region, central Bahia, northeastern Brazil. In gray color -Chapada Diamantina National Park: 1. Boudaries of the National Park; 2. Main Roads; troglobitic species could be a relictual 3. Secondary Roads; 4. Rivers and small drainages; 5. Cities; 6. Locality of occurence of characteristic, being a plesiomorphic Copionodontinae catfishes: Povoado de Igatu, Andaraí county, Bahia state.

International Journal of Speleology, 42 (1), 57-63. Tampa, FL (USA) January 2013 Phototatic behaviour of cave Copionodontinae from NE Brazil 59 by metasedimentary rocks from the Paraguaçu and Chapada Diamantina groups. These rocks were deposited one billion years ago and the dominant lithologies are sandstone, siltstones, argillites, conglomerates and pelites (Schobbenhaus et al., 1999). The altitudes vary from 1,000 to 1,700 m, representing a watershed between the São Francisco and the East Basins. The climate is tropical semi- arid, with distinct wet/dry periods, Aw according to Köppen’s (1948) classification. Medium annual temperature is around 20°C (Nimer, 1989).

Studied species Fig. 2 Glaphyropoma spinosum – alive specimen (Photo: Adriano Gambarini). The troglobitic copionodontines (G. spinosum and Copionodon new species) occur in the following sandstone/quartzite caves: Cobras cave, Parede Vermelha cave, Morro de Alvo System, Rio dos Pombos cave, Torras System (Torras I e II caves), Criminoso cave and Lobo cave. These caves are located inside the Chapada Diamantina National Park. The epigean species, C. pecten, occurs in the rivers Coisa Boa, Xavier and Paraguaçu, in the boundaries of the cave localities. This epigean species was also recorded inside some of the caves where the troglobites occur, showing an interesting case of syntopy. In natural environment, G. spinosum presents eyes and pigmentation regression (showing iridescence), with little variability in these characters, configuring a homogeneous population (Bichuette et al., 2008 Fig. 3 Copionodon sp. n. – alive specimen (Photo: Dante Fenolio). – Fig. 2). Considering Copionodon new species, a higher morphological variability is observed, with not so pronounced regression of eyes (from reduced to normal eyes) and a mosaic of characters concerning pigmentation, varying from pale yellow to light gray, in its natural habitat (Fig. 3). The epigean species C. pecten is homogeneous, showing developed black eyes and typically light brown coloration, with dark and iridescent lines in the body sides (Fig. 4).

Specimens collecting and laboratory maintenance Copionodontinae catfishes were captured with hand nets and transported in three liter gallons, conditioned in thermal boxes, with low temperature achieved with addition of recycled hipergel. Intraspecific groups up to 10 individuals are Fig. 4 Copionodon pecten – alive specimen (Photo: Adriano Gambarini). maintained in 50-liter aquaria with continuous filtration, aeration and available shelters. The 1994; Trajano & Gerhard, 1997; Bichuette, 2003; aquaria are installed at the Laboratório de Estudos Timmermann & Plath, 2009), which simulates Subterrâneos (LES) at the Departamento de Ecologia the interface between epigean and subterranean e Biologia Evolutiva of Universidade Federal de São environments (Trajano & Gerhard, 1997). The tests Carlos (UFSCar), in permanent darkness (except during were performed one month after the catfishes arrived maintenance activities). The epigean Copionodontinae in the laboratory. are also maintained in the LES, but in a separate The test aquarium consisted in a 50-liter tank, compartment, covered with a black shield, where a divided in two sectors by a black plastic screen with lamp attached to a timer simulates the day phase (12 openings in the middle and at the sides. One of these hours per day). The catfishes have been fed once a sectors was illuminated by a white light bulb and the week with commercial food for carnivorous fishes (dry other remained dark, covered with a black cardboard. or frozen Artemia salina) at no fixed days and times. The plastic screen prevented the entrance of light, but did not block the movements of the catfishes across Reaction to light tests the aquarium floor and through the walls. We studied The choice-chamber method was used in the tests the fishes reaction to light under 40 lux (twilight), 170 of light reaction, as in the study of other troglobitic lux (beginning of twilight) and 1,700 lux (cloudy day), catfishes (Langecker, 1992; Trajano & Souza, using a white fluorescent bulb. Twelve specimens of

International Journal of Speleology, 42 (1), 57-63. Tampa, FL (USA) January 2013 60 Rantin & Bichuette each species (G. spinosum, Copionodon new species 170 lux, p=0.01941- Fig. 6). The epigean species, C. and C. pecten) were tested individually. Only the pecten, showed indifference under all light intensities, individuals 13 and 25 were juveniles, the remaining corroborated by the statistical results of the One- being adults. way ANOVA with repeated measures tests, which did In order to determine the acclimation time, we not show significance (40 lux, p=0.9935; 170 lux, made previous observations, which demonstrated p=0.4403; 1,700 lux, p=0.7907 – Fig. 7). that an acclimation time of less than one hour Thus, there is a photophobia degree exhibited by before the beginning of tests was enough, since the the three Copionodontinae species: the troglobite G. individuals showed low and calm swimming and spinosum is the most photophobic one, followed by opercular beating. the new species of Copionodon and finally the epigean At the beginning of the tests, a focal fish was taken species C. pecten, which reacted indifferently under from a stock tank and gently introduced into the test- the three intensities. aquarium, where it had one hour to acclimate to the Comparing light reaction to morphologic characters, new environment and to the light intensity. After this we note that G. spinosum, which has regressed eyes period, the control test was initiated, with also one size and pigmentation, exhibited strong avoidance hour duration, and we counted the total time that the under the three light intensities. Copionodon sp. n. individual spent in the left sector of the aquarium (the only showed response to light in the lower intensities side that would be the lighted one during the actual (40 and 170 lux), and also presents variability for tests). During acclimation period and control, the eyes and pigmentation. The epigean species C. whole aquarium remained illuminated. pecten, clearly homogeneous in relation to eyes and After the control, the right sector of the aquarium pigmentation, showed indifference in the response to was covered with black cardboard (dark chamber), light in all intensities. and the total time spent in the lighted sector of the aquarium was recorded during one hour. The same DISCUSSION procedure was carried on for the other luminosities. Between one test and the other, that is, before Most troglobitic catfishes studied concerning changing light intensity, the light was turned off for phototactic behaviour present epigean relatives at least 20 minutes. The order of the light intensities displaying photophobia (Parzefall & Trajano, was always random, but never in an increasing or 2010). However, for the epigean Copionodontinae, decreasing order, to prevent biases in responses. The C. pecten, we observed an inverse pattern: the phototactic tests were carried on in the same LES troglobitic species, G. spinosum and Copionodon room, between 8 am and 7 pm. sp. n. displayed more photophobic habits than After tests, standard-length and eye diameter the epigean one, which is indifferent to light. This of each specimen were measured with a digital inverted pattern was also observed for Balitoridae caliper, 0.001 millimeters of precision. The eyes were (Eurasian Cypriniformes fishes) from Thailand: considered as reduced when less than 0.1 millimeter while the epigean is generally diurnal and non- of diameter, and as developed when between 0.1 and cryptobiotic/benthonic, the troglobitic species 1.0 millimeter. Also, the pigmentation was observed Schistura oedipus Kottelat 1988 shows indifference under white light and the fishes were divided in four to light and a weak photophobia to lower light classes of coloration: light gray with iridescence, intensities plus strong photophobia to higher light medium gray, dark gray and purple. intensities (Trajano et al., 2002). This inverted Responses to light were divided in four photophobia pattern can raise the hypothesis that the cave degrees: photophilia (more than 31 minutes in lighted species probably originated from an ancestral chamber), photophobia (between 29 and 11 minutes which showed twilight or nocturnal activities. in lighted chamber), marked photophobia (10 minutes Furthermore, some ecological hypotheses can be or less in lighted chamber) and indifference to light considered. Additional data from observation and (30 minutes in each chamber). collecting in the same locality of occurrence of the epigean species indicated the existence of two major Statistical analysis nocturnal predators coexisting with C. pecten: the Statistically significant differences between the trahiras Hoplerythrinus unitaeniatus Spix & Agassiz, total time spent in the illuminated sector in tests and 1829 and the catfish Trachelyopterus galeatus in control were detected by One-way ANOVA Repeated Linnaeus, 1766. Thus, a predation pressure, measures with =5% (Zar, 1996). probably recent (Hoplerythrinus unitaeniatus is an exotic fish and was introduced about 180 years RESULTS ago – R. C. Santos, pers. comm.), must be the principal triggering process for this indifference to The troglobitic species G. spinosum is photophobic light behaviour in C. pecten. Indeed, groups of C. under the three light intensities, confirmed by pecten gather either in exposed and hidden places statistically significant differences between control during the day phase. Thus, it is possible that, if C. and tests (40 lux, p=0.03073; 170 lux, p=0.00014; pecten has once had photophobic behaviour, it is in 1,700 lux, p=0.00495 – Fig. 5). Copionodon sp. n., process of inversion, due to this predation pression the other troglobitic species, is only photophobic at night, which would force them to forage during under the lower light intensities (40 lux, p=0.03732; the day.

International Journal of Speleology, 42 (1), 57-63. Tampa, FL (USA) January 2013 Phototatic behaviour of cave Copionodontinae from NE Brazil 61

Fig. 5 Box-plot with medians and means of time spent in lighted sector of aquarium, under 40 lux. A. Control, B. Test.

Fig. 6 Box-plot with medians and means of time spent in lighted sector of aquarium, under 170 lux. A. Control, B. Test.

Fig. 7 Box-plot with medians and means of time spent in lighted sector of aquarium, under 1,700 lux. A. Control, B. Test. It is also important to point out that we observed manifested in both juvenile and adult specimens. changes in pigmentation pattern in all cave Several authors have suggested that the skin of copionodontine individuals that remained in some cave vertebrates may mediate light reactions, captivity: all specimens of G. spinosum changed for instance, the characidae Astyanax fasciatus from pale yellow to purple 10-15 days after arrival Cuvier, 1819 (Kähling, 1961), the amblyopsidae from the field, indicating that this species may Amblyopsis spelaea DeKay, 1842 (Payne, 1907) and perceive light by the skin, a typical extraocular the salamander Proteus anguineus Laurenti, 1819 photoreception (EOP), very common in vertebrates (Hawes, 1946). For Copionodon sp. n., however, the (Langecker, 1992). Indeed, these pigmentation pigmentation changes were subtle, changing from changes cannot be related to food, since the food light gray to medium or dark gray, suggesting that varies during maintenance in laboratory, nor the eyes must still be the main light receptor in this to changes related to aging, since pigmentation species.

International Journal of Speleology, 42 (1), 57-63. Tampa, FL (USA) January 2013 62 Rantin & Bichuette Moreover, this study reinforces the idea of different Dodonov for help in statistical analysis. This work is reactions to light according to the stimuli context part of the Master degree of Bianca Rantin and part of a (gradation in laboratory and sudden stimuli in natural major research project of Maria E. Bichuette, both were habitat) since Copionodontinae catfishes did not financially supported by the Fundação de Amparo à show any reactions under lantern light in the natural Pesquisa no Estado de São Paulo (FAPESP, processes environment. The same was observed for trichomycterid numbers 2008/08910-8 and 2008/05678-7). Thanks catfishes I. bambui and T. itacarambiensis, heptapterid also to the environmental governmental agency catfishes P. kronei, and armoured loricariid catfishes ICMBIO (Instituto Chico Mendes de Biodiversidade) A. formoso (Bichuette, 2003; Trajano & Gerhard, for the collecting permission document (license 1997), besides the balitoriid cave fishes from Thailand number 20165-1) and to PPGERN/UFSCar (Programa S. oedipus (Trajano et al., 2002). de Pós Graduação em Ecologia e Recursos Naturais da Finally, there is, apparently, some correlation Universidade Federal de São Carlos/ Brasil), for the between morphological traits and reaction to light infrastructure to develop this work. Eleonora Trajano regarding G. spinosum. The fact that this species, (IBUSP), Nivaldo Nordi (UFSCar), Andréa Peripato morphologically homogeneous, reacts similarly under (UFSCar) and Lilian Casatti (UNESP) contributed the three light intensities suggests that the response with suggestions and critical considerations for the is related to its low variation degree of eyes and manuscript. pigmentation. On the other hand, Copionodon sp. n., a heterogeneous population, reacted differently REFERENCES under the three lights intensities, which could be due to individual differences. It suggests that this latter Bessa E. & Trajano E., 2001 - Light reaction and species has been isolated in the hypogean realm for a cryptobiotic habits in armoured catfishes, genus longer time when compared to the more homogenous Ancistrus, from caves in central and northwest Brazil (regarding both morphological and behavioural (Siluriformes: Loricariidae). Mémoires de Biospéleologie, aspects) G. spinosum. 28: 29-37. Bichuette M. E., 2003. 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International Journal of Speleology, 42 (1), 57-63. Tampa, FL (USA) January 2013 64 International Journal of Speleology 42 (1) 65-76 Tampa, FL (USA) January 2013

Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Official Journal of Union Internationale de Spéléologie

Diversity and biosynthetic potential of culturable aerobic heterotrophic bacteria isolated from Magura Cave, Bulgaria Iva Tomova1, Irina Lazarkevich1, Anna Tomova1, Margarita Kambourova1, and Evgenia Vasileva-Tonkova1* 1 Bulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology, Acad. G. Bonchev Str., Bl. 26, 1113 Sof ia, Bulgaria

Abstract: Biocapacity of bacteria inhabiting karstic caves to produce valuable biologically active compounds is still slightly investigated. A total of 46 culturable heterotrophic bacteria were isolated under aerobic conditions from the Gallery with pre-historical drawings in Magura Cave, Bulgaria. Phylogenetic analysis revealed that most of bacterial isolates aff iliated with Proteobacteria (63%), followed by Actinobacteria (10.9%), Bacteroidetes (10.9%), and Firmicutes (6.5%). A strong domination of Gram-negative bacteria (total 81%) belonging to nine genera: Serratia, Pseudomonas, Enterobacter, Sphingobacterium, Stenotrophomonas, Commamonas, Acinetobacter, Obesumbacterium, and Myroides, was observed. Gram-positive isolates were represented by the genera Bacillus, Arthrobacter, and Micrococcus. One isolate showed a signif icant phylogenetic distance to the closest neighbor and could represent а novel species. Heterotrophic bacterial isolates from Magura Cave were investigated for hydrolytic enzymes production, antimicrobial and hemolytic activity. Predominance of producers of protease (87%), followed by xanthan lyase (64%), lipase (40%), β-glycosidase (40%), and phytase (21%) was observed. Over 75% of the isolates demonstrated antimicrobial and hemolytic activity. The results suggest that heterotrophic bacteria isolated from Magura Cave could be a valuable source of industrially relevant psychrotolerant enzymes and bioactive metabolites. This study is a f irst report on the taxonomic composition and biological activity of culturable bacteria inhabiting a cave in Bulgaria.

Keywords: caves; Magura Cave; culturable bacteria; 16S rDNA; enzyme production; antimicrobial activity; hemolytic activity Received 7 June 2012; Revised 3 October 2012; Accepted 1 December 2012

Citation: Tomova I., Lazarkevich I., Tomova A., Kambourova M. and Vasileva-Tonkova E., 2013. Diversity and biosynthetic potential of culturable aerobic heterotrophic bacteria isolated from Magura Cave, Bulgaria. International Journal of Speleology, 42 (1), 65-76. Tampa, FL (USA) ISSN 0392-6672 http://dx.doi.org/10.5038/1827-806X.42.1.8

INTRODUCTION and biodeterioration, and the control, treatment, and preservation of these specific environments is a Caves are nutrient-poor, dark ecosystems with serious worldwide problem (Cañaveras et al., 2001; relatively constant low temperature and high relative Allemand & Bahn, 2005; Fox, 2008). Today, most caves humidity in the dark zone deep within the cave. containing such paintings are closed off to visitors as Although providing extreme conditions for life, a conservative measure to prevent further degradation the caves are colonized by diverse psychrophilic of valuable cultural heritage sites. The literature on (cold-loving) and psychrotolerant (cold-adapted) microbial communities in subterranean environments is microorganisms that are able to grow in these mainly restricted to caves found in Spain, Italy, France, environments (Schabereiter-Gurtner et al., 2002a; Romania, and the USA. A long-term microbiological Barton & Northup, 2007). Processes of disintegration study of the cave microflora is being conducted in and formation of cave saltpeter, mineral oxides Altamira Cave (Spain) and Lascaux Cave (France), both accumulations, coloration, and moonmilk deposits in containing Paleolithic paintings (Schabereiter-Gurtner many karstic caves have been linked with microbial et al., 2002a; Bastian et al., 2009). activity (Castanier et al., 2000). Despite their archaeological importance, our At present, most of the well-known and visited knowledge on the microbiology of caves is still caves containing valuable pre-historical paintings scanty and incomplete. Although geomicrobiological are affected by progressive microbial colonization environmental investigations are widely presented, a

*[email protected] 66 Tomova et al. significant increase in biospeleological research was observed within the last two decades (Urzi et al., 2010). Caves can be considered extreme environments for life providing ecological niches for highly specialized microorganisms (Schabereiter-Gurtner et al., 2002b). These microorganisms, called extremophiles, produce enzymes that are functional under extreme conditions (Van den Burg, 2003). Although many papers have been published on isolation of psychrophilic and psychrotolerant bacteria from cold environments and their enzymatic activities, caves are still under-explored as an object for such investigations. Our knowledge of cave microbial diversity is limited; therefore, great potential exists to find novel microorganisms in caves (Engel, 2010). One of the essential mechanisms for adaptation of microorganisms to cold environments is the activity of their enzymes at low temperatures. In recent years, there is an increased interest in cold- adapted microorganisms and their enzymes because of their great biotechnological potential, offering numerous economic and ecological advantages. Because of the unique properties, cold-adapted enzymes could be extremely useful in different biotechnological fields, for example, in the detergent and food industry, biotransformation, environmental bioremediation, molecular biology, diagnostics, biosensors and bioreporters (Russell, 1998; Gerday et al., 2000; Cabeza et al., 2011; Cavicchioli et al., 2011). Karstic Magura Cave is situated near the village of Rabisha, North-West Bulgaria. Being a monument of culture and a tourist destination, it is one of the biggest and the most beautiful caves in Bulgaria. In one of the Galleries unique prehistorical drawings were discovered that represent carvings into the stone filled with bat guano (Fig. 1). Ancient drawings are multi-layered, dating back from the Late Neolithic and Eneolithic Ages, to the the early Bronze Age, and are unique for the Balkan Peninsula (Stoytchev, 1994; 1995). They represent different religous events and feasts in separate groups. The Solar calendar from the Late Neolith is the earliest solar calendar discovered in Europe; it depicts five festivals and 366 days and may have represented a shrine for the prehistoric settlement. As a result, Magura Cave was placed on the tentative list for consideration as a World Heritage Site by UNESCO in 1984. The cave has been opened for visitors only for three years; however, this resulted in changes in cave’s Fig. 1. Rock wall drawings in the gallery with the drawings in Magura microclimate due mainly to the presence of artificial Cave. light and emitted heat, and in enhanced deterioration of many of the drawings by vandal effects. That is why, in MATERIALS AND METHODS 2008 the so called Gallery with the drawing was closed to the public as a conservative measure to preserve the Sampling site valuable drawings. Geological analysis showed that the formation of No studies have been undertaken until now for Magura Cave has started 15 million years ago on a isolation and cultivation of microorganisms inhabiting calcareous hill (at 461 m above the sea level) composed Bulgarian caves and investigation of their biosynthetic of a succession of subhorizontal decimeter-thickbeds capacity. In this study, culturable aerobic heterotrophic of Cretaceous calcarenitic limestones. Magura Cave bacteria inhabiting the Gallery with the drawings in consists of a main gallery and three side branches Magura Cave were isolated and characterized, and with a current overall length of approximately 3,000 m their biosynthetic abilities were investigated in aim to (http://www.magura-cave.com/1024x768/eng/the_cave.htm). identify promising psychrotolerant bacterial strains, The northern end of “the Landslip” is connected to the producers of industrially relevant enzymes and alley with the drawings. On its main axis, the corridor antimicrobial compounds. measures 240 m in length and covers 3,750 m2 in

International Journal of Speleology, 42 (1), 65-76. Tampa, FL (USA) January 2013 Culturable aerobic heterotrophic bacteria from Magura Cave, Bulgaria 67 area. Its maximum height is 24 m. The unique cave was incubated in a T100 Thermal Cycler (BIO- drawings are situated in this corridor. The isolated RAD) for an initial denaturation at 94 °C for 5 min location of the Gallery with the drawings and its followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 longness contribute to the maintenance of a relatively s, and 72 °C for 60 s, then a final extension step at constant temperature of around 12 oC and relative 72 °C for 5 min. The amplified 16S rRNA genes of humidity above 90%. the isolates were subjected to two separate RFLP analyses, using four base pairs restriction enzymes Sample collection in order to maximize the RFLP resolution. Seven μl of Samples were collected in the beginning of April the amplified PCR products were separately digested 2011 from the Gallery with the drawings in Magura with 5 U of restriction endonucleases MspI and HaeIII Cave. Samples of about 30 to 50 g each were collected (Fermentas) in a final volume of 10 μl for 2 h at 37 °C. in sterile glass jars by gently scrapping by sterile Isolates with identical RFLP profiles were considered scalpels of rock walls (in depth up to 1 mm) in the to be phylogenetically identical. Representatives of vicinity of separated drawings at a level 30-50 cm each RFLP pattern group were chosen for sequencing above the floor. A control sample was also collected (total 24 strains). from rock wall surfaces of a side area of the Gallery The full sequence (8-1,492 nt; E. coli numbering) of without drawings. In parallel, wall samples, sediment the 16S rRNA gene of the strains was determined with samples from the floor (containing bat guano), and Applied Biosystems model 373A DNA sequencer by water samples were also collected by the research using the ABI PRISM cycle sequencing kit (Macrogen, team from Sofia University. Collected samples were South Korea). DNA sequence analyses were performed transferred to the laboratory in a thermostat bag, kept using BLASTn (Altschul et al., 1990) and Ribosomal in a refrigerator for about 18 h and used as inocula Database Project resources (Maidak et al., 1994) for enrichment. to determine their close relatives and approximate phylogenetic affiliations. Multiple sequence alignments Isolation and growth of heterotrophic bacteria were obtained using CLUSTALX (Thompson et al., Enrichment for isolation of bacterial cultures was 1997). The phylogenetic analysis was performed using performed for each sample (about 10 g) aerobically the program PAUP* Version 4.0b10 (Swofford, 2002). at 14 oC during 10 days including two transfers, in The sequence that showed more than 3% divergence 100-ml Erlenmeyer flasks containing 10-ml sterile with the closest phylogenetic neighbor was submitted nutrient broth (NB). After two transfers, a loopful of to a database under accession number HE663456. All each inoculum source was streaked onto nutrient other sequences were not deposited due to the lack of agar (NA) plates. The plates were then incubated at novelty and were used for phylogenetic identification 14 oC and monitored at 24 h intervals for a period of a of the isolates. week in order to isolate a variety of growing bacteria. Different types of colonies were picked after incubation Screening for enzyme and hemolytic activities and stroked onto NA plates to obtain pure cultures. All Hydrolytic enzyme production by bacterial isolates isolates were maintained by monthly transfers onto from Magura Cave was determined qualitatively by slants containing NA and stored at 4 oC. The strains the agar diffusion method. Petri plates contained were stored also at -80 oC in NB supplemented with basal mineral medium with 1.5% agar and a substrate 30% (v/v) glycerol. specific for the corresponding activity. Basal mineral

Growth of bacteria isolated from Magura Cave was medium (pH 7.0) contained (%): KH2PO4 0.1, (NH4)2SO4 o o o o followed at temperatures of 4 C, 10 C, 20 C, 28 C, 0.5, MgSO4 .7H2O 0.01, and NaCl 0.01. A loop of and 40 oC. Isolates were cultivated at the indicated each of 24 h pure bacterial culture grown on NA was temperatures in test-tubes containing NB with spread via a needle on the test media. The plates were added aliquots of each bacterial inoculum, and the then incubated in the dark at 28 ± 2 ºC for 4 to 10 growth was monitored until the stationary phase by days depending on the activity tested. Two replicates measuring the optical density at 570 nm (OD570). were carried out for each sample. The cultures that showed growth, clearing zones, or color-diffusion 16S rDNA amplification and restriction analysis zones on respective specific media were considered as The isolates were subjected to 16S rDNA analysis positive cultures. Extracellular protease production to determine their phylogenetic affiliation. Genomic was determined by using 30% (v/v) skim-milk or 1% DNA from a broth culture of the strains was isolated (w/v) gelatin; a clear zone around the colony indicated using GenElute Bacterial Genomic DNA kit (Sigma). presence of proteolytic activity (PrA). The α-amylase The small-subunit rRNA gene was amplified from activity (AmA) was determined by using 1% (w/v) the extracted DNA using universal bacterial primers insoluble starch and detected as a clear zone around specific to 16S rRNA gene, primer 8F and primer the colony (Dhawale et al., 1982). Ribonuclease 1492R (Weisburg et al., 1991). The PCR reaction (RNAse) or desoxiribonuclease (DNAse) activities were mixture (20 μl) contained: 1 to 10 ng of template, detected in test agars containing 0.5% (w/v) RNA Prime Taq Premix 2x (GENET BIO) containing 1 U/10 or DNA, respectively. Growth with RNAse/DNAse μl DNA Polymerase, 20 mM Tris-HCl, 80 mM KCl, 4 production appeared surrounded by a clear halo as mM MgCl2, enzyme stabilizer, sample, loading dye, a result of the degradation of RNA/DNA to soluble pH 9.0, 0.5 mM of each dATP, dCTP, dGTP, dTTP, nucleotides, which were not precipitated by the and 400 nM of each primer. The reaction mixture added 1N hydrochloric acid. Lipolytic activity (LipA)

International Journal of Speleology, 42 (1), 65-76. Tampa, FL (USA) January 2013 68 Tomova et al. was tested by hydrolysis of 1% Tween 80 in a basal Magura Cave (Table 1). Fourteen bacterial strains agar medium amended with 0.01% CaCl2·2H2O and were recovered from the painted surfaces, 25 from was seen as either a visible precipitate of the calcium adjacent rock wall surfaces, 5 from bat guano (in chloride-lipid complex around a colony, or as a clearing sediment samples), and 2 from water samples. zone around a colony due to complete degradation of Strains No 1-25 were isolated in Faculty of Biology the salt complex (Smilbert, 1994). The strains were at Sofia University; other strains were isolated in the screened for cellulase activity in agar medium with 2% Institute of Microbiology-BAS and marked shortly (w/v) CMC (Carboxymethyl cellulose) as a substrate. as IM. After amplification and restriction of the 16S The plates were incubated and stained with Congo rRNA gene for each isolate their restriction profiles red dye and destained (Teather & Wood, 1982). The were compared and the strains were distributed into positive cellulase activity was shown as a presence of eighteen groups. Representatives of four phylogenetic yellow hallo against red background. Urease activity groups: Proteobacteria, Actinobacteria, Firmicutes, was tested on urea agar plates with 2% urea and phenol and Bacteroidetes were identified with a strong red and detected as a formation of a pink-colored Proteobacteria domination (Fig. 2). After sequencing clear zone around the colonies (MacFaddin, 2000). and phylogenetic analysis, a strong domination Phytase activity (PhyA) was determined after growth of Gram-negative heterotrophic bacteria was of the strains up to 10 days on agar plates containing established (81%), belonging to nine genera: Serratia, phosphate-depleted basal medium supplemented with Pseudomonas, Enterobacter, Sphingobacterium, 0.5% (w/v) sodium phytate (Sigma, USA) as a substrate Stenotrophomonas, Commamonas, Acinetobacter, following procedure described by Bae et al. (1999). Obesumbacterium, and Myroides, with predominance The β-glucosidase activity (β-gluA) was evaluated of Serratia and Pseudomonas strains. Gram-positive on a selective medium containing 0.4% arbutin isolates (19%) were representatives of three genera: (hydroquinone β-d-glucopyranoside) (Santa Cruz, Bacillus, Arthrobacter, and Micrococcus. Isolate IM13 USA); a positive result was indicated by the growth and was determined to share 94% sequence similarity of its dark brown halo around the colonies (Mendes Ferreira 16S rRNA gene to the nearest phylogenetic relative of et al., 2001). Polygalacturonase (PGAse) activity was the genus Myroides and may represent a novel species. detected by the degradation of polygalacturonic acid using Ruthenium red plate assay (Cotty et al., 1990). Growth patterns of bacterial isolates Xanthan lyase activity was detected by the growth of Growth of the bacterial isolates from Magura Cave the strains in liquid basal mineral medium containing was followed at temperatures of 4 oC, 10 oC, 20 oC, 28 o o 0.2% (w/v) xanthan as a sole carbon source. C, and 40 C, and values of OD570 in the beginning Extracellular hemolytic activity was tested by of stationary phase were compared. The growth of dropping of 0.1 ml cell suspensions of each bacterial Magura Cave isolates at different temperatures is culture (grown on NB for 20-h) into wells on blood agar shown in Table 1 and Fig. 3. The majority of isolates plates. After incubation for 48 h to 72 h at 28 ± 2 °C, (33 strains) were psychrotolerant determined by the zones of hemolysis of erythrocytes were measured. their ability to grow at both 4 oC (although too slow) and above 20 oC, highest growth rate observed at Screening for antimicrobial activity temperatures between 20 and 30 oC, and may have Bacterial isolates from Magura Cave were tested upper limits as high as 40 oC (Morita, 1975). Thirteen for their ability to inhibit the growth of four indicator strains appear to be mesophilic, for which the growth cultures: Bacillus subtilis ATCC 6633, Pseudomonas at 40 oC was from 2.5 to 9 times higher (depending on aeruginosa NBIMCC 1390 (National Bank of Industrial the strain) than the growth determined at 4 oC. Microorganisms and Cell Cultures, Bulgaria; http:// www.nbimcc.org/en/about.htm), the phytopathogenic Hydrolytic enzymes and hemolytic activity bacterial strain Xanthomonas oryzae, causing bacterial Heterotrophic bacteria isolated from Magura blight of rice, and the yeast Rhodotorula mucilaginosa Cave were tested for hydrolytic enzymes activity on 6526 (formerly Rhodotorula rubra). Cave bacterial isolates were grown on NB for 20 h (in exponential growth phase) before detection of an antimicrobial 8.7% activity by the agar well diffusion method. Indicator 6.5% cultures were spread on the agar media, followed by hole-punches (6-mm in diameter) that were filled with 0.1 ml cell suspension of each cave organism. After Proteobacteria - 63% overnight incubation at 28 ± 2 °C, clear zones (halos) 10.9% Bacteroidetes - 10.9% Actinobacteria - 10.9% were recorded as a zone diameter (in millimeters) Firmicutes - 6.5% around the wells minus 6-mm diameter of the well. Not determined - 8.7%

RESULTS 10.9%

Isolation and identification of culturable bacteria 63% from Magura Cave A total of 46 aerobic heterotrophic bacterial strains Fig. 2. Phyla distribution of heterotrophic bacteria isolated from the were isolated from the Gallery with the drawings in Gallery with the drawings in Magura Cave.

International Journal of Speleology, 42 (1), 65-76. Tampa, FL (USA) January 2013 Culturable aerobic heterotrophic bacteria from Magura Cave, Bulgaria 69 Table 1. Properties of heterotrophic bacteria recovered from the Gallery with the drawings in Magura Cave.

Growth at 4 oC Growth Nearest relative on the basis Isolate code* Gram staining Sample type o (OD570) at 40 C (OD570) of 16S rDNA sequences similarity Enterobacter sp. 2B1C B1 Gram negative Guano 0.078 0.037 (EU693561), 99% Pseudomonas fragi B2 Gram negative Guano 0.389 0.118 (AB685688), 99% B3 Pseudomonas f luorescens 15834 Gram negative Drawings 0.205 0.123 (B8, G7) (AB680973), 99% B4 Gram negative Drawings 0.127 0.309 ND Serratia sp. a101-87 B5 Gram negative Drawings 0.126 0.338 (HM468088), 100% B6 Enterobacter asburiae JCM6051 Gram negative Guano 0.088 0.327 (B11) (NR_024640), 99% Sphingobacterium sp. Ag8 B7 Gram negative Guano 0.190 0.085 (JN257086), 99% P. f luorescens 15834 B8 Gram negative Water 0.160 0.157 (AB680973), 99% Bacillus safensis KTT-25 B9 Gram positive Rock wall 0.087 0.029 (HQ696447), 98% Enterobacter asburiae JCM6051 B11 Gram negative Drawings 0.055 0.327 (NR_024640), 99% Sphingobacterium sp. Ag8 B13 Gram negative Drawings 0.197 0.089 (JN257086), 99% Serratia sp. L0305 B14 Gram negative Drawings 0.342 0.227 (JN169125), 99% Serratia sp. a101-87 B16 Gram negative Rock wall 0.056 0.013 (HM468088), 100% Micrococcus luteus CJ-G-TSA7 B17 Gram positive Drawings 0.363 0.170 (HM584259), 99% Serratia sp. a101-87 B18 Gram negative Water 0.072 0.435 (HM468088), 100% B19 Gram negative Drawings 0.029 0.078 ND Serratia sp. a101-87 B20 Gram negative Drawings 0.057 0.312 (HM468088), 100% M. luteus CUB12 CВ9 Gram positive Drawings 0.035 0.253 (JF802083), 99% Serratia sp. a101-87 CВ11 Gram negative Guano 0.077 0.322 (HM468088), 100% Bacillus f lexus SL21 CВ12 Gram positive Drawings 0.029 0.271 (JN645852), 99% CВ13 Gram negative Drawings 0.175 0.294 ND CВ15 Sphingobacterium sp. Ag8 Gram negative Drawings 0.029 0.014 (B7, B13, IM3) (JN257086), 99% Bacillus amyloliquefaciens ZP0308 CВ16 Gram positive Drawings 0.023 0.185 (JN021517), 99% Acinetobacter sp. CJ-S-PYD4 G5 Gram negative Rock wall 0.153 0.274 (HM584281), 100% P. f luorescens 15834 G7 Gram negative Rock wall 0.150 0.040 (AB680973), 99% IM1 Serratia sp. 136-2 Gram negative Rock wall 0.322 0 (IM2, IM4, IM16) (EU557341), 99% Serratia sp. 136-2 IM2 Gram negative Rock wall 0.222 0.174 (EU557341), 99% Sphingobacterium sp. Ag8 IM3 Gram negative Rock wall 0.261 0.189 (JN257086), 99% Serratia sp. 136-2 IM4 Gram negative Rock wall 0.514 0 (EU557341), 99% P. f luorescens LMG 14576 IM5 Gram negative Rock wall 0.347 0 (GU198121), 100% Arthrobacter sp. R4 IM6 Gram positive Rock wall 0.037 0 (FN392695), 99% Obesumbacterium proteus 511 IM7 Gram negative Rock wall 0.246 0.206 (FJ492810), 99% Serratia proteamaculans DSM4543 IM8 Gram negative Rock wall 0.403 0 (NR_025341), 99% IM9 Gram negative Rock wall 0.288 0 ND IM10 Arthrobacter sp. R-36193 Gram positive Rock wall 0.312 0 (IM11) (FR682676), 99% Arthrobacter sp. R-36193 IM11 Gram positive Rock wall 0.150 0.183 (FR682676), 99% IM12 P. f luorescens LMG 14576 Gram negative Rock wall 0.400 0.172 (IM5, IM21) (GU198121), 100% Myroides sp. IT-2012 IM13 Gram negative Rock wall 0.155 0.010 (HE663456), 94% IM14 Serratia sp. a101-87 Gram negative Rock wall 0.335 0 (B5, B16, B18, B20, CB11) (HM468088), 100% Pseudomonas putida IM15 Gram negative Rock wall 0.305 0 (AB681704), 99% Serratia sp. 136-2 IM16 Gram negative Rock wall 0.321 0.131 (EU557341), 99% IM17 Stenotrophomonas sp. DIC6JA Gram negative Rock wall 0.042 0 (IM19) (FJ668834), 99% Comamonas sp. BM-9_6 IM18 Gram negative Rock wall 0.244 0.025 (AY635871), 99% Stenotrophomonas sp. DIC6JA IM19 Gram negative Rock wall 0.053 0.023 (FJ668834), 99% Pseudomonas plecoglossicida IM20 Gram negative Rock wall 0.295 0.024 (AY972231), 100% P. fluorescens LMG 14576 IM21 Gram negative Rock wall 0.139 0 (GU198121), 100% ND, not determined. *, strains in the brackets were referred to the same restriction group and were non-sequenced.

International Journal of Speleology, 42 (1), 65-76. Tampa, FL (USA) January 2013 70 Tomova et al. the karstic Magura Cave are inhabited by diverse heterotrophic bacteria. By using NA, we isolated 46 culturable aerobic heterotrophic bacteria referred to four phyla. The relatively low number of isolated bacteria could be due to using nutrient-rich media, which may restrict the growth of bacteria which may be better adapted to the oligotrophic conditions of the cave environment. The observed domination of Proteobacteria is in a good agreement with previous studies in Kartchner Caverns, Arizona (Ikner et al., 2007), which also reported Proteobacteria as the most Fig. 3. Growth of bacterial isolates (IM1-21) from Magura Cave at abundant phylum and suggested that their presence different temperatures is a consequence of increased organic input by cave visitors. Considerable input of organic matter in agar medium using specific substrates. Isolates many caves supported the growth of different types of were screened for production of the main hydrolytic heterotrophic bacteria, led to changes in microclimate enzymes (proteases, lipases, amylases, nucleases) as and increased microbial dispersal and colonization well as for production of some enzymes with potential (Ikner et al., 2007; Bastian et al., 2009; Mulec et al., biotechnological application, such as phytase, xanthan 2012). Bat guano appears to be the major source of lyase, polygalacturonase, and β-glucosidase. We found organic matter in Magura Cave that could contribute that the most often observed hydrolytic enzymes were in increasing the abundance of Proteobacteria in proteases and xanthan lyases than the other enzymes the Gallery with the drawings. The predominance (Table 2). Up to 87% of the isolates showed proteolytic of Serratia and especially Pseudomonas isolates in activity, and skim milk appeared to be more favorable Magura Cave could be considered as an evidence of substrate than gelatin (68% and 51% of isolates, a high degree of contamination of surfaces, probably respectively). About 64% of isolates were able to grow due to insects and bats feces (Jurado et al., 2010), on xanthan mineral medium indicating presence of or could be the result of using rich medium for xanthan lyase activity; 40% of isolates showed lipase isolation. Comparing the bacterial diversity in caves and β-glycosidase activity, and 21% – phytase activity. from Australia, China, Spain and USA, Zhou et al. Polygalacturonic acid was hydrolyzed only by two (2007) have found that a general trend in caves is strains, IM14 and IM15. RNAse and amylase activity that Proteobacteria is the largest group, followed by were detected only for a strain CB16. No hydrolysis of Acidobacteria, Actinobacteria, and Firmicutes. The DNA and CMC was observed. bacteria isolated from several Siberian karstic cavities It was found that about 76% of the cave isolates have been identified as Pseudomonas, Arthrobacter, exhibited hemolytic activity after testing on blood Bacillus, and coryneform bacteria (Khizhnyak et agar plates (Table 3). Among them, 40% showed weak al., 2003). Chelius & Moore (2004) have cultured hemolytic zone up to 1.5 mm; 15% showed zone of Actinobacteria and Proteobacteria isolates from Wind 2-2.5 mm, and 21% of the strains – zone of 3-4 mm. Cave (USA), but have not recovered any Firmicutes. Bacterial communities in the cave Škocjanske Antimicrobial activity Jame (Slovenia) contained Enterobacteriaceae and It was established that about 50% of the isolates Vibrionaceae, low proportions of Gram-positive from Magura Cave exhibited inhibitory activity against bacteria, and practically no culturable Actinobacteria P. aeruginosa and the yeast strain R. mucilaginosa (Gerič et al., 2004). Contrary to our findings for (Table 3). A significant inhibition zone of 20-mm was Magura Cave, Actinobacteria are a dominant group observed for isolate B3 (Pseudomonas fluorescens) in heterotrophic bacterial communities in many against P. aeruginosa, and 16-mm against R. caves (Groth & Saiz-Jimenez, 1999). Most of the mucilaginosa. Isolates IM15 (Pseudomonas sp.), IM16 isolated Actinobacteria could be assigned to the (Serratia sp.), IM18 (Comamonas sp.), and IM20 genus Streptomyces in certain caves such as Grotta (Pseudomonas plecoglossicida) inhibited significantly dei Cervi (Italy), Altamira and Tito Bustillo (Spain), the growth of B. subtilis. About 15% of the strains and 19 karstic caves in Turkey (Groth et al., 1999, exhibited antimicrobial activity against X. oryzae; 2001; Yucel & Yamac, 2010). The low number of the highest inhibition was achieved by isolates B7 isolated Actinobacteria from Magura Cave could be (Sphingobacterium sp.), IM18 (Comamonas sp.), and due to the use of nutrient agar/broth, which is not IM20 (P. plecoglossicida) with a zone of inhibition of 10- routinely used for their isolation. Three of the isolated mm in diameter. Among isolates, IM16 (Serratia sp.), strains from Magura Cave (IM6, IM10, and IM11) were IM18 (Comamonas sp.), and IM20 (P. plecoglossicida) representatives of the genus Arthrobacter. One isolate, could be considered as the most active inhibiting the IM13, belonging to the genus Myroides is a candidate growth of all tested cultures. for a new species (accession number HE663456) suggesting that Magura Cave could be a source of DISCUSSION novel microorganisms. Some of the species belonging to the genera, This study demonstrates that surfaces of drawings identified in Magura Cave: Pseudomonas, Bacillus, and rock walls in the Gallery with the drawings in Sphingobacterium, Stenotrophomonas, Arthrobacter,

International Journal of Speleology, 42 (1), 65-76. Tampa, FL (USA) January 2013 Culturable aerobic heterotrophic bacteria from Magura Cave, Bulgaria 71 Table 2. Extracellular enzyme activities of heterotrophic bacteria isolated from the Gallery with the drawings in Magura Cave.

Isolate Strains referred to the nearest 16S PrA PrA LipA AmA RNAse DNAse CMCase Urease PGAse XanthA* PhytA β-gluA code sequence identity skim-milk gelatin Tween 80

B1 Enterobacter sp. 2B1C - - ++ ------++ - - B2 Pseudomonas fragi ++ ++ ------++ ++ - B3 Pseudomonas fluorescens 15834 +++ +++ + ------+ + ± B4 ND + ------+ - - B5 Serratia sp. + + ------B6 Enterobacter asburiae JCM6051 ± ------++ - - - - B7 Sphingobacterium sp. ± ± ------+ - - B8 Pseudomonas sp. ++ ------++ - ++ - - B9 Bacillus safensis KTT-25 +++ +++ ------+ - ± B11 Enterobacter sp. ± + ------+ - - B13 Sphingobacterium sp. ++ ± - - - - - + - + - - B14 Serratia sp. L0305 + +++ ++ - - - - ++ - ++ + + B16 Serratia sp. + ++ +++ - - - - +++ - - - - B17 Micrococcus luteus CJ-G-TSA7 + ------+++ - ++ - ++ B18 Serratia sp. - - ± ------++ - - B19 ND +++ + ± ------+ - - B20 Serratia sp. - ++ + ------+ + CB9 Micrococcus luteus CUB12 +++ +++ ++ ------+++ + - CB11 Serratia sp. - ++ ± ------+ - + CВ12 Bacillus flexus SL21 +++ ------++ - - CВ13 ND ++ - + ------++ - - CВ15 Sphingobacterium sp. Ag8 +++ +++ ++ ------+ +++ CВ16 Bacillus amyloliquefacien ZP0308 +++ +++ - ++ ++ ------G5 Acinetobacter sp. CJ-S-PYD4 - - ++ ------+ ± ± G7 Pseudomonas sp. ++ + - - - - - ++ - - + - IM1 Serratia sp. 136-2 +++ ± + ------+ IM2 Serratia sp. - ++ + ------+ - - IM3 Sphingobacterium sp. - +++ ------+ - - IM4 Serratia sp. +++ - + ------+ IM5 Pseudomonas fluorescens +++ + + ------+ - - IM6 Arthrobacter sp. R4 ++ ------++ - - IM7 Obesumbacterium proteus 511 - ± ------+ - + IM8 Serratia proteamaculans DSM4543 - - + ------+ - ± IM9 ND - +++ ------± - + IM10 Arthrobacter sp. R-36193 +++ + + ------++ IM11 Arthrobacter sp. + ± ------+ - - IM12 Pseudomonas fluorescens LMG +++ +++ ------± - ± 14576 IM13 Myroides sp. IT-2012 ------+ - - - -

IM14 Serratia sp. a101-87 +++ ++ ------++ - ± ++ IM15 Pseudomonas putida +++ ------+ - IM16 Serratia sp. +++ - + - - - - - ++ ± - ++ IM17 Stenotrophomonas sp. DIC6JA +++ + ------+ IM18 Comamonas sp. BM-9_6 - ++ ------IM19 Stenotrophomonas sp. +++ +++ ------+ IM20 Pseudomonas plecoglossicida - ++ ------± - - IM21 Pseudomonas sp. ------+ - -

Symbols: ±, zone < 1 mm; +, zone 1 to 3 mm; + +, zone 3 to 5 mm; + + +, zone > 5 mm. *Xanthan lyase activity was tested in liquid MSM with xanthan as a sole carbon source.

International Journal of Speleology, 42 (1), 65-76. Tampa, FL (USA) January 2013 72 Tomova et al.

Table 3. Antimicrobial and hemolytic activity of heterotrophic bacteria isolated from the Gallery with the drawings in Magura Cave

Inhibition zone (mm)

Strains referred to the nearest Isolate code Hemolytic activity (mm) 16S sequence identity B. subtilis X. oryzae P. aeruginosa R. mucilaginosa

B1 Enterobacter sp. 2B1C - - - - -

B2 Pseudomonas fragi - - 8 - W Pseudomonas f luorescens B3 2 20 16 W 15834 - B4 ND - - 8 - W B5 Serratia sp. - 4 - - 3.0 B6 Enterobacter asburiae JCM6051 - - - - - B7 Sphingobacterium sp. - 10 10 - W

B8 Pseudomonas sp. - - - 10 2.0 B9 Bacillus safensis KTT-25 - - - - - B11 Enterobacter sp. - - - - W B13 Sphingobacterium sp. - - 12 6 -

B14 Serratia sp. L0305 - - 10 6 W

B16 Serratia sp. - - 10 - 2.0 B17 Micrococcus luteus CJ-G-TSA7 - - - 2 W B18 Serratia sp. - - - - W B19 ND - - 8 - 2.0 B20 Serratia sp. - - - - W

CВ9 Micrococcus luteus CUB12 - - - - - CВ11 Serratia sp. - - - - W CВ12 Bacillus flexus SL21 - - - - W CВ13 ND - - 8 - - CВ15 Sphingobacterium sp. Ag8 - - 10 - W Bacillus amyloliquefaciens CВ16 10 3.0 ZP0308 - - - G5 Acinetobacter sp. CJ-S-PYD4 - - - 4 - G7 Pseudomonas sp. - - - 8 3.0 IM1 Serratia sp. 136-2 - - 10 2 - IM2 Serratia sp. - - - 2 W IM3 Sphingobacterium sp. - - - - 3.0 IM4 Serratia sp. - - 12 - W IM5 Pseudomonas fluorescens - - 12 16 3.0

IM6 Arthrobacter sp. R4 - - 4 6 3.0 IM7 Obesumbacterium proteus 511 - - 10 16 4.0 Serratia proteamaculans IM8 14 2 DSM4543 - - - IM9 ND - - 14 4 W IM10 Arthrobacter sp. R-36193 - - 6 4 - IM11 Arthrobacter sp. - - - - 2.0 Pseudomonas fluorescens LMG IM12 16 W 14576 - - - IM13 Myroides sp. IT-2012 - - - 8 W

IM14 Serratia sp. a101-87 - - - 8 3.0 IM15 Pseudomonas putida 16 6 - 8 W

IM16 Serratia sp. 16 2 12-14 10 2.0

IM17 Stenotrophomonas sp. DIC6JA - - 12-14 - 2.0

IM18 Comamonas sp. BM-9_6 20 10 12-14 8 3.5

IM19 Stenotrophomonas sp. - - 12-14 2 W

IM20 Pseudomonas plecoglossicida 20 10 4 10 3.5

IM21 Pseudomonas sp. - - - - 3.0

Symbols: -, no zone observed; clear inhibition zones minus 6 mm diameter of well are indicated. W, weak clear zone ≤ 1 mm

International Journal of Speleology, 42 (1), 65-76. Tampa, FL (USA) January 2013 Culturable aerobic heterotrophic bacteria from Magura Cave, Bulgaria 73 Acinetobacter, and Enterobacteriaceae family could valuable cold-tolerant enzymes with a potential for be potential pathogens, perhaps translocated into application in various industries - in food and dairy the cave by animals and human visitors (Northup & industry, textile industry, brewing and wine industry, Lavoie, 2001; Campbell et al., 2011). Bastian et al. laundry, etc. High activity of cold-active enzymes (2009) reported that the Lascaux Cave is a reservoir at low and moderate temperatures offers potential of potential pathogenic bacteria. Pathogenic bacteria economic benefits through substantial energy savings frequently isolated from caves are Escherichia coli, in large-scale processes (Russell, 1998; Gerday et al., Staphylococcus aureus, and species of Pseudomonas, 2000; Cavicchioli et al., 2011). Our study is a first Sphingomonas, and Alcaligenes (Ikner et al., 2007). investigation of production of RNAse, urease, phytase, Lavoie & Northup (2006) considered Bacillus spp., E. polygalacturonase, and xanthan lyase enzymes coli, and S. aureus as indicators of human impact in by psychrophilic/psychrotolerant microorganisms the caves. isolated from caves. As promising producers of cold- We found that most of the isolated heterotrophic tolerant enzymes could be accepted: B2 (Pseudomonas bacteria from Magura Cave (about 69%) are fragi) and CB9 (Micrococcus luteus) – for xanthan psychrotolerant (psychrotrophic) by the highest lyase and phytase; B16 (Serratia sp.) – for lipase and growth rate observed between 20 and 30 oC. Similarly, urease; B17 (Micrococcus luteus) – for urease; CB15 Khizhnyak at al. (2003) reported that the amount (Sphingobacterium sp. Ag8) – for protease, phytase of typically mesophilic strains in some Siberian and β-glucosidase; CB16 (B. amyloliquefaciens) – for cavities is many times lower than psychrophilic and protease, amylase and RNAse; Serratia strains IM14 psychrotolerant strains. According to Gounot (1994), and IM16 – for polygalacturonase and β-glucosidase. all autochthonous bacteria in caves situated in Nowadays, there is a need for new antimicrobial temperate regions are psychrotrophic, growing well at agents due to the increase in drug resistance in many 20 oC and above. common bacterial pathogens, and the emergence of Psychrophilic and psychrotrophic microorganisms new infections (Davis & Webb, 1998). Cold-adapted have developed a complex range of structural and microorganisms have been recognized as valuable functional adaptations to thrive successfully in low sources of novel bioproducts including antimicrobial temperature environments (Margesin & Miteva, 2011). metabolites (Sanchez et al., 2009). Information about Poor levels of nutrients in the caves promote the antimicrobial activity of bacteria isolated from karstic competition of microorganisms by production of high caves is rare, related mainly to Actinobacteria (Kim et amounts of exopolymeric substances, enzymes and al., 1998; Yucel & Yamac, 2010). Kim et al. (1998) antimicrobial metabolites that is one of the strategies have shown that up to 78% of Actinobacteria isolated for their survival (Smalås et al., 2000; Burgess et from cave soils in Korea have been antagonistic to al., 1999; Slattery et al., 2001). It is known that plant pathogenic fungi. About 62% of Actinobacteria heterotrophic bacteria and their enzymes play a key isolated from rock wall and speleothems surfaces, and role in the processes of recycling, decomposition and soil samples of 19 karstic caves in Turkey exhibited mineralization of a wide spectrum of organic polymers antimicrobial activity against a panel of four bacteria, in different environments (Munster & De Haan, 1998). two yeasts and four filamentous fungi (Yucel & Yamac, Patterns of enzyme activities can be used to define 2010). We selected several bacterial isolates from the composition of organic matter in the ecosystems Magura Cave as promising producers of antimicrobial (Boschker & Cappenberg, 1998). Heterotrophic compounds such as B3 (P. fluorescens), B7, B13 bacteria isolated from Magura Cave showed varied (Sphingobacterium sp.), IM15, IM18 (Comamonas enzymatic patterns, which expressed different degrees sp.), IM19 (Stenotrophomonas sp.), and IM20 (P. of nutritional and ecological versatility. plecoglossicida). Isolates B5, B7, IM15, IM18, and Only few reports have been published on hydrolytic IM20 exhibiting antibiotic activity against X. oryzae enzymes production by culturable bacteria isolated are prospective strains for future application in the from subterranean environments including caves. agriculture for plant protection. Martinez et al. (2011) considered that measuring Most of heterotrophic bacteria isolated from Magura extracellular enzyme activity is one way to measure Cave exhibited hemolytic activity on blood agar microbial responses to nutrient availability. The plates. Surface activity is often accomplished by authors have assayed alanine aminopeptidase, hemolysis and sometimes by antimicrobial activity, phosphatase, and β-glucosidase activities in samples and both activity assays have been used to search from three limestone caves in New Mexico and have for surfactant-producing bacteria. Due to their determined whether microorganisms are nitrogen, amphiphilic character, surfactants often induce carbon or phosphate limited. Similar to our results, hemolysis at a given concentration (Vinardell & Infante, Khizhnyak et al. (2003) reported that cultures from 1999). Hemolysis, observed on blood agar plates, has several Siberian caves synthesized more exogenous been widely used to screen surfactant-producing proteases compared to amylolytic enzymes because microorganisms (Moran et al., 2002). Production of there were no natural sources of starch there. Rashid biosurfactants is of biotechnological importance due et al. (2001) reported for a low-temperature lipase to their potential application in different industries produced by psychrotrophic Pseudomonas sp. isolated - in food, cosmetics, pharmaceutical, agricultural, from subterranean environments. Psychrotolerant petrochemical industries, etc. (Banat et al., 2010). heterotrophic bacteria isolated from Magura Cave The search for different types of biosurfactants at given conditions could be attractive producers of is important since their use in industry requires

International Journal of Speleology, 42 (1), 65-76. Tampa, FL (USA) January 2013 74 Tomova et al. different physicochemical properties. Bacillus, Boschker H.T.S. & Cappenberg T.E., 1998 - Pseudomonas, and Serratia strains are well-known Patterns of extracellular enzyme activities in littoral producers of lipopeptide biosurfactants having sediments of Lake Gooimeer, The Netherlands. important biotechnological applications, whereas FEMS Microbiology and Ecology, 25: 79-86. http://dx.doi.org/10.1111/j.1574-6941.1998.tb00461.x knowledge about Comamonas, Sphingobacterium Bottos E., 2004 - Screening for Biosurfactant Production or Stenotrophomonas as producers of surface active in Actinomycetes Isolated from Cave and Forest Soils. substances is rare (Banat et al., 2010; Burgos- (Student Poster Presentation). Diaz et al., 2011). Among microorganisms isolated Burgess J.G., Jordan E.M., Bregu M., Mearns- from caves, only Actinobacteria have been screened Spragg A. & Boyd K.G., 1999 - Microbial for biosurfactant production (Bottos, 2004). Our antagonism: A neglected avenue of natural products study is a first report about the hemolytic activity research. Journal of Biotechnology, 70: 27-32. of heterotrophic cave bacteria. Eight of bacterial http://dx.doi.org/10.1016/S0168-1656(99)00054-1 strains isolated from Magura Cave (B5, CB16, IM5, Burgos-Diaz C., Pons R., Espuny M.J., Aranda F.J., Tereul J.A., Manresa A., Ortiz A. & Marqués A.M., IM6, IM7, IM16, IM18, and IM20) expressed high 2011 - Isolation and partial characterization of a hemolytic activity together with antimicrobial activity biosurfactant mixture produced by Sphingobacterium and could be suggested as prospective producers of sp. isolated from soil. Journal of Colloid and Interface biosurfactants. Science, 361: 195-204. 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International Journal of Speleology, 42 (1), 65-76. Tampa, FL (USA) January 2013 International Journal of Speleology 42 (1) 77-96 Tampa, FL (USA) January 2013

Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Official Journal of Union Internationale de Spéléologie

A world review of fungi, yeasts, and slime molds in caves Karen J. Vanderwolf1*, David Malloch2, Donald F. McAlpine2, and Graham J. Forbes3 1 New Brunswick Museum, 277 Douglas Avenue, Saint John, New Brunswick E2K 1E7 and Canadian Wildlife Federation, 350 Promenade Michael Cowpland Drive, Kanata, Ontario K2M 2W1 2 New Brunswick Museum, 277 Douglas Avenue, Saint John, New Brunswick E2K 1E7 3 University of New Brunswick, Fredericton, New Brunswick E3B 6E1

Abstract: We provide a review of fungi, yeasts, and slime molds that have been found in natural solution caves and mines worldwide. Such habitats provide frequent roost sites for bats, and in eastern North America the environmental conditions that support white-nose syndrome, a lethal fungal disease currently devastating bat populations. A list of 1029 species of fungi, slime moulds, and yeasts in 518 genera have been documented from caves and mines worldwide in 225 articles. Ascomycota dominate the cave environment. Most research has been conducted in temperate climates, especially in Europe. A mean of 17.9±24.4SD fungal species are reported per study. Questions remain about the origin and ecological roles of fungi in caves, and which, if any, are cave-specialists. In the northern hemisphere, caves are generally characterized by relatively stable, low temperatures and a lack of organic substrates. This environment favors communities of oligotrophic, psychrotolerant fungi. Data that may help explain how cave environmental features and faunas influence the introduction and transmission of cave fungi remains scant.

Keywords: cave; fungi; yeast; mine; Geomyces destructans; bats Received 11 August 2012; Revised 20 December 2012; Accepted 1 January 2013

Citation: Vanderwolf K., Malloch D., McAlpine D.F. and Forbes G.J., 2013. A world review on fungi, yeasts, and slime molds in caves. International Journal of Speleology, 42 (1), 77-96. Tampa, FL (USA) ISSN 0392-6672 http://dx.doi.org/10.5038/1827-806X.42.1.9

INTRODUCTION literature on cave fungi (127 species in 59 genera) but did not include all relevant papers available at that The sudden and catastrophic appearance of a lethal time, and many articles have since been published. fungal disease, White Nose Syndrome (WNS), in cave- Landolt et al. (1992) summarized the few records of dwelling North American bats in 2006 has stimulated slime molds found in caves, but otherwise there has increased interest in the fungi that occur in caves been no comprehensive review of the microflora in (Wibbelt et al., 2010; Lindner et al., 2011). WNS is cave habitats. Here we organize data from 225 studies caused by the recently described fungus Geomyces dealing with fungi, yeasts, and slime molds occurring destructans Blehert & Gargas 2009 (Lorch et al., in caves, with 130 of these allowing some exploration 2011), but it is still unclear if G. destructans is native of broader patterns of occurrence in relation to to North America or a newly arrived exotic (Gargas the ecology of the cave habitat. Although studies et al., 2009; Puechmaille et al., 2011). The current published before 1965 do provide useful records of limited information suggests the latter and points to occurrence, unfortunately many do not report their Europe as the origin for this infection (Puechmaille et methods in sufficient detail to allow comparisons with al., 2011); an infection now estimated to have killed later work. We also identify gaps in information, but 5.7-6.7 million North American bats and one predicted most importantly, we note the need for adequately to result in North American agricultural losses of $3.7 applied and consistently reported methodologies. Our billion/year (Boyles et al., 2011; U.S. Fish and Wildlife hope is that the review of cave fungi presented here Service news release, January 17, 2012) will prove timely and useful to both speleologists and This paper reviews information on fungi, yeasts, WNS researchers. and slime molds that have been reported from caves and cave-like habitats (i.e. mines) worldwide. Much METHODS of the published literature dealing with fungi, yeasts, and slime molds in caves is found in sources that are This review summarizes data on the fungi, yeasts, scattered, obscure, or not readily available. Rutherford and slime molds of caves from 225 papers published and Huang (1994) presented a short review of the in the peer-reviewed literature of 149 journals and

*[email protected] 78 Vanderwolf et al. 10 books issued in 14 languages (English, French, caution; apparent geographic differences may be a German, Dutch, Russian, Spanish, Italian, Czech, reflection of different methodologies, variations in the Croatian, Romanian, Slovene, Polish, Japanese, and focus of the studies, or the amount of effort expended, Greek). Although we have included much non-English rather than demonstrating underlying biological scientific literature, additional literature likely exists. patterns. Some studies consist of one visit while other We have restricted our review to solution caves (caves studies span several years. Of the 225 papers, 45 formed naturally in limestone and gypsum bedrock) were conducted in North America (including Mexico), and mines, sites most frequently used as hibernacula 18 in Central and South America, 132 in Europe, 25 by bats. Thus, we do not cover fungi, yeasts, or in Asia, 10 in Africa, and 6 in Oceania (Fig. 1). The slime molds reported from sea caves, ice caves, countries with the greatest number of cave mycological lava tubes, tectonic rift caves, or other natural or papers are: United States (37, especially the northeast human-made subterranean habitats (e.g. tombs and with 25), France (25), Italy (23), Spain (19), and Great temples, Agrawal et al., 1988; Saarela et al., 2004; Britain (17). Simonovicova et al., 2004; Kiyuna et al., 2008), some Of the fungal taxa reported (species and genera of which may infrequently support roosting bats. combined) from caves and mines, 69.1% are All mine types except open-pit were included. Mine Ascomycota, 20% Basidiomycota, 6.6% Zygomycota, types studied in the relevant literature include gold, 2.6% Mycetozoa, 1% Oomycota, and 0.8% other asbestos, coal, iron, clay, copper, nickel, phosphate, (Amoebozoa, Chytridiomycota, Microsporidiomycota, aluminum, uranium, and manganese. Histoplasma and Percolozoa). Only to a very limited degree do capsulatum, one of the best studied fungal species these percentages reflect the number of species associated with caves, has often been targeted described for each phylum. Kirk et al. (2008) report exclusively by researchers, and of late, Geomyces 64,163 described species of Ascomycota, 31,515 destructans has become a fungal species of great Basidiomycota, 1,065 Zygomycota, Mycetozoa (there interest. The numerous reports of these species are are 1019 spp. in kingdom Amoebozoa, to which this examined separately. We located papers using the phylum belongs), and 956 Oomycota. Basidiomycota internet search engines Thomson Reuters’ ISI Web of are often associated with nutrient rich substrates such Science and Google Scholar, as well as by scanning as wood and dung in caves (Table 1 available online bibliographies, reading books on cave science, and at http://dx.doi.org/10.5038/1827-806X.42.1.9). drawing on two previous compilations (Rutherford & Wood and dung are generally not significant elements Huang, 1994; Landolt et al., 1992). A fungal species of cave environments, although wooden support list was extracted from each paper and where reported, timbers may be prevalent in old mines and some we recorded details of the methodologies followed in caves contain massive guano piles. Nonetheless, the each study, including location, the number of samples general lack of large, nutrient rich substrates in caves collected, the number and type of caves or mines may explain the comparative rarity of Basidiomycota sampled, whether bats or other fauna were present, versus Ascomycota. However, Basidiomycota are the presence of standing or running water, the length difficult to culture, and particularly to identify in of the cave or mine, cave temperature and humidity, culture, so the methods used by the majority of the substrates sampled, whether culture or genetic cave fungal studies (Table 2) are biased to detecting methods were used, culture media used, incubation Ascomycota. Zygomycota are comparatively easy to temperature and length of incubation period. As used detect due to abundant spore production and fast here, a ‘location’ is a country with two exceptions: growth, so the relative abundance of such taxa in Canada has been divided into eastern and western the cave environment may be overestimated. The halves, and the United States has been divided into disproportionate abundance of Mycetozoa is largely 4 quadrants, although no studies could be found for a reflection of the studies of Landolt et al. (1992), the northwest quadrant. Spearman rank correlation Landolt & Stihler (1998), and Landolt et al. (2006). analysis (rs) was used to explore the relationship of The genera most frequently reported from studies on cave temperature, incubation temperature, and cave mycology, aside from Histoplasma and Geomyces, length of incubation with the number of fungal taxa are: Aspergillus (38 out of 60 locations), Penicillium reported. Other variables were not analyzed due to (36), Mucor (29), Fusarium (27), Trichoderma (25), lack of data. Analysis was conducted on a subset of Cladosporium (23), Alternaria (21), Paecilomyces (21), 130 out of a total 225 papers. Excluded papers lacked Acremonium (19), Chrysosporium (19), Laboulbenia detail on methodology, and generally were published (18), Rhizopus (18), Mortierella (17), Chaetomium (16), before 1965. The earliest published study of fungi in Rhachomyces (16), Trichophyton (15), Humicola (14), caves was by Humboldt (1794) as described in Dobat Isaria (14), Absidia (13), Beauveria (13), Phoma (13), (1967); unfortunately the data could not be used due Verticillium (13), Aureobasidium (12), Gliocladium to outdated and untraceable nomenclature. (12), Coprinus (11), Cunninghamella (11), Epicoccum (11), Geotrichum (11), Microsporum (11), Botrytis (10), Taxa Documented from Caves Candida (10), Mycena (10), Scopulariopsis (10), and A list of 1029 species in 518 genera of fungi, slime Stachybotrys (10) (Table 1). moulds, and yeasts documented from caves is given The species most frequently reported from studies in Table 1, with an average of 17.9±24.4SD species on cave mycology, aside from Histoplasma spp. and reported per study (n=130, range 1-195, mode 1, Geomyces destructans, are: Aspergillus versicolor median 10). Table 1 should be interpreted with (23), Aspergillus niger (20), Penicillium chrysogenum

International Journal of Speleology, 42 (1), 77-96. Tampa, FL (USA) January 2013 A world review of fungi, yeasts, and slime molds in caves 79

Fig. 1. The distribution of 225 studies on fungi, yeasts, and slime molds found in caves or mines. Each location was categorized into either: no studies, 1 study, 2-3 studies, 4-8 studies, or ≥ 9 studies. A study can represent the documentation of one species or many.

(19), Cladosporium cladosporioides (18), Aspergillus (2004) found a core population of 9 fungal species fumigatus (16), Aspergillus ustus (13), Aspergillus present during all three sampling periods. This flavus (12), Fusarium solani (12), Geomyces pannorum pattern is frequent in fungal studies of both cave and (12), Trichoderma viride (12), Beauveria bassiana non-cave environments (Marletto, 1966; Christensen, (11), Cephalotrichum stemonitis (11), Cladosporium 1989; Kuzmina et al., 2012; Vanderwolf et al., 2013). herbarum (11), Alternaria alternata (10), Aureobasidium pullulans (10), Paecilomyces lilacinus (10), Penicillium Geomyces destructans brevicompactum (10), Penicillium simplicissimum (10), Geomyces destructans, only recently described and Rhizopus stolonifer var. stolonifer (10) (Table 1). (Gargas et al., 2009), has been reported from caves in These most commonly reported taxa likely reflect the northeastern United States, eastern Canada, and study methodologies rather than biological patterns multiple European countries (Martinkova et al., 2010; in cave mycology. The use of high-nutrient media and Puechmaille et al., 2010; Wibbelt et al., 2010; Kubatova incubation temperatures over 20°C favor the isolation of et al., 2011; Turner et al., 2011). Knowledge of the many of the most commonly reported fungi. Also, some distribution of this pathogenic fungus is expanding species, such as dermatophytes (Arthrodermataceae; rapidly and has generated increased interest in Table 3), are targeted by investigators. Consequently, cave mycology generally. Attempts to document the other non-target fungal species are unreported or presence of G. destructans in caves have resulted unidentified. Most of the fungal taxa commonly in the inadvertent isolation of other fungi. While reported from caves are widespread and cosmopolitan searching for G. destructans in cave sediments in the opportunistic saprotrophs associated with soils, plant material, or insects (Table 3). Table 2: The percentage of studies reporting selected environmental The isolation of some fungal species from multiple features of cave study sites and the methodologies employed. A study was considered to use genetic methods if genetics were employed for sampling substrates in multiple caves suggests a initial environmental sampling or for identification of fungal cultures. distribution that is ubiquitous both within and among % of studies (n) caves. However, Table 1 shows that 59.1% of the taxa # of samples taken 34.6 (130) documented from caves have only been reported # of caves sampled 93.1 (130) from 1 location. Although to some degree this may Type of cave/mine 56.2 (130) Presence/absence of bats 29.2 (130) reflect varying methodology, it does appear that a core Presence of other fauna 23.1 (130) group of fungi are widespread in cave environments, Presence of standing or running water 16.2 (130) accompanied by many species that are rarely isolated. Size of cave 29.2 (130) In a study of fungi in Israeli caves, Grishkan et al. Cave temperature 41.5 (130) (2004) found that most species were isolated with very Cave humidity 21.5 (130) Substrate sampled 96.9 (130) low abundance and had low frequency of occurrence. Using genetics 30.8 (130) Nearly half the species were represented by one Using culture methods 91.5 (130) isolate (31 out of 68 species) and more than half were Incubation temperature(s) of samples 67.2 (119) recovered from only one sampling point (37 species) Length of incubation 48.7 (119) (Grishkan et al., 2004). However, Grishkan et al. Agar type(s) used 84.9 (119)

International Journal of Speleology, 42 (1), 77-96. Tampa, FL (USA) January 2013 80 Vanderwolf et al. Table 3: The 20 families of mycota most frequently reported from had different susceptibilities to the fungal pathogen caves. Abundance is the number of times genera in each family Coccidioides immitis. They attributed the resistance of were reported from among 225 cave mycological studies. Ecological information taken from Kirk et al. (2008) and Domsch et al. (1980). A. pallidus to the presence of the bacterium Serratia marcescens in the tissues of the bat host. Phylum Family Abundance Ecology While G. destructans causes mass mortality of

Saprotrophic, rarely on living bats in North America, it has not been documented Ascomycota Trichocomaceae 620 plants or animals to cause significant mortality or morbidity among Saprotrophic in soil and European bats (Puechmaille et al., 2010; Puechmaille Zygomycota Mucoraceae 148 organic materials, many species proteolytic et al., 2011). The lack of European mortality may Saprotrophic, mycoparasitic Ascomycota Nectriaceae 106 be due to differences in host immune response or and phytoparasitic other aspects of physiology or behaviour, differences Ascomycota Laboulbeniaceae 105 Entomoparasitic in the cave environment, or variation in associated Phagotrophic on organic microflora or fauna (Cryan et al., 2010; Wibbelt et al., Mycetozoa Dictyosteliaceae 87 debris and other micro- organisms 2010). Unfortunately, not enough is currently known Saprotrophic or necrotrophic about caves or bats in relation to G. destructans such Ascomycota Pleosporaceae 86 on living and dead plants that intercontinental comparisons can be made that Saprotrophic on organic Ascomycota Microascaceae 77 materials might explain the dramatic difference in bat mortality Saprotrophic on plant between North America and Europe. Ascomycota Hypocreaceae 77 materials or mycoparasitic It is interesting to note that although 132 of the 225

Ascomycota Cordycipitaceae 76 Entomoparasitic papers on cave mycology (58.7%) were conducted in Europe, G. destructans was not documented until it Saprotrophic, most species Ascomycota Chaetomiaceae 72 strongly cellulolytic was targeted by researchers after WNS appeared in Saprotrophic, mycoparasitic, North America. It is now believed that G. destructans Ascomycota Bionectriaceae 70 or phytoparasitic is widespread and native in Europe (Puechmaille et Saprotrophic and Ascomycota Davidiellaceae 64 phytoparasitic al., 2010; Wibbelt et al., 2010). This event illustrates the importance of methodology and the inherent Basidiomycota Polyporaceae 50 Saprotrophic on wood difficulties of studying cave fungi.

Ascomycota Myxotrichaceae 50 Saprotrophic or mycorrhizal Histoplasma capsulatum Saprotrophic, often Ascomycota Arthrodermataceae 48 keratinolytic Recent molecular data suggests that Histoplasma Saprotrophic, often Zygomycota Mortierellaceae 44 capsulatum consists of at least eight clades and chitinolytic multiple geographically isolated species (Kasuga et Entomoparasitic, Ascomycota Clavicipitaceae 33 phytoparasitic or al., 2003). However, much of the literature does not endomutualistic on plants reflect this recent finding and we therefore report on Saprotrophic, causing brown Basidiomycota Fomitopsidaceae 30 rot of wood H. capsulatum sensu lato. As the etiological agent of Saprotrophic in soil, dung, histoplasmosis, a potentially fatal mammalian disease Basidiomycota Psathyrellaceae 29 wood acquired by the inhalation of spores, H. capsulatum Saprotrophic in wood and Basidiomycota Mycenaceae 28 leaf litter is certainly the most widely studied fungus found in caves. The fungus is most commonly found in soil enriched with bird or bat guano, both inside and United States, Lindner et al. (2011) documented many outside of caves. Histoplasmosis occurs worldwide Geomyces spp. distinct from G. destructans. Given but is considered endemic to Southeast Asia, India, Geomyces destructans temperature requirements for Australia, Africa, and parts of South America and optimal growth and its known distribution (Blehert North America, particularly the Mississippi-Ohio River et al., 2009; Gargas et al., 2009), it appears to be Valley in the United States. Histoplasma capsulatum adapted to the cave environment. Multiple fungal has been isolated inside caves from guano, sediment, species can grow on dead bats in caves (Voyron et air, water, and bats from the following locations: al., 2011), but G. destructans has the unique ability North America: Alabama (Tesh & Schneidau, 1967), to utilize live, hibernating bats as a nutrient source. Arizona (Disalvo et al., 1969), Florida (Tesh & Schneidau, Although fungi in addition to G. destructans have been 1967; Disalvo et al., 1970; Johnson et al., 1970; cultured from live bats in caves (Larcher et al., 2003; Lottenberg et al., 1979), Indiana (Tesh & Schneidau, Voyron et al., 2011; Vanderwolf et al., 2013), it is not 1967), Maryland (Shacklette & Hasenclever, 1968), known if these fungi are able to utilize live bats as a Mexico (Aguirre Pequeno, 1959; Al-Doory & Rhoades, substrate for growth. Geomyces destructans has the 1968; Galindo Rodriguez & Mendival Salgado, 1984; ability to grow on other cave substrates (e.g. raccoon Taylor et al., 1994, 1999; Ulloa et al., 2006; Gonzalez- dung, see McAlpine et al., 2011) but faster growing Gonzalez et al., 2012), New Mexico (Kajihiro, 1965; fungi seem to outcompete G. destructans in these Lewis, 1974), Oklahoma (Tesh & Schneidau, 1967; microhabitats. Bats are not known to have any innate Bryles et al., 1969), Tennessee (Klite, 1965), Texas defenses against this fungal infection during the (Emmons et al., 1966; Tesh & Schneidau, 1967; Al- winter, although the impact of G. destructans varies Doory & Rhoades, 1968; Disalvo et al., 1970; McMurray among bat species (Turner et al., 2011). Krutzsch & & Russell, 1982), and Virginia (Klite, 1965). Watson (1978) found that in the laboratory two bat South/Central America: Argentina (Gonzalez- species (Antrozous pallidus and Macrotus californicus) Gonzalez et al., 2012), Belize (Quinones et al., 1978),

International Journal of Speleology, 42 (1), 77-96. Tampa, FL (USA) January 2013 A world review of fungi, yeasts, and slime molds in caves 81 Columbia (Lewis, 1974; Castaneda et al., 1981), Costa been documented, though not identified, from plant Rica (Lyon et al., 2004; Neuhauser et al., 2008), Cuba roots that penetrate into shallow caves (Lamont & (Nocedo Pous et al., 1965; Font D’Escoubet & Macola Lange, 1976; Jasinska et al., 1996). Many fungi that Olano, 1976; Fernandez, 1988), El Salvador (Klite, can be cultured from caves may not grow in the cave 1965), Guatemala (Dreux, 1974), Jamaica (Finchman, environment, but are present regularly or rarely as 1978), Panama (Taylor et al., 1962; Klite & Diercks, spores, carried in by water, air currents, or animals. 1965; Klite & Young, 1965; Hasenclever et al., 1967; In many caves, fungi and bacteria probably constitute Shacklette et al., 1967), Peru (Lazarus & Ajello, 1955), the major food sources for nonpredacious troglobitic Puerto Rico (Carvajal-Zamora, 1977a, b, c; Nieves- invertebrates, such as isopods and collembolans, Rivera, 2003), Trinidad (Ajello et al., 1962), Venezuela as well as protozoa (Dickson & Kirk, 1976; Sustr et (Campins et al., 1956; Montemayor et al., 1958; Ajello et al., 2005; Walochnik & Mulec, 2009; Bastian et al., al., 1960a), and the West Indies (Magnaval et al., 1984). 2010). Several fungal species in caves are known to Other: Australia (Hunt et al., 1984), Israel (Ajello parasitize cave insects (Benoit et al., 2004; Santamaria et al., 1977), Malaysia (Ponnampalam, 1963), New & Faille, 2007; Yoder et al., 2009), and cave mycota Guinea (Quilici et al., 1984), Nigeria (Gugnani et al., can contribute to the inorganic nutrient pool by 1994; Muotoe-Okafor & Gugnani, 1997), Tanzania solubilizing bedrock (Cubbon, 1976). Fungi may also (Manson-Bahr, 1958; Ajello et al., 1960b), and contribute to the formation of speleothems, such as Romania (Alteras, 1966). secondary calcium carbonate deposits (needle fiber Due to its detrimental affect on humans, some calcite) (Bindschedler et al., 2012). effort has been made to control H. capsulatum in the Cave microbiota are sensitive to changes in organic environment. Tosh et al. (1966) found that solutions of inputs from external sources (Chelius et al., 2009); formaldehyde and cresol were effective decontaminating several fungal species found in caves will proliferate agents against H. capsulatum in soil, both in the field opportunistically when suddenly presented with and laboratory, although the fungus was again detected a food source (e.g. vegetation inputs, carcasses; after several weeks in field experiments. Increasing Cubbon, 1976). Min (1988) noted that organic debris the quantity of solution used in the field resulted in in caves is often rapidly covered with conidia of more permanent decontamination (Tosh et al., 1967; Aspergillus sp., Penicillium sp., and Mucor sp. Due Bartlett et al., 1982). Morehart and Larsh (1967) to the scarcity of organic matter in caves, fungi are found that H. capulatum was vulnerable to multiple generally nutrient limited and dependent on inputs organic fungicides in the laboratory and Emmons of organic matter from the outside environment and Piggott (1963) found that covering contaminated (Feldhake, 1986; Jurado et al., 2010). However, in soil with 15-20cm of fresh soil suppressed the spread the Lechuguilla Caves, New Mexico, interdependency and growth of the pathogen. However, none of these was observed between bacteria and fungi, with experiments involved caves, nor would these methods chemolithoautotrophic bacteria supporting pop- likely be practical or effective in caves. To that end, ulations of chemoheterotrophic bacteria and Muotoe-Okafor and Gugnani (1997) investigated many varieties of fungi (Cunningham et al., 1995). antibiosis between H. capsulatum var. duboisii and Gherman et al. (2007) documented a similar situation other fungi found in cave environments. Most of the in a Romanian mine. In a Mexican cave, abundant fungi tested were neither antagonistic nor parasitic, chemoautotrophic microbes appear to act as primary and although there was overgrowth and intermingling producers in the cave ecosystem which includes growth by some species, this did not affect the viability fungi, multiple insect species, and cave fish (Hose & of Histoplasma strains (Muotoe-Okafor & Gugnani, Pisarowicz, 1999). 1997). Only Chrysosporium indicum inhibited growth Fauna in caves can represent a considerable, though of Histoplasma strains and Muotoe-Okafor and often inaccessible, nutrient source for fungi. There are Gugnani (1997) mention its potential as a biocontrol. no published studies on the fungi found on fish, reptiles, Flooding may provide a natural control. Shacklette and birds, invertebrates other than insects, or mammals Hasenclever (1968) found that H. capsulatum could other than bats, occurring in caves. Only a single study not be isolated from cave soil for 2-3 months following of the fungi present on amphibians in caves has been a flooding event, although previously it had been published. Rimer & Briggler (2010) tested amphibians easily obtainable. Whether any of these observations in a Missouri cave for the presence of Batrachochytrium have applicability for the control of G. destructans dendrobatidis, the cause of chytridiomycosis; species of has yet to be examined. Taylor et al. (1999) suggested Eurycea and Lithobates tested positive while Plethodon that caves with small ceiling to floor distances and spp. were negative. There are multiple fungal species large guano deposits favor the infection of bats with that can utilize insects in caves as nutrient sources H. capsulatum. Since infection with H. capsulatum (e.g. species of Isaria, Beauveria, Rhachomyces) and occurs through the inhalation of spores, this pattern insects are the best studied host-taxon in caves is likely not applicable to G. destructans, which has a mycologically (Table 1). These infectious fungal spores very different etiology. can be transmitted among and between insect species, or be acquired from the cave environment (Boyer- Mycological cave ecology Lefevre, 1966). However, unlike Geomyces destructans, Fungi in the cave environment generally function as which is usually restricted to bats in caves, active parasites or decomposers, although these roles may insect fungal infections are well known both inside and not be mutually exclusive. Mycorrhizal fungi have outside cave environments.

International Journal of Speleology, 42 (1), 77-96. Tampa, FL (USA) January 2013 82 Vanderwolf et al. Bats can affect the diversity of fungi found in caves highest number of heterotrophic bacteria and fungi in by transporting spores and through the deposition of areas of the study cave with the most human traffic. guano and carcasses. Bats may be vectors for fungal Conversely, Shapiro & Pringle (2010) reported that spores in and out of the cave environment (Vanderwolf caves with high human visitation had lower fungal et al., 2013). Novakova (2009a) found that the greatest diversity compared to caves with low visitation. This diversity of fungal species in a Slovakian cave occurred pattern was also found in Austrailan caves when on bat guano as compared to other cave substrates. samples were cultured, but genetic methods showed Min (1988) reported that guano can be a dominant the reverse (Adetutu et al., 2011). Most of these factor in determining the mycota of caves. Sustr et studies compared high-use show caves to caves al. (2005) found that fungi on bat guano had a higher closed to the public. When caves with a few hundred biomass compared to the rest of the cave. Several human visitors per year were compared to caves papers have assumed that bat guano fuels cave food with <100 visits, no effect was detected (Vanderwolf webs (Beck et al., 1976) but the available evidence et al., 2013). Clearly, human visitors can influence is contradictory (Horst, 1972; Poulson, 1972). In the quantity and diversity of microbiota in caves, tropical caves, especially dry ones, bat guano is the but the threshold(s) at which these influences occur most common source of organic matter and may form is unknown. Changes in the quantity and diversity the trophic base for many invertebrate communities of cave mycota are likely due to the organic inputs (Decu, 1986; Ferreira & Martins, 1999; Fenolio et al., accompanying human visitation (lint, hair, dander), 2006). Invertebrates may eat the guano directly or the as well as the introduction of new spores (Chelius et fungi that grow on the guano (Moulds, 2006; Ribeiro al., 2009). The introduction of organic matter often et al., 2012). Other types of dung, such as that from leads to a proliferation of fast-growing species that birds (e.g. Steatornis caripensis in Venezuela) and can mask slow-growing oligotrophic species. Northup rodents (e.g. Erethizon dorsatum in eastern North et al. (2000) found that organic inputs from human America), can also influence the fauna (Calder & visitation supported the growth of bacteria and fungi Bleakney, 1965; Herrera, 1995; Moseley, 2007) and alien to the oligotrophic cave environment; this mycoflora of caves (Vanderwolf et al., 2013). eliminated native species and reduced biodiversity. Many different types of insects have been associated It is also possible that microclimate changes due to with dung in caves (Calder & Bleakney, 1965; human visitation and lighting systems (Pulido-Bosch Estrada-Barcenas et al., 2010), and this can affect the et al., 1997) could impact fungi in caves. mycoflora. Estrada-Barcenas et al. (2010) found that Fungi are not distributed evenly throughout caves. mites were the most diverse invertebrate taxa in bat The distribution of cave microbiota colonies is guano, with Sancassania sp. representing the most influenced by susceptibility of the host material to be abundant microarthropod. These mites eat microfungi, colonized, by microenvironmental conditions (water including Histoplasma capsulatum, and may provide availability, temperature, pH, and nutrient sources), a natural biological control for this pathogen, as well and by parameters such as mineral composition, as regulating numbers of cave microfungi in general porosity and rock permeability (Gorbushina, 2007). (Estrada-Barcenas et al., 2010). Insects on dung in Dickson and Kirk (1976) noted that more fungi caves are exposed to a high diversity and quantity of and bacteria were found on the cave floor than on fungi, and some species are known to produce anti- the walls or ceiling, likely because organic matter microbial substances to prevent infection (Ribeiro et accumulates on the floor. Bacteria are more uniformly al., 2012). Insects may introduce fungi into caves by distributed than fungi, which are often associated transporting spores externally and internally. Dickson with invertebrates and organic matter (Dickson & (1975) noted that populations of invertebrates were Kirk, 1976; Khizhnyak et al., 2003). Cave bacterial positively correlated with populations of fungi in diversity and biomass is much higher than that of Virginia cave sediments. Cave Crickets (Ceuthophilus fungi, while yeasts and amoeba are the least common gracilipes gracilipes) are believed to be vectors of (Dickson & Kirk, 1976; Pitzalis et al., 1991; Vaughan- dictyostelid cellular slime molds into and within caves Martini et al., 2000; Khizhnyak et al., 2003; Urzi et (Stephenson et al., 2007). al., 2010). However, Mulec et al. (2012) found that the A number of studies have investigated whether quantity of fungi in cave air exceeded that of bacteria, visits to caves by humans has an impact on the except in areas frequented by tourists, where bacteria fungal diversity of subterranean habitats. Vaughan- dominated. Bacteria are more numerous in dry than Martini et al. (2000) found that an Italian cave with in wet cave passages, while fungi show the reverse high human visitation (~400,000 visitors/year) had pattern (Dickson & Kirk, 1976). more yeast strains than two other caves with low Caves generally lack the rich fungal diversity human visitation (a few speleologists/year). Mosca & commonly found elsewhere in nature. An exception Campanino (1962) reported the same pattern for cave to this is the high diversity of fungi found on dung in fungi and Somavilla et al. (1978) for cave bacteria. The caves (Dickson & Kirk, 1976). Dickson and Kirk (1976), quantity of airborne fungal spores in Chinese caves Volz and Yao (1991), and Hsu and Agoramoorthy was positively correlated with human visitor numbers (2001) found a greater diversity of fungal species in (Wang et al., 2010), while Fernandez-Cortes et al. (2011) soils outside of caves compared to inside. Hsu and and Adetutu et al. (2011) found that the microfungal Agoramoorthy (2001), Urzi et al. (2010), Kuzmina species composition was altered with increasing et al. (2012), and Mulec et al. (2012) noted that human visitation. Kuzmina et al. (2012) recorded the microorganism biodiversity and biomass decreased

International Journal of Speleology, 42 (1), 77-96. Tampa, FL (USA) January 2013 A world review of fungi, yeasts, and slime molds in caves 83 from the entrance to deep zones in caves. Conversly, types of fungal spores found indoors are dependent in a South African gold mine, Pohl et al. (2007) on geographic location, season, and outdoor climate determined that the diversity of filamentous fungal (Sterling & Lewis, 1998). Amend et al. (2010) found genera in the air increased from outside to deeper that as one moved away from the equator fungal into the mine, whereas the diversity of yeast genera diversity inside buildings increased (contrary to the showed the opposite trend. In Russia, mesophilic pattern found in nearly all other organisms), and species dominate close to the cave entrance while that distance from the equator was the strongest the ratio of psychrotolerant micromycetes increased predictor of fungal assemblage similarity between with depth (Kuzmina et al., 2012). Koilraj et al. (1999) buildings, followed by mean annual rainfall and found that some fungal genera occurring in Indian mean annual temperature. Amend et al. (2010) states caves were more abundant in the sediment at the that the fungal composition inside buildings is most entrance compared to the interior (e.g. Aspergillus, strongly influenced by local outdoor environmental Penicillium), while other genera were more prevalent factors. However, Sterling & Lewis (1998) found in the interior of the cave (e.g. Absidia, Rhizopus, indoor microclimates are important in the secondary Mucor, Chaetomium, Sepedonium). Fernandez- release and growth of spores within homes, which can Cortes et al. (2011) found that Cladosporium spores alter the fungal assemblage from that found outside. were more abundant in the air at the cave entrance Microclimates and substrates found in caves often compared to the interior, while Penicillium showed differ from the outside environment, and likely these the opposite pattern. These contradictory results of are major factors that drive the formation of unique fungal abundance patterns demonstrate the need for cave fungal assemblages. The absence of sunlight more detailed studies. may also be a factor. The lack of sunlight is often cited It appears that the majority of fungi documented as the cause of deformities that mushrooms growing from caves originate from the non-subterranean in mines occasionally exhibit (Saric-Sabados, 1957; environment. The fungal taxa most commonly Lohwag, 1961; Georgescu & Tutunaru, 1966; Dobat, reported from caves (Table 1) are also commonly 1970; Kuthan, 1977). found in the environment above ground. Mason- Whether true troglobitic fungi exist is currently Williams and Benson-Evans (1967) found that the unknown. Certainly there are species that have been bacteria in the air of many smaller British caves were exclusively isolated from caves {e.g. Aspergillus baeticus directly related to the frequency of disturbance of the (Novakova et al., 2012), Aspergillus spelunceus (Raper cave air by outside influences. Air currents entering & Fennell, 1965; Marvanova et al., 1992), Aspergillus and circulating within caves can distribute spores thesauricus (Novakova et al., 2012), Chrysosporium from the surface environment, suggesting that the chiropterorum (Beguin et al., 2005), Chrysosporium study of cave structure and air flow is important speluncarum (Novakova & Kolarik, 2010), Microascus when determining the origin of cave micro-organisms. caviariformis (Malloch & Hubart, 1987; Vanderwolf Seasonal patterns in the presence of cave microfungi et al., 2013), Mucor troglophilus (Gunde-Cimerman confirm the importance of outside influences. Wang et al., 1998), Ochroconis anomala (Martín-Sánchez et et al. (2010) found a greater diversity and quantity of al., 2012), Ochroconis lascauxensis (Martín-Sánchez microfungi in Chinese cave air in the summer when et al., 2012), Ombrophila speluncarum (Lagarde, compared to the winter and Docampo et al. (2010) 1913), Trichosporon akiyoshidainum (Sugita et al., documented the same pattern in Spanish cave air. 2005), Trichosporon cavernicola (Sugita et al., 2005), Borda et al. (2004) found more colony forming units Trichosporon chiropterorum (Sugita et al., 2005)}, but of bacteria and fungi in the air of Romanian caves in this may reflect insufficient sampling outside the cave the summer than in the winter. This seasonal pattern habitat. Geomyces destructans, the best known ‘cave is also seen among microfungi outside caves. In a fungus’, has been isolated from bats outside caves Spanish cave Docampo et al. (2011) found a signif icant (Dobony, 2012). positive correlation between outside rainfall and concentrations of certain airborne spore types inside Sampling and culturing cave fungi the cave. These spore types have a proven association The most common method of detecting cave between the rainy season and increases in their microfungi is by cultivating spores from cave airborne concentrations in the outside environment substrates on agar plates (Table 2). Studies that (Docampo et al., 2011). Wang et al. (2010) found the do not use this method either employ genetic quantity of airborne fungal spores in Chinese caves environmental sampling or directly sample visible was positively correlated with temperature (inside fungal growth in the cave (e.g. mushrooms). Studies and outside the cave) and negatively correlated with have exposed plates to cave air (gravity settling relative humidity and outside rainfall (water can method), placed cave sediment or other substrates wash away spores that may otherwise be carried into directly onto the plates (usually diluted), or used the cave via air currents). The composition of fungal swabs. A multitude of agar types have been used, genera in these caves was identical to the genera with variations of Malt agar (31.7% of 101 studies found outside (Wang et al., 2010). reporting agar type used), Sabouraud agar (21.8%), Many of the patterns described above are similar to Potatoe Dextrose agar (20.8%), and Czapek (18.8%) those found by investigators studying fungi occurring being the most common. The choice of media can in human homes versus the outside environment significantly affect results since different fungal (Sterling & Lewis, 1998; Shelton et al., 2002). The taxa have different physiological requirements. For

International Journal of Speleology, 42 (1), 77-96. Tampa, FL (USA) January 2013 84 Vanderwolf et al. example, Semikolennykh et al. (2004), in a study slow growing (Vanderwolf et al., 2013), perhaps an of Russian caves, used two media, Wort agar and adaptation to the oligotrophic environment. Culturing Czapek’s agar. They found that Wort agar supported is therefore a time consuming approach, and even a more diverse population of fungi (particularly with long incubation periods and multiple agar types, rare species) than Czapek agar, while the opposite may underestimate the number of taxa present. Some pattern was found for soils outside of their study fungal taxa may not grow in culture (Bass & Richards, cave. Cave mycological studies should use more than 2011), or may be present in numbers too small to one type of medium; in our experience, low nutrient detect by culturing methods. agar that mimics the oligotrophic cave environment, There are other methods that do not involve such as dextrose-peptone-yeast extract agar, yield culturing. One method targets fungi in cave air by the greatest diversity of genera (Papavizas & Davey, trapping spores using sampling machines (Wang et 1958; Vanderwolf et al., 2013). al., 2010; Docampo et al., 2011; Porca et al., 2011). The incubation temperature of agar plates should also Baiting is also used, such as the To-Ka-Va hair be carefully chosen. As a general guideline, samples baiting method which targets keratinophilic fungi and should be incubated close to the temperature of the dermatophytes. A traditional method for the isolation cave they originated from. Of 35 cave mycology studies of Histoplasma capsulatum is to mix sediment or that reported both incubation temperature and cave guano from caves with saline solution and then temperature, 21 (60%) studies cultured samples within inject it into small mammals (usually mice or bats). 4°C of ambient cave temperature. Many investigators After a period of time the mice are euthanized and cultured samples at room temperature which may their organs cultivated for fungi (Taylor et al., 1962). exclude some psychrophilic and psychrotrophic fungal However, this procedure has largely been abandoned species. Of 81 cave mycology papers that reported in favor of genetic methods (Neuhauser et al., 2008). incubation temperature, 49 (60.5%) studies cultured Genetic methods are now widely used to identify their samples ≥25°C, 19 (23.5%) at 20°C − <25°C, 11 cave fungi as they are more accurate and less time (13.6%) at >10°C − <20°C, 12 (14.8%) at ≤10°C, and 5 consuming than culturing. Direct genetic sampling of (6.2%) at ‘room temperature’ (note: a given study may the environment may yield many new fungal taxa from fall into more than one category). Although a greater caves (Lindner et al., 2011). Shapiro and Pringle (2010) diversity of fungal species seemed to be isolated at found that 5 sets of morphologically identical species of lower incubation temperatures, the relationship was fungi in caves each had different ITS locus sequences, not significant (rs= −0.201 p=0.08; 68 studies listed suggesting they may have been different genetic fungal taxa isolated by temperature). Very different species. However, genetic methods have limitations microfungal species spectra were obtained from forest as cultures are required for describing new species, soils in Rhode Island when two different isolation studying ecology, and determining physiological temperatures (0°C, 25°C) were used (Carreiro & requirements. As well, genetic identifications require Koske, 1992). Ivarson (1973) found that the isolation a pre-existing bank of sequences for comparison. frequency of the psychrophilic Geomyces pannorum Given the large number of undescribed fungal taxa increased as incubation of the soil and agar plates (Hawsworth, 2001; Bass & Richards, 2011), and the approached 0°C. The number of microfungi species apparent rarity of many fungal species encountered isolated from a Czech Republic cave wall was 1.5 times in caves and mines, existing gene banks may lack the higher when plates were incubated at 15°C vs. 25°C required sequences. (Marvanova et al., 1992). A similar pattern was found A wide variety of substrates in caves have been for cave bacteria, with fewer colonies growing at 37°C examined for fungi. The substrate that has yielded vs. 30°C vs. 18°C after the plates had been exposed to the greatest number of isolates is sediment/soil (Fig. cave air in England (Mason-Williams & Benson-Evans, 2). Whether this is a true biological pattern cannot 1967). Khizhnyak et al. (2003) and Kuzmina et al. be determined because sediment is also the most (2012) found that psychrophilic and psychrotolerant commonly studied cave substrate (Fig. 2). Precise strains of bacteria and fungi outnumbered mesophilic characterization of what is meant by ‘sediment’ or ‘soil’ species in Russian caves. Conversely, Semikolennykh should be reported by authors given the variability in et al. (2004) found the opposite pattern for fungus in definitions (e.g. sediment type, presence of organic Russian caves (more species at 25°C vs. 15°C vs. 5°C) debris). Air has also been commonly studied, but and attributed this to the stress situation induced by in particular it has been intensively studied (high the atypical temperature stimulating the growth of number of samples taken per study), in part due to dormant species. air quality concerns in show caves and in part due Cave mycological studies should also incubate to the ease with which air sampling devices can be samples for an appropriate length of time. The employed. The fur and skin of live bats, although average length of incubation was 18.2±20.4SD days, infrequently examined, appears to be a surprisingly range 2-90 days (for the 57 studies where reported), diverse substrate for fungi (Fig. 2). However, we have but we have found it can take >30 days for some slow done much of the limited work and believe this result growing fungal species to become visible (Vanderwolf is largely a reflection of methodology. Nonetheless, et al., 2013). However, length of incubation for the differences in methodology do not seem to explain 57 studies was not signif icantly correlated with the the lower diversity of fungal genera encountered by number of fungal species documented (rs =0.236, Lorch et al. (2012) in cave soils from the northeastern p=0.078). Several fungal species found in caves are USA when compared to live bats sampled in eastern

International Journal of Speleology, 42 (1), 77-96. Tampa, FL (USA) January 2013 A world review of fungi, yeasts, and slime molds in caves 85 Canada (Vanderwolf et al. 2013). Differences suggest 22.7°C±10.7SD (n=11 studies that reported sampling that the surface of live bats in caves may actually a mine and mine temperature) while that for caves is harbor a greater diversity of fungi than adjacent 13.4°C±6.4SD (n=23 studies that reported sampling cave soils, perhaps reflecting bat movement through a cave and cave temperature). However, there was surface and subterranean habitats, and emphasizing no significant difference in the number of fungal bat importance as fungal vectors. taxa isolated in caves (n=40) versus mines (n=32) Most of the environmental features of caves listed in (W=1597.5, p=0.121). A Mann-Whitney test was used Table 2 were not reported often enough to determine because the number of taxa isolated per study is not correlations with the number of fungal taxa. For normally distributed, even with transformation. example, of 38 studies reporting the presence/absence of bats, only 3 studies give population estimates, 8 Management of cave fungi studies identify the species present, and 2 studies Although there has been little attention paid to indicate that no bats were present. The average cave the suppression of fungi present in caves generally, temperature was not correlated with the number of fungicides have been suggested as control agents fungal taxa found (n=52, rs =0.177, p=0.209), but the for Geomyces destructans (Chaturvedi et al., 2011). correlation was influenced by which studies were However, limited understanding of the interactions included for analysis. When the two studies reporting among fungal species or associated fauna in caves, the greatest number of fungal species are excluded, or their response to fungicides, suggests that the both done in temperate regions (Novakova, 2009a; success of spraying will be unpredictable. Both Vanderwolf et al., 2013), the correlation moves towards chemical (e.g. Lastanox) and biological control (e.g. significance (n=50, rs =0.278, p=0.05). This suggests use of mycoparasites such as Trichoderma viride) that warmer caves are more speciose than colder have been attempted to preserve wooden supports ones, but results are confounded by varying sampling in mines from degradation by fungi (Prihoda, 1965; intensities and other aspects of study methodology. Fassatiova et al., 1974). However, the results of Fungal diversity in the general environment is thought such efforts are not quantified. Various chemical to be greater in tropical than in temperate regions compounds and ultraviolet light have been used in (Frohlich & Hyde, 1999), although Amend et al. (2010) caves for fungal control but, again, the microbial found the opposite pattern in their global study of response is not quantified (Oba et al., 1985; Garg fungi in buildings. Given that the vast majority of cave et al., 1995). In France, treating prehistoric cave fungal studies have been done in temperate regions paintings with benzalkonium chloride to control to date, a comparison of temperate versus tropical destructive microfungi resulted in the proliferation of caves would not be valid at this time. Conversely, this resistant bacteria and further fungal colonizations of correlation could reflect a difference between caves the cave art (Bastian et al., 2009b; Martin-Sanchez et and mines. The average temperature of mines is al., 2012a). It is believed that antifungal compounds

800 43%

700

600

500 18% 19%

400 16% 18% 4% 300 16% 6%

Number Taxa of Fungal 200 5% 100 2% 6% 5% 1% 1% 1% 0 Insects SedimentAir Cave wallEarthwormWater castsWood Dung Guano Bat fur/skinBat internal(live)Bat fur/skintissueBat bones(dead)Other Unreported

Fig. 2. The number of fungal taxa recorded from various cave substrates. Each taxon in a study is counted as 1 report; therefore the same species may be reported from the same substrate multiple times by different studies. The percentage of papers sampling that substrate is given above the bars; most papers sampled >1 substrate type. n=225 studies for the bars, n=126 for the percentages. Many of the studies excluded from the percentage calculations did not report the substrate sampled.

International Journal of Speleology, 42 (1), 77-96. Tampa, FL (USA) January 2013 86 Vanderwolf et al. produced by cave bacteria (Kim et al., 1998; Groth et and Research Library, and University of New al., 1999) can have a significant effect on cave fungi. Brunswick library staff for tracking down numerous Jurado et al. (2009) found that no fungal growth obscure papers. Our work on the mycology of caves or fungal DNA could be isolated from cave walls has been supported by grants to DFM and KJV from colonized by bacteria. Attempts to eradicate specific the Canadian Wildlife Federation, New Brunswick cave microorganisms may lead to undesired fungal Environmental Trust Fund, New Brunswick Wildlife or bacterial outbreaks since intact communities may Trust Fund, New Brunswick Department of Natural interact as biological control agents (Jurado et al., Resources, National Speleological Society WNS Rapid 2009). 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International Journal of Speleology, 42 (1), 77-96. Tampa, FL (USA) January 2013 I World Karst Science Reviews Cave and Karst Science of the British Cave Research Association Volume 39(2), August 2012

Contents Ostracoda (Crustacea) from freshwater Preparing the ground – new mechanisms caves in the western Black Sea region of for karst and speleogenesis: ‘altération’, Turkey. fantomisation and replacement. Yavuzatmaca M., Külköylüoğlu O., Sari N., Başak Laverty M., 72-76 E., Mengi H., 53-58

Fauna reported from Batu caves, Selangor, Phreatic maze caves, Grinton Moor, Malaysia: annotated checklist and Swaledale, UK: survey of the Devis Hole bibliography. Mine Caves. Moseley M., Lim T.W., Lim T.T., 77-92 Harrison T., 59-62 Forum (Correspondence, Book Review, Thesis Abstract, Karstologia abstracts) The occurrence of Hydra circumcincta (Schulze, 1914) (Hydrozoa: Hydridae) in a 93-96 well in the Dorset Chalk, UK. Knight L.R.F.D., Johns T., 63-65

A new eyeless species of cave-dwelling trechine beetle from northeastern Guizhou Province, China (Insecta: Coleoptera: Carabidae: Trechinae). Tian M., Clarke A., 66-71

Die Höhle of the Verbands Österreichischer Höhlenforscher und Verbands de Deutschen Höhlen- und Karstforscher e. Volume 63(1-4), 2012

Papers Zur Mikrobiologie von Bergmilch Höhlendunkel und Wissbegierde - Eine Microbiology of moonmilk. Kulturgeschichte der Höhlenforschung in Europa von der Antike bis zur Romantik Reitschuler C., Schwarzenauer T., Lins P., Wagner Obscurity and curiosity - The cultural A.O., Spötl C., Illmer P., 3-17 history of cave exploration in Europe from ancient world to romantic era. Strukturgeologie der Karstformen auf der Mattes J., 63-81 Schneealpe (Stmk.) Structural geology of karst features of the Schneealpe (Styria). Bauer H., Hatzenbichler G., Plan L., Decker K., 105 Jahre Forschung am Hölloch im 18-31 Mahdtal und seiner Umgebung, Bayern 105 years of exploration in the Hölloch and its surroundings, Bavaria. Wolf A., 82-85 Fledermausfunde aus dem Gamslöcher- Kolowrat-Salzburgerschacht-System (1339/1) des Untersbergs bei Salzburg Bat f indings from the Gamslöcher-Kolowrat- Die Verbindung von Oberer Salzburgerschacht cave system (1339/1) in Brandgrabenhöhle und Hirlatzhöhle the Untersberg near Salzburg. Connection of Obere Brandgrabenhöhle and Hirlatzhöhle. Spötl C., Zagler G., Bauer K., Mangini A., Bieniok A., 32-37 Buchegger G., Wimmer G., 86-90

Bericht über die Probegrabung (2011) in Forschungen in der Grotte des Chamois der Pendling-Bärenhöhle bei Kufstein (Alpes-de-Haute-Provence, Frankreich) (Nordtirol; 1266/21) Report on the test 2009-2011 Explorations in the Grotte des excavation (2011) in the Pendling bear Chamois 2009-2011. cave near Kufstein (Northern Tyrol; Wielander B., Audra P., 91-101 1266/21). Frischauf C., Mazelis E., Rabeder G., 38-42

Zur Erforschungsgeschichte der Paläotraun (Dachstein) Remarks on the exploration history of the Paläotraun (Dachstein). Trimmel H., 43-62 II World Karst Science Reviews Journal of Cave and Karst Studies of the National Speleological Society Volume 74(1), April 2012

Papers Response of the Karst Phreatic Zone to Flood Events in a Major River (Bohemian Cave Cricket Exit Counts: Environmental Karst, Czech Republic) and its Implications Influences and Duration Surveys. for Cave Genesis. Wekerly F.W., 1-6 Vysoká H., Bruthans J., Žák K., and Mls J., 65-81

A New Species of Nicoletiidae (Insecta: Involvement of Bacteria in the Origin Zygentoma) from Kartchner Caverns State of a Newly Described Speleothem in the Park, Arizona. Gypsum Cave of Grave Grubbo (Crotone, Espinasa L., Pape R.P., Henneberry A., and Italy). Kinnear C., 82-89 Cacchio P., Ercole C., Contento R., Cappuccio G., Preite Martinez M., Del Gallo M., and Lepidi A., 7-18 Regionalization Based on water Chemistry and Physicochemical Traits in the Ring of Cenotes, Yucatan, Mexico. The Prehistoric Cave Art and Archaeology Pérez-Ceballos R., Pacheco-Ávila J., Euán-Ávila of Dunbar Cave, Montgomery County, J.I., and Hernández-Arana H., 90-102 Tennessee. Simek J.F., Blankenship S.A., Cressler A., Douglas J.C., Wallace A., Weinand D., and Welborn H., 19-32 Delineating Protection Areas for Caves Using Contamination Vulnerability Mapping Techniques: The Case of Herrerías Cave, Asturias, Spain. Marín A. I., Andrea B., Jiménez-Sánchez M., Candidate Cave Entrances on Mars. Cushing G.E., 33-47 Dominguez-Cuesta M. J., and Meléndez-Asensio M., 103-115

The First Subterranean Freshwater Microbiological Activities in Planarians from North Africa, with an Moonmilk Monitored Using Isothermal Analysis of Adenodactyl Structure in the Microcalorimetry (Cave of Vers Chez Le Brandt, Neuchatel, Switzerland). Genus Dendrocoelum (Platyhelminthes, Braissant O., Binderschedler S., Daniels A. U., Tricladida, Dendrocoelidae). Verrecchia E. P., and Cailleau G., 116-126 Harrath A.H., Sluys R., Ghala A., and Alwasel S., 48-57

Importance of karst Sinkholes in Preserving Relict, Mountain, and Wet- Micro-Charcoal Abundances in Stream Woodland Plant Species under Sub- Sediments from Buckeye Creek Cave, Mediterranean Climate: A Case Study from West Virginia, USA. Souther Hungary. Springer G.S., Mihindukulasooriya L.N., White Bátori Z., Körmöczi L., Erdös L., Zalatnai M., and D.M., and Rowe H.D., 58-64 Csiky J., 127-134

Journal of Cave and Karst Studies of the National Speleological Society Volume 74(2), August 2012 Editorial Quaternary Alluvial Sinkholes: Record of Environmental Conditions of Karst Development, Examples From The Ebro Karst Environments: Problems, Basin, Spain. Management, Human Impacts, and Soriano M.A., Luzón A., Yuste A., Pocoví A., Pérez Sustainability - An Introduction to the A., Simón J.L., and Gil H., 173-185 Special Issue. Brinkmann R. and Parise M., 135-136 Quantifying Concentrated and Diffuse Recharge in Two Marble Karst Aquifers: Big Papers Spring and Tufa Spring, Sequoia and Kings Canyon National Parks, California, USA. Sinkhole Evolution in the Apulian Karst of Tobin B.W. and Schwartz B.F., 186-196 Southern Italy: A Case Study, with Some Considerations on Sinkhole Hazards. Festa V., Fiore A., Parise M., and Siniscalchi A., Drowned Karst Landscape Offshore the 137-147 Apulian Margin (Southern Adriatic Sea, Italy). Taviani M., Angeletti L., Campiani E., Ceregato A., Foglini F., Maselli V., Morsilli M., Parise M., and Karst Aquifer Draining During Dry Trincardi F., 197-212 Periods. Fiorillo F., Revellino P., and Ventafridda G., 148- 156 Five Blues Lake National Park, Belize: A Cautionary Management Tale. Day M. and Reynolds B., 213-220 Karst of Sicily and its Conservation. Di Maggio C., Madonia G., Parise M., and Vattano M., 157-172 A Sustainability Index for Karst Environments. van Beynen P., Brinkmann R., and van Beynen K., 221-234 III World Karst Science Reviews

Journal of Cave and Karst Studies of the National Speleological Society Volume 74(3), December 2012

Electrical Resistivity Imaging of Cave Div Aska Jama, Slovenia. Mihevc A., and Stepisnik U., 235-242 Diet of the Newt, Triturus Carnifex (Laurenti, 1768), in the Flooded Karst Sinkhole Pozzo Del Merro, Central Italy. Romano A., Salvidio S., Palozzi R., and A Survey of the Algal Flora of Sbordoni V., 271-277 Anthropogenic Caves of Campi Flegrei (Naples, Italy). Archeological District Cennamo P., Marzano C., Ciniglia C., Pinto G., Human Urine in Lechuguilla Cave: The Cappelletti P., Caputo P., and Pollio A., 243-250 Microbiological Impact and Potential for Bioremediation. Johnston M.D., Muench B.A., Banks A.D., and Barton H.A., 278-291 Assessment of Spatial Properties of Karst Areas on a Regional Scale using GIS and Statistics – The Case of Slovenia. Komac M. and Urbanc J., 251-261 A Method to Determine Cover-Collapse Frequency in the Western Pennyroyal Karst of Kentucky. Currens J.C., Paylor R.L., Beck E.G., and Lizards and Snakes (Lepidosauria, Davidson B., 292-299 Squamata) from the Late Quaternary of the State of Ceará in Northeastern Brazil. Schmaltz Hsiou A., De Oliveira P.V., Ximenes C.L., and Somália Sales Viana M., 262-270