original scientific paper UD C 551.442:593.1(450 Vicenza) 
PROTISTS FROM CAVES: PRELIMINARY DATA O N POPULATIONS OF THE 
"COVOL O DELLA GUERRA", BERICI HILLS (VICENZA, ITALY) 

Laura GUIDOLIN 
Department of Biology, University of Padova, IT-35131 Padova, Via G . Colombo 3 E mail: Guidolin@civ.bio.unipd.it 
Olimpia COPPELLOTTI KRUPA 
Department of Bioiogy, University of Padova, iT-35131 Padova, Via G . Colombo 3 E mail; Olimpiak@civ.bio.unipd.it 
ABSTRACT 
Periodic samplings between June 1997 and September 1998 in the karst cave known as the "Covolo deila Guerra" (Vicenza, Italy) allowed the monitoring of some chemico-physicai parameters, such as illumination, air humidity, air and water temperatures, pH and conductivity of water, and concentrations of heavy metals in water. The initial results on populations of Protists from five different sites of the cave indicated a rich variety of organisms belonging to various taxa, such as Mastigophora, Sarcodina and Ciliopbora. Until now about 70 Protist taxa have been detected, 42 belonging to Ciliopbora. 
Key words: cave, karst, Vicenza, Protists, Ctliophora, Mastigophora, Sarcodina 
INTRODUCTION 

Increased interest in spelaeology has produced remarkable results for some Italian caves in the last few decades. However, only some sporadic visits with the aim of collecting faunistic material are reported in the literature (Vandd, 1964). Moreover, the collected ma­terial concerns only some taxonomic groups, such as insecta, Chilopoda and Gastropoda (Saccetti, 1966; Minelii, 1974; Pezzoli, 1990). 
The "Covolo delia Guerra" ("war cave") is one of the most famous caves in the Berici Hills (Vicenza, Italy) (Fig. 1). Its name derives from the fact that it was a shelter for the inhabitants of this area during past wars. An exhaustive description of this cave has been made by Boscolo & Mantovani (1971), 
The Berici Hills are situated south of the town of Vicenza, separated by strips of plain from the nearby mountains and hills. The Berici hills, shaped appro­ximately as a lengthened parallelogram, stretch south for 20 kilometres, in the form of a plateau, cut by valleys. 
These hills are mostly formed of Eocene and Oligocene limestone: consequently, karst morphogenesis is active on their surface (Castiglioni, 1991). In particular, because of the almost flat upper surface, conditions are good for the formation of dolinas, which are very numerous, up to a density of 20 per square kilometre (Mietto & Sauro, 1989). 
Owing to the relative ease of exploration of the karst cave, faunistic studies of some invertebrate phyla and Chiroptera have been made (Boscolo 1968; Vernier, 1996). 
Since the second part of the 19th century, Protists dwelling in caves in many European countries and in North America have been studied (Landolt et al., 1992; Golemansky & Bonnet, 1994) and in japan (Sudzuki & Hosoyama, 1991), Studies on Protists have also con­cerned endoparasites in various caverniculous inver­tebrates (Golemansky, 1973, 1980). 
As regards the protists living in the Covolo del la Guerra, Boscolo (1968) reported only three species of Peritrichida (Intrastylum steinii, Lagenophrys mono 
Laura CUIDOU N & Olimpia COPPRLOTT I KRUPA: PROTISTS FROM CAVES: PRELIMINARY DATA ON POPULATIONS ..., 73-80 
Fig. 1: The karst cave Covofo delta Guerra and location 
of sampling sites (A, B, C, D, E); * indicates the third, 
long, narrow branch of the cave, not easily accessible 
and not yet studied. Modified from Mietto & Sauro 
(1989). 

SI. 1: Jama Covolo della Guerra in lokacija vzorčišč (A, 
B, C, D, E); * ponazarja tretji (dolgi in ozki) odsek jame, 
ki je težko dostopen in tudi še neraziskan. Prirejeno po 
Miettu in Sauru (1989). 

Fig. 2; The cave Covolo della Guerra. a) Entrance of Si. 2; jama Covolo della Guerra. a) Vhod v jamo; b) cave; b) Detail of "Grande Colata", a large concretion "Grande Colata", velik sklop stalaktitov in stalagmitov. of stalactites and stalagmites. 
l.anrs GUIDOU N & Otimpia COPPEU.OTT I KRUPA: PROT1STS EROM CAVES: PRELIMINARY DAT A O N POPULATIONS ..., 73-80 
listrae, Lagenophrys sp.) and one belonging to Suctorida (Acineta sp.). These ciliates were recovered from the gilis or appendages of the amphipod Nipbargus costoz­zae or the isopod Monolistra berica. It seemed particu­larly interesting to investigate Protist populations in this peculiar environment, in view of the value of such or­ganisms in constituting simple models for a monitoring system (Boikova, 1990; Coppellotti 1998). Checking the karst environment was necessary, taking into account the presence of a spring, in connection with the hydro­logical system of this area. 
This paper presents initial results from the northern part of the Covolo delia Guerra, dealing with the corn-position of Protist communities and some parameters of chemico-physicai conditions, including heavy metal determination, 
MATERIALS AN D METHODS 

Five sampling sites were chosen in the Covolo deila Guerra, Berici Hills, Vicenza (45°27'20" N, 0°52' 27" W), indicated on the land registry map as N. 127 V-VI (Fig. 1). The entrance to the cave (Fig. 2a) is 150 metres above sea level; the cave itself has a total length of 652 metres, and a difference in level of 43 metres. Collection sites are indicated in Fig. 1. Site A is at the entrance, site B on the wall opposite the entrance, site C in the left branch of the cave, site D in the right branch, at the base of a large concretion of stalactites and sta­lagmites, called the "Grande Colata" ("great flow") (Fig. 2b), and site L is at the spring. Data regarding the third, long (537 m) and narrow branch of the Covolo delta Guerra, which is not easily accessible, will be collected in the near future. 
Samples and measurements of chemico-physicai pa­rameters were taken every two months in the period June 1997 - September 1998. In every site, when possi­ble, the following parameters were measured: water and air temperatures, using a Cbecktemp 1 Hanna ther­mometer; relative humidity using a psychrometer, water pH (HI270 Hanna pHmeter), illumination (HD 8366 luxmeter), and conductivity (H18733 Hanna conductiv­ity meter). 
Sampling of sediments was carried out by pressing a plexiglass tube 3 cm in diameter into the sediment; the part from 0 cm to 2.0 cm below the surface was col­lected. Samples thus consisted of 14 ml each. They were placed in plastic 250-ml bottles and 100 ml of fresh water taken from the environment were added. Water was collected by sucking into a plastic 500-ml bottle. Material from the wall (site B) was collected by scraping with a plastic spoon. Samples were taken to the labora­tory in a refrigerated container and observations started after 3 hours. 
The material was examined first under a Wild M8 stereomic rose ope, and then on a Leitz Diaplan micro­scope at magnification 31 Ox or 5G0x. Phase contrast at 500x was often used. 
Most Protists were isolated and cultured in the labo­ratory in distinct strains at 12°C in the dark in mineral water supplemented with bactotryptone, yeast extract or proteose-peptone. Subsequently, impregnation by pro­targoi or silver nitrate was used for correct identification of most ciliates, following respectively Wilbert or Chatton-Lwoff procedures, as modified by Foissner (1991). Drawings of protargoi- or silver nitrate-impreg­nated specimens were made with the aid of a camera lucida under a Diaplan Leitz microscope. Flagellates were identified after staining with acid Lugol's iodine solution (Leakey et al., 1994). The taxonomic scheme of Levine et al. (1980) was used. Genus and species de­scriptions by Kahl (1930-1935), Matthes et al. (1988), Foissner & Berger (1996), Foissner et al. (1991, 1992, 1994, 1995), Patterson (1996) and Warren (1986) were also used. 
Metals (Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd) were deter­mined in waters with an inductively coupled plasma atomic emission spectrometer (ICP-AES, Spectro). 
RESULTS AN D DISCUSSION 


Chemico-physicai data 

Periodic samplings between June 1997 and September 1998 in the Covolo deila Guerra karst cave allowed the monitoring of some chemico-physicai pa­rameters. The overall results are shown in Tab. 1. 
There is complete darkness in most of the cave, I.e. the left and right branches and the site where the spring flows. Illumination values are in the ranges of 240-3300 lux and 10-85 lux, respectively at the entrance (site A) and on the opposite (site B). 
Owing to the existence of a small stream which flows from the spring in the right branch of the cave and to the continual dripping of water which percolates from outside, humidity is very high. Indeed, there are puddles both in the two branches and immediately after the en­trance. Water temperatures may reach 6.8°C in winter and 14.4°C in summer. The minimum value (6.8°C) is lower than that (11 °C) recorded by Boscolo (1968) in the period October 1964 - April 1968, whereas the maximum one (14.4°C) is the same. Moreover, relative humidity in the air is always high and may reach 100%, maintaining a value of 85-90% at the spring site (E). 
Air temperatures may reach a maximum of 16.3°C inside the cave, with higher values at the entrance. 
pH measurements of water at sites A, C, D and E al­ways indicated basic values between 7.60 and 8.60; the values in the puddles in the two branches were higher than in the spring. It is noteworthy that the pH values recorded by Boscolo (1968) were always acidic and ranged between 6.5 and 6.7. The higher values 
laura GUSDOU N & Olimpia COPPELLOTTI KRUPA: PROTISTS FROM CAVES: PRELIMINARY DATA O N POPULATIONS .... 73-80 
Tab. 1: Cbemico-physica! data in Covolo defla Guerra ()une 1997-September 1998). 
Tab. 1: Kemijsko-fizikalni podatki iz jame Covolo della Guerra (!uní) 1997 -September 1998). 

Sampling site  Relative hu- Illumination  Air tempera- Water tempera- Water pH  Conductivity  
midity (%)  (lux)  ture (°C>  ture fC )  (pS)  
Entrance (A)  70-100  240-3300  9.0-20.0  7.0-14.0  8:47  *  
Wall (B)  80-100  10-85  9.1-15.7  *  *  *  
Left branch (C)  82-100  0  8.6-16.3  6.8-14.4  8,37-8.60  304-460  
Right branch (D)  78-100  0  10.8-15.5  9.8-13.6  8.42-8.49  460-510  
Spring (E)  85-90  0  8.0-14.8  11.0-12.5  7.6-7.84  475-496  
* Values not determined.  

Tab. 2: Protist tax a recovered from Covolo della Litonotus lamella Guerra (June 1997-September 1998). Litonotus crystallinus Tab. 2: Taksoni enoceliCarjev iz jame Covolo della Colpoda Ínflala Guerra (junij 1997 -September 1998). Colpoda steini 
Colpoda cu cull us 
Chilodonella uncinata


Phylum SARCOMASTIGOPHORA 
Acineta pyriformis 

Subphylum Mastigophora 
Acineta tuberosaAstasia sp. 

Class Oíigohymenophorea 
Entosiphon sulcatum Col pi diurn colpodaPeranema trichophorum Colpidium kleini Polytoma uvella Cinetochilum margaritaceumMonosiga ova fa Glaucoma scintillansBicosoeca sp. 
Tetrahymena pyriformis Bodo putrinus Paramecium caudatum Bodo caudatus Cyclidium heplatricbumRynchomonas nasuta Cyclidium glaucoma

Subphylum Sarcodina 
Philasterides armatus 

Class Lobosea 
Uronema nigricans Hartmanella vermiformis Vórtice!la as ty! i form is Acanthamoeba sp. 
Vórtice!!a aquadulcis Naegleria sp. 
Vorticella campanula Vannella mira Vorticella con vallaría Vexillifera aurea Vorticella infusionum Cryptodifflugia oviformis Vorticella pictaDifflugia sp. 
Vorticella pyriforme Thecamoeba sp. 
Vorticella turgicula Aredia hemisphaerica 

Class Poiyhymenophorea Class Filosea 
Metopus esCypboderia grandis Brachonella spiralisEugiypha sp. 
Ha hería grand'mella Tracheleuglypha sp. 
Strobilidium hum He Thecamoeba sp. 
Holosticha kessleri 

Class Heliozoea 
Holosticha pullaster Actinopbrys sol Oxytricha fall ax Heterophrys sp. 
Oxytricha hymenostoma 
Oxytricha saprobia
Phylum OLiOPHORA 
Sterkiella histriomuscorum 


Class Kinetofragmmophorea 
Stylonychia pustulata Loxodes rostrum Aspidisca lynceus 

Lacrymaria oior 
Amphileptus incurvata Aspidisca sp. 

Laura GUIDOLI N & Olimpia COPPELLOTTI KRUPA: PROTISTS FROM CAVESi PRELIMINARY DAT A O N POPULATIONS ..., 73-80 
• jftSi-i 
•' i. \i. S 

Fig. 3: Coipodid ciliate Co!poda infiata. a) Camera luci­da drawing of silver nitrate impregnated specimen> ventral side; b) Micropbotograph of a specimen. Cy=cytostome; K=kineties; k=kinetosome. 1400x. Si 3: Migetalka Colpoda inflatar iz reda Colpodida. a) Risba (camera lucida) primerka, impregniranega s srebrovim nitratom, trebušna stran; Mikrofotografija primerka. Cy = citostom; K = kmete; k = kinetosom. 1400-kratna povečava. 
recorded in the present work may be due to the great accumulation of guano from the large colonies of various species of Chiroptera, which live in both branches of the cave. 
Analysis of heavy metals in waters indicated no presence of Cr, Mn, Fe, C o or Ni, whereas Cu, Zn and Cd were detected up to a maximum of 33, 38 and 0.54 ppb, respectively. It must be noted that the values of Cu and Zn are in the standard range for freshwaters, whereas that of Cd slightly exceeds the baseline value. The presence of Cd must be carefully considered before using these waters for drinking. 
Fig. 5: Morphology of hypotrichid ciliate Sterkiella his­triomuscorum with protargol impregnation, a) Camera lucida drawing of dorsal side of a specimen, 95Ox; b) drawing of ventral side of same specimen, 950x; c) mi­crophotograph of same specimen. 1400x. AZM=adoral zone of membranelles; BC=buccal cirrus; FC=frontal cirri; k=kinetosome; MC=marginal cirri; MDV=mid­ventral cirri; MA=macronucleus; MI=micronucleus; TC=transverse cirri; UM=undulating membrane. SI. 5; Morfologija migetalkarja Sterkiella histriomusco­rum iz reda Hypotrichida, impregniranega s protar­golom. Risba (camera lucida) hrbtne strani primerka. 950-kratna povečava; risba trebušne strani istega primerka, 950-kratna povečava; c) mikrofotografija istega primerka, 1400-kratna povečava. AZM « obustni predel membranel; BC = obustni čiri; FC ~ prednji ciri; k = kinetosom; MC= marginalni ciri; MDV -sred­njetrebušni ciri; MA = makronukleus; Ml = mikro­nukleus; TC = prečni ciri; UM = valovita membrana. 
Fig. 4: Hymenostomatid Glaucoma scintillans. a) Camera lucida drawing of silver nitrate impregnated specimen, ventral side, b) Drawing of dorsal side, c) Micropbotograph of same specimen. 2000x. OM=oral membranelles; Cp=cytoproct; Ki=kineties. SI. 4: Glaucoma scintillans iz reda Hymenostomatida. a) Risba (camera lucida) primerka, impregniranega s srebrovim nitratom, trebušna stran, b) Risba hrbtne strani, c) Mikrofotografija istega primerka. 2000-kratna povečava. OM = ustne membranele; Cy = citoprokt; K 
-kinete. 
...See %AZft 
•"« ' «J-""2™ 
t H V 
.<• uwf®™" 
f.\A 

Laura GUIDOLI N & Olimpia COPPEUOTT I KRUPA: PROTISTS FROM CAVES: PRELIMINARY DATA O N POPULATIONS .... 73-80 
Fig. 6: Phase contrast images of living protists. a) Naegleria sp. (Sarcodina, Lobosea); b) Acineta tuberosa 
(Ciliophora, Suctorida); c) Vorticella picta (Ciliophora, Peritrichida); d) Oxytricha fallax (Ciliophora, Hypotrichida). 
550x. 
SI. 6: Kontrastne podobe živih enoceličarjev. a) Naegleria sp. (Sarcodina, Lobosea); b) Acineta tuberosa 
(Ciliophora, Suctorida); c) Vorticella picta (Ciliophora, Peritrichida); d) Oxytricha fallax (Ciliophora, Hypotrichida). 
550-kratna povečava. 

Protists tected. Previous work (Boscolo, 1968) reported only 
three species of Peritrichida and one belonging to Initial results on Protist populations indicated a rich Suctorida, living on the gills or appendages of one spe­variety of organisms belonging to various taxa, such as cies of Amphipoda and one of Isopoda. All Protists were Mastigophora, Sarcodina and Ciliophora (see Tab. 2), found in puddles, walls, mud or guano deposited by the and about 70 taxa have been identified until now. large populations of Chiroptera living in the branches of Particular attention was paid to the last class. Indeed, the cave. The Protists identified belong to taxa living in until now, at least 42 species of ciliates have been de-damp soil, moss or a freshwater environment, according 
Laura CUIDOLI N & Olimpla COF'PELLOTTI KRUPA: PROTISTS FROM CAVES: PRELIMINARY DATA O N POPULATIONS .... 73-80 
to previous findings reported by Golemansky & Bonnet (1994) on caverniculous Protists. The extensive presence of bacteria in summer, especially in the bat guano, sus­tains many bacterivorous Protists, such as Colpidium colpoda. However, it must be noted that, according to Foissner (1987), many soil ciliates prey on other ciliates, zooflagellates, and/or naked and shelled amoebae, con­trasting with the widely accepted view that most soil ciliates are bacterivorous. 
In winter, many ciliates are recovered as temporary and protective cysts, developed in laboratory conditions with culture methods favouring the growth of bacteria. This was the case of many Hypotrichida (Oxytricha, Sterkiella, Stylonychia) and Colpodida (Colpoda). Figs. 3-5 show the morphology of some representative Protist taxa, after techniques of impregnation with silver pro­teinate or nitrate, and Fig. 6 shows four species of Protists observed In vivo by phase contrast microscopy. 
The high diversity of Vorticellidae, which are repre­sented by at least eight species, is noteworthy. Owing to the complex systematics of this taxon, it is possible that other species, new to science, are present. Studies are in progress to extend taxonomic studies to other groups of ciliates, especially Hypotrichida. The physiological characteristics of ciliate Protists living in the karst envi­ronment will also be considered, taking into account the fact that ciliates, essential components of nearly all eco­systems, are simple models for ecotoxicological studies, due to their relative ease of culture, short life-cycle, cosmopolitan distribution, and sensitivity to environ­mental changes. Future studies will also concern that part of the Covolo della Guerra cave which branches off to the south and which is not easily accessible. 


ACKNOWLEDGEMENTS 

Financial support was provided by University of Padova. Grateful thanks are due to Gabriel Walton for correcting the English text and to Claudio Friso for drawings. 
JAMSKI ENOCEUČARJI : PREDHODN E RAZISKAV E O NJIHOVI H POPULACIJA H V KRAŠKI JAMI "COVOL O DELL A GUERRA" V HRIBOVJ U BERICI (VICENZA , ITALIJA) 
Laura GUIDOLIN 
Department of Biology, University of Padova, IT-35131 Padova, Via G. Colombo 3 
E mail: Cuidolin@civ.bio.unipd.it 

Olimpia COPPELLOTTI KRUPA 
Department of Biology, University of Padova, IT-35131 Padova, Via G. Colombo 3 
E mail: Olimpiak@civ.bio.unipd.it 

POVZETEK 
Posledica povečanega zanimanj za speleologijo je nekaj prav presenetljivih odkritij iz obdobja zadnjih nekaj let v italijanskih jamah. Toda na podlagi zbranega favnističnega gradiva so bila do danes posebno raziskane le nekatere taksonomske skupine, na primer Insecta, Chilopoda, Gastropoda in Chiroptera. 
Zaradi pomena organizmov, ki živijo v skoraj vseh ekosistemih, se je zdelo še posebno zanimivo raziskati, kot preproste modele za sistem njihovega spremljanja (monitoringa), populacije enoceličarjev (s posebnim poudarkom na migetalkarjih) v italijanski kraški jami "Covolo della Guerra". 
Predhodni rezultati, dobljeni v severnem delu jame, zadevajo nekaj kemijsko-fizikalnih parametrov (ki so glede na tamkajšnji izvir vsekakor vredni pozornosti) in govorijo o precejšnji raznoterosti enoceličarjev, ki pripadajo različnim taksonom, kot na primer Mastigophora, Sarcodina in Ciliophora. Do danes je bilo raziskanih 70 taksonov, 42 od katerih so migetalkarji. 
Ključne besede: jama, kras, Vicenza, enoceličarji, Ciliophora, Mastigophora, Sarcodina 
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