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Objective obstacles such as high water levels that prevent access to caves at certain times of the year or the need to use complex tools and skills such as cave diving make biological research in caves extremely complex and occasionally dangerous. Moreover, to study physiological and behavioral adaptations of cave animals, they must be kept under controlled laboratory conditions. For this reason, we have established an animal facility in our laboratory. The available literature on invertebrate setup, operation, and care was sparse and unsuitable for the species we were studying. Fortunately, many cave biologists provided us with advice and support during the initial phase. Here we present our experiences to provide some guidelines for other researchers undertaking similar efforts. Among other animal groups, we are culturing several morphotypes of the cave snail Physella sp. and two cave and one surface species pair of bivalves: Congeria spp. and Dreissena polymorpha. We discuss good and bad practices in mollusk care - housing, water treatment, different types of food, and small aquarium design. Special attention is given to problems we have encountered with our colonies and changes we have made to address these problems, both successful and unsuccessful. We also address the general protocols required for keeping multiple species in a single facility, including procedures for decontamination of equipment and tools, and quarantine.

Many aspects of cave animal biology cannot be understood through field observations alone. Access to some subterranean habitats is often technically challenging, rendering long-term monitoring of cave-dwelling species or investigating some aspects of their biology difficult. Also, contemporary research questions and methods require sampling individuals at certain life stages, and most cave adapted species have lengthy life spans or are not accessible in their natural habitats year-round. Therefore, we have established an animal facility in our laboratory where we keep cave-dwelling and related surface-dwelling crustacean species under controlled conditions. This allows us to study physiological and behavioral adaptations and the role of phenotypic plasticity in the evolution of these traits in cave dwellers. We are culturing freshwater, marine, and terrestrial isopods from three different families (Asellidae, Sphaeromatidae, and Trichoniscidae), among others. To date, we have established breeding colonies of several different cave and surface species pairs of aquatic asellids, and our most successful colonies have lived for two years and three generations. We have also established colonies of cave (freshwater) and surface (marine) sphaeromatids with varying degrees of success. Among terrestrial trichoniscids, colonies of cave species have persisted for more than two years, but without successful reproduction. Our repeated attempts to maintain surface species of Trichoniscus have been unsuccessful. Here we present our experiences to provide guidance for other researchers making similar attempts. We discuss general care practices such as decontamination of equipment and tools, quarantine, good and bad practices for small aquarium and terrarium design, housing, water treatment and conditioning, various food sources, and breeding approaches. We also review some of the problems we have encountered with our colonies and describe our attempts to solve them with varying degrees of success.

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Biology Students Association – BIUS (BIUS) from Zagreb, Croatia is a non-government and non-profit association founded in 1999 by biology students from the Faculty of Science, University of Zagreb. Aiming to develop future experts in different fields of biology, BIUS is composed of groups that specialize in various subject areas. Besides holding diverse science lectures, workshops, and field trips, the main BIUS' activities are annual student research camps with the participation of up to 130 members. An integral part of BIUS from the very beginning is the Biospeleology group (Group). During these first 23 years of the Group's existence, many members discovered their interest in cave biology through field research, cabinet work, and educational workshops. The Group organized two student research camps and participated in 16 others. In addition, the Group organized seven independent projects. Throughout all of these projects, cave fauna was collected from over 150 caves and pits at different localities of Croatian karst. The Group's long-term existence is facilitated by the guidance of mentors from the Croatian Biospeleological Society. Altogether, the Group provides numerous skills and valuable opportunities to students and is important for developing future scientific experts in cave biology in Croatia. So far, 12 Group members have written their master thesis and four have their doctoral thesis in the field of cave biology. Former members currently work at universities, research institutes, government, and civil sector, or as entrepreneurs on topics closely related to cave biology and karst research.

Terenska istraživanja za Drugu biospeleološku ekspediciju – Pelješac 2019 su provedena između studenog 2018. i prosinca 2020. godine. Ekspediciju su organizirali Hrvatsko biospeleološko društvo i Breganja, a sudjelovalo je 27 biospeleologa i speleologa iz 9 udruga i 5 država.Posjećeno je 46 speleoloških objekata od kojih je 26 speleološki istraženo i izrađen je topografski snimak. Najdublja je Jama u priroju dubine 124 m, a najveća špilja Spilja Rupine duljine 93 m. U Tomasovoj spilji je pronađeno puno ulomaka keramike prapovijesne i antičke starosti.U 40 špilja i jama je sakupljana fauna. Otkriven je 1 novi rod pauka, 1 novi rod kornjaša i 10 novih vrsta iz skupina pauka, jednakonožnih rakova, skokuna i kornjaša.

Biokovo Mountain in Croatia is a Dinaric hotspot of subterranean biodiversity and its area of 196 km 2 is protected as a nature park. Until now, more than 400 caves have been registered here, however, only in about 30% of them biospeleological research has been carried out. It is hypothesized that over 1000 caves exist in highly karstified Biokovo Mt. Biospeleological research of the area started at the beginning of 20th century and deep caves were infrequently explored after the year 1980. Previous research recorded more than 90 troglobiotic species in this area and most of them are endemic. The primary goal of the Expedition was a systematic survey of eight deep caves (depth over 250 m), one of which was never before biospeleologically surveyed. We also surveyed other caves from which material for detailed faunistic analysis was missing. The main part of the expedition took place from 17 th to 25 th June 2017, with 52 participants. Fauna was collected by hand and by pitfall traps with baits, in the total of 26 caves. Five deep caves were completely surveyed (depth up to 710 m), the topographic map was made when missing, and fauna was photographed. Microclimatic conditions were measured and in two caves data loggers were placed. According to preliminary results, around 10 new species for science have been found from following groups: leeches (Hirudinea), spiders (Araneae), harvestmen (Opiliones), woodlice (Isopoda), centipedes (Chilopoda), springtails (Collembola), two-pronged bristletails (Diplura) and beetles (Coleoptera). These results confirm that deep caves of Biokovo Mt are still underexplored and that detailed and systematic research is needed.

Pretpostavlja se da na Biokovu postoji preko 1 000 speleoloških objekata od kojih je nešto više od 400 do sada zabilježeno, a više od 115 je biospeleološki istraženo. Prijašnja istraživanja podzemne faune pokazala su da je Biokovo jedno od najbogatijih područja podzemnom faunom u Hrvatskoj. Navedeni podaci su ukazali na potrebu za dodatnim biospeleološkim istraživanjem speleoloških objekata Biokova. Prvu biospeleološku ekspediciju – Biokovo 2017 je organiziralo Hrvatsko biospeleološko društvo u suradnji s udrugom Breganja i Speleološkim odsjekom Planinarskog društva Imber. Ekspedicija je početno imala dva glavna cilja: biospeleološki istražiti 8 dubokih jama na području Parka prirode Biokovo i ponoviti nacrt Vilimove jame (A-2). Glavni termin ekspedicije je bio od 17. do 25. lipnja 2017. Prije same ekspedicije su napravljena dva terenska izlaska u svibnju i lipnju kada su označeni pristupi do dubokih jama i glavnina objekata se opremila, te jedan teren u listopadu nakon ekspedicije. Na ekspediciji je prema preliminarnim rezultatima pronađeno 10-ak novih vrsta za znanost. Topografski su snimljene 2 duboke jame (Jama pod Kamenitim vratima i Nova velika), Vilimova jama (A-2) do 101 m dubine, A-1 do 110 m dubine i 4 manja speleološka objekta. Biospeleološka istraživanja su provedena u ukupno 26 speleoloških objekata, među kojima je bilo 8 dubokih jama. Ekspedicija je brojila ukupno 52 sudionika među kojima 43 biospeleologa i/ili speleologa iz 20 speleoloških i bioloških udruga i organizacija. Među sudionicima je bilo i 6 stranaca., It is conjectured that there are over 1000 caves on the Biokovo Mountain of which more than 400 have been found so far and 115 have been biologically surveyed. Earlier research suggested that the Biokovo Mountain is home to the richest subterranean fauna in Croatia and that further understanding of the cave biology was necessary. The “1st Biospeleological Expedition - Biokovo 2017” was organised by the Croatian Biospeleological Society in cooperation with the Breganja Association and Imber Speleological Section. The two main goals of the expedition were to biologically survey eight deep caves in the Biokovo Nature Park and to update the survey of Vilimova Jama (A-2). The main part of the expedition took place from 17th to 25th June 2017 but it took two other visits in May and June to mark the approaches to the deep caves and to equip most of them. More work was also done in October after the expedition. According to preliminary results, around 10 new species have been found, two deep caves (Jama Pod Kamenitim Vratima and Nova Velika) and four shallower caves were completely surveyed, and Vilimova Jama (A-2) Cave and A-1 Cave were mapped to depths of 101 m and 110 m, respectively. Biospeleological research was carried out in 26 caves of which eight are deep caves. Altogether 52 people participated in the expedition of which 43 were speleologists or biospeleologists from twenty different speleological and biological associations. Among the participants, six people were from other countries.

Background and Purpose: The signal crayfish Pacifastacus leniusculus (Dana, 1852) is one of the most successful invasive species of crayfish in European freshwaters, an extremely diverse though endangered group of ecosystems. The main goal of this study was to functionally characterize multixenobiotic resistance (MXR) mechanism defense activity in P. leniusculus tissues for the first time. MXR mechanism protects the cell from a wide variety of toxic compounds, and it is mediated by the transport activity of ATP-binding cassette (ABC) proteins. Materials and Methods: MXR transporter activity dye assay was performed by using fluorescent model substrate rhodamine B (RB) in combination with inhibitors of MXR efflux pumps: MK571 and Verapamil, known to inhibit multidrug resistance-associated proteins (MRP) and P-glycoprotein (P-gp), respectively. In this assay, the increase in intracellular fluorescence of the substrate dye, indicates inhibition of MXR efflux protein pumps. The assay was performed in three different tissues (gills, hepatopancreas, tail muscle). Additionally, tissues were exposed to selected heavy metals – mercury (HgCl2) and zinc (ZnCl2), known to occur in open freshwaters as pollutants. Results: Optimal time for RB accumulation in gills and hepatopancreas was determined to be 30 minutes. RB efflux in gills was inhibited by MK571 and in hepatopancreas by Verapamil, suggesting that multidrug resistanceassociated proteins are dominant in gills of P. leniusculus, and P-glycoprotein in hepatopancreas. Finally, inhibitory effect of mercury (HgCl2: 10 and 20 μM) and zinc (ZnCl2: 5–20 μM) on multixenobiotic resistance mechanism activity in gills, and only mercury in hepatopancreas, was detected. Conclusions: The results for the first time demonstrate the presence of multixenobiotic resistance mechanism efflux activity as an important tissue specific defense mechanism in P. leniusculus and provide the basis for future molecular and toxicological studies of this invasive and adaptable species.