A practical guide for the husbandry of cave and surface invertebrates as the first step in establishing new model organisms

Abstract

While extensive research on traditional model species has significantly advanced the biological sciences, the ongoing search for new model organisms is essential to tackle contemporary challenges such as human diseases or climate change, and fundamental phenomena including adaptation or speciation. Recent methodological advances such as next-generation sequencing, gene editing, and imaging are widely applicable and have simplified the selection of species with specific traits from the wild. However, a critical milestone in this endeavor remains the successful cultivation of selected species. A historically overlooked but increasingly recognized group of non-model organisms are cave dwellers. These unique animals offer invaluable insights into the genetic basis of human diseases like eye degeneration, metabolic and neurological disorders, and basic evolutionary principles and the origin of adaptive phenotypes. However, to take advantage of the beneficial traits of cave-dwelling animals, laboratory cultures must be established-a practice that remains extremely rare except for the cavefish Astyanax mexicanus. For most cave-dwelling organisms, there are no published culturing protocols. In this study, we present the results of our multi-year effort to establish laboratory cultures for a variety of invertebrate groups. We have developed comprehensive protocols for housing, feeding, and husbandry of cave dwellers and their surface relatives. Our recommendations are versatile and can be applied to a wide range of species. Hopefully our efforts will facilitate the establishment of new laboratory animal facilities for cave-dwelling organisms and encourage their greater use in experimental biology.

Fig 1. Schematic diagram of steps necessary to establish a culture of wild species to be used in laboratory experiments.

Credit: Iva Čupić, Tin Rožman.

Fig 2. Photos of selected animals used in our studies, one species per genus.

A. Alpioniscus balthasari. B. Titanethes albus. C. Proasellus karamani. D. Asellus aquaticus. E. Ceacidotea pricei. F Monolistra pretneri. G. Lekanesphaera hookeri. H. Physella sp. Scale bars, 5 mm. Credits: Jana Bedek (a), Mike Slay (e), Tin Rožman (b, c, d, f, g, h).

Fig 3. Representation of housing used in our studies.

A. Plastic containers used for aquatic animals. B. Plastic containers with bottom made of plaster used for terrestrial animals. C. Plastic containers with a plaster bottom inclined to form a large pool of water optimal for rearing larger semi-aquatic cave trichoniscids. Credit: Iva Čupić.

Fig 4. Food used in our studies.

A. Food pellets cut into cubes. B. Conditioned leaves packaged in small bags are kept frozen until use. Credit: Tin Rožman.

Fig 5. Diseases and affected colonies.

A. Aquatic isopod Monolistra radjai with fungal overgrowth. B. Dreissenid bivalve (Congeria sp.) with fungal infestation. C. Physid snail (Physella sp.) with bacterial overgrowth on the shell. D. Physid snail (Physella sp.) with colonies of rotifers on the shell. Credit: Tin Rožman.