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

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.

Telomere length and dynamics in Astyanax mexicanus cave and surface morphs.

Background: Telomeres are non-coding DNA repeats at the chromosome ends and their shortening is considered one of the major causes of aging. However, they also serve as a biomarker of environmental exposures and their length and attrition is affected by various stressors. In this study, we examined the average telomere length in Astyanax mexicanus, a species that has both surface-dwelling and cave-adapted populations. The cave morph descended from surface ancestors and adapted to a markedly different environment characterized by specific biotic and abiotic stressors, many of which are known to affect telomere length. Our objective was to explore whether telomere length differs between the two morphs and whether it serves as a biological marker of aging or correlates with the diverse environments the morphs are exposed to. Methods: We compared telomere length and shortening between laboratory-reared Pachón cavefish and Rio Choy surface fish of A. mexicanus across different tissues and ages. Results: Astyanax mexicanus surface fish exhibited longer average telomere length compared to cavefish. In addition, we did not observe telomere attrition in either cave or surface form as a result of aging in adults up to 9 years old, suggesting that efficient mechanisms prevent telomere-mediated senescence in laboratory stocks of this species, at least within this time frame. Our results suggest that telomere length in Astyanax may be considered a biomarker of environmental exposures. Cavefish may have evolved shorter and energetically less costly telomeres due to the absence of potential stressors known to affect surface species, such as predator pressure and ultra-violet radiation. This study provides the first insights into telomere dynamics in Astyanax morphs and suggests that shorter telomeres may have evolved as an adaptation to caves.

The first cavefish in the Dinaric Karst? Cave colonization made possible by phenotypic plasticity in Telestes karsticus.

Cave animals are an excellent model system for studying adaptive evolution. At present, however, little is known about the mechanisms that enable surface colonizers to survive in the challenging environment of caves. One possibility is that these species have the necessary genetic background to respond with plastic changes to the pressures of underground habitats. To gain insight into this process, we conducted a comparative study with the fish species Telestes karsticus, which occurs in a hydrological system consisting of an interconnected stream and a cave. Results showed that T. karsticus resided year-round and spawned in Susik cave, making it the first known cavefish in the Dinaric Karst. Cave and surface populations differed in morphological and physiological characteristics, as well as in patterns of gene expression without any evidence of genetic divergence. To test whether observed trait differences were plastic or genetic, we placed adult fish from both populations under light/dark or constant dark conditions. Common laboratory conditions erased all morphometric differences between the two morphs, suggesting phenotypic plasticity is driving the divergence of shape and size in wild fish. Lighter pigmentation and increased fat deposition exhibited by cave individuals were also observed in surface fish kept in the dark in the laboratory. Our study also revealed that specialized cave traits were not solely attributed to developmental plasticity, but also arose from adult responses, including acclimatization. Thus, we conclude that T. karsticus can adapt to cave conditions, with phenotypic plasticity playing an important role in the process of cave colonization.

Pleiotropic function of the oca2 gene underlies the evolution of sleep loss and albinism in cavefish.

Adaptation to novel environments often involves the evolution of multiple morphological, physiological, and behavioral traits. One striking example of multi-trait evolution is the suite of traits that has evolved repeatedly in cave animals, including regression of eyes, loss of pigmentation, and enhancement of non-visual sensory systems.1,2 The Mexican tetra, Astyanax mexicanus, consists of fish that inhabit at least 30 caves in Mexico and ancestral-like surface fish that inhabit the rivers of Mexico and southern Texas.3 Cave A. mexicanus are interfertile with surface fish and have evolved a number of traits, including reduced pigmentation, eye loss, and alterations to behavior.4-6 To define relationships between different cave-evolved traits, we phenotyped 208 surface-cave F2 hybrid fish for numerous morphological and behavioral traits. We found differences in sleep between pigmented and albino hybrid fish, raising the possibility that these traits share a genetic basis. In cavefish and other species, mutations in oculocutaneous albinism 2 (oca2) cause albinism.7-12 Surface fish with mutations in oca2 displayed both albinism and reduced sleep. Further, this mutation in oca2 fails to complement sleep loss when surface fish harboring this engineered mutation are crossed to independently evolved populations of albino cavefish with naturally occurring mutations in oca2. Analysis of the oca2 locus in wild-caught cave and surface fish suggests that oca2 is under positive selection in 3 cave populations. Taken together, these findings identify oca2 as a novel regulator of sleep and suggest that a pleiotropic function of oca2 underlies the adaptive evolution of albinism and sleep loss.

Collecting eco-evolutionary data in the dark: Impediments to subterranean research and how to overcome them.

Caves and other subterranean habitats fulfill the requirements of experimental model systems to address general questions in ecology and evolution. Yet, the harsh working conditions of these environments and the uniqueness of the subterranean organisms have challenged most attempts to pursuit standardized research.Two main obstacles have synergistically hampered previous attempts. First, there is a habitat impediment related to the objective difficulties of exploring subterranean habitats and our inability to access the network of fissures that represents the elective habitat for the so-called "cave species." Second, there is a biological impediment illustrated by the rarity of most subterranean species and their low physiological tolerance, often limiting sample size and complicating laboratory experiments.We explore the advantages and disadvantages of four general experimental setups (in situ, quasi in situ, ex situ, and in silico) in the light of habitat and biological impediments. We also discuss the potential of indirect approaches to research. Furthermore, using bibliometric data, we provide a quantitative overview of the model organisms that scientists have exploited in the study of subterranean life.Our over-arching goal is to promote caves as model systems where one can perform standardized scientific research. This is important not only to achieve an in-depth understanding of the functioning of subterranean ecosystems but also to fully exploit their long-discussed potential in addressing general scientific questions with implications beyond the boundaries of this discipline.

Phenotypic plasticity as a mechanism of cave colonization and adaptation.

A widely accepted model for the evolution of cave animals posits colonization by surface ancestors followed by the acquisition of adaptations over many generations. However, the speed of cave adaptation in some species suggests mechanisms operating over shorter timescales. To address these mechanisms, we used Astyanax mexicanus, a teleost with ancestral surface morphs (surface fish, SF) and derived cave morphs (cavefish, CF). We exposed SF to completely dark conditions and identified numerous altered traits at both the gene expression and phenotypic levels. Remarkably, most of these alterations mimicked CF phenotypes. Our results indicate that many cave-related traits can appear within a single generation by phenotypic plasticity. In the next generation, plasticity can be further refined. The initial plastic responses are random in adaptive outcome but may determine the subsequent course of evolution. Our study suggests that phenotypic plasticity contributes to the rapid evolution of cave-related traits in A. mexicanus.

The Mexican tetra is a fish that has two forms: a surface-dwelling form, which has eyes and silvery grey appearance, and a cave-dwelling form, which is blind and has lost its pigmentation. Recent studies have shown that the cave-dwelling form evolved rapidly within the last 200,000 years from an ancestor that lived at the surface. The recent evolution of the cave-dwelling form of the tetra poses an interesting evolutionary question: how did the surface-dwelling ancestor of the tetra quickly adapt to the new and challenging environment found in the caves? 'Phenotypic plasticity' is a phenomenon through which a single set of genes can produce different observable traits depending on the environment. An example of phenotypic plasticity occurs in response to diet: in animals, poor diets can lead to an increase in the size of the digestive organs and to the animals eating more. To see if surface-dwelling tetras can quickly adapt to cave environments through phenotypic plasticity, Bilandzija et al. have exposed these fish to complete darkness (the major feature of the cave environment) for two years. After spending up to two years in the dark, these fish were compared to normal surface-dwelling and cave-dwelling tetras. Results revealed that surface-dwelling tetras raised in the dark exhibited traits associated with cave-dwelling tetras. These traits included changes in the activity of many genes involved in diverse processes, resistance to starvation, metabolism, and levels of hormones and molecules involved in neural signaling, which could lead to changes in behavior. However, the fish also exhibited traits, including an increase in the cells responsible for pigmentation, that would have no obvious benefit in the darkness. Even though the changes observed require no genetic mutations, they can help or hinder the fish's survival once they occur, possibly determining subsequent evolution. Thus, a trait beneficial for surviving in the dark that appears simply through phenotypic plasticity may eventually be selected for and genetic mutations that encode it more reliably may appear too. These results shed light on how species may quickly adapt to new environments without accumulating genetic mutations, which can take hundreds of thousands of years. They also may help to explain how colonizer species succeed in challenging environments. The principles described by Bilandzija et al. can be applied to different organisms adapting to new environments, and may help understand the role of phenotypic plasticity in evolution.

The role of gene flow in rapid and repeated evolution of cave-related traits in Mexican tetra, Astyanax mexicanus.

Understanding the molecular basis of repeatedly evolved phenotypes can yield key insights into the evolutionary process. Quantifying gene flow between populations is especially important in interpreting mechanisms of repeated phenotypic evolution, and genomic analyses have revealed that admixture occurs more frequently between diverging lineages than previously thought. In this study, we resequenced 47 whole genomes of the Mexican tetra from three cave populations, two surface populations and outgroup samples. We confirmed that cave populations are polyphyletic and two Astyanax mexicanus lineages are present in our data set. The two lineages likely diverged much more recently than previous mitochondrial estimates of 5-7 mya. Divergence of cave populations from their phylogenetically closest surface population likely occurred between ~161 and 191 k generations ago. The favoured demographic model for most population pairs accounts for divergence with secondary contact and heterogeneous gene flow across the genome, and we rigorously identified gene flow among all lineages sampled. Therefore, the evolution of cave-related traits occurred more rapidly than previously thought, and trogolomorphic traits are maintained despite gene flow with surface populations. The recency of these estimated divergence events suggests that selection may drive the evolution of cave-derived traits, as opposed to disuse and drift. Finally, we show that a key trogolomorphic phenotype QTL is enriched for genomic regions with low divergence between caves, suggesting that regions important for cave phenotypes may be transferred between caves via gene flow. Our study shows that gene flow must be considered in studies of independent, repeated trait evolution.

Behavioural changes controlled by catecholaminergic systems explain recurrent loss of pigmentation in cavefish.

Multiple cave populations of the teleost Astyanax mexicanus have repeatedly reduced or lost eye and body pigmentation during adaptation to dark caves. Albinism, the complete absence of melanin pigmentation, is controlled by loss-of-function mutations in the oca2 gene. The mutation is accompanied by an increase in the melanin synthesis precursor l-tyrosine, which is also a precursor for catecholamine synthesis. In this study, we show a relationship between pigmentation loss, enhanced catecholamine synthesis and responsiveness to anaesthesia, determined as a proxy for catecholamine-related behaviours. We demonstrate that anaesthesia resistance (AR) is enhanced in multiple depigmented and albino cavefish (CF), inversely proportional to the degree of pigmentation loss, controlled by the oca2 gene, and can be modulated by experimental manipulations of l-tyrosine or the catecholamine norepinephrine (NE). Moreover, NE is increased in the brains of multiple albino and depigmented CF relative to surface fish. The results provide new insights into the evolution of pigment modification because NE controls a suite of adaptive behaviours similar to AR that may represent a target of natural selection. Thus, understanding the relationship between loss of pigmentation and AR may provide insight into the role of natural selection in the evolution of albinism via a melanin-catecholamine trade-off.

Characterization of a group I Nme protein of Capsaspora owczarzaki-a close unicellular relative of animals.

Nucleoside diphosphate kinases are enzymes present in all domains of life. In animals, they are called Nme or Nm23 proteins, and are divided into group I and II. Human Nme1 was the first protein identified as a metastasis suppressor. Because of its medical importance, it has been extensively studied. In spite of the large research effort, the exact mechanism of metastasis suppression remains unclear. It is unknown which of the biochemical properties or biological functions are responsible for the antimetastatic role of the mammalian Nme1. Furthermore, it is not clear at which point in the evolution of life group I Nme proteins acquired the potential to suppress metastasis, a process that is usually associated with complex animals. In this study we performed a series of tests and assays on a group I Nme protein from filasterean Capsaspora owczarzaki, a close unicellular relative of animals. The aim was to compare the protein to the well-known human Nme1 and Nme2 homologs, as well as with the homolog from a simple animal-sponge (Porifera), in order to see how the proteins changed with the transition to multicellularity, and subsequently in the evolution of complex animals. We found that premetazoan-type protein is highly similar to the homologs from sponge and human, in terms of biochemical characteristics and potential biological functions. Like the human Nme1 and Nme2, it is able to diminish the migratory potential of human cancer cells in culture.

Melanization in response to wounding is ancestral in arthropods and conserved in albino cave species.

Many species adapted to aphotic subterranean habitats have lost all body pigmentation. Yet, melanization is an important component of wound healing in arthropods. We amputated appendages in a variety of cave-adapted and surface-dwelling arthropods. A dark clot formed at the site of injury in most species tested, including even albino cave-adapted species. The dark coloration of the clots was due to melanin deposition. The speed of wound melanization was uncorrelated with a difference in metabolic rate between surface and cave populations of an amphipod. The chelicerate Limulus polyphemus, all isopod crustaceans tested, and the cave shrimp Troglocaris anophthalmus did not melanize wounds. The loss of wound melanization in T. anophthalmus was an apomorphy associated with adaptation to subterranean habitats, but in isopods it appeared to be a symplesiomorphy unrelated to colonization of subterranean habitats. We conclude that wound melanization i) is an important part of innate immunity because it was present in all major arthropod lineages, ii) is retained in most albino cave species, and iii) has been lost several times during arthropod evolution, indicating melanization is not an indispensable component of wound healing in arthropods.

A potential benefit of albinism in Astyanax cavefish: downregulation of the oca2 gene increases tyrosine and catecholamine levels as an alternative to melanin synthesis.

Albinism, the loss of melanin pigmentation, has evolved in a diverse variety of cave animals but the responsible evolutionary mechanisms are unknown. In Astyanax mexicanus, which has a pigmented surface dwelling form (surface fish) and several albino cave-dwelling forms (cavefish), albinism is caused by loss of function mutations in the oca2 gene, which operates during the first step of the melanin synthesis pathway. In addition to albinism, cavefish have evolved differences in behavior, including feeding and sleep, which are under the control of the catecholamine system. The catecholamine and melanin synthesis pathways diverge after beginning with the same substrate, L-tyrosine. Here we describe a novel relationship between the catecholamine and melanin synthesis pathways in Astyanax. Our results show significant increases in L-tyrosine, dopamine, and norepinephrine in pre-feeding larvae and adult brains of Pachón cavefish relative to surface fish. In addition, norepinephrine is elevated in cavefish adult kidneys, which contain the teleost homologs of catecholamine synthesizing adrenal cells. We further show that the oca2 gene is expressed during surface fish development but is downregulated in cavefish embryos. A key finding is that knockdown of oca2 expression in surface fish embryos delays the development of pigmented melanophores and simultaneously increases L-tyrosine and dopamine. We conclude that a potential evolutionary benefit of albinism in Astyanax cavefish may be to provide surplus L-tyrosine as a precursor for the elevated catecholamine synthesis pathway, which could be important for adaptation to the challenging cave environment.

Evolutionary history of relict Congeria (Bivalvia: Dreissenidae): unearthing the subterranean biodiversity of the Dinaric Karst.

BACKGROUND: Patterns of biodiversity in the subterranean realm are typically different from those encountered on the Earth's surface. The Dinaric karst of Croatia, Slovenia and Bosnia and Herzegovina is a global hotspot of subterranean biodiversity. How this was achieved and why this is so remain largely unresolved despite a long tradition of research. To obtain insights into the colonisation of the Dinaric Karst and the effects of the subterranean realm on its inhabitants, we studied the tertiary relict Congeria, a unique cave-dwelling bivalve (Dreissenidae), using a combination of biogeographical, molecular, morphological, and paleontological information. RESULTS: Phylogenetic and molecular clock analyses using both nuclear and mitochondrial markers have shown that the surviving Congeria lineage has actually split into three distinct species, i.e., C. kusceri, C. jalzici sp. nov. and C. mulaomerovici sp. nov., by vicariant processes in the late Miocene and Pliocene. Despite millions of years of independent evolution, analyses have demonstrated a great deal of shell similarity between modern Congeria species, although slight differences in hinge plate structure have enabled the description of the two new species. Ancestral plesiomorphic shell forms seem to have been conserved during the processes of cave colonisation and subsequent lineage isolation. In contrast, shell morphology is divergent within one of the lineages, probably due to microhabitat differences. CONCLUSIONS: Following the turbulent evolution of the Dreissenidae during the Tertiary and major radiations in Lake Pannon, species of Congeria went extinct. One lineage survived, however, by adopting a unique life history strategy that suited it to the underground environment. In light of our new data, an alternative scenario for its colonisation of the karst is proposed. The extant Congeria comprises three sister species that, to date, have only been found to live in 15 caves in the Dinaric karst. Inter-specific morphological stasis and intra-specific ecophenotypic plasticity of the congerid shell demonstrate the contrasting ways in which evolution in the underground environments shapes its inhabitants.

Evolution of albinism in cave planthoppers by a convergent defect in the first step of melanin biosynthesis.

Albinism, the reduction or loss of melanin pigment, is found in many diverse cave-dwelling animals. The mechanisms responsible for loss of melanin pigment are poorly understood. In this study we use a melanogenic substrate assay to determine the position where melanin synthesis is blocked in independently evolved cave planthoppers from Hawaii and Croatia. In this assay, substrates of enzymes responsible for melanin biosynthesis are added to fixed specimens in vitro and their ability to rescue black melanin pigmentation is determined. L-tyrosine, the first substrate in the pathway, did not produce melanin pigment, whereas L-DOPA, the second substrate, restored black pigment. Substrates in combination with enzyme inhibitors were used to test the possibility of additional downstream defects in the pathway. The results showed that downstream reactions leading from L-DOPA and dopamine to DOPA-melanin and dopamine-melanin, the two types of insect melanin, are functional. It is concluded that albinism is caused by a defect in the first step of the melanin synthesis pathway in cave-adapted planthoppers from widely separated parts of the world. However, Western blots indicated that tyrosine hydroxylase (TH), the only enzyme shown to operate at the first step in insects, is present in Hawaiian cave planthoppers. Thus, an unknown factor(s) operating at this step may be important in the evolution of planthopper albinism. In the cavefish Astyanax mexicanus, a genetic defect has also been described at the first step of melanin synthesis suggesting convergent evolution of albinism in both cave-adapted insects and teleosts.

Taxonomic position of Eunapius subterraneus (Porifera, Spongillidae) inferred from molecular data--a revised classification needed?

The freshwater sponge Eunapius subterraneus was described in 1984 on the basis of its morphology and unique ecological features. It inhabits caves in the Ogulin karst area as the only known stygobitic sponge, and an endangered karst species. We used three genetic markers with different evolutionary rates in phylogenetic analyses of E. subterraneus. All of the markers exclude this sponge from the genus Eunapius. Based on our results, we emphasize the need for revision of the taxonomic classification of E. subterraneus as well as the need for a thorough re-evaluation of freshwater sponge systematics.