Phreatichthys Research Articles

Cavefishes in Chronobiological Research: A Narrative Review

Cavefish are vertebrates living in extreme subterranean environments with no light, temperature changes, and limited food. Circadian rhythms in these fish are suppressed in natural habitats. However, they can be found in artificial light-dark cycles and other zeitgebers. The molecular circadian clock has its peculiarities in cavefish. In Astyanax mexicanus, the core clock mechanism is tonically repressed in the caves due to the overactivation of the light input pathway. A lack of functional light input pathway but rather the entrainment of circadian genes' expression by scheduled feeding were revealed in more ancient Phreatichthys andruzzii. Different evolutionarily determined irregularities in the functioning of molecular circadian oscillators can be expected in other cavefish. The unique property of some species is the existence of surface and cave forms. Along with the ease of maintenance and breeding, it made cavefish a promising model for chronobiological studies. At the same time, a divergence of the circadian system between cavefish populations requires the strain of origin to be indicated in further research.

The subterranean catfish Phreatobius cisternarum provides insights into visual adaptations to the phreatic environment

Vertebrate eyes share the same general organization, though species have evolved morphological and functional adaptations to diverse environments. Cave-adapted animals are characterized by a variety of features including eye reduction, loss of body pigmentation, and enhanced non-visual sensory systems. Species that live in perpetual darkness have also evolved sensory mechanisms that are independent of light stimuli. The subterranean catfish Phreatobius cisternarum lives in the Amazonian phreatic zone and displays a diversity of morphological features that are similar to those observed in cavefish and appear to be adaptations to life in the dark. Here we combine histological and transcriptome analyses to characterize sensory adaptations of P. cisternarum to the subterranean environment. Histological analysis showed that the vestigial eyes of P. cisternarum contain a rudimentary lens. Transcriptome analysis revealed a repertoire of eleven visual and non-visual opsins and the expression of 36 genes involved in lens development and maintenance. In contrast to other cavefish species, such as Astyanax mexicanus, Phreatichthys andruzzii, Sinocyclocheilus anophthalmus and Sinocyclocheilus microphthalmus, DASPEI neuromast staining patterns did not show an increase in the number of sensory hair cells. Our work reveals unique adaptations in the visual system of P. cisternarum to underground habitats and helps to shed light into troglomorphic attributes of subterranean animals.

From the darkness, light

From the darkness, light

The skull osteology of the blind cave cyprinid Phreatichthys andruzzii Vinciguerra, 1924

The present article describes the morphology of the skull of Phreatichthys andruzzii Vinciguerra, 1924, blind cave fish from central Somalia. The main findings concern a general flattening of the skull and the loss of several bones (coronomeckelian, prefrontal, supratemporal, intercalar, supraopercle) belonging to the various regions of the head skeleton; but the more important modifications are shown by the orbital region, consisting in the complete disappearance of lachrymal, suborbital, sclerotics, and the substantial reduction of the other circumorbital bones. Usually the cranial lateral line canals run through the dermal bones; on the contrary in P. andruzzii the canals are nearly exclusively sheathed by thin bony muffs. These are sometimes fused to the underlying dermal bones, but in several regions they remain fully separated and independent, running through the adjoining soft tissues. The considerable degree of osteological regression shown by the skull appears to be related to the high level of a...

Extra-Visual Systems in the Spatial Reorientation of Cavefish

Disoriented humans and animals are able to reorient themselves using environmental geometry (“metric properties” and “sense”) and local features, also relating geometric to non-geometric information. Here we investigated the presence of these reorientation spatial skills in two species of blind cavefish (Astyanax mexicanus and Phreatichthys andruzzii), in order to understand the possible role of extra-visual senses in similar spatial tasks. In a rectangular apparatus, with all homogeneous walls (geometric condition) or in presence of a tactilely different wall (feature condition), cavefish were required to reorient themselves after passive disorientation. We provided the first evidence that blind cavefish, using extra-visual systems, were able i) to use geometric cues, provided by the shape of the tank, in order to recognize two geometric equivalent corners on the diagonal, and ii) to integrate the geometric information with the salient cue (wall with a different surface structure), in order to recover a specific corner. These findings suggest the ecological salience of the environmental geometry for spatial orientation in animals and, despite the different niches of adaptation, a potential shared background for spatial navigation. The geometric spatial encoding seems to constitute a common cognitive tool needed when the environment poses similar requirements to living organisms.

Modulation of DNA Repair Systems in Blind Cavefish during Evolution in Constant Darkness

How the environment shapes the function and evolution of DNA repair systems is poorly understood. In a comparative study using zebrafish and the Somalian blind cavefish, Phreatichthys andruzzii, we reveal that during evolution for millions of years in continuous darkness, photoreactivation DNA repair function has been lost in P.andruzzii. We demonstrate that this loss results in part from loss-of-function mutations in pivotal DNA-repair genes. Specifically, C-terminal truncations in P.andruzzii DASH and 6-4 photolyase render these proteins predominantly cytoplasmic, with consequent loss in their functionality. In addition, we reveal a general absence oflight-, UV-, and ROS-induced expression of P.andruzzii DNA-repair genes. This results from a loss of function of the D-box enhancer element, which coordinates and enhances DNA repair in response to sunlight. Our results point to P.andruzzii being the only species described, apart from placental mammals, that lacks the highly evolutionary conserved photoreactivation function. We predict that in the DNA repair systems of P.andruzzii, we may be witnessing the first stages in a process that previously occurred in the ancestors of placental mammals during the Mesozoic era.

Evolution: A Dark Past

The cavefish Phreatichthys andruzzii has lost the capacity to perform light-mediated DNA repair. This finding provides further support for the 'nocturnal bottleneck' hypothesis which posits that many key mammalian traits originated from adaptations linked to the nocturnal lifestyle of our early evolutionary ancestors.

Mutations in blind cavefish target the light-regulated circadian clock gene, period 2

Light represents the principal signal driving circadian clock entrainment. However, how light influences the evolution of the clock remains poorly understood. The cavefish Phreatichthys andruzzii represents a fascinating model to explore how evolution under extreme aphotic conditions shapes the circadian clock, since in this species the clock is unresponsive to light. We have previously demonstrated that loss-of-function mutations targeting non-visual opsins contribute in part to this blind clock phenotype. Here, we have compared orthologs of two core clock genes that play a key role in photic entrainment, cry1a and per2, in both zebrafish and P. andruzzii. We encountered aberrantly spliced variants for the P. andruzzii per2 transcript. The most abundant transcript encodes a truncated protein lacking the C-terminal Cry binding domain and incorporating an intronic, transposon-derived coding sequence. We demonstrate that the transposon insertion leads to a predominantly cytoplasmic localization of the cavefish Per2 protein in contrast to the zebrafish ortholog which is distributed in both the nucleus and cytoplasm. Thus, it seems that during evolution in complete darkness, the photic entrainment pathway of the circadian clock has been subject to mutation at multiple levels, extending from opsin photoreceptors to nuclear effectors.

Immunocytochemical identification and ontogeny of adenohypophyseal cells in a cave fish, Phreatichthys andruzzii (Cypriniformes: Cyprinidae).

The morphogenesis of the pituitary gland and the chronological appearance of adenohypophyseal cells were investigated for the first time in the Somalian cave fish Phreatichthys andruzzii by immunocytochemistry. The adult adenohypophysis contained: a rostral pars distalis, with prolactin (PRL) cells arranged in follicles and adrenocorticotropic (ACTH) cells, a proximal pars distalis with somatotropic (GH), β-thyrotropic (TSH), β-gonadotropic type I (FSH) and type II (LH) cells and a pars intermedia with α-somatolactin (SL), α-melanotropic (MSH) and β-endorphin (END) cells. All regions were deeply penetrated by neurohypophyseal branches. At hatching (24 h post-fertilization) the pituitary was an oval cell mass, close to the ventral margin of diencephalon. The first immunoreactive cells appeared as follows: PRL at 0·5 days after hatching (dah), GH and SL at 1·5 dah, END at 2 dah, TSH, ACTH and MSH at 2·5 dah, FSH at 28 dah and LH at 90 dah. The neurohypophysis appeared at 5 dah and branched extensively inside the adenohypophysis at 130 dah, but there was no boundary between rostral pars distalis and proximal pars distalis at this stage. The potential indices of prolactin and growth hormone production increased until 28 and 60 dah, respectively. The potential index of growth hormone production correlated positively with total length. Activity of PRL and GH cells, measured as ratio of cell area to nucleus area, was significantly higher in juveniles than in larvae.

Relaxed selective constraints drove functional modifications in peripheral photoreception of the cavefish P. andruzzii and provide insight into the time of cave colonization.

The genetic basis of phenotypic changes in extreme environments is a key but rather unexplored topic in animal evolution. Here we provide an exemplar case of evolution by relaxed selection in the Somalian cavefish Phreatichthys andruzzii that has evolved in the complete absence of light for at least 2.8 million years. This has resulted in extreme degenerative phenotypes, including complete eye loss and partial degeneration of the circadian clock. We have investigated the molecular evolution of the nonvisual photoreceptor melanopsin opn4m2, whose mutation contributes to the inability of peripheral clocks to respond to light. Our intra- and inter-species analyses suggest that the 'blind' clock in P. andruzzii evolved because of the loss of selective constraints on a trait that was no longer adaptive. Based on this change in selective regime, we estimate that the functional constraint on cavefish opn4m2 was relaxed at ∼5.3 Myr. This implies a long subterranean history, about half in complete isolation from the surface. The visual photoreceptor rhodopsin, expressed in the brain and implicated in photophobic behavior, shows similar evolutionary patterns, suggesting that extreme isolation in darkness led to a general weakening of evolutionary constraints on light-responsive mechanisms. Conversely, the same genes are still conserved in Garra barreimiae, a cavefish from Oman, that independently and more recently colonized subterranean waters and evolved troglomorphic traits. Our results contribute substantially to the open debate on the genetic bases of regressive evolution.

Studying the Evolution of the Vertebrate Circadian Clock: The Power of Fish as Comparative Models.

The utility of any model species cannot be judged solely in terms of the tools and approaches it provides for genetic analysis. A fundamental consideration is also how its biology has been shaped by the environment and the ecological niche which it occupies. By comparing different species occupying very different habitats we can learn how molecular and cellular mechanisms change during evolution in order to optimally adapt to their environment. Such knowledge is as important as understanding how these mechanisms work. This is illustrated by the use of fish models for studying the function and evolution of the circadian clock. In this review we outline our current understanding of how fish clocks sense and respond to light and explain how this differs fundamentally from the situation with mammalian clocks. In addition, we present results from comparative studies involving two species of blind cavefish, Astyanax mexicanus and Phreatichthys andruzzii. This work reveals the consequences of evolution in perpetual darkness for the circadian clock and its regulation by light as well as for other mechanisms such as DNA repair, sleep, and metabolism which directly or indirectly are affected by regular exposure to sunlight. Major differences in the cave habitats inhabited by these two cavefish species have a clear impact on shaping the molecular and cellular adaptations to life in complete darkness.

Cavefish eye loss in response to an early block in retinal differentiation progression.

The troglomorphic phenotype shared by diverse cave-dwelling animals is regarded as a classical example of convergent evolution. One unresolved question is whether the characteristic eye loss in diverse cave species is based on interference with the same genetic program. Phreatichthys andruzzii, a Somalian cavefish, has evolved under constant conditions in complete darkness and shows severe troglomorphic characteristics, such as complete loss of eyes, pigments and scales. During early embryonic development, a complete eye is formed but is subsequently lost. In Astyanax mexicanus, another blind cavefish, eye loss has been attributed to interference during eye field patterning. To address whether similar pathways have been targeted by evolution independently, we investigated the retinal development of P. andruzzii, studying the expression of marker genes involved in eye patterning, morphogenesis, differentiation and maintenance. In contrast to Astyanax, patterning of the eye field and evagination of the optic vesicles proceeds without obvious deviation. However, the subsequent differentiation of retinal cell types is arrested during generation of the first-born cell type, retinal ganglion cells, which also fail to project correctly to the optic tectum. Eye degeneration in both species is driven by progressive apoptosis. However, it is retinal apoptosis in Phreatichthys that progresses in a wave-like manner and eliminates progenitor cells that fail to differentiate, in contrast to Astyanax, where lens apoptosis appears to serve as a driving force. Thus, evolution has targeted late retinal differentiation events, indicating that there are several ways to discontinue the development and maintenance of an eye.

Fish don't need eyes for numeracy

![Figure][1] Blind cavefish ( Phreatichthys andruzzii ). Photo credit: Luca Scapoli. Fish tend not to be renowned for their intellect. Their memories weren't thought to be great and until recently no one thought that they could assess quantity. But now we are having to reconsider these

Non-visual numerical discrimination in a blind cavefish (Phreatichthys andruzzii)

Over a decade of comparative studies, researchers have found that rudimentary numerical abilities are widespread among vertebrates. While experiments in mammals and birds have employed a variety of stimuli (visual, auditory and tactile), all fish studies involved visual stimuli and it is unknown whether fish can process numbers in other sensory modalities. To fill this gap, we studied numerical abilities in Phreatichthys andruzzii, a blind cave-dwelling species that evolved in the phreatic layer of the Somalia desert. Fish were trained to receive a food reward to discriminate between two groups of objects placed in opposite positions of their home tank. In Experiment 1, subjects learned to discriminate between two and six objects, with stimuli not controlled for non-numerical continuous variables that co-vary with numbers, such as total area occupied by stimuli or density. In Experiment 2, the discrimination was two versus four, with half of the stimuli controlled for continuous quantities and half not controlled for continuous quantities. The subjects discriminated only the latter condition, indicating that they spontaneously used non-numerical information, as other vertebrates tested in similar experiments. In Experiments 3 and 4, cavefish trained from the beginning only with stimuli controlled for continuous quantities proved able to learn the discrimination of quantities based on the sole numerical information. However, their numerical acuity was lower than that reported in other teleost fish tested with visual stimuli.

Encephalic photoreception and phototactic response in the troglobiont Somalian blind cavefish Phreatichthys andruzzii.

Many physiological and behavioural responses to changes in environmental lighting conditions are mediated by extraocular photoreceptors. Here we investigate encephalic photoreception in Phreatichthys andruzzii, a typical cave-dwelling fish showing an extreme phenotype with complete anophthalmy and a reduction in size of associated brain structures. We firstly identified two P. andruzzii photopigments, orthologues of rod opsin and exo-rod opsin. In vitro, both opsins serve as light-absorbing photopigments with λ(max) around 500 nm when reconstituted with an A(1) chromophore. When corrected for the summed absorption from the skin and skull, the spectral sensitivity profiles shifted to longer wavelengths (rod opsin: 521 nm; exo-rod opsin: 520 nm). We next explored the involvement of both opsins in the negative phototaxis reported for this species. A comparison of the spectral sensitivity of the photophobic response with the putative A(2) absorbance spectra corrected for skin/skull absorbance indicates that the A(2) versions of either or both of these pigments could explain the observed behavioural spectral sensitivity.

Erratum: Can chemical cues act as landmarks in the orientation of the cave fish Phreatichthys andruzzii?

Erratum: Can chemical cues act as landmarks in the orientation of the cave fish <i>Phreatichthys andruzzii</i>?

Non visual discrimination of shapes in the blind cave cyprinid Phreatichthys andruzzii Vinciguerra 1924

Vision is precluded in the darkness of hypogean environment, which is the reason why cave fish have to direct their locomotor activity using information only acquired through alternative sensory channels. The aim of the present study was to evaluate whether the presence of an object or its morphological features can be used as landmark in orienting the swimming activity of the blind cave cyprinid Phreatichthys andruzzii Vinciguerra 1924. We could verify, by means of a food conditioning procedure, that Phreatichthys is capable of (a) recognising and memorising the presence/absence of an object, (b) discriminating between different shapes and (c) utilising such an information to orient its locomotion in space. The results of our tests show that morphological cues can act as landmarks for Phreatichthys, thus supporting the hypothesis that an orienting mechanism based on such type of information can operate in the permanent darkness of cave habitats.

Can chemical cues act as landmarks in the orientation of the cave fishPhreatichthys andruzzii?

In the constant darkness of cave environments fish locomotion has to be directed by nonvisual spatial information. Four series of tests were done to determine the ability of the hypogean cyprinid Phreatichthys andruzzii Vinciguerra, 1924 to memorize a synthetic chemical cue (morpholine, C4H9NO), to associate it with an area, to utilize such information for direct locomotory activity, and to determine how long that association is retained in fish memory. Although morpholine acts as neutral stimulus for P. andruzzii, after acclimation in morpholine-scented areas devoid of food resources specimens showed a clear tendency to avoid waters characterized by the odour of that chemical. We hypothesize that an association between odours and trophic characteristics of an area allows the fish to optimize their exploratory activity, as it allows them to recognize areas already experienced to be devoid of trophic resources and to avoid these as unprofitable places. The above association seems to be memorized for a short time; the behavioural response vanishing between 6 and 18 h after the end of the acclimation period. After a certain time has elapsed, it could be profitable to visit the same areas again to verify the incidental availability of new food sources.

Genetic variability and differentiation of hypogean Cyprinid fishes from Somalia

The genetic structure of populations of two hypogean Somali cyprinid species was compared by analysing genetic variation at 30 allozyme loci. The two species, Phreatichthys andruzzii and Barbopsis devecchii, are of particular interest, representing two different steps in adaptation to cave life, as indicated by several morphological features including eye regression. Phreatichthys is completely anophthalmic, while Barbopsis shows a highly variable microphthalmia. Results showed a close relationship between Phreatichthys and Barbopsis, suggesting their origin from a common epigean ancestor. Population structure of the two species differs and levels of gene flow are much higher between the Barbopsis populations than between the Phreatichthys populations. Two possible scenarios leading to the current situation are hypothesized.

Ultrastructure and distribution of superficial neuromasts of blind cavefish, Phreatichthys andruzzii, juveniles

Transmission and scanning electron microscopy (TEM, SEM) were used to study the ultrastructure of superficial neuromasts in 15 six-month old blind cavefish juveniles, Phreatichthys andruzzii (Cyprinidae). In five specimens examined with SEM, the number of superficial neuromasts over the fish body (480-538) was recorded. They were localized mainly on the head (362-410), including the dorsal surface, the mentomandibular region, and laterally from the mouth to the posterior edge of the operculum. Neuromasts were also present laterally on the trunk and near the caudal fin (116-140). A significantly higher number of neuromasts were present on the head compared to the trunk (t-test, P < 0.05). Superficial neuromasts of the head and those along the trunk were similar in ultrastructure. Each neuromast comprised sensory hair cells surrounded by nonsensory support cells (mantle cells and supporting basal cells) with the whole covered by a cupula. Each hair cell was pear-shaped, 15-21 microm high and 4-6 microm in diameter, with a single long kinocilium and several short stereocilia. Most support cells were elongated, with nuclei occupying a large portion of the cytoplasm. In the margin of the neuromast, mantle cells were particularly narrow. Both types of support cells had well-developed Golgi apparatus and rough endoplasmic reticulum. The number of hair cells and nonsensory support cells of the anterior lateral line (head) did not differ significantly from those of the posterior lateral line (trunk) (t-test, P > 0.05).