Blind Mexican Cavefish Research Articles

Light-sensitive Motile Iridophores and Visual Pigments in the Neon Tetra, Paracheirodon innesi

Although motile iridophores in the longitudinal stripes of neon tetra skin are under control of the sympathetic nervous system, they also respond to light directly and show circadian color changes. Using neon tetra skin, we found that the photoresponse of iridophores depends on light intensity, and that light near 500 nm is most effective. RT-PCR demonstrated the expression of mRNAs encoding rhodopsin and two kinds of cone opsins (Pi-green1 and Pi-green2) in neon tetra skin where the light-sensitive iridophores exist. These mRNAs are also expressed in the lateral eyes. The cone opsin genes, Pi-green1 and Pi-green2, show high similarity with the g101 and g103 genes of unique green cone opsins (belonging to the MWS/LWS group) of the blind Mexican cavefish. These results show that Pi-green1, Pi-green2, and/or rhodopsin may play important roles in the photoresponse of neon tetra iridophores, which are most sensitive to light near 500 nm.

The vertical component of a fish's spatial map

Fish orient by using a memorized map of their surroundings and, as they live in aquatic environments, this map must be three dimensional. Studies have shown that fish are able to encode horizontal information; however, we have little information about the cues that are learnt and remembered in the vertical axis. We used the blind Mexican cave fish, Astyanax fasciatus, to test whether fish can orient in the vertical axis and whether they use hydrostatic pressure to do so. After learning the cues associated with a specific height in the aquarium (evident by a reduction in swimming speed), the fish showed significant dishabituation (their swimming speed increased) when moved vertically upwards. When we controlled for the increase in light intensity from the bottom to the top of the tank the effect persisted, indicating that the most likely cue used by the fish to orient vertically was hydrostatic pressure. A post hoc experiment revealed that the change in swimming speed in response to altered light levels probably represents a change in the level of activity rather than an orientational response. This may define a photokinetic mechanism which operates in conjunction with a negatively phototactic response (revealed in previous experiments) by which the fish orient away from higher light levels and swim faster as light levels increase. This would result in the fish spending more time in lower light levels and would prevent the fish exiting the cave.

Molecules, Biodiversity, and Adaptive Evolution

The American Genetic Association (AGA) is the oldest professional society that is devoted to study the mechanisms and consequences of heredity. The AGA publishes Journal of Heredity and promotes researches in such fields as population/ecological genetics, molecular evolution, bioinformatics, developmental genetics, and physiological/ biochemical genetics (e.g., see Crow 2004). Although it is not widely known, in addition to its annual meetings, the AGA also provides financial support for other scientific meetings as well. On June 17–20, 2004, the AGA cosponsored the international meeting of ‘‘Genomes and Evolution 2004’’ with the International Society of Molecular Biology and Evolution (SMBE) at Pennsylvania State University, in State College, Pennsylvania. In this meeting, the AGA sponsored symposia on ‘‘Adaptive Evolution’’ and ‘‘Molecules and Biodiversity.’’ The AGA has a tradition of recognizing a Wilhemine E. Key Lecturer each year. Professor Walter Gehring from University of Basel in Switzerland was the recipient of the AGA 2004 Key Lecturer award and delivered a keynote lecture on ‘‘Development and Evolution of Eyes and Photoreceptors.’’ The articles collected in this volume, which are derived from oral and poster presentations at the two symposia, give us some hints about exciting new research directions on molecular diversity and adaptive evolution. In the first article, ‘‘New Perspective on Eye Development and the Evolution of Eyes and Photoreceptors,’’ Gehring shows that the master control gene, Pax 6, can induce ectopic eyes in both insects and vertebrates, arguing strongly for a monophyletic orgin of the eye. He also hypothesizes that the origin of photoreceptor cells in metazoa can be traced to a colonial protist and/or to photosynthetic cyanobacteria. In the second article on a similar subject, ‘‘Adaptive Evolution of Eye Degeneration in the Mexican Blind Cavefish,’’ W. R. Jeffery shows that Hedgehog midline signaling inhibits eye formation by inducing lens apoptosis in the Mexican blind cavefish embryos and suggests that eye degeneration in cavefish is caused by adaptive evolution. In ‘‘Phytochrome Evolution in Green and Nongreen Plants,’’ S. Mathews reviews the current level of our understanding of the evolution of light sensing molecules, photochromes, in early angiosperms, Arabidopsis, and a family of parasitic plants. In ‘‘Evolution of New Hormone Function: Loss and Gain of a Receptor,’’ D. M. Irwin and K. Wong reveal that although three glucagon receptor genes (glucagon, GLP-1, and GLP-2) have distinct functions in regulating metabolism in mammals, glucagon and GLP-1 in fish exhibit similar physiological functions by maintaining the functionally important DNA segments. In ‘‘FoxP2 in Song-Learning Birds and Vocal-Learning Mammals,’’ D. M. Webb and J. Zhang show that song-learning birds and vocal-learning whales, dolphins, and bats do not share specific amino acid substitutions, which have caused a selective sweep of speech and language development during human evolution. In ‘‘Retention of Latent Centromeres in the Mammalian Genome,’’ G. C. Ferreri, D. M. Liscinsky, J. A. Mack, M. D. B. Eldridge, and R. J. O’Neill examine the roles of centromere repositioning on the evolution of chromosome structure and syntenic order as well as theories of the permutability of centromere location within a chromosome in mammals using evidence gained in human and the tammar wallaby. In ‘‘To B or Not to B a Flower: The Role of DEFICIENS and GLOBOSAOrthologs in the Evolution of the Angiosperms,’’ L. M. Zahn, J. Leebens-Mack, C. W. dePamphilis, H. Ma, and G. Theissen review the function, phylogeny, and expression of genes involved in the DEFICIENS (DEF) and GLOBOSA (GLO) MADS-box subfamily and suggest that the original function of these genes in the angiosperm could have been different. In ‘‘The Ecological Genetics of Homoploid Hybrid Speciation,’’ B. L. Gross and L. H. Rieseberg show that ecological selection plays a major role for promoting homoploid hybrid speciation and test a set of explicit questions using the sunflower species,Helianthus deserticola. In ‘‘Spatial Dynamics and Molecular Ecology of North American Rabies,’’ L. A. Real, C. Russell, L. Waller, D. Smith, and J. Childs study the temporal and spatial dynamics of a current rabies epidemic in North America by considering local environmental variables, genetic heterogeneity, and host specificity. In ‘‘Interaction of Rearing Environment and Reproductive Tactics on Gene Expression Profiles in Atlantic Salmon,’’ N. Aubin-Horth, B. H. Letcher, and H. A. Hofmann study the effects of rearing environment (either in a natural stream or in laboratory conditions) on

Adaptive Evolution of Eye Degeneration in the Mexican Blind Cavefish

The evolutionary mechanisms responsible for eye degeneration in cave-adapted animals have not been resolved. Opposing hypotheses invoking neural mutation or natural selection, each with certain genetic and developmental expectations, have been advanced to explain eye regression, although little or no experimental evidence has been presented to support or reject either theory. Here we review recent developmental and molecular studies in the teleost Astyanax mexicanus, a single species consisting of a sighted surface-dwelling form (surface fish) and many blind cave-dwelling forms (cavefish), which shed new light on this problem. The manner of eye development and degeneration, the ability to experimentally restore eyes, gene expression patterns, and comparisons between different cavefish populations all provide important clues for understanding the evolutionary forces responsible for eye degeneration. A key discovery is that Hedgehog midline signaling is expanded and inhibits eye formation by inducing lens apoptosis in cavefish embryos. Accordingly, eyes could have been lost by default as a consequence of natural selection for constructive traits, such as feeding structures, which are positively regulated by Hh signaling. We conclude from these studies that eye degeneration in cavefish may be caused by adaptive evolution and pleiotropy.

Cavefish

What are cavefish? Like other cave-dwelling animals, fish that live in caves have often lost their eyes and body colour, but the evolutionary mechanisms underlying these reductions are unknown. There are 86 species of cavefish that belong to 18 different families, and many of them have lost their eyes independently. Comparing cavefish to their free-living ancestors can thus help us understand convergent evolution. However, the ancestors of most cave species are already extinct, with the notable exception of the Mexican Blind Cavefish. What is the Mexican Blind Cavefish? In 1819, the French anatomist Georges Cuvier described the Mexican tetra (Astyanax fasciatus mexicanus). Astyanax belongs to the teleost order Characiformes, which also includes the piranha, the neon tetra or the pencil fish. What is so special about them?Astyanax comprises both eyed, surface-dwelling and eyeless, cave-dwelling populations. Each cavefish population shows a different degree of eye and pigment degeneration. Recent data indicate that some cavefish populations independently evolved from eyed surface fish at different times. Why are they blind? One hypothesis is that losing the eyes could have selective advantages for living in a cave. Darwin thought that reduced eye size and covering the eye with a flap of skin could be advantageous by preventing inflammation and diseases. Others have proposed that eye loss is economical, because the visual system consumes a lot of energy. An alternative explanation suggests that mutations have accumulated in genes required for eye formation, because selection on the visual system is relaxed in darkness. There is also the possibility of an evolutionary trade-off, such that functional eyes were sacrificed to allow the development of more ‘useful’ characters, such as more taste buds and an enhanced lateral line system. What about the other senses? Cavefish have evolved enhanced chemo- and mechanosensory systems. Extra taste buds help them find food more quickly in complete darkness. Their enhanced mechanosensory lateral line system helps them to perceive their environment. These enhanced senses also might have aided the fish in maintaining their complex spawning dance in darkness. How did they lose their eyes? The signaling molecule sonic hedgehog (Shh) is involved in eye, jaw, and taste bud development in the Mexican cavefish. During embryonic development, Shh expression is expanded in the cavefish compared to surface fish, causing eye degeneration and an increase in the number of taste buds. This pleiotropic effect supports the trade-off theory. How did they lose their color? It has been proposed that the loss of dark pigment cells is a consequence of the accumulation of neutral mutations under relaxed selection. A recent study has shown that independently evolved Astyanax cave populations have retained pigment cell precursors but cannot synthesize the pigment, possibly due to damage of the same pigment-synthesizing pathway. Do cavefish sleep at night? The sleeping behavior of cavefish is less obvious than that of their surface relatives, but in the lab they are less active at night. Their pineal organ is functional and can detect light. Although cavefish seem to have a circadian rhythm in the lab, it is not clear if they have a daily rhythm in a dark cave. Is there a cavefish genome project? Not yet, but comparing the genomes of cavefish and surface fish might reveal the causes and consequences of microevolutionary change at the genetic level as well as mechanisms of convergent evolution. A genome project could also help understanding macroevolution. While Astyanax has a ‘generalized’ body shape, its relatives, e.g. piranhas, have evolved a variety of unique morphological characters.

Spatial parameters encoded in the spatial map of the blind Mexican cave fish, Astyanax fasciatus

Although much is now known about the mechanisms that insects, birds and mammals use to orient within familiar areas, our knowledge of such mechanisms in fish is scant. I used the transformational approach to test whether the blind Mexican cave fish can encode shape and size in an internal representation of space. These fish are excellent study animals, as they swim at high velocities (presumably to enhance lateral line organ stimulation) when faced with unfamiliar landmarks or environments. As they are blind, potentially confounding cues from visual global landmarks are unavailable. The fish learnt a square configuration of four landmarks and so must have been be able to encode spatial relationships between the elements within this configuration. After learning landmark arrays, the cave fish showed significant dishabituation (swimming velocity was increased) when exposed to landmark transformations. The fish must therefore have been comparing the environment that they perceived with an internal representation of the environment that they had learnt. The results show that blind Mexican cave fish can encode size (absolute distance between landmarks) and possibly also shape within their spatial maps.

To See or Not to See: Evolution of Eye Degeneration in Mexican Blind Cavefish

The evolutionary mechanisms responsible for the loss of eyes in cave animals are still unresolved. Hypotheses invoking natural selection or neutral mutation have been advanced to explain eye regression. Here we describe comparative molecular and developmental studies in the teleost Astyanax mexicanus that shed new light on this problem. A. mexicanus is a single species consisting of a sighted surface-dwelling form (surface fish) and many blind cave-dwelling forms (cavefish) from different caves. We first review the evolutionary relationships of Astyanax cavefish populations and conclude that eye degeneration may have evolved multiple times. We then compare the mechanisms of eye degeneration in different cavefish populations. We describe the results of experiments showing that programmed cell death of the lens plays a key role in controlling eye degeneration in these cavefish populations. We also show that Pax6 gene expression and fate determination in the optic primordia are modified similarly in different cavefish populations, probably due to hyperactive midline signaling. We discuss the contributions of the comparative developmental approach toward resolving the evolutionary mechanisms of eye degeneration. A new hypothesis is presented in which both natural selection and neutral mutation are proposed to have roles in cavefish eye degeneration.

Multiple photopigments from the Mexican blind cavefish, Astyanax fasciatus: a microspectrophotometric study

The cave-dwelling (hypogean) form of the teleost Astyanax fasciatus is blind, having only subdermal eye rudiments, but nevertheless maintains intact opsin genes. Second generation offspring of a cross between these and the normally sighted surface (epigean) form inherit opsin genes from both ancestries. A study of the expressed hypogean opsins of the hybrids, in comparison to the epigean forms, was undertaken by microspectrophotometry. The hybrid population showed considerable variation in the visual pigments of double cones, with evidence for two groups of cells with λ max intermediate to those of the epigean pigments. Possible explanations for these intermediate pigments are discussed, including the hypothesis that they may represent hybrid genes similar to the genes for anomalous cone pigments in humans. Evidence was also found for ultraviolet-sensitive single cones and for an additional MWS pigment.

Cell migration in the postembryonic development of the fish lateral line.

We examine at the cellular level the postembryonic development of the posterior lateral line in the zebrafish. We show that the first wave of secondary neuromasts is laid down by a migrating primordium, primII. This primordium originates from a cephalic region much like the primordium that formed the primary line during embryogenesis. PrimII contributes to both the lateral and the dorsal branches of the posterior lateral line. Once they are deposited by the primordium, the differentiating neuromasts induce the specialisation of overlying epidermal cells into a pore-forming annulus, and the entire structure begins to migrate ventrally across the epithelium. Thus the final two-dimensional pattern depends on the combination of two orthogonal processes: anteroposterior waves of neuromast formation and dorsoventral migration of individual neuromasts. Finally, we examine how general these migratory processes can be by describing two fish species with very different adult patterns, Astyanax fasciatus (Mexican blind cavefish) and Oryzias latipes (medaka). We show that their primary patterns are nearly identical to that observed in zebrafish embryos, and that their postembryonic growth relies on the same combination of migratory processes that we documented in the case of the zebrafish.

Toxicity of Co2+: implications for lateral line studies.

It has been reported that superficial neuromasts, a type of lateral line organ, mediate rheotaxis in fish. These studies used Co2+ at 2 mmol l(-1) for 3 h to ablate the entire lateral line system. The recommended concentration is 0.1 mmol l(-1). The present study shows that at 2 mmol l(-1) Co2+ is highly toxic to blind Mexican cave fish. Fish exposed to this concentration died in less than 17 h, and produced copious mucus. No control fish died. No cobalt treated fish died after 3 h exposure, but cobalt-treated fish swam about their aquaria faster than control fish and tended to swim at the surface. Hence, both survival and behavior were changed by the excessive concentration of Co2+.

The sensory basis of rheotaxis in the blind Mexican cave fish, Astyanax fasciatus

The sensory basis of rheotaxis (orientation to currents) was investigated in the blind Mexican cave fish, Astyanax fasciatus. An unconditioned rheotactic response to uniform velocity flows was exhibited, with a threshold of less than 3 cm s−1. Disabling the entire lateral line or the superficial neuromast receptor class increased the rheotactic threshold to greater than 9 cm s−1. A pharmacological block of the lateral line canal system alone had no effect. These results demonstrate that the superficial lateral line system controls rheotaxis at low current velocities. The effect of pairing an odor stimulant with the water current dropped the rheotactic threshold to less than 0.4 cm s−1. This study provides a clear behavioral role for the superficial neuromasts where none previously existed, and also establishes a link between the mechanosensory lateral line and olfactory systems in the olfactory search behavior of the cave fish.

BLIND MEXICAN CAVE FISH (ASTYANAX HUBBSI) RESPOND TO MOVING VISUAL STIMULI

In apparatus for measuring optomotor behaviour, blind Mexican cave fish, Astyanax hubbsi, increase their swimming velocity upon rotation of a striped cylinder, i.e. in response to a solely visual stimulus. The fish follow the movements of the stripes at (i) rotation velocities between 60 degrees s-1 and 80 degrees s-1, (ii) light intensities of less than 20 lx and, (iii) stimulus widths subtending an angle of less than 1 °. Extirpation of the vestigial eye structures does not affect the response to the moving visual stimulus, which indicates that the response is mediated by extra-ocular photoreceptors. An optomotor response can be reliably evoked in a round test aquarium. Fish do not respond when the test aquarium contains environmental cues, such as bars on the wall or when a section of the round aquarium is divided off. This indicates that the fish obtain information about their environment from different sensory sources and that the visual stimulus is effective only when no other means of orientation are available. We suggest a modified theory of the optomotor response, which emphasizes the crucial role of the environment in eliciting the response and which permits behaviours more complex than just following the stimulus.

Studies on the effects of Ca2++ and Co++ on the swimming behavior of the blind Mexican cave fish

The hypothesis that the blind cave fish (Astyanax hubbsi) adjusts the level of stimulation to its lateral line system (LLS) by varying its own velocity was examined. When the sensitivity of the LLS sense organs was reduced by lowering the Ca2+ concentration in the water or by adding Co2+ the fish compensated for this by swimming at a higher velocity.

Sensory performance of blind mexican cave fish after destruction of the canal neuromasts

Sensory performance of blind mexican cave fish after destruction of the canal neuromasts

Isolation, DNA sequence and evolution of a color visual pigment gene of the blind cave fish Astyanax fasc1atus

Visual pigment genes have been isolated from a Mexican blind cave fish, Astyanax fasciatus. We report here the DNA sequence of one of these genes, which has 70% nucleotide similarity with both the human red and green pigment genes. This gene appears capable of encoding a functional protein and is probably responsible for the green-sensitive pigment found in the pineal organ of the blind cave fish ( Tabata, 1982). The pattern of nucleotide substitutions most likely reflects functional adaptation of the visual pigment gene during the past 400 million years of fish evolution.

Collision with and avoidance of obstacles by blind cave fish Anoptichthys jordani (Characidae).

Blind Mexican cave fish (Anoptichthys jordani) were released into unknown surroundings and their swimming tracks were recorded. During the first 24 h in a new tank, i.e., in unfamiliar environments, the average swimming velocity of the fish was higher than during the remaining time in the tank. Collision with obstacles was recorded by an electrical contact detection method. Single frame analysis of video-recordings revealed that collision can be correlated with tail movements when the fish is close to an obstacle, whereas avoidance correlates with gliding during approach to an obstacle. These behavior patterns are discussed in the context of the flow field hypothesis and inhibition of the lateral line organ during movement.

Discrimination between stationary objects by the blind cave fishAnoptichthys jordani (Characidae)

Blind Mexican cave fish were trained to discriminate between grids of vertical bars placed at various distances from each other. Discrimination was possible only if the freely swimming fish was allowed to glide past the grids in close proximity.

Detection of stationary objects by the blind Cave FishAnoptichthys jordani (Characidae)

It was noticed that the blind Mexican Cave Fish repeatedly passes along objects new to it at a short distance. Observations and experiments are reported which support the hypothesis that water movements occurring between the stationary object and the fish as it passes by convey information about the location and possibly the shape of the object, which is detected by the lateral line organ of the fish. Water movements of the expected type were recorded with the help of a model fish equipped with a mechano-electric analogue of a free neuromast.

The morphology of the Weberian ossicles of two species of the genus Astyanax (Ostariophysi: Characidae)

AbstractThe morphology of the Weberian ossicles of Astyanax mexicanus, an eyed river fish, and A. jordani, the Mexican blind cave fish, was investigated and the results were correlated with behavioral data on the auditory capacities of the two species. Several characteristics of the ossicles of the two species were inter‐specifically significantly different while other characteristics varied as much intra‐specifically as inter‐specifically. The ascending arm of the scaphium was shorter in the blind fish than in the river fish and the ascending arm of the intercalarium was further from the distal end of the base of the bones in the blind fish than in the river fish.A previously unreported ligament between the scaphium and the sinus atrium impar was found in Astyanax. It was hypothesized that this connective tissue retained perilymphatic fluid within the inner ear, helped the ossicles return to an anterior position after moving posteriorly, and might protect the inner ear during intense acoustical stimulation.

Auditory capacities of the Mexican blind cave fish ( Astyanax jordani) and its eyed ancestor ( Astyanax mexicanus)

The auditory capacities of two species of characid fish, Astyanax mexicanus and A. jordani, were determined using avoidance conditioning techniques. Pressure thresholds for both species were as low as those reported for other ostariophysines and the minimum threshold for both species was at 1000 Hz. Both species of fish could hear a wider range of sounds than that reported for any other species of fish tested in a quantitative manner. There is no evidence that the Mexican blind cave fish can detect pressure stimuli any better or more poorly than its ancestral eyed relative. There is no obvious selective advantage for maintenance of a wide auditory range upon entry into the cave since no source of sounds above about 1000 Hz are present there. There may be selective pressures for maintenance of the ear for less obvious reasons, such as the need for the equilibrium information that the ear provides. Although not enough ostariophysines have yet been investigated, these data substantiate earlier findings that they can hear a wider range of sounds and lower intensities than non-ostario-physines. The behaviour of the fish in the avoidance apparatus differs strikingly during initial training and during testing, and this is probably due to the different orientation of these two species.