Mexican Cavefish Research Articles

Lateral line research: Recent advances and new opportunities

Computational fluid dynamics, digital particle imaging velocimetry, and engineered micro flow sensors, along with emerging collaborations between neurobiologists and engineers, have opened up new vistas for lateral line research in recent years. Engineering advances have increased our ability to measure the spatiotemporal characteristics of biologically significant, more complex flow signals and how these signals are sensed and processed by the lateral line. Following in the rich tradition of Art Myrberg’s work on sound communication in fishes, studies on hydrodynamic signal production and use by fish during interactive social behaviors are now feasible. In addition to these new opportunities, several recent studies have enriched the lateral line landscape. The lateral line sensing and encoding of vortex trails shed by swimming fish or obstacles in a stream have been investigated, along with associated behaviors (e.g., vortex trail following and obstacle entrainment). Blind Mexican cavefish have been shown to form cognitive maps of their surroundings using lateral‐line based, hydrodynamic cues. Finally, the responses of goldfish auditory and lateral line systems to the same stimulus source have been documented to reveal how these two closely related systems might work in concert to extract complementary but different types of information from nearby hydroacoustic sources.

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.

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.

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.

Transcription of the opsin gene in degenerate eyes of cave-dwelling Astyanax fasciatus (Teleostei, Characidae) and of its conspecific epigean ancestor during early ontogeny

The early morphogenesis of the degenerate eyes of the Mexican cave fish Astyanax fasciatus and of its conspecific epigean ancestor has been studied comparatively using light- and electron-microscopical techniques; the transcription of the opsin gene has been analysed during early ontogeny in both populations by in situ hybridization. The opsin protein is an integral component of the disk membranes of the photoreceptor cells. In epigean specimens, its expression is correlated with the beginning of outer segment formation on the third day of development. Morphogenesis of the cave fish eye is similar to that of the epigean eye until end of the third day. However, eye growth and morphogenesis of the retina are delayed and sporadic cell death occurs in all retinal layers at the beginning of the second day. Retinal cytodifferentiation breaks off at the point of outer segment formation. Cave specimens are not able to develop regular outer segments at any stage, but the opsin gene is nevertheless expressed in the outer nuclear layer of the developing retina for a limited period of time. On the basis of the comparative morphology and transcriptional studies of epigean and cave specimens, it is suggested that the eye regression of cave fish is primarily the result of mutations of developmental control genes and not of structural genes.

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

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.

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.

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.

GENETIC VARIABILITY IN THE KENTUCKY CAVE BEETLE NEAPHAENOPS TELLKAMPFII (COLEOPTERA: CARABIDAE).

This paper reports data on genetic variability and genetic similarity in five populations of Neaphaenops tellkampfii, a troglobitic carabid beetle which occurs in many caves of the Central Kentucky Karst, including those of Mammoth Cave National Park. Previous studies have found that cave species generally show lower genetic variability than similar surface species. In the Central Kentucky Karst Region (Quinlan, 1970), Laing et al. (1976a) reported average frequencies of heterozygous loci per individual, H, of 0.008 and 0.056, respectively, for two populations of the obligate cave dwelling (i.e., troglobitic) millipede Scoterpes copei. Likewise, Laing et al. (1976b) reported average H values of 0.048 for six populations of the troglobitic scavenger beetle Ptomaphagus hirtus (Leiodidae) another central Kentucky species. Avise and Selander (1972) compared genetic variability of surface and cave populations of the Mexican cavefish Astyanax mexicanus. Whereas surface populations maintained average H values of 0. 112, cave populations of the same species were much less variable with H values averaging only 0.036. In addition to showing low variability, comparisons of genetic similarity between sets of populations of each of the above species showed low levels of similarity, particularly when contrasted with comparable surface species. An exception to the pattern of low variability and similarity in populations of troglobitic species emerges from preliminary data on Rhadine subterranea (Coleoptera: Carabidae) (Avise and Selander, 1972). This species is a predator in terrestrial communities of a Texas cave system (Mitchell, 197 la; 1971b; 1971c), preying on the eggs of cave crickets. Calculations of H from the data for three loci presented by Avise and Selander (1972) yield values :0.20 for R. subterranea. In addition, the two populations sampled appear to be nearly identical genetically.

Lactate dehydrogenase gene function in the blind cave fish, anoptichthys jordani, and other characins.

The functions of the genes encoded for lactate dehydrogenase (LDH) in six genera of characins (teleost) were examined by electrophoretic and immunochemical analyses of the LDH isozymes. The characins possess the LDH A and B loci present in all vertebrates. The eyeless Mexican cave fish (Anoptichthys jordani) and other characins possessing normal eyes, e.g., Mexican tetra (Astyanax mexicanus, which is able to hybridize with the cave fish), blue tetra (Mimagoniates microlepis), sailfin tetra (Crenuchus spilurus), head and tail light tetra (Hemigrammus ocellifer), and the piranha (Serrasalmus spilopleura), all lack the function of a third LDH locus (the E locus) present in many teleosts which codes for a distinctive isozyme synthesized in the nervous system, particularly in the neural retina.

The ACTH cells in the adenohypophysis of the Mexican cave fish Anoptichthys jordani, as identified by metopirone (SU 4885) treatment.

Administration of the anti-adrenal drug metopirone (SU 4885) allows for the identification of the ACTH cells in the adenohypophysis of the teleost Anoptichthys jordani. The drug causes a correlated stimulation of the interrenal cells and one cell type in the pituitary gland, i.e. the amphiphilic pro-2 cells, situated in a palisade like layer between the acidophils of the pro-adenohypophysis and the neurohypophysis.

The cell types in the adenohypophysis of the blind Mexican cave fish, Anoptichthys jordani (Hubbs and Innes).

Staining sagittal sections of the pituitary of the Blind Mexican Cave Fish, Anoptichthys jordani, with a number of methods including Herlant's tetrachrome, Herlant's Alcian Blue (AB)-periodic acid Schiff (PAS)-orange G, Gabe's aldehyde fuchsin and lead hematoxylin according to Mac Conaill, made it possible to distinguish seven tinctorial cell types in the adenohypophysis: in the pro-adenohypophysis erythrosin-positive acidophils, and lead hematoxylin-positive amphiphils, lining branches of the neural lobe; in the meso-adenohypophysis orange G-positive acidophils, large, weakly AB- and AF-positive, strongly PAS- positive basophils, and smaller strongly AB- and AF-positive basophils; in the metaadenohypophysis lead hematoxylin-positive and -negative cells. These cell types are compared with those in other teleosts.

THE SELECTIVE VALUE OF EYE AND PIGMENT LOSS IN MEXICAN CAVE FISH.

Theories regarding the underlying basis of "regressive evolution" have fallen into two main categories. One school maintains that the degeneration of an organ is the result of disuse combined with mutation pressure and "relaxed selection." The second group suggests that selection pressure is of primary importance in bringing about "regressive evolution." This study was made in the hope of distinguishing between these theories experimentally. Mexican cave fish, which have lost their eye and body pigmentation, were chosen for study. These cave fish provide the only known instance where a recently evolved species can be crossed with its ancestral type and studied genetically. Crosses between the blind, depigmented cave fish and eyed, pigmented river fish revealed that there is a polygenic basis for generalized eye loss and body pigmentation loss (Sadoglu, 1956, 1957a; Kosswig, 1963). In addition, two independent factors have been found which have a major effect on both eye pigment and body pigment reduction. Still another factor has been detected which apparently is responsible for normal lens fibre formation (unpublished data). In the present paper another approach has been undertaken in order to test directly whether pigmentation and sight, or the absence of them are selected in photic and aphotic environments.

The Selective Value of Eye and Pigment Loss in Mexican Cave Fish

The Selective Value of Eye and Pigment Loss in Mexican Cave Fish