Research Article |
Corresponding author: Luis Espinasa ( luis.espinasa@marist.edu ) Academic editor: Horst Wilkens
© 2023 Luis Espinasa, Kayla-Ann Lewis.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Espinasa L, Lewis K-A (2023) Eye convergence is evoked during larval prey capture (LPC) without visual stimulus and in blind cavefish. Subterranean Biology 46: 147-60. https://doi.org/10.3897/subtbiol.46.105707
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In zebrafish larvae, the first response when detecting prey is an oculomotor behavior; eye convergence. Eye convergence increases the overlap between the visual fields of the left and right eyes to prepare for tracking prey. A high vergence angle is maintained throughout the prey-tracking and capture swim phases, enhancing binocular depth. Since the discovery of eye convergence, hundreds of articles reporting on this behavior in zebrafish have been published. In this study, we found that the larvae of blind tetra cavefish, Astyanax mexicanus, despite being adapted to the absence of visual stimuli due to the lack of light in the cave, have retained the oculomotor behavior of eye convergence in their vestigial eyes. In Astyanax, eye convergence responses can be triggered singlehandedly by vibrations elicited with a glass rod at frequencies similar to those generated by its prey (10–35 Hz). The blind cave tetra offers an intriguing combination of regression of the eye structure, while retaining several of the physiological functions and actions performed in the eye, including light-entrained retinomotor rhythms and eye convergence.
Astyanax, behavior, binocular vision, eye convergence, larval prey capture, ocular vergence, troglobite
Often, natural selection cannot eliminate vestigial structures because they have retained some essential function. For example, the human embryo has gills slits like a fish. Why are the gill slits retained? During development, these gills become various structures essential for survival, such as the ear cavities, middle ear bones, muscles for chewing, the lower jaw, and certain parts of the neck including the thymus and thyroid (
Cave animals are excellent models which can provide insight into the general principles of regressive evolution. Many organisms in caves retain features that can no longer serve their ancestral purpose. For example, the Cholevid beetle, Ptomaphagus hirtus, common in Mammoth Cave, Kentucky, has tiny vestigial eyes that retain light perception and have a complete circadian clock gene network (
Studies of regressed structures generally assume them to be nonfunctional (
Adult Astyanax cavefish have minute optic capsules buried deep beneath the integument, which are not responsive to visual stimuli (
Astyanax mexicanus has two morphs; a surface, eyed morph (A) and a blind, cave morph (B) whose nonfunctional optic capsules are buried deep beneath the integument. At birth, cavefish have eyes and respond to light, but soon after, the eye degenerates. At 16–21 dpf, both the surface (C) and the cavefish larvae (D) have eyes. However, while the eye capsule and retina of the surface larvae (E) are well suited for vision, the eye capsule of the cavefish (F) has degenerated, the lens has undergone apoptosis, the outer nuclear layer of the retina is not completely differentiated, and there are essentially no photoreceptors. Cavefish larvae may detect light and darkness at this stage, but they lack central visual acuity and are thus blind to form perception. The scale in the right column is the same for the left column.
For this study, we concentrated on another physiological function and actions performed by the eyes of fish; eye convergence during Larval Prey Capture (LPC) behavior. LPC is characterized by a fast-striking motion toward the prey within tens of milliseconds. Serial time-lapse images of single prey capture events have revealed that in Astyanax (
Analysis of zebrafish conducting LPC while hunting paramecia uncovered a novel oculomotor behavior, eye convergence, which constitutes the first response of larvae to their prey (
This study aims to establish if fry from cavefish, despite being adapted to living in an environment characterized by perpetual darkness, have retained eye convergence when conducting LPC.
For this study we used the recordings of LPC used by
Approximately 24 hours before behavioral experiments, Brine shrimp cysts (Artemia salina) were added to a plastic container with 1.2 L of water at a salinity of 25–30 ppt, pH of 7.5–8.5, and a temperature of 28 °C, with constant aeration. Immediately prior to testing, Artemia were rinsed with fresh water and placed into recording chambers. Only newly hatched Artemia nauplii, of the 1st instar stage, were used in behavioral experiments to ensure consistency of vibrational stimuli.
As mentioned before, recordings of LPC were the same as the ones used by
For recordings of LPC behavior on live prey, single fish were placed in a 9 cm diameter petri dish filled with ~20 mm of water to constrict the larvae into a single focal plane. Fry were allowed to acclimate for 2 minutes before the experiment began. Approximately 30 Artemia nauplii were used to record feeding behavior, and fish were imaged until they completed at least four successful strikes.
For recordings of LPC behavior on a vibrating glass rod, microinjection needles were made from glass capillaries with a Narishige’s PC-10 Dual-Stage Glass Micropipette Puller. Borosilicate glass capillaries were heated and pulled to get fine needles, like those used for cell injection. The tip of the glass rod had a diameter of ~0.15 mm, about half the size of an Artemia nauplii. The vibration stimulus was generated using the ~0.15 mm diameter glass rod attached to an audio speaker (8ohm 0.1W 38 mm speaker) that produced 10 Hz with a TTI TG210 2MHz Function Generator. The peak-to-peak voltage was set to 21V. The axis of the vibration was in the horizontal plane. Individual fish were placed in a 9 cm diameter petri dish or a 3.5 cm diameter petri dish with water to a depth of ~3 mm. Fry acclimated in the experimental room for at least 2 hours. They were then transferred gently to the Petri dishes, where they further acclimated for 2 minutes before introducing the glass rod. The age of the fry tested was 16–21 dpf (Fig.
An analysis frame by frame of the recording was done starting 2 seconds before the initiation of movement toward the prey or vibrating glass rod until 2 seconds after LPC ended. Eye vergence angles were measured before, during, and after responses to the stimuli by drawing two lines along the width of each eye until the line from one eye converged with the line drawn from the other (Figs
A line was also drawn perpendicular to the eye’s width, passing through the center of the pupil, in the direction of the center of that eye’s visual field (Fig.
Eye vergence in surface fish stimulated by a vibrating (10 Hz) glass rod (A, B). Larval prey capture (LPC) behavior is characterized by a fast-striking motion toward the vibrating glass rod (yellow arrow) within tens of microseconds. Red asterisks highlight instances when the eyes converged. Higher magnification to highlight the changes in eye position during a strike (C–F). Freely swimming larvae have eyes pointing sub-perpendicular to their body in which the binocular overlap (blue) region of their visual space is minimal (C, D). During LPC, the mean eye vergence angle changes, largely expanding the binocular area of visual space (E, F).
Eye vergence in surface fish while in the dark (A). Higher magnification to emphasize that despite being in the dark and without visual stimuli, the eyes change position (B, D) which, if illuminated, would have largely expanded the binocular proportion of visual space shown in blue (C, E). Notice that eyes converged when prey is detected at a distance (A:0.43 and D), followed by a strike (A:0.86). Soon after, eyes return to normal position (A:1.14). Yellow arrows highlight the prey and red asterisks highlight instances when the eye converged.
Blind cavefish Astyanax larvae have ocular vergence during LPC in response to vibrations from a glass rod, which elicits a strike behavior. Freely swimming larvae have eyes pointing sub-perpendicular to their body (A). When the source of a vibration stimulus is over the head, eyes turn upward (B). This was followed by a strike in which the cavefish larvae bit the glass rod (C). Eyes vergence remains for a few moments after a strike (D). Dotted arrows highlight eye angle before vergence to show the change of eye position.
Larval prey capture (LPC) behavior is characterized by a fast-striking motion toward the prey within tens of microseconds. Our first experiment tested if surface Astyanax larvae have ocular convergence during LPC when presented with a source of vibrations under light conditions that are not the stereotypical image of prey, such as a microcrustacean. For this, we used a vibrating glass rod at a frequency of 10 Hz. Ten Hz is a frequency similar to the one generated by Artemia nauplii that preferentially trigger successful strikes by Astyanax larvae (
Our second experiment tested if surface Astyanax larvae have ocular convergence during LPC with no visual stimuli. For this, we recorded LPC with an infrared LED light source. In trials, vergence of the eyes responded to the non-visual stimulus (Fig.
As the introduction mentions, Astyanax cavefish and surface fish are initially born with equivalent eye structures, and both respond actively to light stimuli. While up to adulthood, cavefish may have some type of detection and response to light, cavefish larvae become effectively blind to patterns other than shadows early on. As reviewed in the introduction, in the 16–21 dpf cavefish larvae used for this study there is an overall degeneration of the eye capsule and almost complete regression of the outer nuclear layer that contains the cell bodies of the photoreceptor cells (Fig.
Our third experiment tested if blind cavefish Astyanax larvae have ocular vergence during LPC when presented exclusively with vibrations in the range generated by their prey. For this, we used a vibrating glass rod at a frequency of 10 Hz. Recordings for experiments showed that freely swimming cavefish larvae have eyes in a lateral-oriented position, but when a stimulus is above them, their eyes move upward, as reflected by the position of the pupil (Fig.
Scale drawings showed that, just as in surface fish, freely swimming larvae have eyes pointing sub-perpendicular to their body, with the region of binocular overlap (blue) being minimal (Fig.
Each eye can have its own vergence. Depending on the position of the source of the vibrating stimulus, a single eye may move forward while the other remains laterally pointing (Fig.
Despite being blind, the eyes of cavefish larvae tracked the position of the source of vibrations (A, B). During LPC, the mean vergence angle changes in the cavefish, advancing the binocular field to close to the front of the mid-point of the eyes (C, D). Each eye can have its own and different angle of vergence (E–H). Depending on the position of the source of the vibrating stimulus, a single eye may move forward, while the other remains laterally pointing (E). At another position of the stimulus, both eyes may converge forward (G). Soon after, eyes return to normal position (F, H). Eye vergence may represent a vestigial behavioral character, left-over in the evolution of Astyanax cavefish, since changes in the binocular field may be irrelevant due to the blindness of larvae by this stage of development and because of living in the dark with no visual stimuli.
Astyanax
cavefish have been reported to be blind and lack physiological response to light in the tectum (
Our behavioral data suggest that surface Astyanax fish may have several prey-capture specific motor programs. One may start with a visual stimulus that activates ocular convergence for enhanced binocular processing of visual information. This is followed by the appropriate J-turns of the tail, or C-bend turns movement for a strike towards the prey. Another motor program may start with non-visual stimuli, such as a 10 Hz vibration. This may activate in synchrony the J- or C- turns while positioning the eyes in convergence. The advantage of this synchronous activation is that under light conditions when the fish has positioned itself for the final strike motion based on vibration information, binocular optimization can then occur. This is supported by observations in zebrafish (
For cavefish, the second motor program may be at work. LPC may start with a non-visual stimulus, such as a 10 Hz vibration, smell, or sound. This may activate in synchrony the C- or J- turns while positioning the eyes in convergence. In the case of the blind cavefish, the vergence of the eyes may currently serve no function while in the dark environment of the cave and be a left-over of evolution, where natural selection or other evolutionary forces have not regressed this behavior. The activity of premotor neurons producing eye convergence commands is assumed to have been a fundamental component of the activity pattern underlying all behavioral responses to prey-like stimuli in the ancestral surface fish that gave rise to the cavefish. Just as it may be in the existing surface Astyanax fish. This activity has not regressed at the same pace as structural eye degeneration.
Retinomotor rhythms and ocular vergence may have been preserved fortuitously in the degenerating cavefish eye evolution. The persistence of the retinomotor movements in response to a circadian rhythm, and the eye vergence during LPC, suggest that both are controlled by genetic and physiologic signals independent of degenerating cavefish eye. The expression of sonic hedgehog and tiggy-winkle hedgehog genes is enhanced along the anterior midline of cavefish embryos (
The blind tetra, Astyanax mexicanus, despite being adapted to the absence of visual stimuli due to the lack of light in the cave, have retained the oculomotor behavior of eye convergence in their vestigial eyes as a response to prey stimuli. In Astyanax, eye convergence responses can be triggered singlehandedly by vibrations elicited with a glass rod at frequencies similar to those generated by its prey (10–35 Hz).
Ehud Vinepinsky helped with the set-up for recording behavior. Most research was performed at the laboratory of German Sumbre at the Institut de Biologie de l’ENS (IBENS), CNRS, France, during a sabbatical to LE from Marist College. Some of the specimens used were provided by Sylvie Rétaux at the Paris-Saclay Institute of Neuroscience, CNRS, and University Paris-Saclay, France. Jordi Espinasa reviewed the manuscript.