Introduction

Similar to right- and left-handedness, humans exhibit lateral differences in eye usage. Typical visual lateralization appears in sighting dominance1,2 such as the preference of an individual to use either eye to look through a microscope, keyhole, or gunsight. This preference is traditionally attributed to the tendency of the brain to prioritize monocular input, resulting in a single visual image being consciously perceived by suppressing input from the other eye3,4. Approximately 65% of humans with normal vision prefer to use their right eye as the dominant eye, whereas 35% prefer their left eye5 and eye dominance remains consistent at the retest conducted a year later6. Visual lateralization has been demonstrated in many animals, and its function has attracted considerable attention7.

Several hypotheses have been proposed to explain why these animals exhibit eye dominance. One hypothesis states that the eye with better visual acuity and resolution is used more often than the other8 as it receives relatively more precise information to perceive or recognize subjects. Differences in pupil size or photoreceptor innervation density have been reported to contribute to visual sensitivity9,10. For example, chicks (Gallus domesticus) use their left eye when inspecting potential predators and exhibit higher ganglion cell densities in the left eye than in the right eye11,12. Another hypothesis posits that preferential eye use results from hemispheric specialization, suggesting that eye dominance is a consequence of functional differentiation within the brain. A wide range of taxa, including mammals13,14,15 birds16,17,18,19 reptiles20,21 and fish22,23,24,25,26,27 show an object or context-dependent use of either eye. For example, chicks prefer to use the right eye to search for food and the left eye to respond to predators or attacking conspecifics28,29,30. Functional visual lateralization in pigeons is thought to be associated with asymmetry of visual pathways within the brain31,32. Such visual lateralization has been observed in cephalopods such as octopuses and squid33,34,35,36. The preferential use of one eye may share a common evolutionary principle favoring asymmetric information processing, enabling more efficient complex sensory inputs. It is believed that relatively more lateralized individuals can outperform non-lateralized conspecifics under certain biological circumstances37,38,39. Monitoring a particular object with one eye might result in left-right differences in the efficiency of the resulting behavior, as visual information is transmitted and processed predominantly by the contralateral side of the brain, which may then initiate lateralized behavior. However, the relationship between visual lateralization and behavioral choice has not been directly tested, except in birds40 and the potential role of visual lateralization remains obscure in other animals7.

The scale-eating cichlid fish Perissodus microlepis from Lake Tanganyika in Eastern Africa provides an excellent model for studying lateralized behavior41,42,43,44. In the field, adult P. microlepis exhibits clear lateralized predation, utilizing their asymmetric mouth morphology, as lefty fish exclusively attack the left side of the prey fish, whereas righty fish attack the right side45. Experimental evidence shows that this lateralized behavior is as pronounced as human handedness41,46,47. The water in Lake Tanganyika is very clear, allowing scale-eating fish to visually search and target prey fish48. Figure 1A shows the typical predatory action sequences of P. microlepis observed in a laboratory tank. The scale-eating fish utilized the eye on the mouth-opening side (Fig. 1B) to assess the position, posture, and distance of the prey fish and target scales, and to decide when it should turn its head to attack the side of the prey at the preshape phase of the attack. Based on this lateralized behavior, we speculate that the eye on the mouth-opening side may play a crucial role in predation. Most fish, including P. microlepis, have large monocular with small binocular visual fields because of their laterally positioned eyes, and the optic nerves from the eyes project mainly to the contralateral side of the optic tectum, known as an important sensorimotor center that processes visual information in fish49,50,51,52,53,54. Owing to this circuit organization, blocking one-eye vision eliminates direct visual input to the hemispheres and enables us to examine the functional role of the hemisphere of visual information in the lateralized predation behavior of this fish.

Fig. 1
figure 1

Visually guided predation behavior of Perissodus microlepis. (A) The typical predation behavior consisted of five behavioral components: (1) approach dash, (2) stealthy swimming, (3) S-shaped posture for readiness to attack, (4) body flexion of attack, and (5) twisting to tear off scales. After approaching the prey fish, the scale-eating fish positioned itself for an attack, presumably relying on monocular visual input at that time (frame #3). Time in milliseconds is represented on each frame. (B) Dorsal view of the heads of lefty and righty fish individuals. Arrows indicate the direction of mouth opening. Red ellipses indicate the eye corresponding to the mouth-opening side, and blue dot ellipses indicate the eye on the contralateral side. Scale bar = 1 cm.

Full size image

In the present study, we aimed to elucidate the functional significance of visual lateralization in the scale-eating fish P. microlepis through two behavioral experiments. First, we compared behavioral responsiveness to visual stimuli to test whether they responded equally to the same visual stimulus presented to either eye. Fish generally exhibit body bending and escape behaviors in response to rapidly looming stimuli, with quick shifting of their heads to the contralateral side55,56. Second, to elucidate how visual input from each eye contributes to lateralized behavior, we examined the effects of impaired vision on the mouth-opening side or the contralateral side on predation behavior, focusing on attack-side preference, motion kinetics, and predation success. The results revealed that both visually evoked escape and voluntary predation behaviors were driven more effectively by the dominant eye. The consistent use of the eye on the mouth-opening side to target their prey or avoid nociceptive stimuli suggests the presence of a dominant eye with higher sensitivity or more efficient visual system in the brain than that of the other eye. Visual lateralization appears to confer a significant survival advantage by enhancing the ability to efficiently predate and escape potential threats.

Results

Dominant eye is present on the mouth-opening side

Escape responses to visual stimuli are common among many animals7. To observe the escape behavior of P. microlepis in response to a visual stimulus through one eye, we applied an image of a looming black disk to the left or right side of individuals. We observed that P. microlepis typically moved away from the visual stimulus before the black circle on the display reached its maximum size (Fig. 2, A and B). It is known that the fish exhibited a startle response with quick body flexion as C-bend in response to sudden visual stimulus57. In the present study, a startle response was rarely observed in response to the looming stimulus, but the fish retreated from the stimulus by shifting or turning the head to the opposite side. Lefty P. microlepis individuals that predominantly attacked the prey fish present on their left side (Fig. 1) retreated from the stimulus at a significantly higher rate when their right eye (on the mouth-opening side) was stimulated (Fig. 2B; Supplementary Movie 1) than when their left eye was stimulated (Fig. 2C; one-sample Wilcoxon signed rank test, P < 0.001). Similarly, the righty fish showed a higher escape rate when their left eye was exposed to the visual stimulus than when their right eye was exposed to the stimulus (Fig. 2C; P < 0.001). Thus, there was a significant difference in response direction between the two groups (Fig. 2C; Wilcoxon rank sum test, Z = − 2.85, P = 0.004). Therefore, the visual stimulus applied to the eye on the mouth-opening side was more effective in eliciting an escape response, indicating that the eye or visual system input by the eye was more effective than that by the contralateral eye in eliciting behavioral responses. Hereafter, we refer to the eye on the mouth opening side as the dominant eye. Notably, the dominant eye viewed the scales on the side of the prey during predation.

Fig. 2
figure 2

Visually evoked escape response. (A) Schematic diagram of the visual stimulation setup. A subject scale-eating fish was placed in the narrow tank (blue) between two screen monitors. A dark looming disk (black) applied from the screen monitors elicited escape responses in the fish. The responses of the fish were monitored with a video camera positioned above the tank. (B) Sequence of the responses of a lefty scale-eater to a visual looming stimulus from the right side. The fish retreated to the side of the tank opposite the stimulus. (C) Boxplots (mean, median and interquartile values) of direction of the visually evoked retreat response [(R − L)/(R + L), see Materials and Methods] in lefty and righty scale-eaters (N = 19 fish in each category). Dotted lines indicate chance levels. The visual responsiveness was tested using a one-sample Wilcoxon signed-rank test for deviation from the chance level (0). Wilcoxon rank sum test was used to compare the response preference of the lefty and righty fish. **P < 0.01; ***P < 0.001.

Full size image

To examine how visual inputs from the dominant eye contribute to predation behavior and efficiency in P. microlepis, we impaired the vision of either eye through artificial cataract induction by injecting methanol into the anterior chamber between the cornea and lens of one eye (Fig. 3, A and B; see Materials and Methods). After manipulation, the P. microlepis individuals were healthy enough to consume the artificial feed. To confirm visual impairment, visually evoked behavioral responses were tested before and after the induction of artificial cataracts. After one eye was impaired, either on the mouth-opening side or contralateral side, the probability of response to the visual stimulus on the impaired side stimulus was significantly reduced (Fig. 3, C and D; Wilcoxon rank sum test results for impairment in the eye on the mouth-opening side: Z = − 2.70, P = 0.007; impairment in the eye on the contralateral side: Z = − 2.51, P = 0.012). In contrast, the responses to visual stimuli in the intact eyes remained unchanged.

Fig. 3
figure 3

Effects of visual impairment (artificial cataracts) in either eye on the visually evoked response. (A) Methanol was injected into the anterior chamber between cornea and lens in either eye. (B) Lens was clouded after methanol injection, resulting in an artificial “cataract.” (C, D) Boxplots (median and interquartile values) of visually evoked responses before and after the induced “cataract”. Artificial cataracts significantly reduced the probability of response to the stimulus applied to the cataracted side, whereas no change was observed in the probability of response to the stimulus applied from the intact side. (C) Fish with cataracts in the eye on the mouth-opening side (N = 7 fish). (D) Fish with cataracts in the eye on the contralateral side (N = 6 fish). Wilcoxon rank-sum test was used to compare the number of visually evoked responses before and after the visual impairment. *: P < 0.05, **: P < 0.01, ns: not significant.

Full size image

Dominant eye is required for attack side preference

Even after visual impairment in either eye, P. microlepis individuals could attack their prey fish and feed on scales (Supplementary Movie 2). To test the role of visual information from each eye in predation behavior, we compared the total number of predatory attacks before and after the induction of artificial cataracts (Fig. 4A and B). After the induction of artificial cataract in the dominant eye, there was no significant change in the number of predatory attacks (before cataract induction: 19.3 ± 2.4 times/h; after cataract induction: 18.9 ± 2.5 times/h; mean ± standard deviation [SD], Wilcoxon signed-rank test, S = − 1.5, P = 0.844). Similarly, artificial cataract in the contralateral eye also did not result in any significant change in the number of attacks (before manipulation: 25.3 ± 4.4 times/h; after manipulation: 19.0 ± 2.8 times/h; S = − 8.0, P = 0.125). Thus, as long as one eye was available, the fish could initiate and actively engage in predation behavior.

Fig. 4
figure 4

Effects of visual impairment on the predation behavior. (A) Boxplots (median and interquartile values) of total number of predatory attacks by individuals with cataract in the eye on the mouth-opening side or those with cataract in the eye on the contralateral side. (B) Number of attacks on the non-dominant side by individuals with cataract in the eye on the mouth-opening side and those with cataract in the eye on the contralateral side. Wilcoxon rank-sum test was used to compare these values before and after the visual impairment. *: P < 0.05, ns: not significant. Seven fish were visually impaired on the mouth-opening side, and six fish were visually impaired on the contralateral side. The numbers of fish remain the same in Figs. 5 and 6.

Full size image

However, the attack direction was greatly affected by visual impairment (Fig. 5). Visual impairment of the eye on the mouth-opening side drastically reduced the number of attacks on the dominant side and significantly increased the number of attacks on the non-dominant side, whereas visual impairment in the eye on the contralateral side did not induce any change in the number of attacks on either the dominant or non-dominant side (impairment of the eye on the mouth-opening side: S = 13.5, P = 0.031; impairment of the eye on the contralateral side: S = 6.5, P = 0.250).

Fig. 5
figure 5

Rate of predatory attacks on the dominant side and the success rate of predation before and after visual impairment. (A) Boxplots (median and interquartile values) of rate of predatory attacks on the dominant side by individuals with the cataracted eye on the mouth-opening side and those with the cataracted eye on the contralateral side. (B) Success rate of predation by the individuals after cataract induction. Wilcoxon signed-rank test and Wilcoxon rank sum test were used to compare the rate of predatory attacks on the dominant side and the success rate of predation before and after the visual impairment, respectively. *: P < 0.05, **: P < 0.01, ns: not significant.

Full size image

Owing to the change in the number of predatory attacks on the dominant vs. non-dominant side, the percentage of attacks on the dominant side decreased from over 90% before manipulation to less than 50% after visual impairment in the eye on the mouth-opening side (Fig. 5A; Wilcoxon signed-rank test: S = − 14.0, P = 0.016). Furthermore, the success rate of predation on the mouth-opening side was greatly reduced compared with that before the visual impairment of the eye on this side (Fig. 5B; Wilcoxon rank sum test, Z = − 3.07, P = 0.002). In contrast, when the vision of the contralateral eye was inhibited, neither the percentage of predatory attacks on the dominant side nor the success rate of predation was affected (percentage of attacks on the dominant side: S = 8.5, P = 0.094; success rate of predation: Z = − 1.20, P = 0.229). These results revealed that the attack-side preference was highly dependent on the dominant eye.

Visual impairment in the dominant eye affects predation kinetics

Attack motion during predation also changed significantly after the dominant eye was impaired (Supplementary Movies 2 and 3; Fig. 6A). We compared the kinetics of the attack motion in P. microlepis individuals before and after visual impairment in either eye. Prior to the manipulation, both the body flexion angles and maximum angular velocities during predatory attacks were significantly greater in attacks on the dominant side (i.e., on the mouth-opening side) than in attacks on the non-dominant side (i.e., on the contralateral side) (flexion angle in attacks on the dominant side: 53.86 ± 14.85°; flexion angle in attacks on the non-dominant side: 44.90 ± 16.05°; mean ± SD, Z = − 2.26, P = 0.024) (angular velocity in attacks on the dominant side: 6,378.68 ± 2,591.55 degree/s; angular velocity in attacks on the non-dominant side: 4,736.05 ± 2,004.65 degree/s; Z = − 2.90, P = 0.004). After the vision of the eye on the mouth-opening side was impaired, the body flexion angle during predatory attacks on the dominant side (i.e., the mouth-opening side) decreased to approximately half of the pre-manipulation level (Fig. 6B; Z = 3.07, P = 0.002), and the maximum angular velocity of flexion during the attack also decreased by half (Fig. 6C, Z = 2.94, P = 0.003). In contrast, both kinetics were kept as before for the non-dominant side attacks (Fig. 6, B and C; flexion angle: Z = − 0.42, P = 0.676; angular velocity: Z = − 0.21, P = 0.835). Neither the body flexion angle nor the maximum angular velocity of the predatory attacks on the dominant side changed (Fig. 6, B and C, right columns; flexion angle: Z = 1.20, P = 0.230; angular velocity: Z = 1.52, P = 0.128).

Fig. 6
figure 6

Effects of cataract in either eye on the behavioral kinetics of predation. (A) Sequence of predatory attacks on the dominant side by individuals before and after visual impairment in the eye on the mouth-opening side or contralateral side. Time in milliseconds is represented on each frame. Single dagger symbol indicates the moment of maximum body flexion angle. (B) Boxplots (median and interquartile values) of body flexion angle and (C) maximum angular velocity during predatory attacks on the dominant and non-dominant sides by the individuals with cataracted eyes on the mouth-opening side before and after cataract induction. The kinetics of predatory attacks on the dominant side after visual impairment in the eye on the contralateral side are shown in the right column. **: P < 0.01, ns: not significant.

Full size image

Discussion

The results obtained in the present study showed that P. microlepis has a dominant eye on its mouth-opening side and that the dominant eye plays a key role in lateralized scale-eating behavior. The lefty P. microlepis individuals that opened their mouths to the right to attack the prey fish present on their right side responded more sensitively to the visual stimuli applied to the right eye than to the left eye, indicating that the right eye input was more effective in evoking a behavioral response than the left eye input. The opposite was observed in the righty P. microlepis individuals. When the dominant eye (the right eye in lefty or left eye in righty individuals) was impaired, the frequency of predatory attacks on the dominant side decreased, whereas the total number of attacks on either side remained unchanged. However, the number of attacks on the non-dominant side increased, thereby indicating a loss of the original preference in the attack direction. The typical predation behavior of P. microlepis consists of a sequence of five components: approaching the prey, stealthy swimming to sneak around the side of the prey fish, acquiring an S-shaped posture to aim for the scales on the flanks of the prey, fast body flexion to attack, and twisting to tear off the scales of the prey (Fig. 1A)41. In the present study, it was observed that after visual impairment, scale eaters were able to track their prey, but their motor performance decreased, with a significant reduction in body flexion angle and maximum angular velocity during quick body bending of the attack motion. Consequently, the predation success rate decreased significantly. In contrast, impairment of the non-dominant eye did not affect the kinetics of scale eating or success rate of predation. These results suggest that the dominant eye of the scale eater plays a crucial role in targeting the scales of prey fish in the later phases of predation motion. It has been previously shown that in scale-eating fish, the size of the eye on the mouth-opening side is significantly larger than that of the eye on the contralateral side, and that of the optic tectum processing the visual input from that eye58. In general, a larger eye size or higher photoreceptor density contributes to visual acuity59. The preference for using the eye with higher ganglion cell density in visual discrimination tasks has been reported in chickadees and starlings11,12,60,61. Similarly, the dominant eye of P. microlepis is expected to have better visual acuity, enabling it to acquire external information clearly and quickly, thus providing an advantage for targeting prey scales. It has also been suggested that P. microlepis exhibits anatomical or functional lateralization of the visual system, as observed in birds. Thus, the present findings highlight the broader biological significance of eye dominance in complex predatory behaviors.

Motor, sensory, or cognitive lateralization has been reported in various vertebrates and invertebrates42,62,63,64,65,66,67. Sensory lateralization is believed to correlate with behavioral laterality. For instance, vision lateralization is expected to affect the lateralized hand or foot motion, particularly in diurnal animals40. However, few studies have investigated the association between different lateralization modalities, such as the correlation between eye preference and hand use in humans5. In the present study, we observed that, in P. microlepis, reliance on the dominant eye greatly affected the choice of attack direction and enhanced fine-tuned movements through accurate visual information. These findings provide valuable evidence for the relationship between sensory lateralization and behavioral laterality in fish.

To survive, animals must quickly detect nociceptive stimuli or threats to their surroundings and take appropriate actions, such as escaping or freezing to survive68. Numerous independent studies across a wide range of animals have provided strong evidence that animals with laterally placed eyes possess visual fields on either side for predator avoidance and response69,70,71,72. Furthermore, some animals use a specific eye based on the context of visual information because they may exhibit specific cognitive processes that are partitioned into separate hemispheres. For example, when toads are presented with a snake on the side of their left monocular eye, their escape and defense responses are stronger than when a snake is introduced on the side of their right monocular eye73. In contrast, the predatory responses of toads occurred relatively more frequently when insects moved to the right visual field74. However, having a specific eye to respond to a potential predator or prey seems ecologically disadvantageous, because it makes it relatively simple for other animals, including predators, prey, and competitors, to predict the next action24,75. In the present study, P. microlepis showed left-right differences in behavioral responses elicited by visual stimuli provided to either eye, which corresponded to the lateral morphs of visual sensitivity (lefty and righty). Similar two-directional escape behaviors associated with lateral morphs have also been observed in other fish species, such as gobies, killifish and livebearing fish25,27,76. Assuming that the prey and predators interact, the mathematical model estimates that prey are relatively more difficult to capture when escaping in several directions77. Thus, these fish species would take advantage of intraspecific dimorphism in their visual responsiveness and corresponding escape direction to confound the prediction of predators. Similarly, dimorphism in predation behavior must disperse the vigilance of prey animals toward predatory attacks45,78. Therefore, P. microlepis exhibits intraspecific dimorphism in both visual sensitivity and attack motion. Collectively, intraspecific dimorphism in visual responsiveness may offer adaptive advantages for both escape and predation strategies.

Our results suggest that the dominant eye is essential for precise targeting during the later stages of predation and attacks by P. microlepis to forage on the scales of other fish. Visual lateralization and behavioral laterality, which are closely related to the morphological asymmetry of the mouth, are well matched, raising the question of how this efficient relationship is acquired. There are several possible explanations for these findings. First, a genetic link can induce the coincidence of lateralization in different modalities such as the visual system and mouth morphology. The left-right difference in mouth morphology in the scale eater, which correlates with lateralized predation behavior, has been suggested to be regulated by multiple gene loci79,80. Morphological asymmetry of the mouth is observed in fish larvae with yolk in the 1st week of age79indicating that the laterality of mouth morphology is genetically determined before the start of scale eating. Thus, the existence of a genetic link between the dominant eye and lateralized motor performance must be verified. Additionally, it is worth investigating whether experience-dependent learning refines the visual capabilities necessary for accurate predation, as it does for the reinforcement of attack side preference in scale-eating fish46,81. These possibilities linking visual and behavioral lateralization are critical for studying the mechanisms underlying lateralization. In addition to the higher accuracy or resolution in the dominant eye, differences in circuit architecture and function between the left and right sides of the brain may play a role. Investigating neuroanatomical connectivity and signal transmission along neuronal circuits from the retina to the central nervous system, including the pretectal, tectal, and reticulospinal pathways, could shed light on how lateralized behaviors are fine-tuned by visual inputs from the dominant and non-dominant eyes. Another important finding of this study is that attack side preference was significantly altered by visual impairment on the mouth-opening side. Predatory attacks from the nondominant side mechanically stimulate unusual craniofacial skeletal sites, and such feeding experiences can affect mouth morphological asymmetry of the mouth82. Thus, manipulation can modify the original behavioral laterality and asymmetric mouth morphology of P. microlepis.

In the present study, we identified the dominant eye in the scale-eating fish P. microlepis. The dominant eye is closely correlated with specific lateralized behaviors that directly enhance the predation efficiency. Collectively, these findings highlight the role of the dominant eye in the predation success of P. microlepis and offer valuable insights into the potential functions and evolutionary significance of laterality.

Limitations of the study

This study demonstrates a clear concordance between visual and behavioral laterality in P. microlepis, a relationship that likely enhances predation efficiency. This concordance suggests the potential involvement of genetic linkages or plastic changes driven by learning, although the precise mechanisms remain unexplored. Further experimental investigations are necessary to validate these hypotheses and elucidate the implications of sensory lateralization and behavioral flexibility.

Methods

Experimental animals

The scale-eating fish P. microlepis is endemic to Lake Tanganyika and is widely distributed throughout the lake83. The scale-eating fish (scale eaters) used in this study were bred in our laboratory and at the World Freshwater Aquarium Aquatotto Gifu (Gifu Prefecture, Japan). In the laboratory tank system, water temperature and pH were maintained at 27 °C and 8.3, respectively, while the light/dark cycle was kept at 12 h:12 h.

Visually elicited behavioral response

To assess the left-right differences in responsiveness to visual stimuli, we examined the behavioral response of the scale-eating fish to a looming stimulus that rapidly increased the size of a black disk, as if an object was approaching either eye. A 10.1-inch screen monitor was positioned on both the left and right sides of a narrow test aquarium (width, length, and height: 4.5, 22, and 20 cm, respectively) to which individual fish were introduced (Fig. 2A). The looming stimulus increased in diameter from 0.5 to 10 cm during 3 s. A digital video camera (FDR-AX45; SONY, Tokyo, Japan) and two LED lights (36 W) were placed above the tank to capture the behavioral responses of the fish. The entire setup was enclosed in a black curtain to maintain constant surrounding conditions during the experiment. In addition to the individuals used in the predation experiment described below, 19 fish aged 11–12 months were used in the visual response experiment. The fish were housed individually in isolated tanks (25 × 8 × 15 cm) and fed granular food once daily. These were included in the analysis because their conditions were comparable to those of the individuals used in the predation experiment.

Before the visual response experiment, each P. microlepis individual was introduced into the test aquarium and allowed to acclimate to the experimental environment for 30 min. Visual stimuli were provided when the fish was positioned parallel to the screens to stimulate either eye. To minimize habituation, looming stimuli were randomly applied to either the left or right side of the fish at intervals of more than a minute. Ten presentations were given on each side. Based on the video images, we determined whether the fish exhibited a retreat behavior from its original position within 500 ms of stimulation onset (Fig. 2B). To quantify the left-right difference in responsiveness in the visually evoked responses, we calculated the response bias using the following formula: (R − L)/(R + L), where “R” represents the number of trials when the fish responded to the visual stimulus on the right, and “L” represents the number of trials when the fish responded to the visual stimulus on the left.

Artificial cataract induction

To assess the role of visual input from each eye in foraging, we examined the effects of visual inhibition in either eye on the foraging behavior of fish. This has been studied by stitching eyelids or securing adhesive patches to the eyes of birds, lizards, and mammals84,85. However, these methods are impractical for fish and amphibians in aquatic environments. To address this issue, we created artificial cataracts in either eye by injecting methanol beneath the cornea86.

Scale-eating fish were anesthetized with 0.01% ethyl m-aminobenzoate (MS222; Sigma-Aldrich, Poole, UK) and observed under a stereomicroscope. Approximately 0.5 µL of 100% methanol was injected between their cornea and lens with a 27G syringe (NN-2719 S; TERUMO, Tokyo, Japan) (Fig. 3, A and B). Once the anterior chamber and lens were clouded, the fish were transferred to an isolated tank. All individuals with visual impairment in either eye remained in good health and actively foraged when fed. One week after surgery, we checked whether vision was inhibited by testing the visually evoked behavioral response using a looming stimulus on the side of the eye subjected to artificial cataract induction (Fig. 3C and D).

Predation experiment

The scale eaters used in the predation experiment were fed only granulated feed once daily in a rearing tank (90 × 45 × 45 cm) from hatching until the predation experiment, which was performed at four months of age. They reached approximately 35 mm in standard length (SL) at that time, which corresponds to the size at which P. microlepis starts eating scales in the field47,87. During the following 4 months, the scale eaters were fed daily by introducing goldfish (Cyprinus carpio; SL: 50–70 mm) into the tank as prey for feeding on their scales for 10 min. Goldfish were replaced daily, and their scales recovered within one month.

After 4 months of predation training, we investigated the predation behavior of 8-month-old subadult scale-eaters (SL: 43.6 ± 2.6 mm, N = 23) in an arena tank (45 cm × 30 cm × 35 cm, water depth approximately 10 cm) with a hiding place at a corner. To accurately capture predation events, a high-speed video camera system (500 fps, 1,024 × 1,024 pixels, NR4-S3; IDT Japan, Tokyo, Japan) was mounted above the tank, and a digital video camera (30 fps, 3,840 × 2,160 pixels, FDR-AX45; SONY, Japan) was set within a meter in front of the tank. Two halogen lights (HVC-SL; Photron, Tokyo, Japan) were positioned at an angle to the tank, and the entire setup was enclosed by a light-shielding curtain.

One day before the experiment, scale-eating P. microlepis individuals were starved. One individual was introduced into the arena tank on the day of the experiment. Following an hour of acclimatization, the predation experiment was initiated by introducing one prey fish into the tank for an hour. After the experiments, the scale eaters and goldfish were gently captured and returned to their respective home tanks. Perissodus microlepis individuals with a low number of predatory attacks (less than five) were checked for anorexia by feeding on artificial food. Although most individuals resumed feeding, those who did not exhibit feeding behavior were excluded from subsequent experiments. Subsequently, video images were analyzed to determine the number of predatory attacks, attack rate from the dominant side, and predation success rate. The angular velocity of body flexion during the attack and the change in flexion angle were acquired from high-speed camera images. For each predation event, the attack direction (left or right) and success or failure (hit or miss) of the predation were measured. Successful predation was defined as contact between the mouth of P. microlepis and the body of the prey fish during the attack.

The attack rate from the dominant side and the predation success rate were calculated for each individual using the following equations:

$$\:Attack\:rate\:from\:the\:dominant\:side=Ad/(\:Ad\:+An)$$
(1)
$${\text{Success rate of predation}} = (S_{d} + S_{n} )/(A_{d} + A_{n} )$$
(2)

where Ad is the number of attacks from the dominant side, An is the number of attacks from the nondominant side, Sd is the number of successful attacks from the dominant side, and Sn is the number of successful attacks from the nondominant side.

Of the 23 individuals trained for predation for four months, the predation experiment revealed a significant attack-side preference in 15 individuals (binomial test, P < 0.05). Five individuals had no clear lateral bias, whereas three others showed a notably low predation frequency and were excluded from further experiments. Two of the 15 individuals who displayed an attack-side preference were excluded from further analysis because of health issues. After visual impairment, predation experiments were conducted on the remaining 13 individuals.

The recorded images of predation behavior were analyzed using kinematic analysis software (DIPP-Motion Pro 2D; Direct Co. Ltd., Tokyo, Japan) to calculate the body flexion angle and angular velocity during the attack. Body flexion angle and angular velocity were measured at three points on the midline of the body: the snout, caudal peduncle, and center of mass. Based on previous data41 the fulcrum of flexion was set at 38.3% of the body length headward on the midline of the body of the scale-eating fish. The body flexion angle was measured during the attack phase, from the preshaped posture for an attack to the moment the scale-eater’s mouth contacted the prey to forage on its scales (Figs. 1A and 6A). Angular velocity was calculated from the angular change per shooting frame.

Morphological left-right differences in scale-eating fish were determined based on the direction of mouth opening41,45. After the behavioral experiments, P. microlepis samples were anesthetized with 0.01% MS-222 and their mouth and craniofacial morphologies were observed under a stereomicroscope. Lefty fish were identified based on three characteristics: the left mandible was larger than that of righty fish, the left side of the head faced forward, and the mouth was opened to the right. Righty fish were identified based on opposite traits88. All specimens used in the experiment had their mouths open on either side.

A binomial test (P < 0.05) was used to determine whether the attack direction exhibited a significant bias in individual scale-eating fish. Only individuals demonstrating a statistically significant bias in the attack direction were selected for subsequent artificial cataract experiments in which the vision of either the mouth opening or the contralateral side was impaired. A second predation experiment was performed on individuals with artificial cataracts to assess alterations in predation behavior, including attack-side preference, predation success ratio, and scale-eating motion.

This study was approved by the Committee on Animal Experiments of Hokkaido University (approval number: 20–0141) and complied with all relevant Japanese regulations. All handling was performed using MS-222, and utmost efforts were made to minimize suffering.