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Article

Non-Dose-Dependent Relationship between Antipredator Behavior and Conspecific Alarm Substance in Zebrafish

by
Yaxi Li
1,2,
Zhi Yan
3,
Ainuo Lin
1,2,
Xiaodong Li
1 and
Ke Li
1,*
1
Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China
2
College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100190, China
3
School of Ocean, Yantai University, Yantai 264005, China
*
Author to whom correspondence should be addressed.
Fishes 2023, 8(2), 76; https://doi.org/10.3390/fishes8020076
Submission received: 10 December 2022 / Revised: 17 January 2023 / Accepted: 27 January 2023 / Published: 28 January 2023
(This article belongs to the Section Biology and Ecology)
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Abstract

:
A series of behavioral detection paradigms have been developed for zebrafish (Danio rerio) to examine anxiety-like behavioral responses. Among them, the novel tank diving test is rapidly gaining popularity in translational neuroscience and behavioral research for the investigation of psychopharmacological activity focusing on stress. Zebrafish respond to conspecific epidermal-released alarm substances with antipredator reactions. Although the alarm responses of zebrafish were well characterized in a novel tank diving experiment, the relationship between the intensity of the alarm behavior and the concentration of the alarm substance needed to be understood more adequately. In the current paper, we investigated the behavioral phenotypes and potencies of zebrafish elicited by the serial dilution of an alarm substance in the novel tank diving test. Using a video-tracking assisted behavioral quantification approach, we demonstrated no linear concentration-dependent relationship between antipredator behavior and skin extracts, suggesting that an optimal concentration induced each typical behavioral response. The results showed that the freezing duration (%) significantly increased when stimulated with 104-fold times dilutions of skin extract (equivalent 5 × 10−5 fish/L), while erratic movements (%), time in the bottom half (%), and latency to the upper half (s) significantly elevated when stimulated with 103-fold times dilutions (equivalent 5 × 10−4 fish/L). Therefore, the concentration threshold for an alarm substance that elicited innate fear behavior in zebrafish was presumed to be an equivalent concentration of approximately 5 × 10−4 fish/L. The conclusions may fill a knowledge gap between the innate fear response triggered by injured skin and a novel tank diving paradigm that provides insights into the characterization of alarm substance, behavioral responses, and physiological response mechanisms in zebrafish.

Graphical Abstract

1. Introduction

Chemical alarm cues are ubiquitous in aquatic predator–prey systems [1,2,3]. Aquatic animals use waterborne chemical signals to assess the predation risk [4,5]. When encountering a predator, the prey actively or passively releases warning signals to remind nearby shoal members to avoid potential risks, thereby improving the population’s adaptability [6,7]. The skin has been documented to be the origin of the alarm substances in numerous fish species [6]. Conspecifics exhibited innate defensive responses, including dashing, decreased activity, freezing, increased shoaling, and area avoidance, in response to the alarm signals, resulting in improved survival [1]. When challenged in different contexts, the behavioral responses to fear-evoking chemical cues can serve as valuable tools for investigating the underlying mechanisms of alarm reactions in vertebrates.
Zebrafish have been developed as a promising model animal to use to study stress- and anxiety-like behavior, as environmental and pharmacological manipulations affect their behavioral phenotypes [8,9]. In experiments of chronic exposure to fluoxetine, acute exposure to caffeine, and acute and chronic exposure to ethanol, alarm pheromones were utilized as counterparts to reflect the innate fear of fish [9,10]. Numerous researchers followed the procedure by extracting the alarm pheromones and creating dilutions [9,11]. This protocol conducted various experiments with minor modifications [12,13,14,15]. Because the chemical nature of the alarm substance is unknown, the absolute concentration of the substance could not be determined. Therefore, the alarm substance, without a known concentration used as a comparable treatment, could not manifest the outcomes accurately.
Zebrafish have a well-documented antipredator response to chemical alarm cues in a laboratory [11,12]. Waldman demonstrated that the alarm substances caused fish to move closer to each other and to the bottom of the extensive experimental tank [11]. Speedie et al. investigated the response using observation-based and video-tracking-aided behavior quantification methods [12]. The results suggested that using alarm substances on zebrafish allowed the development of high-throughput behavioral paradigms for drug and mutation screening to analyze the biological mechanisms of fear in vertebrates.
A battery of behavioral assay paradigms has been established for zebrafish to examine their anxiety-like behavioral responses [16,17,18,19]. The novel tank diving test [20] is the most utilized and has been considered analogous to thigmotaxis in rodents [8,21]. Commonly used behavioral endpoints measured in the novel tank diving test include erratic movements, time spent in the bottom (top), freezing, latency to enter the top, number of entries to the top (bottom), distance traveled (top, bottom, or total), average velocity, and the ratio of the above two endpoints (such as entries top/bottom ratio, time spent top/bottom ratio, average entry duration, distance traveled top/bottom ratio, etc.) [20,22]. However, due to the differences in experimental conditions and the adaptability of zebrafish, the behavioral phenotypes of different laboratories are not completely the same. The behavioral phenotypes with the highest adoption rates are the first four listed.
Currently, there are three main approaches to the study of the anxiety-like behaviors of zebrafish in the novel tank diving test [9]. Exposure to novelty [23] and alarm substances [24] are the methods used to evoke anxiety while pharmacological modulation can either enhance or diminish the anxiety response through anxiolytic or anxiogenic drugs. Usually, more time spent at the bottom, a longer latency to the top, increased erratic movements, and freezing indicated heightened anxiety [8]. Anxiolytic drugs, such as ethanol [9,25], citalopram [26], and fluoxetine [27], can cause more time in the top, and less freezing and erratic movements. Conversely, anxiogenic drugs (caffeine) have been shown to increase anxiety-like behaviors in this paradigm, resulting in a longer latency to the top and more erratic movements [9].
Previous research has shown that zebrafish skin extracts were dose-independent with behavioral phenotypes in alarm substance influx experiments [12]. It is necessary to elaborate on the relationship between stimulus dose and response for typical behavioral phenotypes in novel tank diving tests, which may expand their usage in discovering pheromone chemistry properties and provide comparable evidence for anxiogenic and anxiolytic drug screening. In this report, we applied serially diluted skin extracts to the novel tank diving tests to investigate the relationship between dose and behavioral phenotypes, including vertical drifts, burst swimming, freezing, and exploring.

2. Materials and Methods

2.1. Animals and Housing

The animal experiments in this study fully complied with the guidelines of the Animal Care Ethics Committee of Yantai Institute of Coastal Zone Research (2021R001), Chinese Academy of Sciences. Adult zebrafish (Danio rerio) were purchased from a commercial distributor (Pinduoduo, Fuzhou, Fujian, China). Fish were adapted to the aquarium laboratory for at least ten days before the experiment. Experiments used 4–6 month-old animals (half male and half female) with an average weight of 0.2 g and an average body length of 3.1 cm. The experiments were carried out in September 2020. Fish were maintained in aquaria in groups of 10–20 fish per 40 L tank using a continuous recirculating system (Haisheng, Shanghai, China). Non-chlorinated water was processed through a water filtration system and filled all tanks (Haier, Qingdao, China), with sea salt added to maintain about 750 ppm. The water temperature was maintained at 25–27 °C. Illumination was provided by ceiling-mounted fluorescent light tubes on a 12 h cycle (on: 08.00, off: 20.00). Experimental naive animals were fed brine shrimp (Jingdong, Beijing, China) twice a day.

2.2. Novel Tank Diving Test Procedure

The novel tank test was used to analyze the exploratory and locomotion activity of the zebrafish in a novel environment (Figure 1A). The experiments were carried out in the morning, 1 h after feeding. According to the validated method [20], zebrafish (n = 15 per treatment and control) were exposed to the alarm substance in preconditioning beakers for 5 min [9] and were individually placed in a new trapezoidal prism aquarium (upper 27 × 7 cm, lower 22 × 7, depth 15 cm), virtually divided into two horizontal portions (bottom and top). The swimming behavior of zebrafish in the novel tank was recorded. Freezing was defined as cessation of movement (except for gills and eyes), while the fish exhibited increased opercular movements when immobile for at least 2 s. To assess fear/anxiety-related behaviors and vertical exploration, duration (%) of erratic movements, duration (%) of freezing bouts, time spent (%) in the bottom area, and latency (s) to enter the top have been quantified according to the behavior parameter definition, calculation formula, and statistical method [28].

2.3. Alarm Substance Extraction and Administration

The alarm substance was extracted from fresh zebrafish skin, as described in the protocol [9,11]. An anesthetized 10 donor fish was placed in ice water and euthanized quickly by decapitation (Figure 1B). We used a razor blade to cut about 10–15 shallow slices on one side of the fish’s body, and each incision was approximately 0.8 cm long. The alarm substance was extracted from injured epidermal cells by water. Since blood would contaminate the solution, we controlled the blade carefully. Subsequently, the donor fish was placed in a Petri dish. Then, the body was washed with 10 mL of distilled water and placed on ice. We gently shook the animal with water in the Petri dish to fully extract the alarm substance. The entire process was then repeated on the other side of the donor fish to produce a stock solution at a concentration of 0.1 fish/mL, consistent with most common experiments [10].
The stock solution (100 mL, 0.1 fish/mL) was prepared from 10 doner fish and stored in aliquots at −80 °C (Figure 1B). Each aliquot was serially diluted 10, 102, 103, 104, 105, and 106-fold. The preconditioning treatments were prepared by adding 5.0 mL of stock solution to 1 L of water. Therefore, a 10-fold dilution of the stock solution yielded a treatment concentration equivalent to 0.05 fish/L.

2.4. Quantification of Behavioral Responses

The bioassay was recorded by a camera (Canon EOS Rebel T3) immediately at the start of the transfer and continued for 7 min. We used the software Format Factory to digitally transfer recorded video to a computer and store the file for later playback and analysis. Video tracking was performed using an EthoVision XT10 (Noldus Information Technology, Wageningen, the Netherlands). Tracking analysis was configured to start after the subject fish was detected for more than 1 s. Detection settings were selected to acquire zebrafish alarm behavior accurately [22]. Movement tracks were smoothed (across ten points) and examined for abnormalities by EthoVision XT10. Then, the standard 2D swim track of zebrafish was generated. Erratic movement and freezing were quantified and analyzed as two locomotion patterns known to change in response to alarm substance treatment or other fear-inducing stimuli. Erratic movements were defined as sharp changes in direction or velocity and repeated rapid darting behavior [29]. The time fish spent at the bottom section of the observation tank has been quantified. The duration for which the fish performed the above behaviors was measured, and values were expressed as a percentage of the total session length. The latency to explore the upper segment was defined as the first entry into the upper segment, and the value was expressed as time duration. The control fish were treated in the same way as the experimental fish according to the protocol. The stimulating solution was aquaculture water in the control group, while in the experimental group it was different dilutions of AS.

2.5. Statistical Analysis

Data were expressed as mean ± standard error of the mean (SEM). All data are tested for normality before statistical analysis and compared to the control group only. For data conforming to a normal distribution, independent samples Student’s t-test was used. Conversely, for data that do not conform to a normal distribution, a non-parametric test (Kruskal–Wallis Test) was adopted. A p-value < 0.05 was considered to indicate significance.

3. Results

In all descriptions of the results, the controls were the behavioral responses of the zebrafish treated with cultured water. In terms of erratic movement, the skin extract in zebrafish showed a dose-independent manner (Figure 2A). Compared with the control (n = 15), the duration of erratic movement was significantly increased at the dose of 10-fold (equivalent to 5 × 10−2 fish/L), 100-fold (equivalent to 5 × 10−3 fish/L), and the dose of 1000-fold diluted stock solution (equivalent to 5 × 10−4 fish/L). Analysis of the variance showed that the alarm substance effect of 10-fold dilution was significant (Mann–Whitney U test, experiment n = 15, p < 0.05), while 100-fold and 1000-fold dilutions were highly significant (100-fold, Mann–Whitney U test, experiment n = 15, p < 0.01 and 1000-fold, independent samples t-test, and experiment n = 7, p < 0.01).
Contrary to our expectations, the amount of time spent at the bottom did not change distinctively with the alarm substance serial dilution process (Figure 2B). No significant effect of alarm substance concentration was observed until the 1000-fold dilution treatment (5 × 10−4 fish/L). The zebrafish spent significantly more time at the bottom of this dilution compared to the controls (Mann–Whitney U test, control n = 15, experiment n = 12, and p < 0.01). Although higher concentration treatments (100-fold and 10-fold dilution) also showed significant differences from the control, there was no dose–response relationship.
There was an increasing trend in the freezing duration as the alarm substance serial dilution process decreased from 104-fold to 10-fold (Figure 2C), although the 100-fold dilution treatment showed a slightly lower freezing duration. Notably, the duration of freezing was very short in the control group, suggesting that the freezing duration is a significant behavioral parameter reflecting innate fear. The duration of the freezing behavior was significantly increased (p < 0.01) at doses 104, 103, 102, and 10-fold dilution compared to the control group.
The effects of alarm substances on the latency to move into the upper half for the zebrafish tended to increase with increasing doses (Figure 2D). At the treatment concentration below 1000-fold dilution, the latency to the upper half was not significantly different from the control group. At a 1000-fold dilution, the latency to the upper half was significantly increased compared to the control (independent samples t-test, control n = 9, experiment n = 6, and p < 0.01). Higher concentrations also showed a significant increasing trend (100-fold dilution, independent samples t-test, control n = 9, experiment n = 6, and p < 0.01; 10-fold dilution, independent samples t-test, control n = 9, experiment n = 6, and p < 0.01).
Overall, erratic motion and bottom dwell time showed a dose-independent manner and significantly differed from controls when treated with only the 1000-fold dilution (Figure 2A,B). The duration of freezing and latency to the upper elevated with increasing stimulus dose (Figure 2C,D) and had a positive correlation with dilution (r = 0.329, p = 0.021 for freezing and r = 0.435, p = 0.003 for latency to the upper half). The lowest concentration triggered a significant duration of freezing increase at the 104-fold dilution treatment (Figure 2C), which is an order of magnitude below the concentration threshold evoking a significant increase in latency to the upper (1000-fold dilution treatment) (Figure 2D).

4. Discussion

Although considerable progress has been made in understanding the role of alarm signals, one of the most pressing tasks is to explore the chemistry and nature behind these interactions. The zebrafish is a cyprinid schooling fish with a complex behavioral repertoire [29,30]. Apart from visual and auditory signals, the damage-released alarm substance of conspecifics alone is sufficient to induce significant and robust alarm reactions [12]. Individuals swimming in such groups or alone exhibit typical startle responses and zigzagging. These overt behavioral responses were well-documented and consisted of multiple behavior patterns, including increased shoal cohesion, area avoidance, dashing, freezing, and reduced foraging and mating [1].
The relationship between the stimulus dose and the behavioral response has been explored in zebrafish [31,32,33]. As the mixture is difficult to quantify, there are few quantitative studies on the fear response elicited by epidermally released alarm substances. We calculated the equivalent concentrations of donor fish extract per unit volume, such as 0.1 fish/mL [12,15] and 0.001 fish/mL [34]. Expressing the cue equivalent concentration as the skin area/mL or g of tissue/mL is also practical for experimental reproducibility [35,36,37].
In the experiments, we observed a tendency for fish to freeze prior to erratic movement after being transferred to the test tank, an instinctual behavior of zebrafish to seek protection in an unfamiliar environment by diving and remaining at the bottom until they feel safe enough to explore [9,38]. Only the optimal concentration (equivalent 5 × 10−4 fish/L) treatment resulted in a significant increase in the duration of erratic movement, although higher concentration treatment showed a somewhat elevation. Appropriate stimulus concentrations, such as 100-fold or 1000-fold dilutions, can induce significant differences in all four behavioral parameters, suggesting that fear-like and activity phenotypes in zebrafish can be reliably dissected in novelty-based behavioral paradigms.
Our findings differ from those obtained in an inflow model that delivers alarm substances through aquarium pipes and records behavioral responses in observation tanks [12]. In terms of the bottom dwell time, the 1000-fold dilution showed a significant difference with the control in the novel tank diving test, while ANOVA found no significant effects at all on the dose levels in the inflow model [12]. We also found that the freezing behavioral phenotype and the upper half latency were weakly related to the dose administered. Therefore, we suggested that the duration of freezing and latency to the upper half are more appropriate behavioral parameters to reflect innate fear in the novel tank diving test.
The detection of an alarm substance can serve as an unconditioned stimulus in the paradigm, which causes the recipient to exhibit an innate fear response. When the alarm substance has been used as a positive control in the novel tank diving test for anxiogenic drug screening, the stimulant dose of the alarm substance should be very cautious. The alarm signaling released from the skin might be very potent. Experiments based on threshold levels of responses in fathead minnows indicated that each club cell contains enough alarm pheromones to create a functional space of about 80 L of water [39]. Therefore, one square centimeter of the skin creates a functional space of approximately 58,000 L of water [39]. This volume is equivalent to a sphere with a radius of 2.4 m or a cylinder 1 m deep with a radius of 4.3 m.
In contrast, one square centimeter of zebrafish skin can activate more than 10,000 L of water [1], a volume equivalent to a cube of approximately 2.2 m per side. In addition, alarm cues generated by 2 cm2 of cyprinid skin from fathead minnows and redbelly dace produced functional space in the field that was demonstrably more remarkable than 2 m over a 2 h period [40]. In our experience, an average of 1 cm2 of skin can be harvested per zebrafish. Consequently, the alarm substance released from the 1 cm2 can be diluted around 103 or 104-fold to serve as warning signals, triggering behavioral responses in novel tank diving tests, indicating that zebrafish have highly susceptible chemosensory responses to alarm cues.

5. Conclusions

In conclusion, we assessed the intensities of behavioral responses to the serial dilutions of skin extracts. The result showed that when stimulated with the skin extract at an equivalent concentration of 5 × 10−5 fish/L, the freezing duration increased significantly, while erratic movements, time in the bottom half, and latency to the upper half were significantly elevated at an equivalent concentration of 5 × 10−4 fish/L. Investigating the relationship between the skin extract administered doses and the alarm behavioral endpoints, especially in the novel tank diving test, contributes to translational research on stress and pheromone activity tracing.

Author Contributions

Y.L. and K.L designed the study. K.L. obtained funding. Z.Y. and Y.L. performed the behavioral analysis. Z.Y., Y.L. and X.L. maintained the aquarium. A.L. harvested the skin. K.L. and Y.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Science Foundation of China (32270533). The corresponding author K.L. thanks the Taishan Scholar Program from Shandong Province of China (tsqn20190403), the National Overseas High-level Talent Program, and the Shuangbai Plan from Yantai Municipal City (2018020) for their support.

Institutional Review Board Statement

All animal experiments in the present study were approved by the guidelines of the Animal Care Ethics Committee of Yantai Institute of Coastal Zone Research (2021R001), Chinese Academy of Sciences, and they were performed following the institutional ethical guidelines for experimental animals.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Schematic diagram of behavior test and data analysis of zebrafish. (A) Alarm substance (AS) dilution strategy from zebrafish skin. (B) The orange boxes represent the treatment with behavioral activities; the grey boxes represent inactive fractions; and the blue boxes present dilution steps. The black arrows represent the dilution procedure of AS in the experiments. Finally, the equivalent concentrations in the pretreatment beaker have been expressed in the green box.
Figure 1. Schematic diagram of behavior test and data analysis of zebrafish. (A) Alarm substance (AS) dilution strategy from zebrafish skin. (B) The orange boxes represent the treatment with behavioral activities; the grey boxes represent inactive fractions; and the blue boxes present dilution steps. The black arrows represent the dilution procedure of AS in the experiments. Finally, the equivalent concentrations in the pretreatment beaker have been expressed in the green box.
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Figure 2. Zebrafish behavioral phenotypes, (A) erratic movements duration (%), (B) time in the bottom half (%), (C) freezing duration (%), and (D) latency to upper half (s), triggered by a serial dilution of the conspecific skin extract (stock solution equivalent with 0.5 fish/L, 10-fold dilution represented the equivalent concentration of 0.05 fish/L, and so on). Zebrafish treated with water in the aquarium were used as the control. Asterisks indicate significant differences between treatment and control (* p < 0.05, ** p < 0.01).
Figure 2. Zebrafish behavioral phenotypes, (A) erratic movements duration (%), (B) time in the bottom half (%), (C) freezing duration (%), and (D) latency to upper half (s), triggered by a serial dilution of the conspecific skin extract (stock solution equivalent with 0.5 fish/L, 10-fold dilution represented the equivalent concentration of 0.05 fish/L, and so on). Zebrafish treated with water in the aquarium were used as the control. Asterisks indicate significant differences between treatment and control (* p < 0.05, ** p < 0.01).
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MDPI and ACS Style

Li, Y.; Yan, Z.; Lin, A.; Li, X.; Li, K. Non-Dose-Dependent Relationship between Antipredator Behavior and Conspecific Alarm Substance in Zebrafish. Fishes 2023, 8, 76. https://doi.org/10.3390/fishes8020076

AMA Style

Li Y, Yan Z, Lin A, Li X, Li K. Non-Dose-Dependent Relationship between Antipredator Behavior and Conspecific Alarm Substance in Zebrafish. Fishes. 2023; 8(2):76. https://doi.org/10.3390/fishes8020076

Chicago/Turabian Style

Li, Yaxi, Zhi Yan, Ainuo Lin, Xiaodong Li, and Ke Li. 2023. "Non-Dose-Dependent Relationship between Antipredator Behavior and Conspecific Alarm Substance in Zebrafish" Fishes 8, no. 2: 76. https://doi.org/10.3390/fishes8020076

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