Use of acoustic stimulus to recognize abnormal zebrafish behavior in water quality warning systems

doi:10.21203/rs.3.rs-2074531/v1

Download PDF

Research Article

Use of acoustic stimulus to recognize abnormal zebrafish behavior in water quality warning systems

https://doi.org/10.21203/rs.3.rs-2074531/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Fish behavioral responses have impeded the development of water quality warning technology due to high false alarm rates. The causes of abnormal behavior in fish such as zebrafish (Danio rerio), are often unknown. In this study, acoustic stimuli were tested in recognizing abnormal zebrafish behavior under cadmium exposure. Acoustic startle response sensitivity curves were obtained and analyzed to determine the loudness and frequencies at which the zebrafish responded to the stimuli. The standard stimulus duration was 30 s, and the interval between two stimulations was 20 min. It was found that Cd exposure can weaken zebrafish acoustic startle responses, particularly at 100 Hz and 97 dB, and was significantly correlated with the reduction of the response. After 30 min, the average rate of swimming activity decreased to 24% in 0.25 TU and to 17.6% in 2 TU exposure. This indicates that the acoustic stimulation elicits a response that could help determine a false alarm. Based on this data, we designed a model for testing the abnormal behavior. This study can offer insights for improving the accuracy and stability of water quality warning systems, and is beneficial to the development and application of early-warning systems with biological monitoring of water quality.

Bio-monitoring

Avoidance reaction

Swimming speed

Acoustic stimulus

Behavioral responses

Water quality

Biological monitoring can reflect the changes in the entire water environment caused by chemical pollution. For example, compared to others, an online water quality monitoring system based on zebrafish (Danio rerio) behavioral responses has rich toxicology data and low operating cost, thus attracting extensive attention (Huang et al. 2014; Jesus et al. 2021; Xia et al. 2016). Zebrafish showed obvious behavioral responses to chemical exposure, and there was a good dose-effect relationship between exposure level and abnormal behavior characteristics (Ren et al. 2013)[9]. However, zebrafish behavior is also disturbed by non-chemical factors, such as water temperature (Raza et al. 2022)^[33], turbidity (Huang 2014), pH, light (Shafiei Sabet et al. 2016),^[34] and influent flow rate (Huang et al. 2020)[8], resulting in a large dispersion of behavioral responses and affecting the stability of the system (Mwaffo et al. 2017; Suriyampola et al. 2016; Teles et al. 2015)[10-12]. Even though the interference of these factors on behavior can be effectively controlled (Huang 2014)[13], random changes in the natural behavior of zebrafish are inevitable and resemble the abnormal behavior characteristics exhibited when they are under chemical exposure (Li et al. 2019)^[32]. These abnormal behaviors are directly introduced into warning model calculations without testing, which is the main cause of system false alarms.

The startle response is an unconditioned and relatively simple brainstem reflex that can be produced by a variety of stimuli. Fish’s auditory responses rely mainly on the inner ear and lateral lines, which is located on both sides of the fish body and can feel stimulation such as water pressure, low-frequency vibrations, and temperature change in the water body (Farrell 2011; Xing et al. 2018). The combination of Weberian apparatus ossicles and swim bladder of auditory auxiliary organs further increases the stimulation of auditory hair cells, which in turn leads to higher auditory sensitivity in zebrafish than in other fish (Wang et al. 2018). Studies have shown that zebrafish exhibit a startle response to sudden acoustic stimuli, but its intensity is significantly affected by chemical exposure. This is mainly because the chemical substances can enrich and accumulate in the fish’s body, destroying its nervous regulation system or damaging tissues and organs, thereby hampering its acoustic startle response.

Ramcharitar et al (2013) demonstrated that the acoustic startle response of zebrafish to sudden changes in light while exposed to 2% alcohol is significantly different from that of the control group. Pavlidis et al (2013) showed that zebrafish failed to exhibit the acoustic startle reaction to the mutation of background color due to chemical exposure on the hypothalamus-pituitary-interrenal (HPI) axis, which ultimately decreased the cortisol levels in zebrafish. In the past ToxProtect 64 toxicity analyzers (BBE, Germany) have been used to identify true or false alarms by detecting goldfish movement activity under a sudden change of brightness. Therefore, varying degrees of behavior to acoustic stimulus was considered an effective way to recognize the interference of chemical exposure or other non-toxic factors (Coffin et al. 2010; Johnson et al. 2007; Wang et al. 2017).

Based on its abnormal behavioral characteristics, this study investigated the effects of Cd exposure on the acoustic startle response of zebrafish to test for acoustic stimuli as a viable way to determine false alarms in water monitoring systems. We used acoustically evoked behavioral responses (AEBRs) to track sound sensitivity by determining the decibels and frequencies the zebrafish would respond to. The levels of AEBRs before and after Cd exposure were compared to establish a warning model for the recognition of abnormal behavior in zebrafish. It can offer reference for improving the accuracy and stability of water quality warning systems, and is beneficial for the development and the application of early-warning systems with biological monitoring of water quality.

2.1 Zebrafish husbandry

Adult (6-month-old) AB zebrafish (Danio rerio) were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. Before the test, the subjects were housed in groups of 100 to 120 fish/100 L aquarium at 22-26 °C with constant filtration and aeration of water. The aquarium was kept under a light/dark cycle of a 10:14 h ratio. The subjects were fed three times per day (Shelled brine shrimp eggs: ≥60% protein, ≥22% fat, ≤3% crude fiber, ≤8% moisture content), except for the 24 h before the commencement, and duration of the experiment. For the experiment, only healthy zebrafish with intact body shape were selected. The test time was from 8:00 a.m. to 8:00 p.m. every day. Test operation standards were in accordance with GB/T13267-91 Water quality—Determination of the acute toxicity of substance to a freshwater fish (Brachydanio rerio Hamilton-Buchanan).

2.2 Experimental apparatus

The device setup consisted of a 510×100×420 mm transparent test box (material: polymethyl methacrylate; Shenzhen, China), a pair of JOYO AC-20 (Shenzhen, China), sound power 40 W, and RS -217-4 mm high-definition CCD camera composition. Before starting the experiment, six zebrafish were put into the test chamber with a 1 L/min continuous flow rate, DO > 6 mg/L, residual chlorine < 0.03 mg/L, and the temperature at 22-26 ℃. A high-definition CCD camera connected to a computer (Lenovo, China) was used to capture the behavioral trajectories of zebrafish in the test chamber, and an EthoVision XT 15 (Nodas, Netherlands) was used for online analysis. The measurement point was the center of the test chamber’s front surface (Fig.1). For the acoustic stimulation, an audio signal program (SweepGenTM, China) was used to adjust the sound’s frequency, and then the sound pressure level (SPL) was adjusted by a Bada DC-811 power amplifier.

2.3 Measurement intervals and cadmium concentrations

Swimming activity is defined as the distance the object's center point moved per unit time, and is recognized as an effective index to evaluate the behavioral responses of zebrafish[12][23]. In this study, the swimming activity was calculated every 0.1 s to evaluate the changes caused by Cd exposure, and a total of 240 min of swimming behavior was recorded. For the first 60 min, pure water was delivered, then a Cd solution was introduced into the chamber. Based on our previous research, the 24 h median lethal concentration (LC50-24 h) of Cd to zebrafish was 7 mg/L[20]. Five concentration gradients (0.1 TU, 0.25 TU, 0.5 TU, 1 TU, and 2 TU) were set up to evaluate the effect of Cd on the swimming behavior of zebrafish. The average and standard deviation of swimming activities were calculated every 10 min.

2.4 Acoustic Stimulus thresholds and durations

Stimulus initial parameters were set to Sine Waveform, Log Sweep Mode, and Fast (smooth) Sweep Speed. The stimulus frequencies were 50, 100, 200, 400, 800 and 1000 Hz, and the acoustic behavioral response corresponding to each frequency was measured. The SPL range was determined as 75 dB to 115 dB— with its lower end limited by noise in the test environment, and its upper end by the available placement positions of the JOYO AC-20 loudspeaker. The SPL intervals were set at every 5 dB (HT-825 decibel meter - precision type). The SPL was the sensitivity threshold of the corresponding frequency until the increased rate of swimming activity was greater than 50% after stimulation. To test repeatability, the tests at all SPL and frequencies were measured in three separate trials. In order to reflect the sensitivity of zebrafish to the acoustic stimulus, the average rate of swimming activity (that is, the ratio of the increase in swimming activity in the acoustic stimulus within 1 min to 1 min before the stimulus) was used to record the experimental results.

To determine the stimulus duration and intervals, long-duration stimuli (1 min) at 100 Hz and 97 dB were delivered to the test chamber. The time required for the continuous increase of swimming activity (Sampling rate: 10 samples per second) was analyzed to determine the duration of stimulus; the time required for swimming activity (Sampling rate: 1 per minute) to return to the pre-stimulation level was analyzed to determine the interval of two stimuli. Finally, five concentration gradients (0.1 TU, 0.25 TU, 0.5 TU, 1 TU, 2 TU) were set up to evaluate the effects of Cd exposure on acoustic startle response.

2.5 Data analysis

EthoVision XT 15 (an automated video tracking and motion analysis system; Noldus, Netherlands) was used to smooth the pixels of the captured video[1] and to correct for frame drops[2]. EthoVision XT 15 video pixel acquisition rate is 10 fps. If the video frame rate is too high, the amount of data will be too vast for efficient data processing. Therefore, the original data was processed by MS-Excel with an average value per minute, and then the data were plotted by Origin (Origin 2022, USA). All experimental data were analyzed by one-way analysis of variance (ANOVA), followed by post hoc comparisons between the experimental groups. Significance was set at p<0.05. Data are presented as mean ± SEM.

[1] The noise will make the neighboring pixels’ gray scale values differ greatly from the actual value; EthoVision XT 15 can smooth the video pixels, thus reducing the influence of fine-grained noise on detection.

[2] The quality of USB camera may cause some frames in the video to be lost, EthoVision XT 15 can interpolate and correct the missing sample coordinates

3.1 Effects of Cd on zebrafish swimming activity

In the control group, there were no obvious changes in swimming activity, which ranged from 30 to 50 mm/s. However, swimming activity immediately increased after Cd was introduced into the tank, but subsided back to equal to or lower activity than that of the unexposed conditions in both exposure tests after a period of maintenance (Fig.2). The maximum swimming activity values were 59.37 ± 17.39, 53.17 ± 15.68, and 67.47 ± 12.57 mm/s in the 0.25 TU, 0.5 TU, and 1 TU exposure tests, respectively (Table 1); but decreased to 36.91 mm/s in 2 TU exposure test. However, the average swimming activity decreased by 36.33%, 26.57%, 42.25%, and 62.95% in those same respective exposure tests. These data match previous results reported by Wang et al (2015)[4]. However, swimming activity and duration of hyperactivity differed with exposure concentration. The higher exposure concentration, the greater swimming activity intensity, but the shorter duration of hyperactivity (p<0.05). In 2 TU exposures, the duration of hyperactivity was only 3.7 ± 2.2 min.

Table 1 The average and the maximum of swimming activity of Zebrafish after Cd exposure

Exposure concentration (TU)	The average of swimming activity (mm/s)		The maximum of swimming activity (mm/s)		Duration of hyperactivity (min)
Exposure concentration (TU)	before Cd exposure	after Cd exposure	before Cd exposure	after Cd exposure	Duration of hyperactivity (min)
Control	41.16±3.72	45.55±8.15	42.56±2.24	49.02±4.87	/
0.1	40.77±7.69	40.83±15.16	42.63±5.20	56.38±12.21^*	94.3±9.4
0.25	46.44±9.18	29.57±17.96^*	47.83±9.53	59.37±17.38^*	45.1±4.0
0.5	40.72±7.75	29.90±17.59^*	41.34±8.69	53.17±15.68^*	32.3±3.9
1	46.20±10.79	26.68±17.73^*	49.34±11.25	67.47±12.57^*	16.7±2
2	45.86±3.98	16.99±7.83^*	47.62±3.89	36.91±8.52^*	3.7±2.2

Asterisk indicates statistically significant differences (p<0.05, Dunnett) relatively to control

3.2 Acoustically evoked startle response of Zebrafish

3.2.1 Sensitivity frequency and sound pressure for stimulus

Zebrafish respond to a frequency stimulus only when the SPL reaches a certain value. As shown in Fig.3, zebrafish showed behavioral reactions at 75 dB when the frequency is 50 Hz. Behavioral responses increased with increasing stimulus SPL from 75 dB to 115 dB, matching previously demonstrated results (Ling 2013). For example, the average rate of swimming activity was 28.1 ± 7.35% at 75 dB and 100 Hz, but as much as 62.4 ± 1.93% at 100 dB and 100 Hz. There was no discernable pattern in behavioral response to the stimulus frequency increasing from 50 to 1000 Hz.

It is worth noting that when the frequency and SPL are too low, the stimulus is not enough to generate distinguishable abnormal behavior. However, if the frequency and SPL are too high, the stimulus may cause physiological damage to zebrafish (Saeed et al. 2016). Thus, the lowest level at which a positive response (50% increase) was obtained was considered the hearing threshold, as it most closely approximated the levels that cause physiological damage. The upper part of the AEBRs curve is therefore the sound that can cause significant changes in zebrafish behavior, while the lower part causes no obvious change (Fig.4). The zebrafish hearing threshold showed an initial decrease, reaching the lowest value at 100 Hz and 97 dB, but increased as the frequency rose from 100 Hz and stabilizing between 400 to 1000 Hz. The sensitive hearing range of zebrafish is in the low-frequency region of 50 to 200 Hz. From this data, a standard frequency of 100 Hz and SPL of 97 dB were selected.

3.2.2 Duration and interval for stimulus presentation

As shown in Fig.5(a), zebrafish showed obvious acoustic startle responses at 100 Hz and 97 dB, and there was no difference in response intensity in the three trials of sound stimulation. Swimming activity increased immediately up to 52.45 ± 1.16 mm/s, and then slowly returned to the pre-stimulation levels, which is consistent with the findings of previous studies. As shown in Fig.5(b), the duration of hyperactivity only lasted 28.46 ± 5.9 s in the long-duration (60 s) stimuli, so a standard stimulus duration of 30 s was selected. The average interval time required for zebrafish to return to pre-stimulation activity level was 14.8 min, and it presented with no significant difference between three successive stimuli. The interval between stimulations needed to be as short as physiologically feasible to maximize the number of responses that could be measured during the experiment. However, overly frequent stimulation could cause the fish to adapt to the stimulus, a standard interval of 30 min was selected.

3.3 Effect of Cd exposure on acoustic startle response

With an abrupt acoustic stimulation at 100 Hz frequency and 97 dB, 100% of the unexposed zebrafish responded within 60 s, while those exposed to Cd displayed a significant decrease in acoustic startle response (p<0.05). As shown in Fig.6, the rate of swimming activity significantly decreased from 52.97%–60.65% to 17.75–25.52%. The average rate of swimming activity decreased from 55% to 24% after 30 min and to 17% after 120 min in the 0.25 TU exposure group.Whereas in the 2 TU exposure group, average rate of swimming activity reduced to 17.6% after 30 min exposure.

4.1 Effects of Cd on the acoustic startle response of Zebrafish

The behavioral responses of zebrafish are effective indicators for heavy metals (Wang et al. 2018)^[28], pesticides (Hama Aziz et al. 2019; Huang 2014; Severo et al. 2020)^{[13, 26, 27]}, and other organic pollutants (Bownik and Wlodkowic 2021; Chen et al. 2011) ^{[24, 25]}in water. Those studies have shown that there is a significant dose-effect relationship between the exposure concentration and swimming activity of zebrafish (Liao et al. 2011)[30]. It has been found that the swimming activity of zebrafish in an environmental stress model with the stages of no effect, stimulation action, adaptation, adjustment, and toxic effects,is almost identical under different environmental stresses induced by those different pollutants. Changes in behavior have been detected at concentrations of toxic compounds as low as 0.1% to 5% of the LC₅₀value (Garaventa et al. 2010; Ren et al. 2013).[9] [29]Therefore, taking the behavioral response of zebrafish as the early warning of the impact of pollutants on the aquatic ecological environment is a valuable asset for water quality monitoring (Nüßer et al. 2016)^[31].

Auditory threshold, as the basic acoustic characteristic of fish, is the minimum sound pressure at which fish can just hear the sound. In the auditorily evoked behavioral response (AEBRs) threshold curve, where the horizontal axis is frequency and the vertical axis is the threshold, the lowest point is the highest acoustic sensitivity at the corresponding frequency. Previous studies determined that the auditory sensitive area of zebrafish was located in the low-frequency region of 20 to 200 Hz (Ling 2013)^[41], which was consistent with the frequency used in this study. The startle response to 100 Hz and 97 dB under Cd exposure was indistinguishable to pre-stimulation, can subside to normal behavior in a short time, and showed a similar response during subsequent stimulation intervals of 20 min. Therefore, we determined the optimal sensitive acoustic thresholds for zebrafish in this study to be 100 Hz and 97 dB.

Swimming activity changes are more vulnerable to short-term sound stimulation; however, the zebrafish’s perception of sound is not continuously stable (Saeed et al. 2016). Too frequent a sustained sound stimulus will cause it to adapt to the stimulus. Xing et al (2018) have shown that persistent sound stimulation can cause temporary or permanent hearing loss. Therefore, this paper tries to determine the appropriate duration and interval of stimulation to ensure the obvious response without forming the adaptability of zebrafish to the sound. The acoustic startle response caused by the stimulus began to decline after 30 s, and returned to the normal level within 20 min. Moreover, there was no significant difference in the response of multiple stimuli, so the standard stimulus duration of 30 s and interval of 20 min were selected. Similarly, Murchy et al (2017)[40] found that the swimming behaviors of silver carp and bighead carp returned to normal after 20 s of sound stimulus.

Due to the toxic effect of Cd on zebrafish, there is a marked difference in acoustic startle response between Cd exposed zebrafish and those of the control. With an increase in exposure time, greater the harm to zebrafish, and greater the difference in acoustic startle response. Cadmium can bind to proteins in fish and accumulate in organs such as the liver, causing damage and reducing the response to sound (Lacave et al. 2020). The higher concentration, the greater effect. Studies with Cd suggested that toxicants often moved along the olfactory system, the gills and skin and into the fish, causing cell death and damage of those organs, thus leading to behavioral deficits[42]. Liu et al (2011)[43] found that gill damage caused by Cd exposure can lead to decreased oxygen consumption and damage to osmoregulatory function. It simultaneously inhibits the activity of related enzymes[44] and the transcription of related liver genes[45], which in turn leads to the decrease of acoustic startle response in zebrafish. This decrease may be a result of deafness caused by damaged auditory organs. The lateral line, as a part of the auditory organ of zebrafish, contains sensory hair cells. Studies by Coffin et al (2010) have shown that hair cells are sensitive to heavy metals and can lead to loss or death of hair cells. The study of Hernandez[46] et al (2006) showed that heavy metals caused irreversible damage to lateral lines and sensory hair cells. Therefore, compared with the control group, it is possible to identify abnormal behavior of zebrafish using acoustic stimulation, which can effectively distinguish whether the alarm system is a true alarm or a false alarm.

4.2 Model for recognizing abnormal behavior of zebrafish by acoustic stimulus

Based on the characterizations of acoustic startle responses of zebrafish, we designed the process of testing the abnormal behaviors. As shown in Fig.7, the testing process include two modules: (1) Preprocess Module: the environmental standards of computer vision monitoring were established to eliminate the influence of environmental factors on abnormal zebrafish behavior. The temperature was 22–26 °C, the turbidity was 10 NTU, the illumination intensity of background lamp was 50 lx, and the SPL of the noise in the experimental environment was controlled to be less than 50 dB. In addition, acoustic stimulation can be used to detect acoustic response defects in zebrafish for preliminary behavioral screening (2) Acoustic Stimulus Module: When the system displayed an alarm, a sound of 100 Hz and SPL of 97 dB was produced for 30 s. If there was less than 50% increase in swimming activity then the alarm was true. If there was more than 50% increase in swimming activity then the alarm was false and the system could proceed normally.

Swimming behavior of zebrafish is an effective indicator of water quality monitoring. However, chemical pollutant exposure and other non-toxic factors can disrupt avoidance behavior in zebrafish. Abnormal behavior responses are directly introduced into warning model calculations without testing for false alarms. Based on the sufficient knowledge of the acoustically evoked startle response of zebrafish, the most sensitive acoustic frequency and SPL for the zebrafish domesticated in this study were 100 Hz and 97 dB, respectively. To prevent the adaptation of zebrafish to the acoustic stimulation, the duration of a single stimulation was kept to 30 s, and the interval between two stimulations was 20 min. Under those conditions of stimulus, Cd exposure can weaken the zebrafish acoustic startle response. Furthermore, the exposure to Cd was significantly correlated with the reduction of startle response. It is, therefore, feasible to use acoustic stimulation to identify the presence or absence of chemically induced abnormal behavior in zebrafish, and the research has established a model for identifying them. This study examines an important relationship between pollutants and the acoustic startle response in zebrafish that could ultimately help improve flag false alarms in a monitoring system.

Acknowledgements: We would like to thank Editage (www.editage.cn) for English language editing.

Funding This research was funded by the National Natural Science Foundation of China (51909203), Project of Shaanxi Provincial Department of Education (19JK0392).

Competing Interests The authors have no interest to declare.

Author Contributions Conceptualization: [Yi Huang], [Junxu Wu]; Methodology: [Yi Huang]; Formal analysis and investigation: [Junxu Wu], [Yuetong Lu], [Pingge Zhao], [Ning Jia]; Writing—original draft preparation: [Yi Huang], [Junxu Wu]; Writing—review and editing: [Junxu Wu], [Yuetong Lu], [Ning Jia]; Funding acquisition: [Yi Huang]; Resources: [Yi Huang], [Pingge Zhao]; Supervision: [Pingge Zhao]; Project administration: [Yi Huang], [Pingge Zhao]. All authors have read and agreed to the published version of the manuscript.

Ethical Approval All procedures were conducted in accordance with the “Guiding Principles in the Care and Use of Animals” (China) and were approved by the Laboratory Animal Ethics Committee of Shaanxi Academy of Chinese Medicine Sciences.

Consent to Participate Not applicable

Consent to Publish Not applicable

Availability of data and materials Not applicable

Bownik A, Wlodkowic D (2021) Advances in real-time monitoring of water quality using automated analysis of animal behaviour. Science of The Total Environment 789: 147796
Chen T, Wang Y, Wu Y (2011) Developmental exposures to ethanol or dimethylsulfoxide at low concentrations alter locomotor activity in larval zebrafish: Implications for behavioral toxicity bioassays. Aquatic Toxicology 102: 162-166
Coffin AB, Ou H, Owens KN, Santos F, Simon JA, Rubel EW, Raible DW (2010) Chemical screening for hair cell loss and protection in the zebrafish lateral line. Zebrafish 7: 3-11
Farrell A (2011) Encyclopedia of Fish Physiology: From Genome to Environment. Academic Press, an imprint of Elsevier, Academic Press, an imprint of Elsevier
Garaventa F, Gambardella C, Di Fino A, Pittore M, Faimali M (2010) Swimming speed alteration of Artemia sp. and Brachionus plicatilis as a sub-lethal behavioural end-point for ecotoxicological surveys. Ecotoxicology 19: 512-519
Hama Aziz KH, Omer KM, Hamarawf RF (2019) Lowering the detection limit towards nanomolar mercury ion detection via surface modification of N-doped carbon quantum dots. New journal of chemistry 43: 8677-8683
Hernández PP, Moreno V, Olivari FA, Allende ML (2006) Sub-lethal concentrations of waterborne copper are toxic to lateral line neuromasts in zebrafish (Danio rerio). Hearing Research 213: 1-10
Huang Y (2014) Research on early-warning of water quality by monitoring behavioral responses of zebrafish (Danio rerio) to acute stress. Xi'an University of Architecture and Technology
Huang Y, Zhang J, Han X, Huang T (2014) The Use of Zebrafish (Danio rerio) Behavioral Responses in Identifying Sublethal Exposures to Deltamethrin. International Journal of Environmental Research and Public Health 11: 3650-3660
Huang Y, Zhang JS, Mi FJ, Zhang GH, Sun J (2020) Monitoring low-level mercury contamination by zebrafish school behavioral responses. IOP conference series. Earth and environmental science 612: 12077
Jesus J, Cortes R, Teixeira A (2021) Acoustic and Light Selective Behavioral Guidance Systems for Freshwater Fish. Water 13: 745
Johnson A, Carew E, Sloman K (2007) The effects of copper on the morphological and functional development of zebrafish embryos. Aquatic Toxicology 84: 431-438
Lacave JM, Bilbao E, Gilliland D, Mura F, Dini L, Cajaraville MP, Orbea A (2020) Bioaccumulation, cellular and molecular effects in adult zebrafish after exposure to cadmium sulphide nanoparticles and to ionic cadmium. Chemosphere 238: 124588
Li X, Zhang B, Li N, Ji X, Liu K, Jin M (2019) Zebrafish neurobehavioral phenomics applied as the behavioral warning methods for fingerprinting endocrine disrupting effect by lead exposure at environmentally relevant level. Chemosphere 231: 315-325
Liao Y, Xu J, Wang W (2011) A Method of Water Quality Assessment Based on Biomonitoring and Multiclass Support Vector Machine. Procedia Environmental Sciences 10: 451-457
Ling R (2013) Abnormal swimming behavior under physical stimulation in zebrafish (Danio rerio) and control measures. Harbin Institute of Technology, pp 91. https://kns.cnki.net/kcms/detail/detail.aspx?FileName=1015022385.nh&DbName=CMFD2015
Liu XJ, Luo Z, Li CH, Xiong BX, Zhao YH, Li XD (2011) Antioxidant responses, hepatic intermediary metabolism, histology and ultrastructure in Synechogobius hasta exposed to waterborne cadmium. Ecotoxicology and Environmental Safety 74: 1156-1163
Murchy KA, Cupp AR, Amberg JJ, Vetter BJ, Fredricks KT, Gaikowski MP, Mensinger AF (2017) Potential implications of acoustic stimuli as a non-physical barrier to silver carp and bighead carp. Fisheries Management and Ecology 24: 208-216
Mwaffo V, Peng Z, Cruz SR, Porfiri M (2017) Zebrafish swimming in the flow: a particle image velocimetry study. Peerj 5: e4041
Nüßer LK, Skulovich O, Hartmann S, Seiler T, Cofalla C, Schuettrumpf H, Hollert H, Salomons E, Ostfeld A (2016) A sensitive biomarker for the detection of aquatic contamination based on behavioral assays using zebrafish larvae. Ecotoxicology and Environmental Safety 133: 271-280
Pavlidis M, Digka N, Theodoridi A, Campo A, Barsakis K, Skouradakis G, Samaras A, Tsalafouta A (2013) Husbandry of Zebrafish,Danio Rerio, and the Cortisol Stress Response. Zebrafish 10: 524-531
Ramcharitar J, Ibrahim RM (2013) Ethanol modifies zebrafish responses to abrupt changes in light intensity. Journal of Clinical Neuroscience 20: 476-477
Raza SHA, Abdelnour SA, Alotaibi MA, AlGabbani Q, Naiel MAE, Shokrollahi B, Noreldin AE, Jahejo AR, Shah MA, Alagawany M, Zan L (2022) MicroRNAs mediated environmental stress responses and toxicity signs in teleost fish species. Aquaculture 546: 737310
Ren Z, Liu L, Fu R, Miao M (2013) The Stepwise Behavioral Responses: Behavioral Adjustment of the Chinese Rare Minnow (Gobiocypris rarus) in the Exposure of Carbamate Pesticides. BioMed Research International 2013: 1-9
Saeed SS, Kees W, James C, Peter S, Hans S (2016) Behavioural responses to sound exposure in captivity by two fish species with different hearing ability. Animal Behaviour 116: 1-11
Severo ES, Marins AT, Cerezer C, Costa D, Nunes M, Prestes OD, Zanella R, Loro VL (2020) Ecological risk of pesticide contamination in a Brazilian river located near a rural area: A study of biomarkers using zebrafish embryos. Ecotoxicology and Environmental Safety 190: 110071
Shafiei Sabet S, Van Dooren D, Slabbekoorn H (2016) Son et lumière: Sound and light effects on spatial distribution and swimming behavior in captive zebrafish. Environmental Pollution 212: 480-488
Suriyampola PS, Sykes DJ, Khemka A, Shelton DS, Martins EP (2016) Water flow impacts group behavior in zebrafish (Danio rerio). Behavioral Ecology 27: 1-7
Teles LO, Fernandes M, Amorim J, Vasconcelos V (2015) Video-tracking of zebrafish (Danio rerio) as a biological early warning system using two distinct artificial neural networks: Probabilistic neural network (PNN) and self-organizing map (SOM). Aquatic toxicology (Amsterdam, Netherlands) 165: 241
Wang C, Zhong Z, Sun P, Zhong H, Li H, Chen F (2017) Evaluation of the Hair Cell Regeneration in Zebrafish Larvae by Measuring and Quantifying the Startle Responses. Neural Plasticity 2017: 1-8
Wang L, Ren Z, Kim H, Xia C, Fu R, Chon T (2015) Characterizing response behavior of medaka (Oryzias latipes) under chemical stress based on self-organizing map and filtering by integration. Ecological Informatics 29: 107-118
Wang Y, Wang S, Tang J, Li L, Zhang J (2018) Study on behavioral variation of zebra fish under stress of Cu²⁺and its application on water monitoring and early alarming. Yangtze River 49: 20-26
Wang Y, Yu J, Zhuang X, Li H, Liu H, Yu X, Li J, Zhang G (2018) Auditory sensitivity in crucian carp Carassius auratus Linnaeus. Journal of Dalian Ocean University 33: 775-781
Xia C, Chon T, Liu Y, Chi J, Lee J (2016) Posture tracking of multiple individual fish for behavioral monitoring with visual sensors. Ecological Informatics 36: 190-198
Xing B, Yin L, Zhang G, Zhuang X, Wang Y, Wang Z, Li H, Liu J, Liu H, Xu L (2018) Progress on the auditory characteristics of fish and their application. Marine Fisheries 40: 495-503

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Use of acoustic stimulus to recognize abnormal zebrafish behavior in water quality warning systems

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Zebrafish husbandry

2.2 Experimental apparatus

2.3 Measurement intervals and cadmium concentrations

2.4 Acoustic Stimulus thresholds and durations

2.5 Data analysis

3. Results

3.1 Effects of Cd on zebrafish swimming activity

3.2 Acoustically evoked startle response of Zebrafish

3.2.1 Sensitivity frequency and sound pressure for stimulus

3.2.2 Duration and interval for stimulus presentation

3.3 Effect of Cd exposure on acoustic startle response

4. Discussion

4.1 Effects of Cd on the acoustic startle response of Zebrafish

4.2 Model for recognizing abnormal behavior of zebrafish by acoustic stimulus

5. Conclusions

Declarations

References

Additional Declarations

Status:

Version 1