A small fish circadian rhythm behavior high-throughput observation device and an observation method

Abstract

The application discloses a small fish diurnal rhythm behavior high-throughput observation device and an observation method, wherein the high-throughput observation device comprises a fish observation device; the fish observation device comprises a liquid inlet pipeline, an observation chamber, a liquid outlet pipeline and a water storage and purification pool; the observation chamber comprises a water inlet buffer area, an observation area and a water outlet buffer area, one end of the observation area is communicated with the water inlet buffer area, and the other end of the observation area is communicated with the water outlet buffer area; a plurality of swimming chambers are arranged in the observation area, and adjacent two swimming chambers are communicated with each other; the liquid inlet pipeline is communicated with the water storage and purification pool and the water inlet buffer area, and the liquid outlet pipeline is communicated with the water outlet buffer area and the water storage and purification pool, so as to form a water flow circulation loop. The high-throughput observation device can be used for observing abnormal fish diurnal rhythm behaviors under the influence of chemicals or water quality, can realize high analysis throughput under single observation, and can ensure the effectiveness of single observation under high throughput.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical environmental risk assessment and water quality evaluation, and particularly relates to a small fish circadian rhythm behavior high-throughput observation device and an observation method. BACKGROUND

[0002] It is reported that more than 350,000 chemicals have been registered for production and use worldwide [1] Massive chemicals can be gradually released into the environment in the whole life cycle of production, use and disposal. Water is a good solvent for chemicals, and it has strong mobility, wide connectivity and fast cycle interaction, which quickly leads to serious chemical pollution problems in global water environment. Chemical hazard and water quality risk assessment is an important scientific basis for water pollution control. Current research has proved that fish can be more sensitive to the influence of chemical and water quality changes and make behavioral changes [2] . Therefore, fish behavior test is considered to be an effective tool for risk assessment [3] .

[0003] Circadian rhythm behavior refers to the biological oscillation of physiological and biochemical processes of organisms with a 24-hour cycle. The most important circadian rhythm is the sleep-wake rhythm. Circadian rhythm behavior is jointly regulated by biological and environmental influences. For example, research reports that bisphenol A can inhibit the expression of core circadian rhythm genes per2 and Cry1 in the brain and liver of goldfish and thus disrupt circadian rhythm [4] . Neurological drugs detected in hospital and urban wastewater can cause abnormal changes in the circadian rhythm of fish [5,6] . It can be seen that circadian rhythm is crucial for fish to maintain normal physiological activities, and its changes will comprehensively affect fish foraging, predator avoidance and reproductive opportunities, and thus trigger changes in population structure. Therefore, evaluating chemical hazards and water quality risks based on circadian rhythm has high ecological significance.

[0004] Current attention to fish circadian rhythm behavior is not high, and the corresponding test device and method are relatively lacking. In particular, in terms of devices, although there are currently observation devices for high-attention fish behaviors such as running behavior and predatory behavior, direct application of these devices to fish circadian rhythm behavior observation still has many defects. For example, existing other behavior observation devices such as fish courage and exploration behavior experiment devices are often designed for single observation and only allow one group or a single individual to be observed [7,8]Since the circadian behavior cycle of fish is usually 24 h, and fish still need to adapt for a period of time after entering the device, if it is directly applied to the chemical hazard identification and water quality evaluation process, and multiple groups are sequentially observed for a single observation, there is a problem of too long overall observation period and low efficiency for long-period circadian behavior. Especially, the chemical hazard identification and water quality evaluation process requires multiple groups / multiple individuals to be tested and observed simultaneously in the same observation environment to obtain convincing conclusions, which has a higher demand for single high-throughput observation. In addition, since the above process is not single high-throughput observation, there is a problem of insufficient convincing observation results caused by abnormal behavior of fish due to inconsistent aquaculture water quality in the observation environment such as the observation device. If the same aquaculture water quality is loaded into the observation device before each group observation to ensure consistency of the observation environment between different groups, this method has the problem of tedious operation steps and low efficiency.

[0005] [1] GONG Y, YANG D, BARRETT H, et al. Building the Environmental Chemical-protein Interaction Network (eCpIN): An Exposome-Wide Strategy for Bioactive Chemical Contaminant Identification [J]. Environmental Science & Technology, 2023, 57(9): 3486-95.

[0006] [2] ZHANG J S, HUANG Y, HAN X B, et al. Fish behavior changes in water quality monitoring [J]. Water and Wastewater, 2013, 49(07): 166-70.

[0007] [3] FUKUDA S, KANG I J, MOROISHI J, et al. The Application of Entropy for Detecting Behavioral Responses in Japanese Medaka (Oryzias latipes) Exposed to Different Toxicants [J]. Environmental Toxicology, 2010, 25(5): 446-55.

[0008] [4] CHOI J Y, CHOE J R, LEE T H, et al. Effects of bisphenol A and light conditions on the circadian rhythm of the goldfish Carassius auratus[J]. Biological Rhythm Research, 2018, 49(4): 502-14.

[0009] [5] WU M, LIU S, HU L, et al. Global transcriptomic analysis of zebrafish in response to embryonic exposure to three antidepressants, amitriptyline, fluoxetine and mianserin[J]. Aquatic Toxicology, 2017, 192: 274-83.

[0010] [6] GUO R Y. Effects of tributyltin on the circadian rhythm of zebrafish (Danio rerio) and its mechanism[D];Hainan Normal University, 2024.

[0011] [7] ZHANG H Q, QIU N, LIU H Y, et al. Fish boldness and exploration behavior experimental device and test method, CN112753623B [p / OL].

[0012] [8] SUN M, FENG D J, GUI F K, et al. Fish behavior observation device, CN110800651B [p / OL]. SUMMARY

[0013] The technical problem to be solved by the first aspect of the present application is to provide a small fish circadian rhythm behavior high-throughput observation device, which can realize high-throughput analysis of small fish circadian rhythm behavior through single observation, evaluate the ecological hazards and water quality risks of chemicals, ensure the effectiveness of single high-throughput observation, and improve the observation efficiency and ensure the persuasiveness of the observation results.

[0014] In order to solve the above technical problems, the application discloses a small fish diel rhythm behavior high-throughput observation device, which comprises a fish observation device.

[0015] Specifically, the observation chamber comprises an observation chamber body, a first transverse water uniformity partition, a second transverse water uniformity partition, a transverse observation area partition, and a longitudinal observation area partition, and the observation chamber body is a long rectangular water tank structure with an open top. The first transverse water uniformity partition, the observation chamber body, and the second transverse water uniformity partition form the observation area. The first transverse water uniformity partition connects the observation chamber body to form the water inlet buffer area. The second transverse water uniformity partition connects the observation chamber body to form the water outlet buffer area. The transverse observation area partition and the longitudinal observation area partition divide the observation area to form the plurality of swimming chambers.

[0016] Specifically, the observation chamber comprises a first communication hole, a second communication hole, and a third communication hole, and the transverse observation area partition and the longitudinal observation area partition are each provided with a plurality of first communication holes for connecting adjacent swimming chambers. A plurality of second communication holes are formed in the first transverse water uniformity partition for connecting the water inlet buffer area and the observation area. A plurality of third communication holes are formed in the second transverse water uniformity partition for connecting the water outlet buffer area and the observation area.

[0017] Specifically, the observation chamber comprises a first overflow slit and a second overflow slit, the first transverse water uniformity partition is provided with a first overflow slit, and the second transverse water uniformity partition is provided with a second overflow slit. The first overflow slit and the second overflow slit are located at the same height.

[0018] Specifically, the observation chamber comprises a water inlet and a water outlet, the water inlet is arranged on the side wall opposite to the first transverse water uniformizing partition of the water inlet buffer zone, the water outlet is arranged on the side wall opposite to the second transverse water uniformizing partition of the water outlet buffer zone, the inlet port of the liquid inlet pipeline is connected with the water inlet, the inlet port of the liquid outlet pipeline is connected with the water outlet, and the outlet port of the liquid outlet pipeline is used for guiding the water flow into the water storage and purification pool.

[0019] Further, the fish observation device further comprises a transparent cover plate, which is arranged on the observation chamber body and used for preventing the fish from jumping into the adjacent swimming chamber or jumping out of the observation chamber body.

[0020] Specifically, the water storage and purification pool comprises a pool body, a filter device and a second water pump, the filter device is arranged in the pool body, and the fluid outlet of the filter device is connected with the inlet of the second water pump.

[0021] Specifically, the high-throughput observation device comprises a first water pump, which is used for providing power for the liquid conveyed in the liquid inlet pipeline.

[0022] Further, the water storage and purification pool further comprises a heating rod and an aeration device, which are arranged below the liquid surface of the pool body and used for adjusting the temperature of the water in the water storage and purification pool.

[0023] The second aspect of the present application discloses a small fish circadian rhythm behavior high-throughput observation method, which is implemented by using the small fish circadian rhythm behavior high-throughput observation device.

[0024] The observation method comprises the following steps:

[0025] Step 1: An appropriate amount of fish culture water is added into the observation chamber and the water storage and purification pool of the high-throughput observation device, so that the fish culture water circulates in the water flow circulation loop.

[0026] Step two, under the premise of observing 3 parallel experiments in each exposure group and control group, after the high-throughput observation device enters a stable running state, simultaneously put the control group of live fish and 3 exposure groups of live fish into the high-throughput observation device for adaptation for several hours, wherein the control group of live fish refers to normally cultured live fish, and the exposure group of live fish refers to live fish exposed to a to-be-tested chemical or waste water sample. The exposure group is an observation individual or a single-chamber group. When the exposure group is an observation individual, the control group is an observation individual. When the exposure group is a single-chamber group, the control group is a single-chamber group.

[0027] Step three, after the adaptation is completed, the camera is used to shoot video images of the behavior of live fish in 12 chambers of the observation area according to a preset observation period. The preset observation period at least includes a continuous uninterrupted day and night cycle of 24 hours.

[0028] Step four, after the video images are obtained, an analysis software capable of tracking animal motion trajectory is used to calculate the motion speed in a self-defined extremely short time interval Δt. The calculation principle is that the motion speed V of the fish in the time interval Δt is calculated based on the spatial distance S obtained by image analysis that the fish moves from the position at time t to the position at time t+Δt. The calculation formula of the fish motion speed V is: V=S / Δt.

[0029] Step five, the time period in which the fish motion speed V in the whole observation period is less than 1.5 cm / s and lasts for more than 5 seconds is defined as the sleep time. Based on the fish motion speed V in the whole observation period, the proportion of sleep time in each hour in the day and night cycle of more than 24 hours is calculated, and a curve of the proportion of sleep time in each hour in the day and night cycle with time is drawn.

[0030] Beneficial effects:

[0031] (1) The small fish diurnal rhythm behavior high-throughput observation device and observation method disclosed by the application provide a test target and method for reflecting the comprehensive physiological and biochemical hazards of chemicals and water quality changes on fish for the identification of the increasingly serious water pollution health hazards.

[0032] (2) In the small fish diurnal rhythm behavior high-throughput observation device disclosed in this invention, the fish observation device improves the analytical throughput of a single test by setting up multiple swimming chambers, that is, it realizes single high-throughput observation. This single high-throughput observation is suitable for chemical hazard identification and water quality assessment processes that require multiple groups / individual tests and observations, and can obtain convincing conclusions. By setting up a water circulation loop, the high-throughput observation device can purify the water and recycle the water, avoiding abnormal fish behavior caused by non-test factors due to water quality deterioration caused by high-throughput observation, which could interfere with the observation results. The observation chamber is divided into an inlet buffer zone, an observation zone, and an outlet buffer zone. The observation zone is located between the inlet and outlet buffer zones, and the swimming pools are located in the observation zone. The aquaculture water in the water purification tank first flows into the inlet buffer zone through the inlet pipe to reduce the flow rate. The decelerated aquaculture water then flows into the swimming pools in the observation zone. The aquaculture water in the observation zone first flows into the outlet buffer zone and then is discharged back to the water purification tank through the outlet pipe. This avoids the fish being affected by the eddy current at the outlet, thus ensuring the stability of the water environment in the observation zone and preventing abnormal fish behavior caused by non-test factors due to water environment disturbance from interfering with the observation results. This ensures the overall effectiveness of the results of a single high-throughput observation.

[0033] (3) In one embodiment of the high-throughput observation device for the diurnal rhythm behavior of small fish disclosed in this invention, a heating rod is installed in the water storage and purification tank to regulate the water temperature, thereby controlling the water temperature. Furthermore, an aeration device is installed in the water storage and purification tank to oxygenate the water and create the necessary dissolved oxygen environment for fish life.

[0034] (4) In one embodiment of a high-throughput observation device for the diurnal rhythm behavior of small fish disclosed in this invention, a transparent cover is added to the fish observation device to avoid test failure due to fish jumping. Attached Figure Description

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0036] Figure 1 This is a three-dimensional structural diagram of a fish observation device in a high-throughput observation device for the diurnal rhythm behavior of small fish, as provided in an embodiment of the present invention.

[0037] Figure 2 for Figure 1 A top view of the observation chamber in the fish observation device shown.

[0038] Figure 3 For Figure 2 A cross-sectional view along line AA in the middle.

[0039] Figure 4 For Figure 2 Cross-sectional view along the line B-B.

[0040] Figure 5 For Figure 1 Schematic diagram of the internal structure of the water storage and purification tank.

[0041] Figure 6 The circadian behavior observation live view taken by the data acquisition device of an embodiment of the present application.

[0042] Figure 7 The contrastive diagram of the proportion of sleep time in each hour in the circadian cycle of zebrafish after exposure to different concentrations of typical antidepressants citalopram.

[0043] Figure 8 For Figure 7 The contrastive diagram of the inter-diurnal sleep time of zebrafish after exposure to different concentrations of typical antidepressants citalopram, where * represents significant difference, i.e. p<0.05; ns represents no significant difference.

[0044] The legend of the figures is as follows:

[0045] 1, fish observation device; 100, liquid inlet pipeline; 200, observation chamber; 210, water inlet buffer zone; 220, observation area; 221, swimming chamber; 222, first communication hole; 230, water outlet buffer zone; 240, observation chamber main body; 241, water inlet; 242, water outlet; 250, first transverse water uniformity separator; 251, second communication hole; 252, first overflow slit; 260, second transverse water uniformity separator; 261, third communication hole; 262, second overflow slit; 270, transverse observation area separator; 280, longitudinal observation area separator; 300, liquid outlet pipeline; 400, water storage and purification tank; 410, tank body; 420, filtration device; 440, heating rod; 450, aeration device; 500, first water pump; 2, liquid level. DETAILED DESCRIPTION

[0046] Circadian behavior is an important periodic behavior pattern for maintaining normal physiological life of fish, which is jointly regulated by external environmental interference and self-response, can be more sensitive to perceive external chemical exposure or water quality changes and make corresponding changes, and is an ideal target for environmental risk assessment test.

[0047] Therefore, the application provides a small fish diel behavior high-throughput observation device and an observation method, which can realize high-throughput analysis of diel behavior of small fish through single observation, ensure effectiveness of single high-throughput observation, and improve observation efficiency and persuasiveness of observation results.

[0048] The first aspect of the application provides a small fish diel behavior high-throughput observation device, which comprises a data acquisition device, a data analysis device and a fish observation device 1.

[0049] Referring to Figure 1 The fish observation device 1 comprises a liquid inlet pipeline 100, an observation chamber 200, a liquid outlet pipeline 300 and a water storage and purification pool 400. The observation chamber 200 comprises a water inlet buffer zone 210, an observation zone 220 and a water outlet buffer zone 230. One end of the observation zone 220 is in communication with the water inlet buffer zone 210, and the other end of the observation zone 220 is in communication with the water outlet buffer zone 230. A plurality of swimming chambers 221 are arranged in the observation zone 220, and adjacent two swimming chambers 221 are in communication with each other. Figure 2 The liquid inlet pipeline 100 is in communication with the water storage and purification pool 400 and the water inlet buffer zone 210, and the liquid outlet pipeline 300 is in communication with the water outlet buffer zone 230 and the water storage and purification pool 400, forming a water flow circulation loop.

[0050] In use, the aquaculture water can circulate in the water flow circulation loop. The water storage and purification pool 400 is used for purifying the aquaculture water in the water flow circulation loop to remove fish feces and food residues in the water body. The aquaculture water in the water storage and purification pool 400 flows into the water inlet buffer zone 210 through the liquid inlet pipeline 100, and then flows into each swimming chamber 221 in the observation zone 220. The aquaculture water in the observation zone 220 flows into the water outlet buffer zone 230 and then is discharged back to the water storage and purification pool 400 through the liquid outlet pipeline 300.

[0051] The application sets a plurality of swimming chambers 221 in the observation zone 220 of the observation chamber 200, which improves the analysis throughput of single observation compared with a single swimming chamber in the prior art, that is, single high-throughput observation is realized.

[0052] In order to avoid abnormal behaviors of fish caused by non-test factors due to deterioration of water quality caused by high-throughput observation to interfere with observation results, the application sets a water flow circulation loop comprising the water storage and purification pool 400, which can realize purification of water quality and recycling of water body in the high-throughput observation device, avoid abnormal behaviors of fish caused by non-test factors due to deterioration of water quality caused by high-throughput observation to interfere with observation results, and ensure effectiveness of results of single high-throughput observation.

[0053] In order to avoid the abnormal behaviors of fish caused by the disturbance of water environment from interfering with the observation results, the observation chamber 200 is divided into the water inlet buffer area 210, the observation area 220 and the water outlet buffer area 230, and the swimming chamber 221 is arranged in the observation area 220. The aquaculture water in the water storage and purification tank 400 is first flowed into the water inlet buffer area 210 through the liquid inlet pipeline 100 to reduce the flow rate, and then flowed into each swimming chamber 221 in the observation area 220. The aquaculture water in the observation area 220 is first flowed into the water outlet buffer area 230, and then discharged back to the water storage and purification tank 400 through the liquid outlet pipeline 300, so as to avoid the influence of the water outlet vortex on the fish. Thus, the water environment in the observation area can be ensured to be stable, and the observation results can be ensured to be effective.

[0054] Thus, the single high-throughput observation can be realized, and the observation efficiency can be improved as a whole and the effectiveness of the single high-throughput observation results can be ensured.

[0055] Optionally, the liquid inlet pipeline 100 and the liquid outlet pipeline 300 are flexible pipelines, such as silica gel pipes.

[0056] In the embodiment, the liquid inlet pipeline 100 and the liquid outlet pipeline 300 are flexible pipelines, which can be arbitrarily bent and adjusted in position. The pipelines are used to connect the observation chamber 200 and the water storage and purification tank 400, so that the relative positions between the observation chamber 200 and the water storage and purification tank 400 can be flexibly changed according to the conditions of the experimental site.

[0057] Specifically, referring to Figures 2 to 4 , the observation chamber 200 includes an observation chamber main body 240, a first transverse water uniformizing partition 250, a second transverse water uniformizing partition 260, a transverse observation area partition 270 and a longitudinal observation area partition 280. The observation chamber main body 240 is a rectangular water tank structure with an open top. The first transverse water uniformizing partition 250, the second transverse water uniformizing partition 260, the transverse observation area partition 270 and the longitudinal observation area partition 280 are all plate structures. The first transverse water uniformizing partition 250, the observation chamber main body 240 and the second transverse water uniformizing partition 260 together form the observation area 220. The first transverse water uniformizing partition 250 is connected to the observation chamber main body 240 to form the water inlet buffer area 210. The second transverse water uniformizing partition 260 is connected to the observation chamber main body 240 to form the water outlet buffer area 230. The transverse observation area partition 270 and the longitudinal observation area partition 280 divide the observation area 220 to form a plurality of swimming chambers 221. As can be seen from Figure 2 , the swimming chambers 221 are arranged in a matrix array. The matrix array arrangement of the plurality of swimming chambers 221 has a compact structure, minimizes the space range to be photographed, reduces the difficulty of the data acquisition equipment in collecting behavior images, and makes the activity environment of a single swimming chamber as consistent as possible. In a specific embodiment, as shown in Figure 2As shown, two transverse observation area dividers 270 are provided, and three longitudinal observation area dividers 280 are provided, and the transverse observation area dividers 270 and the longitudinal observation area dividers 280 divide the observation area 220 into twelve swimming chambers 221, for simultaneously observing twelve individuals or twelve test groups, so as to realize single high-throughput observation.

[0058] Specifically, the observation chamber main body 240, the first transverse water uniformity divider 250, the second transverse water uniformity divider 260, the transverse observation area divider 270, and the longitudinal observation area divider 280 are all made of high-transparency materials, such as acrylic transparent plates, to facilitate observation of the behavior of small fish in the swimming chambers 221.

[0059] Specifically, referring to Figure 2 and Figure 3 , the observation chamber 200 includes first communication holes 222, and the transverse observation area dividers 270 and the longitudinal observation area dividers 280 are all provided with a plurality of first communication holes 222, and the plurality of first communication holes 222 are used to communicate adjacent two swimming chambers 221.

[0060] Specifically, referring to Figure 3 , the observation chamber 200 includes second communication holes 251 and third communication holes 261, and the first transverse water uniformity divider 250 is provided with a plurality of second communication holes 251, and the plurality of second communication holes 251 are used to communicate the water inlet buffer area 210 and the observation area 220. The second transverse water uniformity divider 260 is provided with a plurality of third communication holes 261, and the plurality of third communication holes 261 are used to communicate the water outlet buffer area 230 and the observation area 220.

[0061] Specifically, referring to Figure 3 and Figure 4 , the observation chamber 200 includes first overflow slits 252 and second overflow slits 262, and the first transverse water uniformity divider 250 is provided with the first overflow slits 252, and the second transverse water uniformity divider 260 is provided with the second overflow slits 262. The first overflow slits 252 and the second overflow slits 262 are located at the same height position. More specifically, the first overflow slits 252 and the second overflow slits 262 both extend in the transverse direction. The height position of the first overflow slits 252 and the second overflow slits 262 determines the height of the liquid surface 2 of the observation area 220.

[0062] The sizes of the first communication holes 222, the second communication holes 251, the third communication holes 261, the first overflow slits 252, and the second overflow slits 262 should only allow water to pass through and not allow fish to pass through.

[0063] Specifically, referring to Figure 3, the observation chamber 200 comprises a water inlet 241 and a water outlet 242, the water inlet 241 is arranged on the side wall of the water inlet buffer zone 210 opposite to the first transverse water uniformizing partition 250. The water outlet of the liquid inlet pipeline 100 is communicated with the water inlet 241, and the water inlet of the liquid inlet pipeline 100 is below the liquid level of the water storage and purification tank 400. The water outlet 242 is arranged on the side wall of the water outlet buffer zone 230 opposite to the second transverse water uniformizing partition 260, the water inlet of the liquid outlet pipeline 300 is connected with the water outlet 242, and the water outlet of the liquid outlet pipeline 300 is used for guiding the flow into the water storage and purification tank 400. More specifically, referring to Figure 3 , the water inlet 241 is higher than the water outlet 242, so that the water flow obtains a height difference, and can realize self-flow under the action of gravity.

[0064] Specifically, referring to Figure 1 , the high-throughput observation device comprises a first water pump 500, which is used for providing power for the liquid transported in the liquid inlet pipeline 100. Specifically, referring to Figure 1 , the first water pump 500 is arranged in the water storage and purification tank 400, and the outlet of the first water pump 500 is connected with the water inlet of the liquid inlet pipeline 100. The liquid outlet pipeline 300 has no external power, and the aquaculture water in the observation chamber 200 flows into the water storage and purification tank 400 by gravity.

[0065] Further, the fish observation device 1 further comprises a transparent cover plate, which is arranged on the observation chamber main body 240 and is used for preventing the fish from jumping into the adjacent swimming chamber 221 or jumping out of the observation chamber main body 240. Specifically, the transparent cover plate is made of a material with high transparency, for example, acrylic transparent plate, so that the data acquisition equipment can directly shoot from above the transparent cover plate. The transparent cover plate is not shown in the figure.

[0066] Specifically, referring to Figure 5 , the water storage and purification tank 400 comprises a tank body 410, a filtering device 420 and a second water pump, and the filtering device 420 is installed in the tank body 410. The second water pump can be a small water pump, and the fluid outlet of the filtering device 420 is communicated with the inlet of the second water pump. The second water pump is used for sucking the water flow in the tank body 410 into the filtering device 420 for filtering treatment, so as to remove the fish excrement and food residues in the water body. More specifically, the cross section of the tank body 410 is square, the filtering device 420 is installed on one side of the inner cavity of the tank body 410, and filtering cotton is arranged in the filtering device 420.

[0067] Further, referring to Figure 5 , the water storage and purification tank 400 further comprises a heating rod 440 for adjusting the water temperature in the water storage and purification tank 400. The heating rod 440 is arranged below the liquid level of the tank body 410 and is used for maintaining the water temperature in the device within a certain range.

[0068] In the present embodiment, since how to maintain the water temperature by controlling the heating rod 440 belongs to the prior art and is not the main inventive point, it is not described herein.

[0069] Further, referring to Figure 5 , the water storage and purification tank 400 comprises an aeration device 450, which is equipped below the liquid surface of the tank body 410, for oxygenating the water body and creating the necessary dissolved oxygen environment required for fish life.

[0070] In the present embodiment, since how to maintain the dissolved oxygen environment by controlling the aeration device 450 belongs to the prior art and is not the main inventive point, it is not described herein.

[0071] Specifically, the data acquisition device is a camera, and the data analysis device is a computer, and the computer is installed with analysis software capable of tracking animal movement trajectory. More specifically, the camera is arranged at a certain height directly above the fish observation device 1, and the camera is configured to shoot video images of the entire observation area 220 according to a preset observation period. The camera and the computer are not shown in the figure.

[0072] The second aspect of the present application provides a high-throughput observation method for diel rhythm behavior of small fish, which is implemented by using the high-throughput observation device for diel rhythm behavior of small fish described above. The data acquisition device of the high-throughput observation device is a camera, the data analysis device of the high-throughput observation device is a computer, the computer is installed with analysis software capable of tracking animal movement trajectory, and the camera is arranged at a certain height directly above the fish observation device 1 in the high-throughput observation device.

[0073] The observation method comprises the following steps:

[0074] Step one, add an appropriate amount of fish farming water to the observation chamber 200 and the water storage and purification tank 400 of the high-throughput observation device, so that the fish farming water circulates in the water flow circulation loop; aerate the fish farming water in the water storage and purification tank 400, and control the fish farming water to maintain a certain temperature;

[0075] Specifically, the high-throughput observation device comprises a first water pump 500, a heating rod 440, an aeration device 450, and a second water pump. The first water pump 500 is turned on to make the fish farming water circulate in the water flow circulation loop. The aeration device 450 and the second water pump are turned on to aerate the fish farming water in the water storage and purification tank 400. The fish farming water is controlled to maintain a certain temperature by controlling the heating rod 440.

[0076] Step two, under the premise of observing 3 parallel experiments in each exposure group and control group, after the high-throughput observation device enters a stable running state, simultaneously put the control group of live fish and 3 exposure groups of live fish into the high-throughput observation device for adaptation for several hours, wherein the control group of live fish refers to normally cultured live fish, and the exposure group of live fish refers to live fish exposed to a to-be-tested chemical or waste water sample. Among them, the exposure group is an observation individual or a single-chamber group. When the exposure group is an observation individual, the control group is an observation individual; when the exposure group is a single-chamber group, the control group is a single-chamber group.

[0077] In the present application, the stable running state refers to that the water flow circulation, the filter device and the aeration pump are not abnormal, and the water temperature reaches the set state.

[0078] Step three, after the adaptation is completed, set the camera at a certain height above the high-throughput observation device, and use the camera to shoot video images of the behavior of live fish in 12 chambers 221 of the observation area 220 at a preset observation period. The preset observation period at least includes a continuous uninterrupted day and night with a period of 24 hours.

[0079] Step four, after obtaining the video images, use an analysis software capable of tracking animal motion trajectory to calculate the motion speed in a self-defined extremely short time interval Δt. The calculation principle is to calculate the fish motion speed V in the time interval Δt based on the spatial distance S obtained by image analysis that the fish moves from the t time position to the t+Δt time position. The calculation formula of the fish motion speed V is: V=S / Δt.

[0080] Step five, define the time period in which the fish motion speed V in the entire observation period is less than 1.5 cm / s and lasts for more than 5 seconds as the sleep time. Based on the fish motion speed V in the entire observation period, calculate the sleep time proportion in each hour in more than one day and night cycle, and draw a curve of the sleep time proportion in each hour in the day and night cycle with time. In the present application, one day and night cycle refers to 24 consecutive hours.

[0081] By drawing the curve of the sleep time proportion in each hour in the day and night cycle with time, the diurnal rhythm behavior change of the fish can be intuitively reflected.

[0082] The observation method further comprises:

[0083] Step six, the observation data of sleep time and the proportion of sleep time in each hour of the diurnal cycle of each group are counted into the same set, and statistical methods such as single factor variance analysis method and T-test method are used to compare and analyze the diurnal rhythm behavior data of the exposed group and the control group, so as to obtain the evaluation parameter p value. Based on the evaluation parameter p value, it is judged whether there is a significant difference between the exposed group and the control group.

[0084] When the evaluation parameter p value is less than 0.05, it is judged that there is a significant difference between the two, so as to evaluate the influence of the test chemical or waste water sample on the abnormal diurnal rhythm behavior of fish, and provide a reference basis for environmental health risk identification and control.

[0085] Optionally, the analysis software for tracking animal movement trajectory is Image J software.

[0086] In a specific embodiment of the observation chamber 200, the outer contour size of the observation chamber 200 is 41 cm x 26.5 cm x 11 cm. The length, width and height of each swimming chamber 221 are 12 cm x 6 cm x 7 cm respectively. The diameter of the second communication hole 251 and the diameter of the third communication hole 261 are both 1.4 cm. The second communication hole 251 and the third communication hole 261 are arranged in a matrix array, and the row distance and the column distance are both 2 cm. The first overflow slit 252 and the second overflow slit 262 are both slits with a length of 5 cm and a height of 3.25 cm. The water inlet 241 of the observation chamber 200 is 5.7 cm high from the bottom, and the water outlet 242 is 1.7 cm high from the bottom.

[0087] Next, taking a typical model organism zebrafish as an example, the zebrafish is exposed to two environmental concentrations of citalopram, and the changes of the diurnal rhythm behavior of the zebrafish before and after exposure are measured to evaluate the water environmental health risk of citalopram. Specifically, 4-month-old adult zebrafish are exposed to 0.25 μg / L and 2.5 μg / L fluoxetine for 28 days, and the control group is cultured in normal zebrafish culture water.

[0088] Figure 6 The live footage of the control group and the exposed group of zebrafish after exposure being placed into the observation chamber 200 disclosed in the specific embodiment for observation is illustrated. Before the test fish are placed, about 10 liters of zebrafish culture water are added to the high-throughput observation device, and the first water pump 500, the second water pump, the aeration device 450, the heating rod 440 and other supporting equipment are started. The water temperature is maintained at 28°C by using the heating rod 440, the aeration device 450 such as an aeration pump supplements the dissolved oxygen in the water, the filter device filters impurities in the water, and the first water pump 500 circulates the culture water in the water circulation loop to ensure the water quality in the observation chamber.

[0089] According to the embodiment of the present application, after the zebrafish is adapted to the high-throughput observation device for 4 hours, a camera is used to continuously collect behavior images for 24 hours at a height of 75 cm vertically above the high-throughput observation device.

[0090] According to the embodiment of the present application, image analysis is performed by using an analysis software capable of tracking animal movement trajectory, such as Image J software. First, the actual size of the device is calibrated on the image, and a region for image analysis is selected. The time interval Δt is set to 1 s. By analyzing the spatial distance S of the zebrafish moving from a specific position to a position at the next moment, the movement speed in each time interval is calculated.

[0091] According to the embodiment of the present application, the period of time during which the fish movement speed is less than 1.5 cm / s and lasts for more than 5 s in the whole observation period is defined as the sleep time. According to this, the proportion of sleep time in the 24-hour day-night cycle is calculated, and a curve of the proportion of sleep time in each hour in the day-night cycle, the sleep time in the day, and other parameters / indicators reflecting the circadian behavior are drawn, as shown in Figure 7 and Figure 8 . Figure 7 A comparison chart of the proportion of sleep time in each hour in a day-night cycle of zebrafish exposed to different concentrations of citalopram for 28 days is shown, wherein the first to the 14th hours are the light period, and the 15th to the 24th hours are the dark period. Figure 8 A comparison chart of the sleep time in the day of zebrafish exposed to different concentrations of citalopram for 28 days is shown. The different concentrations of citalopram include 0.25 μg / L of citalopram and 5 μg / L of citalopram. In the chart, * indicates that the two groups have significant difference, i.e. p < 0.05, and ns indicates that the two groups have no significant difference.

[0092] According to the embodiment of the present application, by comparing the differences in the circadian rhythm patterns of the exposure group and the control group and performing significant analysis, the interference effect of the to-be-tested chemical or waste water sample on the circadian rhythm behavior of fish is evaluated, thereby providing a reference basis for the environmental health risk identification and control.

[0093] Specifically, first, refer to Figure 7The exposure group exposed to 0.25 μg / L citalopram is referred to as a low concentration group, and the exposure group exposed to 5 μg / L citalopram is referred to as a high concentration group. The result analysis shows that the exposure to 5 μg / L citalopram in the daytime greatly reduces the sleep time of the zebrafish in each hour, and the exposure to 0.25 μg / L citalopram slightly reduces the sleep time of the zebrafish in each hour. In the night, the reduction effect is still observed in the high concentration group, but there is no obvious effect in the low concentration group. It is worth noting that the sleep time of the zebrafish is sharply reduced from the 14th hour to the 16th hour when the environmental condition changes from the daytime to the night, that is, the zebrafish is in the stage of adapting to the environmental change. The control group is reduced the most, followed by the low concentration group, and finally the high concentration group, which to some extent reflects that the exposure to citalopram reduces the reaction ability of the zebrafish. Secondly, see Figure 8 The comparison result of the daytime sleep time also shows that the high concentration group significantly inhibits the sleep of the zebrafish, and the low concentration group has no significant effect.

[0094] The present application provides a small fish circadian rhythm behavior high-throughput observation device and observation method, and the technical scheme is implemented by many methods and ways. The above description is only the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the principle of the present application, some improvements and refinements can be made, which should be regarded as the protection scope of the present application. The components not explicitly described in the embodiment can be realized by the existing technology.

Claims

1. A small fish circadian rhythm behavior high-throughput observation device, characterized in that, The fish observation device (1) comprises a liquid inlet pipeline (100), an observation chamber (200), a liquid outlet pipeline (300) and a water storage and purification pool (400); the observation chamber (200) comprises a water inlet buffer zone (210), an observation zone (220) and a water outlet buffer zone (230), one end of the observation zone (220) is communicated with the water inlet buffer zone (210), and the other end of the observation zone (220) is communicated with the water outlet buffer zone (230); a plurality of swimming chambers (221) are arranged in the observation zone (220), and adjacent two swimming chambers (221) are communicated with each other; the liquid inlet pipeline (100) is communicated with the water storage and purification pool (400) and the water inlet buffer zone (210), and the liquid outlet pipeline (300) is communicated with the water outlet buffer zone (230) and the water storage and purification pool (400), so as to form a water flow circulation loop; The observation chamber (200) comprises an observation chamber main body (240), a first transverse water uniformizing partition (250), a second transverse water uniformizing partition (260), a transverse observation zone partition (270) and a longitudinal observation zone partition (280), the observation chamber main body (240) is a long rectangular water tank structure with an open top; the first transverse water uniformizing partition (250), the observation chamber main body (240) and the second transverse water uniformizing partition (260) are enclosed to form the observation zone (220); the first transverse water uniformizing partition (250) is connected with the observation chamber main body (240) to form the water inlet buffer zone (210); the second transverse water uniformizing partition (260) is connected with the observation chamber main body (240) to form the water outlet buffer zone (230); the transverse observation zone partition (270) and the longitudinal observation zone partition (280) divide the observation zone (220) to form the plurality of swimming chambers (221); The observation chamber (200) comprises a first communication hole (222), a second communication hole (251) and a third communication hole (261), the transverse observation zone partition (270) and the longitudinal observation zone partition (280) are both provided with a plurality of first communication holes (222), and the plurality of first communication holes (222) are used for communicating adjacent two swimming chambers (221); a plurality of second communication holes (251) are arranged on the first transverse water uniformizing partition (250), and the plurality of second communication holes (251) are used for communicating the water inlet buffer zone (210) and the observation zone (220); a plurality of third communication holes (261) are arranged on the second transverse water uniformizing partition (260), and the plurality of third communication holes (261) are used for communicating the water outlet buffer zone (230) and the observation zone (220); The observation chamber (200) comprises a first overflow slit (252) and a second overflow slit (262), the first overflow slit (252) is arranged on the first transverse water uniformity partition (250), and the second overflow slit (262) is arranged on the second transverse water uniformity partition (260); the first overflow slit (252) and the second overflow slit (262) are arranged at the same height.

2. The small fish circadian rhythm behavior high-throughput observation device according to claim 1, wherein, The observation chamber (200) comprises a water inlet (241) and a water outlet (242), the water inlet (241) is arranged on the side wall opposite to the first transverse water uniformity partition (250) of the water inlet buffer area (210), the water outlet (242) is arranged on the side wall opposite to the second transverse water uniformity partition (260) of the water outlet buffer area (230), the water inlet (241) of the liquid inlet pipeline (100) is communicated with the water inlet (241), the water inlet (241) of the liquid inlet pipeline (100) is arranged below the liquid level of the water storage and purification pool (400), the water outlet (242) of the liquid outlet pipeline (300) is connected with the water outlet (242), and the water outlet (242) of the liquid outlet pipeline (300) is used for guiding the water to flow into the water storage and purification pool (400); and the water inlet (241) is higher than the water outlet (242).

3. The small fish circadian rhythm behavior high-throughput observation device according to claim 2, characterized in that, The fish observation device (1) further comprises a transparent cover plate, which is arranged on the observation chamber main body (240) and used for preventing the fish from jumping into the adjacent swimming chamber (221) or jumping out of the observation chamber main body (240).

4. The small fish circadian rhythm behavior high-throughput observation device according to claim 3, characterized in that, The water storage and purification pool (400) comprises a pool body (410), a filter device (420) and a second water pump, the filter device (420) is arranged in the pool body (410), and the fluid outlet of the filter device (420) is communicated with the inlet of the second water pump; the second water pump is used for sucking the water in the pool body (410) into the filter device (420) to remove the fish excrement and food residues in the water.

5. The small fish circadian rhythm behavior high-throughput observation device according to claim 4, characterized in that, The high-throughput observation device further comprises a first water pump (500), which is used for providing power for the liquid conveyed in the liquid inlet pipeline (100).

6. The small fish circadian rhythm behavior high-throughput observation device according to claim 5, characterized in that, The water storage and purification pool (400) further comprises a heating rod (440) and an aeration device (450) used for adjusting the water temperature in the water storage and purification pool (400), the heating rod (440) is arranged below the liquid level of the pool body (410), and the aeration device (450) is arranged below the liquid level of the pool body (410) and used for oxygenating the water.

7. A small fish circadian rhythm behavior high-throughput observation method, characterized in that, The high-throughput observation device comprises 12 swimming chambers (221), and the high-throughput observation device further comprises a computer and a camera, the computer is installed with analysis software capable of tracking the motion trajectory of animals, and the camera is arranged at a certain height above the fish observation device (1) in the high-throughput observation device. The observation method comprises the following steps: Step one, add appropriate amount of fish farming water into the observation chamber (200) and the water storage and purification tank (400) of the high-throughput observation device, so that the fish farming water circulates in the water flow circulation loop; aerate the fish farming water in the water storage and purification tank (400), and control the fish farming water to maintain a certain temperature; Step two, under the premise of observing 3 parallel experiments in each exposure group and control group, after the high-throughput observation device enters a stable running state, simultaneously put the control group of live fish and three exposure groups of live fish into the high-throughput observation device for adaptation for several hours, wherein the control group of live fish refers to normally cultured live fish, and the exposure group of live fish refers to live fish exposed to the tested chemical or waste water sample; wherein the exposure group is an observation individual or a single-chamber group; when the exposure group is an observation individual, the control group is an observation individual; when the exposure group is a single-chamber group, the control group is a single-chamber group; Step three, after the adaptation is completed, use the camera to simultaneously shoot video images of the behavior of live fish in 12 chambers (221) of the observation area (220) according to a preset observation period; the preset observation period at least includes a continuous uninterrupted day and night cycle of 24 h; Step four, after acquiring the video image, the analysis software capable of tracking animal motion trajectory is used to calculate the motion speed in the extremely short time interval Δt defined by the user, the calculation principle is that the fish motion speed V in the time interval Δt is calculated based on the spatial distance S of the fish moving from the position at time t to the position at time t+Δt acquired by image analysis, the calculation formula of the fish motion speed V is: ; Step five, define the time period in which the fish movement speed V is lower than 1.5 cm / s and lasts for more than 5 s as sleep time in the whole observation period; calculate the sleep time proportion per hour in the 24 h or more day and night cycle based on the fish movement speed V in the whole observation period, and draw a curve of the sleep time proportion per hour in the day and night cycle changing with time.

Images