阅读说明：本技术 一种基于斑马鱼模型对水体中毒物进行快速预警的方法 (Method for quickly early warning poison in water body based on zebra fish model ) 是由 吴元钊 徐帆 张安慧 王培淋 刘晋熙 王嘉雯 王帅 俞逸娴 祝鑫宇 王斌杰 许中 于 2021-04-15 设计创作，主要内容包括：本发明公开了一种基于斑马鱼模型对水体中毒物进行快速预警的方法,包括：(1)向96孔板中加入待测水体,然后加入斑马鱼幼鱼；(2)采集斑马鱼幼鱼的自发运动情况,并进行行为学数据处理；(3)根据行为学数据处理结果,判断水体中是否存在毒物。本发明采用斑马鱼作为生物模型,能够快速高效地获得水环境中的毒物情况,尤其是低浓度毒物的情况。(The invention discloses a method for quickly early warning toxicants in a water body based on a zebra fish model, which comprises the following steps: (1) adding a water body to be detected into a 96-well plate, and then adding zebra fish juvenile fish; (2) collecting the spontaneous movement condition of the zebra fish juvenile fish, and performing behavioural data processing; (3) and judging whether the water body has poison or not according to the behavioral data processing result. The method adopts zebra fish as a biological model, and can quickly and efficiently obtain the condition of toxicants in the water environment, particularly the condition of low-concentration toxicants.)

1. A method for quickly early warning toxicants in a water body based on a zebra fish model is characterized by comprising the following steps:

(1) adding a water body to be detected into a 96-well plate, and then adding zebra fish juvenile fish;

(2) collecting the spontaneous movement condition of the zebra fish juvenile fish, and performing behavioural data processing;

(3) and (4) obtaining toxicity data of the water body according to the behavioral data processing result.

2. The zebra fish model-based method for quickly warning toxicants in a water body according to claim 1, wherein the zebra fish juvenile fish is zebra fish with the age of 6 days.

3. The zebra fish model-based method for quickly warning toxicants in water according to claim 1, wherein in the step (1), the toxicants in the water to be tested comprise: potassium cyanide, methyl parathion, fluoroacetamide, diphacinone, flocoumafen, clomazone, chlorfenapyr, difenoconazole, brodifacoum, bromadiolone, hyoscyamine or scopolamine.

4. The method for quickly warning toxicants in a water body based on the zebra fish model as claimed in claim 1, wherein in the step (2), the spontaneous movement conditions of the zebra fish juvenile fish are collected and are respectively carried out in a full-dark environment, a full-bright environment and an alternate light and dark environment.

5. The zebra fish model-based method for quickly warning toxicants in water according to claim 1, wherein in the step (2), Ethovision XT 14 software is adopted to track and collect the spontaneous movement of the zebra fish larvae in a DanioVision high-throughput fish behavior tracking system.

6. The zebrafish model-based method for rapid early warning of toxicants in water body according to claim 1, wherein in the step (2), the spontaneous motion conditions comprise a swimming track, a swimming average distance, a swimming maximum speed, a maximum acceleration, a morphological change or a death characteristic point.

7. The zebra fish model-based method for quickly warning toxicants in a water body according to claim 6, wherein in the step (2), the spontaneous movement condition is the moving distance within 60-120 min;

the spontaneous movement condition of the zebra fish juvenile fish is collected under the environment of light and shade alternation.

8. The zebra fish model-based method for quickly warning toxicants in a water body according to claim 7, wherein in the step (2), the light and shade alternating environment specifically comprises: and (3) performing a full bright environment for 10min, then performing a full dark environment for 10min, and repeating the cycle.

9. The zebrafish model-based method for quickly warning toxicants in water according to claim 1, wherein in the step (3), the toxicity data of the water comprises the existence and magnitude of toxicants in the water.

Technical Field

The invention belongs to the field of poison analysis, and particularly relates to a method for quickly early warning poisons in a water body based on a zebra fish model.

Background

The problems of food safety and environmental pollution are always hot spots of public discussion, and various poisoning events emerge endlessly, which poses great threat to the life safety of people. The method for researching the acute toxicity evaluation of the highly toxic substances is beneficial to rapidly distinguishing the types of the highly toxic substances and locking the toxic source. Since the 70 s of the 20 th century, zebra fish as a novel model animal is widely used in scientific research in the fields of ecological toxicology, environmental monitoring, biological pathology, drug screening and the like, has abundant toxic reaction indexes due to strong reproductive capacity, easy feeding and management and rapid development, achieves 87% of similarity with human genes, and is very suitable for establishing a toxicity screening model by simulating the ecological influence of various highly toxic substances in the environment through observing the growth and development conditions of the zebra fish. The method is used for detecting highly toxic substances based on the zebra fish model, can widen the detection range of the traditional detection technology, improves the detection flux and has the advantage of low cost. In recent years, more and more researches indicate that the zebra fish model can be used for detecting the cumulative effect and the toxic effect of carcinogens such as harmful heavy metal salts, bisphenol A, phenol, cyclohexylamine, organic chlorine, halogenated aromatic compounds and the like in the environment. Since zebra fish enters the sight of researchers, the excellent potential and unique advantages of zebra fish have better experimental conditions compared with other animal models, and a new research idea is widened for the development of toxicity evaluation of highly toxic substances.

At present, the conventional toxicity and other experimental techniques of the zebra fish and the transgenic zebra fish are mature, and the aim of increasing the number of zebra fish is fulfilledThe toxicological-like evaluation indexes evaluate related highly toxic substances, but the defects of incomplete evaluation of highly toxic substances, non-uniform toxic evaluation indexes, long toxicity experiment time and the like still exist. Traditional toxicity test half-lethality LC of zebrafish in 24, 48, 72 and 96h contaminated environments₅₀Toxicity of the compounds is very well evaluated, but few studies have conducted rapid assessments of toxicity within 2h after exposure to a toxicant. The toxicity evaluation of the water body is obtained in a short time, and the method has a very important effect on the related fields of public security. For example, in the food security of major activities, the potential safety hazard that the food is poisoned and polluted by unknown poisons needs to be eliminated; in the event of large-scale leakage of highly toxic substances or pollutants, the toxicity data of the water environment to organisms needs to be obtained as early as possible.

Disclosure of Invention

The invention provides a method for quickly early warning toxicants in a water body based on a zebra fish model, which can quickly obtain toxicity data of a water environment to organisms.

The technical scheme of the invention is as follows:

a method for quickly early warning toxicants in a water body based on a zebra fish model comprises the following steps:

(1) adding a water body to be detected into a 96-well plate, and then adding zebra fish juvenile fish;

(2) collecting the spontaneous movement condition of the zebra fish juvenile fish, and performing behavioural data processing;

(3) and (4) obtaining toxicity data of the water body according to the behavioral data processing result.

Preferably, the zebra fish juvenile fish is a zebra fish with the fish age of 6 days.

Preferably, in the step (1), the poison in the water body to be tested comprises: potassium cyanide, methyl parathion, fluoroacetamide, diphacinone, flocoumafen, clomazone, chlorfenapyr, difenoconazole, brodifacoum, bromadiolone, hyoscyamine or scopolamine.

Preferably, in the step (2), the spontaneous movement of the zebra fish juvenile fish is collected and is respectively carried out in a full dark environment, a full bright environment and a light and dark alternating environment.

Preferably, in the step (2), in a DanioVision high-throughput fish behavior tracking system, adopting Ethovision XT 14 software to track and collect the spontaneous movement condition of the zebra fish juvenile fish within 60-120 min;

collecting the spontaneous movement condition of the zebra fish juvenile fish, and respectively carrying out the spontaneous movement condition in a light and dark alternating environment.

Preferably, in the step (2), the light and dark alternating environment specifically includes: and (3) performing a full bright environment for 10min, then performing a full dark environment for 10min, and repeating the cycle.

Preferably, in the step (2), the spontaneous movement condition includes a swimming trajectory, a swimming mean distance, a swimming maximum speed, a swimming maximum acceleration, a form change, and a death feature point.

Preferably, in step (3), the toxicity data of the water body includes the presence or absence of toxicants in the water body and the magnitude of toxicity. Further, the toxicity is determined according to the length of the moving distance.

Furthermore, the water body to be detected may contain unknown toxicants, and whether the toxicants exist or not and the size of the toxicants can be estimated according to the length of the moving distance.

Compared with the prior art, the invention has the beneficial effects that:

the method adopts zebra fish as a biological model, and can quickly and efficiently obtain the condition of toxicants in the water environment, particularly the condition of low-concentration toxicants.

Drawings

FIG. 1 is a graph showing the effect of different concentrations of potassium cyanide solution on the average distance traveled by zebra fish in the dark environment of example 1;

FIG. 2 is a graph showing the effect of different concentrations of potassium cyanide solution on the average distance traveled by zebra fish in the full bright environment of example 1;

FIG. 3 is the effect of potassium cyanide solution with different concentrations on the average swimming distance of zebra fish in alternate light and dark environment in example 1;

FIG. 4 is the effect of potassium cyanide solution with different concentrations on the behavioral ability (swimming track, average swimming distance, maximum swimming speed, maximum acceleration, morphological change, death characteristic point) of zebra fish in 60-120min in example 1;

FIG. 5 is a graph of the effect of potassium cyanide solution on the distance traveled by zebrafish at different time periods;

FIG. 6 is a graph of the effect of the spinosad solution on the behavioral ability of zebrafish in example 2 at different time periods;

FIG. 7 is a graph of the effect of brodifacoum solution on the behavioral ability of zebrafish in example 3 at different time periods;

FIGS. 8 to 20 are graphs showing the influence of different poison solutions in examples 4 to 16 on the behavior of zebra fish in the detection environment with alternating light and shade in the period of 60 to 120 min.

Detailed Description

The invention is further described below by means of specific examples, the materials and equipment used in the invention being as follows.

Experimental animals:

the parental zebra fishes used in the research are wild type AB strain zebra fishes (WT/AB) bred in a laboratory, purchased from Shanghai Ji fluorescence biotechnology Limited, and supplemented species fishes are purchased from the national zebra fish center in the experiment to avoid inbreeding. All embryos and juvenile fishes required by the experiment are obtained by adopting a healthy 6-month-old breeding fish spawning and hatching mode.

The zebra fish culture condition is characterized in that the pH value of the water environment is controlled to be 7.0-8.0, the water temperature is controlled to be about 28 ℃, the ionic strength is 500-1500, and the light-dark time ratio in one day is 7: 5. Feeding brine shrimp 2-3 times a day.

Instruments and equipment:

ZW-H3000 microscope (zhong wei kou, shenzhen), Ethovision XT 14 behavior detection system (nodaxy, the netherlands), Pacific RO ultrapure water machine (seymelaeishi, usa), thermostated incubator (longyun, shanghai), automatic water circulation culture system (homemade), autoclave (seymelaeishi, usa), electronic analytical balance (sydoris, germany), incubator (haisheng, shanghai), pipette gun (abyde, germany), MS1 minshaker (ika) type vortex shaker, BSA224S-cw sartorius) type analytical balance, 96-well round-plate, 96-well square-plate, culture dish, pipette, and the like.

Reagent:

e3 culture solution: weighing NaCl 17.2g, KCl 0.76g and CaCl respectively₂ 2.91g、MgSO₄·7H₂O4.9 g, using double distilled water H₂The dissolved O was dissolved to prepare 1L of E3 culture medium.

A virulent solution: e3 solutions of highly toxic substances were prepared at 0.1mg/L,1mg/L,10mg/L, 100mg/L, and 500 mg/L). Wherein, DMSO cosolvent is added aiming at the water-insoluble virulent, so as to ensure that the DMSO content in the final solution is 0.1 percent.

General experimental methods:

in the case of a 96-well plate, 270uL of a blank control E3 solution containing a young zebra fish was pipetted into the plate by syringe. 12 replicate wells were set for each concentration. And finally uniformly adding 30uL of hypertoxic solution with corresponding concentration into each hole, mixing, standing, and immediately observing through a system.

Obtaining zebra fish juvenile fish:

the observation of the zebra fish juvenile fish can realize high flux, and the experiment is carried out by using the zebra fish with the fish age of 6 days (6dpf) in combination with the developmental situation of the behavioral ability of the zebra fish juvenile fish reported by related documents. The zebra fish embryo is obtained by adopting a spawning mode of breeding fish. Selecting adult male and female zebra fish of WT/AB line which is healthy and mature for about 6 months at night of seventh day before using the zebra fish juvenile fish, placing the zebra fish into a spawning tank (the male and female zebra fish are separated by a clapboard) according to the male-female ratio of 2:1, transferring the mating tank into a dark environment, removing the clapboard after 14h, transferring an incubation box into a light environment, and mating the female fish with the male fish. Embryos were harvested after about 20min and transferred to petri dishes. Washing embryo with E3 solution for 2-3 times, and placing collected embryo in 28 deg.C incubator. And (5) removing the white dead fish eggs in time.

The treatment method of the zebra fish juvenile fish comprises the following steps:

the 96-hole plate is placed in a DanioVision high-throughput fish behavior tracking system, and the spontaneous movement condition of the zebra fish juvenile fish within 2h is tracked and collected by adopting Ethovion XT 14 software, so that the situation is taken as a characteristic point for counting the swimming track, the swimming average distance, the swimming maximum speed, the maximum acceleration, the form change and the death. Recording the death number, recording abnormal behaviors, and collecting the action tracks of all groups of juvenile fishes by using software to perform ethological data processing.

Data processing method

The experimental data are processed by using the single-factor variance of SPASS 16.0 software, and the concentration of each toxic substance causing the remarkable difference of the behavior ability of the zebra fish is calculated.

Example 1

270uL of the blank control E3 solution containing a young zebrafish was pipetted into the wells of a 96-well plate using a syringe. And then adding 30uL of KCN aqueous solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L and 50mg/L) with different concentrations into each hole, setting 12 repeated holes for each concentration, mixing, standing, immediately observing by a DanioVision high-throughput fish behavior tracking system, respectively tracking and collecting the spontaneous movement condition of the zebra fish juvenile fish within 2h by adopting Ethovion XT 14 software in a completely dark environment, a completely bright environment and a light and dark alternating environment, and taking the spontaneous movement condition as a characteristic point for counting the swimming trajectory, the swimming average distance, the swimming maximum speed, the maximum acceleration, the form change and the death according to the result. Recording the death number, recording abnormal behaviors, collecting the action tracks of each group of juvenile fishes by using software, and performing behavioral data processing, wherein the test results are shown in fig. 1-4, wherein fig. 1 is the influence of potassium cyanide solutions with different concentrations on the average swimming distance of the zebra fishes in a dark environment, fig. 2 is the influence of potassium cyanide solutions with different concentrations on the average swimming distance of the zebra fishes in a bright environment, fig. 3 is the influence of potassium cyanide solutions with different concentrations on the average swimming distance of the zebra fishes in a bright and dark environment (the interval of light and dark alternation time is 10 minutes), fig. 4 is the influence of potassium cyanide solutions with different concentrations on the behavior ability (the swimming track, the average swimming distance, the swimming maximum speed, the maximum acceleration, the morphological change and the death characteristic point) of the zebra fishes at 60-120min, and fig. 5 is the influence of the potassium cyanide solutions on the moving distance of the zebra fishes at different time periods.

The experimental result shows that after the zebra fish juvenile fish is exposed to the highly toxic potassium cyanide solution, the performance difference is obvious under different ethological detection environments. In a dark environment, the activity of the zebra fish in the blank group is obviously higher than that of the zebra fish in other concentrations within 120 minutes, the moving distance is always kept to be about 50mm every 2min, but the activity of the zebra fish in each concentration is greatly changed along with time, and the moving distance distinction degree between different concentrations is not obvious. Under the full-bright environment, after different potassium cyanide solutions are contaminated by young zebra fish, the moving distance is greatly changed, and the situation that the high-concentration moving is higher than the low-concentration moving occurs in a plurality of time periods. In the detection environment with alternating light and shade (10min light +10min dark, repeated circulation), stable periodic change of the zebra fish juvenile fish is observed, and in the later observation period, the zebra fish juvenile fish presents a concentration-related ethological ability, so that the detection environment with alternating light and shade is determined. Within 0-60min of contacting potassium cyanide, we find that the result shows a rule that the moving distance is reduced along with the increase of the concentration. When the concentration of potassium cyanide exceeds 1mg/L, the behavioral ability of the zebra fish juvenile fish is obviously reduced, and the statistical significance is achieved. The higher concentration potassium cyanide solution has more obvious reduction of the behavioral ability of the zebra fish juvenile fish. The rule also exists within 60-120min after the zebra fish juvenile fish is exposed to the poison, which shows that the toxic reaction in the early stage (0-60min) is the same as that in the later stage (60-120min) after the zebra fish juvenile fish is exposed to the potassium cyanide solution.

Example 2

The operation method is basically the same as that of example 1, except that a clomazone solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L and 50mg/L) is adopted to replace a potassium cyanide solution, and the behavior change of zebra fish caused by the clomazone in different time periods is measured, and the result is shown in figure 6. In 0-60min after the zebra fish is contacted with the cloxacillin, the moving distance of the zebra fish under different concentrations is counted, and the result shows that the moving distance changes in an inverted U shape along with the increase of the concentration. When the concentration of the cloxacarb solution is 1mg/L, the ethological ability of the zebra fish juvenile fish is obviously improved, and statistical difference exists. The ethological ability of the zebra fish juvenile fish is obviously improved by a cloxacillin solution with higher concentration, such as 10mg/L, and statistical differences exist. When the concentration is 50mg/L, no obvious difference from the blank exists. The rule does not exist within 60-120min of contacting poison, which shows that after the zebra fish juvenile fish contacts the clomazone solution, the early (0-60min) toxic reaction is different from the later (60-120min), the early takes the violent emergency reaction as the main part, and the later can correctly reflect the toxicity rule.

Example 3

The operation method is basically the same as that of example 1, except that a muriatin solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L and 50mg/L) is adopted to replace a potassium cyanide solution, and the behavior ability change of zebra fish caused by the lordotoxin is measured at different time periods, and the result is shown in figure 7. Within 0-60min after the zebra fish is contacted with the bromelin, the moving distance of the zebra fish under different concentrations is counted, and the result shows that the moving distance does not show a rule of obvious change along with the increase of the concentration. The rule does not exist within 60-120min of exposure to poison, which shows that after the zebra fish juvenile fish is exposed to the potassium cyanide solution, the early (0-60min) toxic reaction is different from the later (60-120min), the early stage mainly takes the adaptive reaction as the main part, and the later stage can correctly reflect the toxicity rule.

Examples 4 to 16

The operation of this example was substantially the same as that of example 1, except that potassium cyanide solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), diphacinone solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), flocoumafen solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), clomazone solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), desmopressin solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), rodenticide solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), bromadiolone solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), dipheny bromide solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), fluoroacetamide solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), chlorfenadone solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), methyl parathion solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L), scopolamine solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L,50mg/L) and a hyoscyamine solution (0.01mg/L, 0.1mg/L,1mg/L,10mg/L and 50mg/L) are used as test poisons, the test environment is under a detection environment with alternate light and shade (10min light +10min dark, repeated circulation), the detection time is 60-120min, the behavior ability change of the zebra fish caused by different poisons in different time periods is measured, the result is shown in figures 8-20, and the obtained statistical early warning model is shown in a table 1.

TABLE 1 behavioral competence of zebra fish larvae after 60-120min of exposure to different toxicants

Representative significance p-value <0.01, representative significance p-value <0.001, representative significance p-value < 0.0001.

Through the normalization processing of the data, we found that when the concentration of common toxicants exceeds 1mg/L (except for diphacinone and clomazone), the moving distance of the zebra fish juvenile fish is remarkably reduced. When the concentration reaches 10mg/L, all poisons cause a significant behavioral decline in the fish, reflecting the sensitivity of our model.