CN108401978A - The method for detecting environmental contaminants development toxicity based on tropical Xenopus laevis tadpole motor behavior - Google Patents
The method for detecting environmental contaminants development toxicity based on tropical Xenopus laevis tadpole motor behavior Download PDFInfo
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- CN108401978A CN108401978A CN201810068199.XA CN201810068199A CN108401978A CN 108401978 A CN108401978 A CN 108401978A CN 201810068199 A CN201810068199 A CN 201810068199A CN 108401978 A CN108401978 A CN 108401978A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/02—Breeding vertebrates
Abstract
The invention discloses a kind of methods detecting environmental contaminants development toxicity based on tropical Xenopus laevis tadpole motor behavior, and this approach includes the following steps：Select the tropical Xenopus Embryo that the stage of development is uniform, healthy；By environmental contaminants according to equal series exposure liquid for being configured to various concentration than gradient；Embryo is placed in environmental contaminants exposure liquid, is cultivated 48 72 hours in 25 DEG C of constant incubators；Tadpole sport video in continuous acquisition 15 minutes, movement locus is visualized, measure include move distance, average speed move, the motor behavior parameter of maximum movement speed；Each exposure group is compared with the motor behavior parameter of control group using variance analysis, judges whether pollutant has development toxicity and development toxicity degree according to inspection result；Development toxicity simple by the quantification motor behavior parameter of tropical Xenopus laevis tadpole, enzyme rapidly and sensitively evaluating environmental contaminants, the supplement Testing index of pollution water sample bio-toxicity is can be used as, the potential information requirement for environmental contaminants in water body provides reference frame.
Description
Technical field
It is specifically a kind of based on tropical Xenopus laevis tadpole the present invention relates to the assessing developmental toxicity technical field of environmental contaminants
The method that motor behavior detects environmental contaminants development toxicity.
Technical background
Current global amphibian animal population decay is very serious, and to be exactly amphibian animal population decline environmental contaminants
Move back one of the immediate cause of even extinction.Amphibian completes breeding and larval development usually in water environment, and amphibious
The ovum of animal easily absorbs organic pollution, therefore its larval development is probably done by environmental contaminants in water
It disturbs.Numerous studies have found that the environmental contaminants such as heavy metal, pesticide can cause amphibian early development poisonous effect, and hold
It is continuous to act on growth and development process, cause amphibian animal population quantity to largely reduce.This requires must pay attention to environmental contaminants
To the development toxicity of amphibian, the development toxicity of exploitation simple effective method to environmental pollutants is assessed.
Amphibian development toxicity research to environmental pollutants mainly uses Xenopus embryos tertogenicity test at present
（The Frog Embryo Teratogenesis Assay Xenopus, FETAX）, i.e., by the Africa xenopus embryo of mesoderm growing early stage
Tire exposure 96h in chemical solutions, by detecting rate of embryonic death, abnormal rate and teratogenic index etc. come the hair of evaluating chemical product
Educate toxicity.However, causing the chemical concentration of embryonic death or deformity usually relatively high, it is not consistent with actual environment residual quantity,
And the pollutant under environmental correclation concentration will not usually cause significant Xenopus Embryo dead or deformity.This makes Africa xenopus embryo
Tire tertogenicity test can not fully meet the demand of the assessing developmental toxicity caused by low concentration environmental contaminants.Therefore, in order to comment
Development toxicity and ecological risk caused by valence actual environment pollutant, exploitation one kind can quickly, sensitively reflect close to ring
The detection method of the pollutant development toxicity of border actual concentration seems particularly necessary.
Invention content
For disadvantages mentioned above of the existing technology, the object of the present invention is to provide one kind based on tropical Xenopus laevis tadpole movement
The method of behavioral value environmental contaminants development toxicity, can simple, enzyme rapidly and sensitively qualitative assessment environmental contaminants developments
Toxicity.
What the object of the invention was achieved through the following technical solutions：
A method of environmental contaminants development toxicity being detected based on tropical Xenopus laevis tadpole motor behavior, this method includes following tool
Body step：
Step 1：Select the tropical Xenopus Embryo that the stage of development is uniform, healthy；
Step 2：By environmental contaminants according to equal series exposure liquid for being configured to various concentration than gradient；
Step 3：The tropical Xenopus Embryo of health is placed in environmental contaminants series exposure liquid, is connected in 25 DEG C of constant incubators
Continuous culture 48-72 hours；
Step 4：Tadpole sport video in continuous acquisition 15 minutes, tadpole movement locus is visualized, measurement include move away from
From the movement of, average speed, the motor behavior parameter of maximum movement speed；
Step 5：Each exposure group is compared with the motor behavior parameter of control group using variance analysis, according to inspection result
Significance judges whether with development toxicity, judges development toxicity journey according to relative motion distance and speed of related movement
Degree.
The step 1 specifically includes：It is embraced using the tropical Xenopus laevis of method induction of manual injection's human chorionic gonadotrophin
To oviposition, is selected under the microscope from same parent, in primitive gut early metaphase, normotrophic tropical Xenopus Embryo, set
It is spare in ringer's solution.
It is specifically included in the step 2：Environmental contaminants can be insecticide, bacteria remover etc.；Pollutant to be measured is dissolved in
In water, then liquid is exposed at required concentration gradient series with ringer's solution successively proportional diluted in proportion；It is insoluble in the to be measured of water
Pollutant first uses cosolvent etc. than gradient dilution, then pollutant solution is taken to be mixed into ringer's solution, is configured to required concentration
Series exposure liquid, series expose final concentration of 0.1% of cosolvent in liquid；Series exposure liquid is now with the current, and wherein maximum concentration is
The 50% of half lethal concentration, dilution ratio 1/5-1/2.
The step 3 specifically includes：The tropical Xenopus Embryo selected is randomly divided into multiple exposed groups, is respectively labeled as sudden and violent
Dew group 1-exposure group n and control group；Control group uses ringer's solution, and the control group for being insoluble in the pollutant of water is used containing 0.1%
The ringer's solution of cosolvent；Embryo is placed in constant incubator, 25 DEG C, continuously cultivate under dark condition, is replaced within 24 hours sudden and violent
Reveal liquid, chooses except dead embryo.
The step 4 specifically includes：After exposure, the tadpole of survival is transferred in 48 orifice plates, one is placed per hole
Corresponding pollutant exposure liquid or 500 μ L of ringer's solution is added in tadpole；After tadpole adapts to 2 minutes in 48 orifice plates, utilize
Tadpole sport video under the conditions of CCD camera continuous acquisition recording light photograph, in 15 minutes, cameras frame rate should be greater than 30 frames/s,
Image size is more than 640 pixel *, 480 pixels；Applied behavior analysis system visualizes the movement locus of every tadpole, obtains fortune
Move distance, average speed moves, the quantitative result of the behavioral parameters of maximum movement speed.
The step 5 specifically includes：Each exposure group and the kinematic parameter of control group are subjected to variance analysis, compared according between group
Compared with significance judge pollutant whether have development toxicity, and according to the variation multiple of move distance, movement velocity come
Judge development toxicity degree caused by environmental contaminants.
Compared with prior art, improved based on tropical Xenopus laevis tadpole fortune on the basis of Xenopus embryos tertogenicity test
The method of dynamic behavioral value environmental contaminants development toxicity, is biological model using tropical Xenopus laevis, passes through the kinematic parameter of tadpole
The development toxicity for carrying out quantitative assessment environmental contaminants has unique advantage.Its main feature and advantage have：Selection had both had non-
The advantages of continent Xenopus laevis, but with egg laying amount bigger, it is ripe faster, the smaller tropical Xenopus laevis of build be biological model, required exposure
Time is short, more simply, quickly；Amphibian is considered as most sensitive one of the species of the response to chemical substance, and behavior
Learn one of Early warning indicator as environmental contaminants ecological risk, can reflect in dynamic and integral level vital movement by
The motor behavior parameter response of the influence arrived, amphibian is more direct and sensitive, for evaluating the pollution of environmental correclation concentration
Object development toxicity has apparent advantage；The variation of motor behavior directly affects microorganism predation and escapes the ability of enemy, to
Species long-term development and existence are influenced, the caused ecological risk of long-term exposure has instruction well to make to environmental pollutants
With therefore, tropical Xenopus laevis tadpole motor behavior parameter can be used as supplement detection and the evaluation index of pollution water sample bio-toxicity, be
The potential information requirement assessment of environmental contaminants provides reference frame in water body.
Description of the drawings
Fig. 1 is flow chart of the present invention；
Fig. 2 is dose-effect relationship figure of the Biphenthrin to tropical Xenopus laevis tadpole move distance；
Fig. 3 is dose-effect relationship figure of the Biphenthrin to tropical Xenopus laevis tadpole maximum movement speed.
Specific implementation mode
With reference to specific embodiment, the present invention is described in detail.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
The present invention includes the following steps：
Step 1：Select the tropical Xenopus Embryo that the stage of development is uniform, healthy；
Step 2：By environmental contaminants according to equal series exposure liquid for being configured to various concentration than gradient；
Step 3：The tropical Xenopus Embryo of health is placed in environmental contaminants series exposure liquid, is connected in 25 DEG C of constant incubators
Continuous culture 48 hours；
Step 4：Tadpole sport video in continuous acquisition 15 minutes, tadpole movement locus is visualized, measurement include move away from
From the movement of, average speed, the motor behavior parameter of maximum movement speed；
Step 5：Each exposure group is compared with the motor behavior parameter of control group using variance analysis, according to inspection result
Significance judges whether with development toxicity, judges development toxicity journey according to relative motion distance and speed of related movement
Degree.
Preferably, in the step 1, the formula of ringer's solution is sodium chloride 0.7889g/L, potassium chloride 0.0186g/L,
Calcium chloride dihydrate 0.0294g/L, 4- hydroxyethyl piperazineethanesulfonic acid 0.2383g/L；The tropical Xenopus Embryo selected should be at primitive gut
Embryo early metaphase, i.e. NF10-11 stages, are not to be exceeded the neural plate phase.
Preferably, in the step 2, the cosolvent for being insoluble in the pollutant to be measured of water is dimethyl sulfoxide (DMSO)（DMSO）；
Final concentration of the 0.1% of DMSO in series exposure liquid, to ensure that cosolvent will not cause development toxicity to tropical Xenopus laevis tadpole；It waits for
Survey pollutant final concentration suggestion is 0.01-1000 μ g/L, and maximum concentration is no more than the 50% of half lethal concentration, exposure group concentration mistake
Height can cause the deviation of motor behavior experimental result.
Preferably, in the step 4, corresponding pollutant exposure liquid or 500 μ of ringer's solution are added in the every hole of 48 orifice plates
L, liquid volume added is very few to have potential hazard, the excessive tadpole of liquid volume added to be easy to happen vertical trip phenomenon, movement is caused to go tadpole survival
For the deviation of parameter；Control group and each exposure group should separately include more than 20 tadpoles, reduce tadpole motor behavior individual difference
The different deviation brought；When acquiring sport video, cameras frame rate is more than 30 frames/s, and image size is more than 640 pixel *, 480 pixels,
Ensure the credibility of each parameter when motor behavior analysis.
Embodiment 1
The development toxicity of Biphenthrin is evaluated using the motor behavior of tropical Xenopus laevis tadpole
Model organism：Tropical Xenopus laevis.
Tested pollutant：Biphenthrin（bifenthrin）, it is purchased from sigma companies of the U.S..
Preparation method：Standard items are dissolved in DMSO and are configured to 2000 mg/L mother liquors, are stored in -20 DEG C, DMSO is used before laboratory
It is diluted to required concentration.
Capture apparatus and analysis system：CCD camera is purchased from Hangzhou collection of illustrative plates Electro-optical Technology, INC. (US) 62 Martin Road, Concord, Massachusetts 017, and camera lens are purchased from west
Pacify telecentric optical system Co., Ltd, analysis system is NouldsEthoVision 8.0.
Experimental method is as follows：
1, tropical Xenopus Embryo is selected：Sexually matured tropical 2-3 pairs of Xenopus laevis is selected, is swashed using manual injection's human chorionic gonadotropin's gland
The method induction of element is embraced to oviposition.Wait that 10-12h collects embryo after laying eggs, cleaning removes unfertilized embryo and impurity, micro-
Sufficient amount is picked out under mirror and reaches NF10-11 stages, normotrophic tropical Xenopus Embryo, is placed in spare in ringer's solution.
2, environmental contaminants exposure liquid is prepared：Before exposure experiment, extremely with DMSO gradient dilutions by Biphenthrin mother liquor
0.064-1000 mg/L, then 1000 times are diluted with ringer's solution liquid, it is configured to the Biphenthrin of 0.064-1000 μ g/L respectively
Series exposure liquid, exposes final concentration of 0.1% of DMSO in liquid.
3, environmental contaminants expose：Experiment altogether be arranged 1 0.1%DMSO control group and 0.064,0.32,1.6,8,40,
200, the Biphenthrin exposure group of 1000 μ g/L7 concentration.Add 40 mL solution and 25 in the glass culture dish of 90 mm of diameter
Only normotrophic embryo is positioned in 25 ± 0.5 DEG C of constant incubators.To avoid Biphenthrin from decomposing, incubator keeps black
Secretly.Exposure liquid is replaced when 24 h of exposure, and is chosen except dead embryo.
4, tadpole sport video quantitative analysis：The tadpole of survival is transferred in 48 orifice plates, a tadpole is placed per hole,
And the corresponding pollutant exposure liquid of 500 μ L or ringer's solution is added.48 orifice plates are placed on above fixed light source, environment peace is kept
It is quiet, after tadpole adapts to 2 minutes, utilize the sport video of tadpole in CCD camera continuous acquisition 15 minutes.Applied behavior analysis system
System visualizes the movement locus of every tadpole using each hole as survey region, obtain tadpole in 15 minutes it is total move away from
From, the quantitative result of average speed, maximum speed.
5, environmental contaminants assessing developmental toxicity：All tadpole kinematic parameters are collected, one-way is utilized between each group variable
ANOVA carries out variance analysis, and the standard of significant difference is set as p between data<0.05.Biphenthrin is drawn to tadpole kinematic parameter
Dose-effect relationship figure, assess Biphenthrin caused by development toxicity effect.
The result shows that：By dose-effect relationship result（Fig. 1, Fig. 2）It is found that when bifenthrin concentration is higher than 0.32 μ g/L
When, the move distance and maximum movement speed of tadpole are reduced there have been significant, and Biphenthrin is to tropical Xenopus laevis tadpole
There is dose-dependences for move distance and maximum movement speed, with the increase of bifenthrin concentration, the movement row of tadpole
For by being inhibited more apparent, with microscopically observation to the phenomenon that be consistent.By it is each exposure organize tadpole kinematic parameter with compare
Group carries out the result of variance analysis（Table 1）It has been shown that, when bifenthrin concentration be 0.32 μ g/L when, exposure group tadpole movement away from
It is reduced to the 26.7% of control group respectively from, average movement velocity and maximum movement speed（p<0.001）、27.4%（p<0.05）With
43.0%（p<0.001）, i.e. to tropical Xenopus laevis tadpole, there are development toxicities for the Biphenthrin for illustrating more than 0.32 μ g/L, and with
Exposure concentrations increase, development toxicity is reinforced.
A. relative motion distance organizes the ratio of tadpole and control group tadpole mean motion distance for each exposure；
B. with respect to average movement velocity, the ratio of average movement velocity in tadpole and control group tadpole 15 minutes is organized for each exposure；
C. relative maximum movement velocity organizes the ratio of maximum movement speed in tadpole and control group tadpole 15 minutes for each exposure；
D. data are expressed as average value ± standard error, and * indicates result compared with the control group, p<0.05, * * * indicate result with it is right
It is compared according to group, p<0.001.
Claims (6)
1. a kind of method detecting environmental contaminants development toxicity based on tropical Xenopus laevis tadpole motor behavior, which is characterized in that should
Method includes step in detail below：
Step 1：Select the tropical Xenopus Embryo that the stage of development is uniform, healthy；
Step 2：By environmental contaminants according to equal series exposure liquid for being configured to various concentration than gradient；
Step 3：The tropical Xenopus Embryo of health is placed in the series exposure liquid of step 2, it is continuous in 25 DEG C of constant incubators
Culture 48-72 hours；
Step 4：Tadpole sport video in continuous acquisition 15 minutes, tadpole movement locus is visualized, measurement include move away from
From the movement of, average speed, the motor behavior parameter of maximum movement speed；
Step 5：Each exposure group is compared with the motor behavior parameter of control group using variance analysis, according to inspection result
Significance judges whether with development toxicity, judges development toxicity journey according to relative motion distance and speed of related movement
Degree.
2. the side according to claim 1 for detecting environmental contaminants development toxicity based on tropical Xenopus laevis tadpole motor behavior
Method, which is characterized in that the step 1 specifically includes：The torrid zone is induced using the method for manual injection's human chorionic gonadotrophin
Xenopus laevis is embraced to oviposition, is selected under the microscope from same parent, in primitive gut early metaphase, normotrophic tropical Xenopus laevis embryo
Tire is placed in spare in ringer's solution.
3. the side according to claim 1 for detecting environmental contaminants development toxicity based on tropical Xenopus laevis tadpole motor behavior
Method, which is characterized in that in the step 2, environmental contaminants are insecticide, bacteria remover；Pollutant to be measured is soluble in water, then press
Ratio exposes liquid with ringer's solution successively proportional diluted at required concentration gradient series；Being insoluble in the pollutant to be measured of water first makes
With cosolvent etc. than gradient dilution, then pollutant solution is taken to be mixed into ringer's solution, is configured to the series exposure liquid of required concentration,
Final concentration of the 0.1% of cosolvent in series exposure liquid；Series exposure liquid is now with the current, and wherein maximum concentration is half lethal concentration
50%, dilution ratio 1/5-1/2.
4. the side according to claim 1 for detecting environmental contaminants development toxicity based on tropical Xenopus laevis tadpole motor behavior
Method, which is characterized in that the step 3 specifically includes：The tropical Xenopus Embryo selected is randomly divided into multiple exposed groups, is marked respectively
It is denoted as 1-exposure group n of exposure group and control group；Control group uses ringer's solution, and the control group for being insoluble in the pollutant of water uses
Ringer's solution containing 0.1% cosolvent；Embryo is placed in constant incubator, 25 DEG C, continuously cultivate under dark condition, 24 hours
Exposure liquid is replaced, is chosen except dead embryo.
5. the side according to claim 1 for detecting environmental contaminants development toxicity based on tropical Xenopus laevis tadpole motor behavior
Method, which is characterized in that the step 4 specifically includes：After exposure, the tadpole of survival is transferred in 48 orifice plates, is put per hole
A tadpole is set, corresponding pollutant exposure liquid or 500 μ L of ringer's solution is added；After tadpole adapts to 2 minutes in 48 orifice plates,
Tadpole sport video under the conditions of being shone using CCD camera continuous acquisition recording light, in 15 minutes, cameras frame rate should be greater than 30
Frame/s, image size are more than 640 pixel *, 480 pixels；Applied behavior analysis system visualizes the movement locus of every tadpole,
Obtain move distance, average speed moves, the quantitative result of the behavioral parameters of maximum movement speed.
6. the side according to claim 1 for detecting environmental contaminants development toxicity based on tropical Xenopus laevis tadpole motor behavior
Method, which is characterized in that the step 5 specifically includes：Each exposure group and the kinematic parameter of control group are subjected to variance analysis, according to
The significance of comparison among groups judges whether pollutant has development toxicity, and according to move distance, the variation of movement velocity
Multiple is come development toxicity degree caused by judging environmental contaminants.
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| CN109329123A (en) * | 2018-10-12 | 2019-02-15 | 广东工业大学 | A kind of collection method of aquatic animal enteron aisle mucus |
| CN113311144A (en) * | 2021-05-20 | 2021-08-27 | 江南大学 | Method for evaluating toxicity of micro-plastic based on behavioral indexes of daphnia magna |
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