CN110024921B - Application of biological stain xylene aniline in thrips insect membrane feeding - Google Patents
Application of biological stain xylene aniline in thrips insect membrane feeding Download PDFInfo
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- CN110024921B CN110024921B CN201910339402.7A CN201910339402A CN110024921B CN 110024921 B CN110024921 B CN 110024921B CN 201910339402 A CN201910339402 A CN 201910339402A CN 110024921 B CN110024921 B CN 110024921B
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- thrips
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/179—Colouring agents, e.g. pigmenting or dyeing agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/90—Feeding-stuffs specially adapted for particular animals for insects, e.g. bees or silkworms
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides application of a biological stain xylene aniline in film feeding of thrips insects. Dissolving dimethylbenzene aniline biological staining agent powder in 0.01mol/L phosphate buffer solution with the pH value of 7.0-7.4 to obtain 1-10mg/ml biological staining solution, and carrying out film feeding on the thrips nymphs fed through a single tube to obtain the stained thrips nymphs. The invention takes dimethylbenzene aniline as an indicator when feeding the thrips insect membrane, can non-specifically combine with biological protein, can provide direct evidence for accurately judging feeding of the thrips insect under safe concentration, and quickly and portably establishes a RAN interference technology system of the thrips insect.
Description
Technical Field
The invention belongs to the field of insect organism dyeing, and particularly relates to application of a biological dyeing agent xylene aniline in thrips insect film feeding.
Background
Thrips palmi Karny belongs to the Thrips genus (Thrips) of the Thrips family (Thripidae) of the order Thysanoptera (Thysanoptera). Thrips palmi is a dangerous worldwide quarantine pest, can harm more than 600 plants and is more serious to various important agricultural and economic crops. Thrips palmi was first discovered in Taiwan of China in the seventies of the last century, and then successively harmed in Guangdong, hunan, sichuan, yunnan, tibet, shandong, beijing and other areas.
At present, the research on the aspect of thrips breeding is less, the research is only limited to basic research such as gene cloning, protein induced expression and gene space-time distribution, the research on membrane feeding is not reported, and the research of applying xylyl aniline as the thrips membrane staining indicator is initiated. Xylenylaniline, known as xylenylcyan FF, is used as a biological stain; xylene blue, molecular formula C 25 H 27 N 2 NaO 6 S 2 The appearance (property) was a bluish black powder with a purity of about 75% (shown in FIG. 1). Xylene aniline is a substance with very low toxicity in acute toxicity test of fish, and can be used asRedox indicators (orange yellow to yellow), nucleic acid electrophoresis indicators or biological dyes, but the usage amount needs to be accurately controlled so as to avoid toxic action on insects and influence scientific research.
The invention combines the advantages of xylene aniline biological dye, namely biological protein can be non-specifically combined, and the influence of the biological dye on the vital signs of insects is controlled within a safe range. The inventor comprehensively considers the effect of the xylyl aniline applied to indicate the feeding effect of the thrips membrane, successfully solves the problem of poor feeding effect caused by low feeding rate in the feeding process of the thrips membrane, and promotes the development of the fields of fluorescence labeling analysis technology, life science and the like.
Disclosure of Invention
The invention well solves the application of the biological stain xylene aniline in the film feeding of thrips insects, can provide technical guidance for dyeing and marking of the thrips insects on one hand, and can also detect the evaluation of the feeding effect of the film feeding method on the rasping type mouthpart insects such as thrips and the like on the other hand.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of the biological stain xylene aniline in the film feeding of thrips insects comprises the following steps:
(1) Dissolving dimethylbenzene aniline biological staining agent powder in 0.01mol/L phosphate buffer solution to obtain biological staining solutions with different concentrations;
(2) Membrane feeding is carried out on the thrips nymphs by the biological staining solution in the step (1), and the membrane feeding is carried out for 24h under the conditions that the temperature is 25 +/-2 ℃, the humidity is 75%, and the illumination is 14L;
(3) And (3) photographing and imaging the staining condition of the marked thrips nymphs in the step (2) by using a microscope.
The pH value of the 0.01mol/L phosphate buffer solution is 7.0-7.4.
The insect age range of the thrips nymphs is 1-2 years.
The concentration of the biological staining solution is 1-10 mg/ml.
According to the thrips obtaining method in the step (2), the method comprises the following steps: starving thrips nymphs for 2h, picking the thrips nymphs into a feeding pipe with openings on two sides by using a writing brush, sealing two ends of the feeding pipe by using a Parafilm membrane, dropwise adding a prepared dye solution into one end of the feeding pipe, and covering the dye solution by using the Parafilm membrane for moisture preservation.
The invention has the advantages that:
the invention discloses an application of a biological staining agent xylene aniline in membrane feeding of thrips insects, which comprises the steps of mixing xylene aniline biological staining agent powder and 0.01mol/L phosphate buffer solution to obtain a biological staining solution, and carrying out membrane feeding on thrips nymphs fed through a single tube to obtain the stained thrips nymphs. Under the condition of ensuring safety and harmlessness to thrips, the effect of dimethylbenzene aniline as a thrips insect membrane feeding indicator is evaluated through two indexes of concentration of a coloring agent and acidity and alkalinity of a buffer solution, the feeding monitoring feasibility of the biological dye indicator to thrips and other thysanoptera insects is successfully realized for the first time, the biological dye indicator can be non-specifically combined with biological protein, direct evidence can be provided for accurately judging feeding of the thrips and other insects, and a thrips insect RAN interference technology system can be quickly and portably established.
Drawings
FIG. 1 chemical structural formula of xyleneaniline.
FIG. 2 shows the staining of thrips with different concentrations of biological staining solution.
FIG. 3 is a graph of the effect of xylidine feeding on thrips survival.
FIG. 4 shows the membrane feeding of thrips by adding biological staining agent and water, respectively.
FIG. 5 shows single-tube feeding of thrips, small kidney beans in the tube.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples. The xylenylaniline used in this example is a known compound and is commercially available.
Example 1
(1) Dissolving xylene aniline biological stain powder in 0.01mol/L phosphate buffer solution to obtain 100mg/ml biological stain solution;
(2) Mixing 100mg/ml xyleneaniline biological stain and 0.01mol/L phosphate buffer solution with the pH value of 7.0 according to the volume ratio of 1:9, mixing to obtain a biological staining solution with the concentration of 10mg/ml;
(3) Membrane feeding is carried out on the thrips nymphs by the biological staining solution in the step (1), and the membrane feeding is carried out for 24h under the conditions of 25 ℃, humidity 75% and illumination 14L at 10D, so as to obtain the stained thrips nymphs;
(4) And (3) photographing and imaging the staining condition of the marked thrips nymphs in the step (2) by using a microscope.
Example 2
(1) Mixing 100mg/ml xylene aniline biological stain and 0.01mol/L phosphate buffer solution with the pH value of 6.5 according to the volume ratio of 1:9, mixing to obtain a biological staining solution with the concentration of 10mg/ml;
(2) Membrane feeding is carried out on the thrips nymphs by the biological staining solution in the step (1), and the membrane feeding is carried out for 24h under the conditions of 28 ℃, humidity 75% and illumination 14L 10D, so as to obtain the stained thrips nymphs;
(3) And (3) photographing and imaging the staining condition of the marked thrips nymphs in the step (2) by using a microscope.
Example 3
(1) Mixing 100mg/ml xyleneaniline biological stain and 0.01mol/L phosphate buffer solution with the pH value of 7.8 according to the volume ratio of 1:9, mixing to obtain a biological staining solution with the concentration of 10mg/ml;
(2) Membrane feeding is carried out on the thrips nymphs by the biological staining solution in the step (1), and the membrane feeding is carried out for 24h under the conditions of 28 ℃, humidity 75% and illumination 14L at 10D, so as to obtain the stained thrips nymphs;
(3) And (3) photographing and imaging the staining condition of the marked thrips nymphs in the step (2) by using a microscope.
Example 4
(1) Mixing 10mg/ml xyleneaniline biological stain and 0.01mol/L phosphate buffer solution with the pH value of 7.2 according to the volume ratio of 1:9, mixing to obtain a biological staining solution with the concentration of 1 mg/ml;
(2) Membrane feeding is carried out on the thrips nymphs by the biological staining solution in the step (1), and the membrane feeding is carried out for 24h under the conditions of 26 ℃, humidity 75% and illumination 14L;
(3) And (3) photographing and imaging the staining condition of the marked thrips nymphs in the step (2) by using a microscope.
Example 5
(1) Mixing 100mg/ml xyleneaniline biological stain and 0.01mol/L phosphate buffer solution with the pH value of 7.0 according to the volume ratio of 1:5, mixing to obtain a biological staining solution with the concentration of 16 mg/ml;
(2) Membrane feeding is carried out on the thrips nymphs through the biological staining solution in the step (1), and the membrane feeding is carried out for 24 hours under the conditions that the temperature is 25 ℃, the humidity is 75%, and the illumination is 14L;
(3) And (3) photographing and imaging the staining condition of the marked thrips nymphs in the step (2) by using a microscope.
Comparative example
(1) Directly taking 0.01mol/L phosphate buffer solution with the pH value of 7.0 as a feeding raw material, and not adding a biological stain xylene aniline to obtain a biological staining solution with the concentration of 0mg/ml;
(2) Membrane feeding the thrips nymphs with the biological staining solution obtained in the step (1), and feeding for 24 hours at the temperature of 25 ℃ and the humidity of 75% to obtain stained thrips nymphs;
(3) And (3) photographing and imaging the staining condition of the marked thrips nymphs in the step (2) by using a microscope.
As can be seen from FIG. 2, compared with the control thrips, the biological stain xylene aniline has better staining effect on thrips under different concentrations (1 mg/mL-10 mg/mL) of the invention, and is easily observed morphologically. The biological stain xylene aniline has larger difference of thrips morphology and slow action and part of thrips death under the concentration and parameters (10 mg/mL, pH value of phosphate buffer is 6.5 and 28 ℃) and (10 mg/mL, pH value of phosphate buffer is 7.8 and 28 ℃), while xylene aniline has slow action and most of thrips death under the conditions of 16mg/mL and pH value of phosphate buffer is 7.0. When the concentration of the staining agent is lower than 1mg/ml, the staining effect is poor, and the experimental observation and monitoring are not facilitated.
In conclusion, the survival rate of the biological staining reagent xylene aniline and the control group in the thrips membrane feeding is similar, which shows that the biological staining reagent xylene aniline has no significant influence on the thrips membrane feeding and can play a role in assisting the thrips membrane feeding effect.
The above description is only a preferred embodiment of the present invention, and all the equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.
Claims (1)
1. The application of the biological stain xylene aniline in the film feeding of thrips insects is characterized by comprising the following steps:
(1) Dissolving dimethylbenzene aniline biological staining agent powder in 0.01mol/L phosphoric acid buffer solution to obtain dimethylbenzene aniline biological staining solutions with different concentrations;
(2) Membrane feeding is carried out on the thrips nymphs by the xylene aniline biological staining solution in the step (1), and the membrane feeding is carried out for 24h under the conditions that the temperature is 25 +/-2 ℃, the humidity is 75%, and the illumination is 14L;
(3) Photographing the dyeing condition of the marked thrips nymphs in the step (2) by using a microscope for imaging;
the pH value of the 0.01mol/L phosphate buffer solution is 7.0 to 7.4;
the insect age range of the thrips nymphs is 1-2 years old;
the concentration of the xylene aniline biological staining solution is 1-10mg/ml;
the film feeding method comprises the following steps: starving thrips nymphs for 2h, picking the nymphs to a feeding pipe with an opening on two sides by using a writing brush, sealing two ends of the breeding pipe by using a Parafilm film, dripping a prepared dimethylbenzene aniline biological dyeing solution on one end of the breeding pipe, and covering the breeding pipe with the Parafilm film for moisturizing.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102524188A (en) * | 2012-01-20 | 2012-07-04 | 福建省农业科学院植物保护研究所 | Insect breeding bottom and breeding method of gynaikothrips ficorum |
| CN102640729A (en) * | 2012-04-06 | 2012-08-22 | 山东省农业科学院植物保护研究所 | Method for breeding frankliniella occidentalis |
| CN104012472A (en) * | 2014-03-12 | 2014-09-03 | 云南农业大学 | Indoor breeding method of thrips |
| CN106975084A (en) * | 2017-04-07 | 2017-07-25 | 中国农业科学院植物保护研究所 | A kind of colouring method of tetranychid digestive system |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102524188A (en) * | 2012-01-20 | 2012-07-04 | 福建省农业科学院植物保护研究所 | Insect breeding bottom and breeding method of gynaikothrips ficorum |
| CN102640729A (en) * | 2012-04-06 | 2012-08-22 | 山东省农业科学院植物保护研究所 | Method for breeding frankliniella occidentalis |
| CN104012472A (en) * | 2014-03-12 | 2014-09-03 | 云南农业大学 | Indoor breeding method of thrips |
| CN106975084A (en) * | 2017-04-07 | 2017-07-25 | 中国农业科学院植物保护研究所 | A kind of colouring method of tetranychid digestive system |
Non-Patent Citations (1)
| Title |
|---|
| 高温对西花蓟马卵巢发育及卵黄蛋白含量的影响;马亚斌 等;《昆虫学报》;20160220;第59卷(第2期);第128-129页的1.2 * |
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