CN104406829A - Sample making method of armyworm larva peritrophic membrane - Google Patents
Sample making method of armyworm larva peritrophic membrane Download PDFInfo
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- CN104406829A CN104406829A CN201410618025.8A CN201410618025A CN104406829A CN 104406829 A CN104406829 A CN 104406829A CN 201410618025 A CN201410618025 A CN 201410618025A CN 104406829 A CN104406829 A CN 104406829A
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Abstract
The invention relates to a sample making method of an armyworm larva peritrophic membrane. The method is characterized in that the method comprises the following steps: 1, placing armyworm larvae in a culture dish provided with paraffin, and respectively fixing each of the armyworm larvae from the head end and the belly end by using insect needles to make the belly face up; 2, slitting along the center line of the belly of each of the armyworm larvae, taking out a peritrophic membrane, and cleaning to remove contents in the peritrophic membrane; 3, placing the peritrophic membrane on the surface of a wood disk, and fixing the peritrophic membrane on the wood disk by needle tips along the periphery of the peritrophic membrane; and 4, putting a fixed peritrophic membrane sample in a small penicillin bottle provided with a glutaraldehyde fixative liquid, immersing, taking out the peritrophic membrane sample, flushing, dehydrating, replacing, drying, and carrying out metal membrane spraying in a vacuum spraying instrument to obtain the armyworm larva peritrophic membrane. The method well solves the problem that the observation effect is severely influenced by difficult peritrophic membrane observation surface unfolding in the production of insect peritrophic membrane electron microscope samples, allows the high-quality armyworm larva peritrophic membrane to be obtained, and is simple, economic and practical.
Description
Technical field:
The present invention relates to the peritrophic method for making sample of a kind of armyworm larvae, belong to the biological control applied technical field of insect.
Background technology:
Funnel (Peritrophic membrane) is the uniform long tubular film of one deck thickness of insect midgut emiocytosis, is extended in hindgut by middle intestines front end always.One of peritrophic major function is as the effective barrier between midgut epithelial cell and food, and protection midgut epithelial cell is from the mechanical damage of roughage particle absorbed and the invasion of the pathogenic microorganism such as virus, bacterium.If funnel is destroyed will lose its barrier function, easily cause the infection of causal organism.
Mythimna separata Mythimna separata (Walker) is a kind of omnivorousness Important Agricultural insect, and its host comprises the multiple gramineous crops such as wheat class, corn, millet, Chinese sorghum, paddy rice, causes harm serious, loses huge.Along with the raising of living standards of urban and rural residents level, change from heat type to auxotype the demand of food, the demand of people to pollution-free food also grows with each passing day simultaneously.At present, the main method of control mythimna separata is still chemical prevention, but the idea of some chemical pesticide in chemical prevention and green agriculture is disagreed, simultaneously chemical pesticide use caused " 3R " problem in a large number, and of common occurrence to phenomenons such as the poisoning that cereal crops produce, therefore the research and development of the non-environmental pollution control method of mythimna separata are imperative.
The status of biological control in insect innoxious governance process of insect is very important, biological control method mainly includes natural enemy insect and beneficial microorganism utilizes, and the application of beneficial microorganism exploitation is more general, wherein market is widely used Dipel Bt, nuclear polyhedrosis virus (PseudaletiaNuclear Polyhedrosis Virus, PsNVP) etc.But the interior first barrier faced of alimentary canal that these microbial insecticides enter insect is exactly funnel, due to peritrophic effect, cause similar microbial insecticide to there is virulence lower, insecticidal effect shows shortcoming slowly, land for growing field crops is applied and is restricted.Therefore find effective synergy approach and just seem very important.
Destroying peritrophic integrality makes it produce space and hole is the important indicator that effective synergistic agent found by a lot of microbial insecticide.Because insect funnel film inside surface morphosis naked eyes are difficult to observe, study different synergistic agent and just must observe under Electronic Speculum the impact of insect funnel morphosis, and then determine its potentiality as synergistic agent.But, the funnel of insect is very thin, be difficult to be in extended position under Electronic Speculum, slightly careless its is organized and is just destroyed, therefore, obtain a high-quality funnel photo not a duck soup, if do not have the method for making sample of the effective scanning electron microscope of complete set to be difficult to take desirable funnel electromicroscopic photograph, cause the screening experiment of follow-up synergistic agent to implement.Given this, study the peritrophic SEM sample preparation method of a set of simple and effective armyworm larvae to be of great significance with regard to tool.
Summary of the invention:
In order to overcome the deficiencies in the prior art, the object of the present invention is to provide the peritrophic method for making sample of a kind of armyworm larvae, be difficult to obtain funnel when can solve making insect funnel electron microscopic sample preferably unfold sightingpiston and have a strong impact on the problem of observing effect, high-quality armyworm larvae funnel sample can be obtained, have the advantages that method is simple, economical and practical.
The present invention realizes above-mentioned purpose by following technical solution.
The peritrophic method for making sample of a kind of armyworm larvae provided by the present invention, comprises the steps:
1, armyworm larvae is placed in the double dish filling paraffin wax, fixes armyworm larvae polypide from head and abdomen end respectively with No. 3 insect needles, make its outside of belly upward; Double dish is 6.5 × 1.5cm, and paraffin wax accounts for 4/5 of its volume;
2, cut open with the ventrimeson of medical knife along armyworm larvae, take out funnel and be placed in clear water, the content in cleaning removing funnel;
3, the funnel after cleaning is placed in wooden disk surfaces, intercepts the tip portion of No. 0 insect needle, funnel is fixed on wooden disk along funnel periphery with needle point; Wooden disk diameter is 0.8cm, and thickness is 1mm, through soaked in absolute ethyl alcohol 15-20 days; The length intercepting the tip portion of No. 0 insect needle is 3mm;
4, the funnel sample fixed is put into the penicillin bottle filling glutaraldehyde immobile liquid and soak 12-16 hour; 3 times are rinsed with phosphate buffer, each 10 minutes after taking-up; Then each once with 30%, 50%, 75%, 80%, 90%, 95% acetone dehydration, each 10 minutes; The dehydration of alcohol of 100% 2 times, each 20 minutes; Isoamyl acetate displacement twice, each 20 minutes; CO
2pull out needle point gently after critical point drying, the metal film of spraying plating 10-20nm in vacuum evaporating instrument, namely obtain armyworm larvae funnel sample, funnel sample is placed in observed under electron microscope funnel surface morphological structure and takes a picture.
The present invention compared with prior art, be difficult to obtain funnel when can solve making insect funnel electron microscopic sample preferably unfold sightingpiston and have a strong impact on the problem of observing effect, high-quality armyworm larvae funnel sample can be obtained, have the advantages that method is simple, economical and practical.
Accompanying drawing illustrates:
Fig. 1 is the peritrophic stereoscan photograph figure of normal armyworm larvae.
Fig. 2 is the peritrophic stereoscan photograph figure of the armyworm larvae after artificial destruction process.
Fig. 3 is the peritrophic stereoscan photograph figure of the armyworm larvae after artificial destruction process.
Embodiment:
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment 1
Implement the present invention, can carry out according to the following steps:
1, first, armyworm larvae is placed in the double dish (6.5 × 1.5cm) filling paraffin wax, paraffin wax accounts for 4/5 of double dish volume; Fix armyworm larvae polypide from head and abdomen end respectively with No. 3 insect needles, make its outside of belly upward;
2, cut open along the ventrimeson of armyworm larvae with medical knife (length is 12.5cm), take out funnel and be placed in clear water, the content in cleaning removing funnel, for subsequent use;
3, the funnel after cleaning is placed in gently wooden disk surfaces (the thickness 1mm soaking more than 15 days through absolute ethyl alcohol, diameter 0.8cm), then intercept the tip portion of No. 0 long insect needle of 3mm, funnel is fixed on gently on wooden disk along funnel periphery with needle point; Wooden disk at least will change 5 absolute ethyl alcohols in immersion process; Wooden disk timber used is taken from the component of the old wood furniture of use more than 3 years, density should be selected large and the timber that tangent plane is smooth;
4, the funnel sample fixed is put into the penicillin bottle soaked overnight filling glutaraldehyde immobile liquid; Next day rinses 3 times with phosphate buffer, each 10 minutes after taking out; Then each once with 30%, 50%, 75%, 80%, 90%, 95% acetone dehydration, each 10 minutes; The dehydration of alcohol of 100% 2 times, each 20 minutes; Isoamyl acetate displacement twice, each 20 minutes; CO
2pull out needle point gently after critical point drying, the metal film of spraying plating 10-20nm in vacuum evaporating instrument, namely obtain armyworm larvae funnel sample, finally, funnel sample is placed in observed under electron microscope funnel surface morphological structure and takes a picture.
Embodiment 2
Electromicroscopic photograph effect identification
Can know from Fig. 1 and see, the funnel smooth surface of normal armyworm larvae, compact structure, does not have hole and gap.
As can be seen from Fig. 2 and Fig. 3, the funnel after artificial destruction process, its surface becomes coarse, and structure is not tight, has occurred some chases and finedraw.
Show thus, by method for making sample of the present invention, the peritrophic stereoscan photograph clapped all unfold clear, and the change of the funnel film inside surface morphosis after process can be observed, therefore, the method can be used in the screening process of the observational study of mythimna separata funnel surface morphological structure and the synergistic agent of microbial insecticide.
Claims (4)
1. the peritrophic method for making sample of armyworm larvae, is characterized in that comprising the steps:
(1), armyworm larvae is placed in the double dish filling paraffin wax, fixes armyworm larvae polypide from head and abdomen end respectively with No. 3 insect needles, make its outside of belly upward;
(2), with the ventrimeson of medical knife along armyworm larvae cut open, take out funnel and be placed in clear water, the content in cleaning removing funnel;
(3), by the funnel after cleaning be placed in wooden disk surfaces, intercept the tip portion of No. 0 insect needle, funnel is fixed on wooden disk along funnel periphery with needle point;
(4), the funnel sample fixed is put into the penicillin bottle filling glutaraldehyde immobile liquid and soak 12-16 hour; 3 times are rinsed with phosphate buffer, each 10 minutes after taking-up; Then each once with 30%, 50%, 75%, 80%, 90%, 95% acetone dehydration, each 10 minutes; The dehydration of alcohol of 100% 2 times, each 20 minutes; Isoamyl acetate displacement twice, each 20 minutes; CO
2pull out needle point gently after critical point drying, the metal film of spraying plating 10-20nm in vacuum evaporating instrument, namely obtain armyworm larvae funnel sample, funnel sample is placed in observed under electron microscope funnel surface morphological structure and takes a picture.
2. the peritrophic method for making sample of a kind of armyworm larvae according to claim 1, it is characterized in that double dish is 6.5 × 1.5cm, paraffin wax accounts for 4/5 of its volume.
3. the peritrophic method for making sample of a kind of armyworm larvae according to claim 2, it is characterized in that wooden disk diameter is 0.8cm, thickness is 1mm, through soaked in absolute ethyl alcohol 15-20 days.
4. the peritrophic method for making sample of a kind of armyworm larvae according to claim 3, is characterized in that the length of the tip portion of intercepting No. 0 insect needle is 3mm.
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| CN201410618025.8A CN104406829A (en) | 2014-11-04 | 2014-11-04 | Sample making method of armyworm larva peritrophic membrane |
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| CN201410618025.8A CN104406829A (en) | 2014-11-04 | 2014-11-04 | Sample making method of armyworm larva peritrophic membrane |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105628466A (en) * | 2015-12-24 | 2016-06-01 | 北京农业信息技术研究中心 | Corn stalk Micro-CT scanning sample and preparing method and application thereof |
| CN109239112A (en) * | 2018-08-07 | 2019-01-18 | 重庆师范大学 | A kind of sample preparation methods for electron-microscope scanning |
| CN109613034A (en) * | 2018-12-12 | 2019-04-12 | 江苏省农业科学院 | The method that planthopper saliva sheath scanning electron microscope example quality can be greatly improved |
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| CN103060261A (en) * | 2012-12-29 | 2013-04-24 | 中国农业科学院植物保护研究所 | Method for large-scale quick separation of insect peritrophic membranes |
| CN103115809A (en) * | 2013-01-23 | 2013-05-22 | 浙江大学 | Transmission electron microscope processing method for insect antenna samples |
| CN103947609A (en) * | 2014-04-18 | 2014-07-30 | 青岛农业大学 | Method for observing wax gland orifices of eriosoma lanigerum |
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| CN103060261A (en) * | 2012-12-29 | 2013-04-24 | 中国农业科学院植物保护研究所 | Method for large-scale quick separation of insect peritrophic membranes |
| CN103115809A (en) * | 2013-01-23 | 2013-05-22 | 浙江大学 | Transmission electron microscope processing method for insect antenna samples |
| CN103947609A (en) * | 2014-04-18 | 2014-07-30 | 青岛农业大学 | Method for observing wax gland orifices of eriosoma lanigerum |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105628466A (en) * | 2015-12-24 | 2016-06-01 | 北京农业信息技术研究中心 | Corn stalk Micro-CT scanning sample and preparing method and application thereof |
| CN109239112A (en) * | 2018-08-07 | 2019-01-18 | 重庆师范大学 | A kind of sample preparation methods for electron-microscope scanning |
| CN109613034A (en) * | 2018-12-12 | 2019-04-12 | 江苏省农业科学院 | The method that planthopper saliva sheath scanning electron microscope example quality can be greatly improved |
| CN109613034B (en) * | 2018-12-12 | 2021-06-01 | 江苏省农业科学院 | Method for preparing rice planthopper salivary sheath scanning electron microscope sample |
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Application publication date: 20150311 |
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