CN107478669A - The method that Predatory Mites spermatophore is observed under transmission electron microscope - Google Patents
The method that Predatory Mites spermatophore is observed under transmission electron microscope Download PDFInfo
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- CN107478669A CN107478669A CN201710808939.4A CN201710808939A CN107478669A CN 107478669 A CN107478669 A CN 107478669A CN 201710808939 A CN201710808939 A CN 201710808939A CN 107478669 A CN107478669 A CN 107478669A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
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Abstract
The present invention relates to the method that Predatory Mites spermatophore is observed under transmission electron microscope, it is characterized in that after spermatophore is obtained on the Phytoseiidae Predatory Mites hero mite gnathosoma to mate under transmission electron microscope from spermatophore method, include the acquisition of male and female mite in mating, the sizing of male and female mite, the separation of male and female mite, the acquisition of spermatophore, spermatophore transmission electron microscope sample are prepared and 5 parts of observation.The present invention is obtained with Phytoseiidae Predatory Mites hero mite spermatophore using very simple cleverly method, and using transmission electron microscope ultra micro observation can be carried out to the internal structure and material of the outer spermatophore of Predatory Mites, by observation, we can further to define whether the form of sperm, quantity and sperm in outer spermatophore change before reaching in female mite body（Such as：Generation heterogeneousization）Deng, this be advantageous to determine Phytoseiidae Predatory Mites hero mite whether served during Sex Determination it is conclusive.
Description
Technical field
The present invention relates to the method that Predatory Mites spermatophore is observed under transmission electron microscope, it is biologically-based to belong to Phytoseiidae Predatory Mites
Plinth research field.
Background technology
Phytoseiidae Predatory Mites are gonochorism animals, must can just be laid eggs by male and female mite mating after fertilization and breed offspring,
Offspring no matter from development of fertilized ova come by male and female.
Available data mates for Phytoseiidae Predatory Mites male and female mite to be formed the understanding of embryonated egg and is：Male mite sperm is entering
It is stored in before in female mite body in spermatophore, spermatophore is filled in female mite copulatory opening by female mite（Bibliography：Liang Lairong, 1979, plant protected
How shield, mite class live）, sperm comes out from spermatophore slow release again, is transferred to spermatheca and ovum combines, form fertilization
Ovum.But how to enter in female mite body for Predatory Mites hero mite sperm and male mite sperm is not reported with the presence or absence of dimorphism, if
Research and solve this series of problems, it is necessary first to obtain spermatophore, observational study is carried out to spermatophore.
Due to very small only hundreds of microns of Predatory Mites individual, its spermatophore just occurs when there was only the mating of male and female mite, handed over
Male and female mite being superimposed together closely during matching somebody with somebody, mating time is also very short only 1 hour or so, and spermatophore size is only
There is more than ten micron and be hidden between the outside of belly of male and female mite body close contact, be intuitively hardly visible, available data does not have yet
There is the method for how obtaining spermatophore.Therefore want obtain spermatophore it is very difficult, to spermatophore carry out transmission electron microscope observing it is more difficult, it is necessary to
Open up a kind of method for obtaining Predatory Mites spermatophore and transmission electron microscope observing being carried out to spermatophore.
The content of the invention
It is an object of the invention to provide the method that Phytoseiidae Predatory Mites spermatophore is observed under transmission electron microscope, using this method
It will be seen that in Predatory Mites spermatophore sperm growth course and sperm morphology, the ultra microstructure in spermatophore can also be grasped, to be bright
Effect of the true Predatory Mites hero mite in Sex Determination provides reference.
The present invention is obtained from the Phytoseiidae Predatory Mites hero mite gnathosoma to mate after spermatophore under transmission electron microscope
The method for observing spermatophore, includes the acquisition of male and female mite, the sizing of male and female mite, the separation of male and female mite, the acquisition of spermatophore, essence in mating
Prepared by bag transmission electron microscope sample is with 5 parts of observation, concrete operations：
（1）The acquisition of male and female mite in mating：Under the microscope, out of Predatory Mites raising population, the ovum for choosing Predatory Mites is individually raised
Support into male and female mite pairing is carried out after mite, 10 minutes or so male and female mites of pairing can mate, so as to male and female mite in being mated；
（2）The sizing of male and female mite in mating：Within Predatory Mites post-coitum 0-120 minutes, the male and female that will be in mating process
Individual is positioned over 3-5 minutes in -80 DEG C of ultra low temperature freezers or liquid nitrogen together with breeding apparatus, carries out male and female mite sizing；
（3）The separation of male and female mite after sizing：Predatory Mites male and female mite is taken out together with breeding apparatus after sizing, then in microscope
Under, the male and female mite of mating is chosen on slide from breeding apparatus with fine, soft fur pen, recycles the work such as No. 0 insect needle and fine, soft fur pen
Tool gently separates the male and female mite of mating, prevents from destroying spermatophore；
（4）The acquisition of spermatophore：After male and female mite successfully separates, under the microscope, Hubei Province body of male mite is scaled off using scalpel, protected
Gnathosoma is stayed, the spermatophore in spermatodactyl top is readily observed on gnathosoma, then with fine, soft fur nib by under the light picking of spermatophore
It is put into 70%-100% alcohol and preserves or directly utilize；
（5）Spermatophore transmission electron microscope sample prepares and observation：
1）It is fixed：The outer spermatophore removed is put into the fixer that concentration is 3.5% glutaraldehyde, in order that fixed effect is more preferable, can
Tween 80 is added into glutaraldehyde（1L:600mL）And sodium chloride（1L:0.9g）, 48 hours are fixed, is then with concentration
0.1mol/l, the phosphate buffer that pH value is 7.2 rinse（10 minutes/time）, rinse 3 hours, then 2 hours are fixed with osmic acid, most
Rinsed 3-4 times with phosphate buffer afterwards；
2）Dehydration：Serial dehydration is first carried out, is individually placed to be dehydrated in 30%, 50%, 70%, 80%, 90%, 100% alcohol, often
Individual serial dehydration 5 minutes, then be dehydrated 15 minutes with acetone, 4-5 times, stay a little acetone soln；
3）Embedding：Resin solution is added, sample and resin are sufficiently mixed, soaks 12 hours, then fitly puts outer spermatophore
Enter and posture is ajusted in resin die, drive bubble in resin away；
4）Solidification：Mould is put into 35 degree of oven for baking 2-3 weeks；
5）Section：Utilize ultramicrotome section 50-60nm；
6）Dyeing：Dyed 1 hour or so with acetic acid uranium and lead citrate；
7）Observation：Observe and take pictures under transmission electron microscope.
Ultra micro observation can be carried out to the internal structure and material of the outer spermatophore of Predatory Mites, pass through observation using transmission electron microscope
We can further to define whether the form of sperm, quantity and sperm in outer spermatophore become before reaching in female mite body
Change（Such as：Generation heterogeneousization）Deng, this be advantageous to determine Predatory Mites hero mite conclusive work whether has been played during Sex Determination
With.
Embodiment
Embodiment is by taking Amblyseius persidolongispinosus as an example, the method for observation Predatory Mites spermatophore under transmission electron microscope
This method be after spermatophore is obtained on the Amblyseius persidolongispinosus hero mite gnathosoma to mate under transmission electron microscope from essence
The method of bag, include the acquisition of male and female mite, the sizing of male and female mite, the separation of male and female mite, the acquisition of spermatophore, spermatophore transmission in mating
Prepared by electron microscopic sample is with 5 parts of observation, concrete operations：
（1）The acquisition of male and female mite in mating：Under the microscope, from Amblyseius persidolongispinosus raising population in, choose plan become mildewed it is blunt pacify
Individually raising is into progress male and female mite pairing after mite for the ovum of mite, and 10 minutes or so male and female mites of pairing can mate, so as to be handed over
With middle male and female mite；
（2）The sizing of male and female mite in mating：Within Amblyseius persidolongispinosus post-coitum 0-120 minutes, it will be in mating process
Male and female individual be positioned over 3-5 minutes in -80 DEG C of ultra low temperature freezers or liquid nitrogen together with breeding apparatus, carry out male and female mite and determine
Type；
（3）The separation of male and female mite after sizing：Amblyseius persidolongispinosus male and female mite is taken out together with breeding apparatus after sizing, then
Under microscope, the male and female mite of mating is chosen on slide from breeding apparatus with fine, soft fur pen, recycles No. 0 insect needle and fine, soft fur
The instruments such as pen gently separate the male and female mite of mating, prevent from destroying spermatophore；
（4）The acquisition of spermatophore：After male and female mite successfully separates, under the microscope, Hubei Province body of male mite is scaled off using scalpel, protected
Gnathosoma is stayed, the spermatophore in spermatodactyl top is readily observed on gnathosoma, then with fine, soft fur nib by under the light picking of spermatophore
It is put into 70%-100% alcohol and preserves or directly utilize；
（5）Spermatophore transmission electron microscope sample prepares and observation：
1）It is fixed：The outer spermatophore removed is put into the fixer that concentration is 3.5% glutaraldehyde, in order that fixed effect is more preferable, can
Tween 80 is added into glutaraldehyde（1L:600mL）And sodium chloride（1L:0.9g）, 48 hours are fixed, is then with concentration
0.1mol/l, the phosphate buffer that pH value is 7.2 rinse（10 minutes/time）, rinse 3 hours, then 2 hours are fixed with osmic acid, most
Rinsed 3-4 times with phosphate buffer afterwards；2）Dehydration：First carry out serial dehydration, be individually placed to 30%, 50%, 70%, 80%, 90%,
It is dehydrated in 100% alcohol, each serial dehydration 5 minutes, then is dehydrated 15 minutes with acetone, 4-5 times, stays a little acetone molten
Liquid；3）Embedding：Resin solution is added, sample and resin are sufficiently mixed, soaks 12 hours, is then fitly put into outer spermatophore
Posture is ajusted in resin die, drives bubble in resin away；4）Solidification：Mould is put into 35 degree of oven for baking 2-3 weeks；5）Cut
Piece：Utilize ultramicrotome section 50-60nm；6）Dyeing：Dyed 1 hour or so with acetic acid uranium and lead citrate；7）Observation：
Observe and take pictures under transmission electron microscope.
Claims (1)
1. the method for Predatory Mites spermatophore is observed under transmission electron microscope, it is characterized in that male from the Phytoseiidae Predatory Mites to mate
Obtain the method for observing spermatophore after spermatophore under transmission electron microscope on mite gnathosoma, include the acquisition of male and female mite in mating, male and female mite
Prepared by sizing, the separation of male and female mite, the acquisition of spermatophore, spermatophore transmission electron microscope sample is with 5 parts of observation, concrete operations：
（1）The acquisition of male and female mite in mating：Under the microscope, out of Predatory Mites raising population, the ovum for choosing Predatory Mites is individually raised
Support into male and female mite pairing is carried out after mite, 10 minutes or so male and female mites of pairing can mate, so as to male and female mite in being mated；
（2）The sizing of male and female mite in mating：Within Predatory Mites post-coitum 0-120 minutes, the male and female that will be in mating process
Individual is positioned over 3-5 minutes in -80 DEG C of ultra low temperature freezers or liquid nitrogen together with breeding apparatus, carries out male and female mite sizing；
（3）The separation of male and female mite after sizing：Predatory Mites male and female mite is taken out together with breeding apparatus after sizing, then in microscope
Under, the male and female mite of mating is chosen on slide from breeding apparatus with fine, soft fur pen, recycles the work such as No. 0 insect needle and fine, soft fur pen
Tool gently separates the male and female mite of mating, prevents from destroying spermatophore；
（4）The acquisition of spermatophore：After male and female mite successfully separates, under the microscope, Hubei Province body of male mite is scaled off using scalpel, protected
Gnathosoma is stayed, the spermatophore in spermatodactyl top is readily observed on gnathosoma, then with fine, soft fur nib by under the light picking of spermatophore
It is put into 70%-100% alcohol and preserves or directly utilize；
（5）Spermatophore transmission electron microscope sample prepares and observation：
1）It is fixed：The outer spermatophore removed is put into the fixer that concentration is 3.5% glutaraldehyde, in order that fixed effect is more preferable, can
Tween 80 is added into glutaraldehyde（1L:600mL）And sodium chloride（1L:0.9g）, 48 hours are fixed, is then with concentration
0.1mol/l, the phosphate buffer that pH value is 7.2 rinse（10 minutes/time）, rinse 3 hours, then 2 hours are fixed with osmic acid, most
Rinsed 3-4 times with phosphate buffer afterwards；
2）Dehydration：Serial dehydration is first carried out, is individually placed to be dehydrated in 30%, 50%, 70%, 80%, 90%, 100% alcohol, often
Individual serial dehydration 5 minutes, then be dehydrated 15 minutes with acetone, 4-5 times, stay a little acetone soln；
3）Embedding：Resin solution is added, sample and resin are sufficiently mixed, soaks 12 hours, then fitly puts outer spermatophore
Enter and posture is ajusted in resin die, drive bubble in resin away；
4）Solidification：Mould is put into 35 degree of oven for baking 2-3 weeks；
5）Section：Utilize ultramicrotome section 50-60nm；
6）Dyeing：Dyed 1 hour or so with acetic acid uranium and lead citrate；
7）Observation：Observe and take pictures under transmission electron microscope.
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| CN201710808939.4A CN107478669A (en) | 2017-09-09 | 2017-09-09 | The method that Predatory Mites spermatophore is observed under transmission electron microscope |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6058878A (en) * | 1998-06-17 | 2000-05-09 | California Polytechnic State University Foundation | Protozoan free colonies of lepidoptera |
| CN102607907A (en) * | 2012-02-24 | 2012-07-25 | 东北农业大学 | Paraffin section method for fern gametophytes |
| CN103868768A (en) * | 2014-02-14 | 2014-06-18 | 河南省农业科学院植物保护研究所 | Treatment method of scanning electron microscope samples of insect tentacles and appendages |
| CN105486554A (en) * | 2015-11-19 | 2016-04-13 | 新疆农业大学 | Formula and method for immobilizing tick sample for scanning electron microscopy |
-
2017
- 2017-09-09 CN CN201710808939.4A patent/CN107478669A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6058878A (en) * | 1998-06-17 | 2000-05-09 | California Polytechnic State University Foundation | Protozoan free colonies of lepidoptera |
| CN102607907A (en) * | 2012-02-24 | 2012-07-25 | 东北农业大学 | Paraffin section method for fern gametophytes |
| CN103868768A (en) * | 2014-02-14 | 2014-06-18 | 河南省农业科学院植物保护研究所 | Treatment method of scanning electron microscope samples of insect tentacles and appendages |
| CN105486554A (en) * | 2015-11-19 | 2016-04-13 | 新疆农业大学 | Formula and method for immobilizing tick sample for scanning electron microscopy |
Non-Patent Citations (1)
| Title |
|---|
| 王月明等: "《粉尘螨生殖系统超微结构的透射电镜观察》", 《昆虫学报》 * |
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Application publication date: 20171215 |