CN107515315A - Phyloseiulus nersimilis hero mite spermatophore transmission electron microscope observing method - Google Patents
Phyloseiulus nersimilis hero mite spermatophore transmission electron microscope observing method Download PDFInfo
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- CN107515315A CN107515315A CN201710808933.7A CN201710808933A CN107515315A CN 107515315 A CN107515315 A CN 107515315A CN 201710808933 A CN201710808933 A CN 201710808933A CN 107515315 A CN107515315 A CN 107515315A
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- spermatophore
- male
- mite
- female
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
- G01Q30/00—Auxiliary means serving to assist or improve the scanning probe techniques or apparatus, e.g. display or data processing devices
- G01Q30/20—Sample handling devices or methods
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
- G01Q30/00—Auxiliary means serving to assist or improve the scanning probe techniques or apparatus, e.g. display or data processing devices
- G01Q30/02—Non-SPM analysing devices, e.g. SEM [Scanning Electron Microscope], spectrometer or optical microscope
Abstract
The present invention relates to Phyloseiulus nersimilis hero mite spermatophore transmission electron microscope observing method, it is characterized in that after spermatophore is obtained on the Phyloseiulus nersimilis 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 Phyloseiulus nersimilis 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 Phyloseiulus nersimilis, 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 Phyloseiulus nersimilis hero mite whether served during Sex Determination it is conclusive.
Description
Technical field
The present invention relates to Phyloseiulus nersimilis hero mite spermatophore transmission electron microscope observing method, belong to Phyloseiulus nersimilis biology
Basic research field.
Background technology
Phyloseiulus nersimilis is gonochorism animal, must can just be laid eggs by male and female mite mating after fertilization and breed offspring, after
In generation, no matter male and female were come from development of fertilized ova.
Available data mates for Phyloseiulus nersimilis male and female mite to be formed the understanding of embryonated egg and is：Male mite sperm is female in entrance
It is stored in before in 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 Phyloseiulus nersimilis hero mite sperm and male mite sperm does not have with the presence or absence of dimorphism
Report, to 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 Phyloseiulus nersimilis individual, its spermatophore just goes out when there was only the mating of male and female mite
Existing, male and female mite being superimposed together closely in mating process, mating time is also very short only 1 hour or so, and spermatophore
Size only has more than ten micron and is hidden between the outside of belly of male and female mite body close contact, is intuitively hardly visible, existing money
The method for expecting also how not obtain spermatophore.Therefore think that acquisition spermatophore is very difficult, it is more tired to carry out transmission electron microscope observing to spermatophore
Hardly possible is, it is necessary to open up a kind of method for obtaining Phyloseiulus nersimilis 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 a kind of method using transmission electron microscope observing Phyloseiulus nersimilis hero mite spermatophore, application
This method will be seen that the growth course and sperm morphology of sperm in Phyloseiulus nersimilis spermatophore, can also grasp super in spermatophore
Micro-structural, reference is provided for effect of the clear and definite Phyloseiulus nersimilis hero mite in Sex Determination.
The present invention is to be seen after spermatophore is obtained from the Phyloseiulus nersimilis hero mite gnathosoma to mate under transmission electron microscope
The method for examining spermatophore, 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 in mating
Prepared by 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 Phyloseiulus nersimilis raising population, choose small plant of Chile and 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 Phyloseiulus nersimilis 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：Phyloseiulus nersimilis 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：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 Phyloseiulus nersimilis, lead to using transmission electron microscope
Cross observe we can further to define in outer spermatophore the form of sperm, quantity and sperm before reaching in female mite body whether
Change（Such as：Generation heterogeneousization）Deng, this be advantageous to determine Phyloseiulus nersimilis hero mite whether risen during Sex Determination
Conclusive effect.
Embodiment
Embodiment Phyloseiulus nersimilis hero mite spermatophore transmission electron microscope observing method
This method be after spermatophore is obtained on the Phyloseiulus nersimilis 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, out of Phyloseiulus nersimilis raising population, choose small plant of Chile and 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 Phyloseiulus nersimilis 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：Phyloseiulus nersimilis 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. Phyloseiulus nersimilis hero mite spermatophore transmission electron microscope observing method, it is characterized in that from the Phyloseiulus nersimilis to mate
The method for observing spermatophore on male mite gnathosoma after acquisition spermatophore under transmission electron microscope, includes the acquisition of male and female mite, male and female mite in mating
Sizing, the separation of male and female mite, the acquisition of spermatophore, spermatophore transmission electron microscope sample prepare with 5 parts of observation, concrete operations are：
（1）The acquisition of male and female mite in mating：Under the microscope, out of Phyloseiulus nersimilis raising population, choose small plant of Chile and 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 Phyloseiulus nersimilis 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：Phyloseiulus nersimilis 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：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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| CN201710808933.7A CN107515315A (en) | 2017-09-09 | 2017-09-09 | Phyloseiulus nersimilis hero mite spermatophore transmission electron microscope observing method |
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| CN201710808933.7A CN107515315A (en) | 2017-09-09 | 2017-09-09 | Phyloseiulus nersimilis hero mite spermatophore transmission electron microscope observing method |
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Citations (5)
| 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 |
| CN104406834A (en) * | 2014-11-04 | 2015-03-11 | 中国水产科学研究院东海水产研究所 | Marine fish sperm staining method |
| 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 CN201710808933.7A patent/CN107515315A/en active Pending
Patent Citations (5)
| 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 |
| CN104406834A (en) * | 2014-11-04 | 2015-03-11 | 中国水产科学研究院东海水产研究所 | Marine fish sperm staining method |
| 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 (2)
| Title |
|---|
| 吴桂华等: "《粉尘螨生殖***形态学研究》", 《昆虫学报》 * |
| 王月明等: "《粉尘螨生殖***超微结构的透射电镜观察》", 《昆虫学报》 * |
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Application publication date: 20171226 |
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