CN107515314A - A kind of method for obtaining and small new neoseiulus californicus spermatophore being observed under ESEM - Google Patents

A kind of method for obtaining and small new neoseiulus californicus spermatophore being observed under ESEM Download PDF

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Publication number
CN107515314A
CN107515314A CN201710808924.8A CN201710808924A CN107515314A CN 107515314 A CN107515314 A CN 107515314A CN 201710808924 A CN201710808924 A CN 201710808924A CN 107515314 A CN107515314 A CN 107515314A
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China
Prior art keywords
male
mite
spermatophore
female
mating
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CN201710808924.8A
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Chinese (zh)
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王伯明
杨静逸
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Hefei Home Of Natural Enemies Biotechnology Co Ltd
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Hefei Home Of Natural Enemies Biotechnology Co Ltd
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Priority to CN201710808924.8A priority Critical patent/CN107515314A/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01QSCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q30/00Auxiliary means serving to assist or improve the scanning probe techniques or apparatus, e.g. display or data processing devices
    • G01Q30/20Sample handling devices or methods
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01QSCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q30/00Auxiliary means serving to assist or improve the scanning probe techniques or apparatus, e.g. display or data processing devices
    • G01Q30/02Non-SPM analysing devices, e.g. SEM [Scanning Electron Microscope], spectrometer or optical microscope

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Radiology & Medical Imaging (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

The present invention relates to a kind of method for obtaining and small new neoseiulus californicus spermatophore being observed under ESEM, it is characterized in that first obtain the small new neoseiulus californicus hero mite it is readily seen that spermatophore, then the method for making and spermatophore being observed under ESEM, prepared and 5 parts of observation comprising the acquisition of male and female mite in mating, the sizing of male and female mite, the separating of male and female mite, the storage of male mite, spermatophore ESEM sample.The present invention is obtained with small new neoseiulus californicus hero mite spermatophore using very simple cleverly method, and the purpose that the trickle external structure of spermatophore is observed under ESEM can be realized, the acquisition of spermatophore can provide raw material to observe spermatophore structure under ESEM, and the research that the result of ESEM is advantageous to the other directions of spermatophore lays the first stone, provides enlightenment.

Description

A kind of method for obtaining and small new neoseiulus californicus spermatophore being observed under ESEM
Technical field
The present invention relates to a kind of method for obtaining and small new neoseiulus californicus spermatophore being observed under ESEM, it is new to belong to California Small peaceful mite Basic of Biology research field.
Background technology
Small new neoseiulus californicus 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 small new neoseiulus californicus 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 small new neoseiulus californicus 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 small new neoseiulus californicus 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 that electron microscopic observation is scanned to spermatophore Hardly possible is, it is necessary to open up a kind of method for obtaining small new neoseiulus californicus spermatophore and electron microscopic observation being scanned to spermatophore.
The content of the invention
, should it is an object of the invention to provide a kind of method for obtaining and small new neoseiulus californicus spermatophore being observed under ESEM With this method can be trickleer observe small new neoseiulus californicus spermatophore external structure.
The present invention is to obtain the small new neoseiulus californicus hero mite it is readily seen that spermatophore, then makes and is observed under ESEM The method of spermatophore, swept comprising the acquisition of male and female mite in mating, the sizing of male and female mite, the separating of male and female mite, the storage of male mite, spermatophore Retouch electron microscope specimen and prepare and be with 5 parts of observation, concrete operations:
(1)The acquisition of male and female mite in mating:Under the microscope, out of small new neoseiulus californicus raising population, it is new small peaceful to choose California 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 small new neoseiulus californicus 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:Male and female mite is taken out together with breeding apparatus after sizing, then under the microscope, used Fine, soft fur pen chooses the male and female mite of mating on slide from breeding apparatus, recycles the instrument such as No. 0 insect needle and fine, soft fur pen will The male and female mite of mating gently separates, and prevents from destroying spermatophore;
(4)The storage of male mite:After male and female mite successfully separates, male mite is chosen with fine, soft fur nib and is put into 70% alcohol in dactylethrae Preserve;
(5)Spermatophore ESEM sample prepares and observation:Comprising dehydration, dry, 5 steps of viscous platform, metal spraying and observation, specifically For:1)Dehydration:Gradient alcohol dehydration is carried out to the male mite being stored in dactylethrae, the concentration of alcohol is respectively:80%, 90%, 100%, first upper strata alcohol is suctioned out with rubber head dropper, then adds the alcohol of next gradient, so circulation is until 100% Alcohol;2)Dry:Sample is placed on sample stage, is dried 2~3 hours using instrument is dried in vacuo;3)Viscous platform:In microscope It is lower to be chosen male mite to viscous platform with fine, soft fur pen, make its outside of belly upward, the outside of belly is pressed lightly on insect needle, to ensure polypide with leading Electric glue can be in close contact with excellent conductive performance;4)Metal spraying:It is gold-plated to mite whole body using ion sputtering instrument;5)Electronic Speculum is seen Examine:Spermatophore is observed under ESEM, spermatophore is located at below the spermatodactyl of male mite.
The present invention is obtained with small new neoseiulus californicus hero mite spermatophore using very simple cleverly method, and can realize The purpose of the trickle external structure of spermatophore is observed under ESEM, the acquisition of spermatophore can provide to observe spermatophore structure under ESEM Raw material, and the research that the result of ESEM is advantageous to the other directions of spermatophore lays the first stone, provides enlightenment.
Embodiment
A kind of method for obtaining and small new neoseiulus californicus spermatophore being observed under ESEM of embodiment
This method is after artificially obtaining spermatophore from the small new neoseiulus californicus hero mite gnathosoma to mate, to be seen under ESEM 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, scanning in mating The part of electron microscopic observation 5, concrete operations are:
(1)The acquisition of male and female mite in mating:Under the microscope, out of small new neoseiulus californicus raising population, it is new small peaceful to choose California 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 small new neoseiulus californicus 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:Male and female mite is taken out together with breeding apparatus after sizing, then under the microscope, with thin Writing brush chooses the male and female mite of mating on slide from breeding apparatus, recycles the instrument such as No. 0 insect needle and fine, soft fur pen to hand over The male and female mite matched somebody with somebody gently separates, and prevents from destroying spermatophore;
(4)The storage of male mite:After male and female mite successfully separates, male mite is chosen with fine, soft fur nib and is put into 70% alcohol in dactylethrae Preserve;
(5)Spermatophore ESEM sample prepares and observation:Comprising dehydration, dry, 5 steps of viscous platform, metal spraying and observation, specifically For:1)Dehydration:Gradient alcohol dehydration is carried out to the male mite being stored in dactylethrae, the concentration of alcohol is respectively:80%, 90%, 100%, first upper strata alcohol is suctioned out with rubber head dropper, then adds the alcohol of next gradient, so circulation is until 100% Alcohol;2)Dry:Sample is placed on sample stage, is dried 2~3 hours using instrument is dried in vacuo;3)Viscous platform:In microscope It is lower to be chosen male mite to viscous platform with fine, soft fur pen, make its outside of belly upward, the outside of belly is pressed lightly on insect needle, to ensure polypide with leading Electric glue can be in close contact with excellent conductive performance;4)Metal spraying, it is gold-plated to mite whole body;5)Spermatophore is observed under ESEM, Spermatophore is located at below the spermatodactyl of male mite.

Claims (1)

1. a kind of method for obtaining and small new neoseiulus californicus spermatophore being observed under ESEM, spermatophore is produced it is characterized in that first obtaining Small new neoseiulus californicus hero mite, a series of processing are then carried out to male mite and are fabricated under ESEM direct observation spermatophore Method, swept among these comprising the acquisition of male and female mite, the sizing of male and female mite, the separating of male and female mite, the storage of male mite, spermatophore in mating Retouch electron microscope specimen and prepare and be with 5 parts of observation, concrete operations:
(1)The acquisition of male and female mite in mating:Under the microscope, out of small new neoseiulus californicus raising population, it is new small peaceful to choose California 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 small new neoseiulus californicus 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:Male and female mite is taken out together with breeding apparatus after sizing, then under the microscope, used Fine, soft fur pen chooses the male and female mite of mating on slide from breeding apparatus, recycles the instrument such as No. 0 insect needle and fine, soft fur pen will The male and female mite of mating gently separates, and prevents from destroying spermatophore;
(4)The storage of male mite:After male and female mite successfully separates, male mite is chosen with fine, soft fur nib and is put into 70% alcohol in dactylethrae Preserve;
(5)Spermatophore ESEM sample prepares and observation:Comprising dehydration, dry, 5 steps of viscous platform, metal spraying and observation, specifically For:1)Dehydration:Gradient alcohol dehydration is carried out to the male mite being stored in dactylethrae, the concentration of alcohol is respectively:80%, 90%, 100%, first upper strata alcohol is suctioned out with rubber head dropper, then adds the alcohol of next gradient, so circulation is until 100% Alcohol;2)Dry:Sample is placed on sample stage, is dried 2~3 hours using instrument is dried in vacuo;3)Viscous platform:In microscope It is lower to be chosen male mite to viscous platform with fine, soft fur pen, make its outside of belly upward, the outside of belly is pressed lightly on insect needle, to ensure polypide with leading Electric glue can be in close contact with excellent conductive performance;4)Metal spraying:It is gold-plated to mite whole body using ion sputtering instrument;5)Electronic Speculum is seen Examine:Spermatophore is observed under ESEM, spermatophore is located at below the spermatodactyl of male mite.
CN201710808924.8A 2017-09-09 2017-09-09 A kind of method for obtaining and small new neoseiulus californicus spermatophore being observed under ESEM Pending CN107515314A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
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

Patent Citations (5)

* Cited by examiner, † Cited by third party
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 (5)

* Cited by examiner, † Cited by third party
Title
吴桂华等: "《粉尘螨生殖系统形态学研究》", 《昆虫学报》 *
吴聪明: "《蠕形螨扫描电镜样本的制备方法》", 《动物学杂志》 *
忻介六: "《蜱螨学刚要》", 31 March 1984, 高等教育出版社 *
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》 *
赵修复: "《害虫生物防治》", 31 May 1999, 中国农业出版社 *

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Application publication date: 20171226