CN114250150A - Method for extracting in-vivo symbiotic microorganisms from coleoptera insects - Google Patents
Method for extracting in-vivo symbiotic microorganisms from coleoptera insects Download PDFInfo
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- CN114250150A CN114250150A CN202110450757.0A CN202110450757A CN114250150A CN 114250150 A CN114250150 A CN 114250150A CN 202110450757 A CN202110450757 A CN 202110450757A CN 114250150 A CN114250150 A CN 114250150A
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- 241000254173 Coleoptera Species 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 15
- 244000005700 microbiome Species 0.000 title claims abstract description 13
- 238000001727 in vivo Methods 0.000 title claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims abstract description 41
- 208000031513 cyst Diseases 0.000 claims abstract description 15
- 210000001015 abdomen Anatomy 0.000 claims abstract description 9
- 238000001514 detection method Methods 0.000 claims abstract description 7
- 206010011732 Cyst Diseases 0.000 claims abstract description 5
- 238000012258 culturing Methods 0.000 claims abstract description 5
- 238000000227 grinding Methods 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 5
- 238000007865 diluting Methods 0.000 claims abstract description 4
- 241000238631 Hexapoda Species 0.000 claims description 28
- 241000233866 Fungi Species 0.000 claims description 21
- 210000001672 ovary Anatomy 0.000 claims description 8
- 238000002474 experimental method Methods 0.000 claims description 6
- 210000000056 organ Anatomy 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 230000001568 sexual effect Effects 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 3
- 230000000249 desinfective effect Effects 0.000 claims description 3
- 230000002779 inactivation Effects 0.000 claims description 3
- 238000005096 rolling process Methods 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 241001247821 Ziziphus Species 0.000 claims 1
- 241001072256 Boraginaceae Species 0.000 description 3
- 235000007689 Borago officinalis Nutrition 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000002224 dissection Methods 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000024241 parasitism Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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Abstract
A method for extracting in-vivo symbiotic microorganisms from coleoptera insects comprises the steps of selecting a plurality of sexually mature female coleoptera insects, enabling the female coleoptera insects to have an upward belly, pressing the rear section in the belly to enable the female coleoptera insects to extend out of a symbiotic bacteria sac (an oval shape has a small cyst type structure, and the interior is mostly yellow or white tissues), completely picking the female coleoptera insects by using a pair of tweezers under a microscope, immersing the female coleoptera insects into a vessel containing a PBS solution with a certain concentration, grinding the female coleoptera insects by using a grinding rod to enable the content of the female coleoptera insects to be fully dissolved into the solution, diluting the female coleoptera insects with the concentration, transferring the female coleoptera insects to a culture vessel containing YNB, culturing the female coleoptera insects in a constant temperature box at a proper temperature (25 ℃) for 2-3 days, carrying out PCR detection if white punctiform or strip-shaped tissues or blocky tissues appear, comparing the female coleoptera gene sequences with known symbiotic bacteria, and evolving the female coleoptera gene sequences to indicate that the female coleoptera in vivo symbiotic microorganisms are successfully extracted.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for extracting symbiotic microorganisms in fungal sacs in insects.
Background
As the largest number of insects in nature, various threats such as natural enemies, pathogenic microorganisms, or adversities in the environment are faced. Researches show that the insect symbiotic bacteria can protect the host from predation and parasitism of natural enemies, resist the invasion of pathogenic microorganisms and assist the environment with poor tolerance. The protozobacteria (i.e. the endosymbionts) are necessary for the survival of the host insects, and have a long-term coevolution relationship, are mainly distributed in the specialized cell-containing tissues (such as coleoptera cysts) of the host insects, and strictly carry out vertical propagation. The function of the endosymbionts is closely related to the distribution positions of the endosymbionts in insect tissues, and some symbionts are mainly stored in the fungal sac and participate in improving the survival of eggs and the growth conditions of larvae. The symbiotic bacteria are vertically spread in coleoptera insects in a mode of coating the symbiotic bacteria nearby the coleoptera insects after spawning, and are spread from parents to offspring, so that larvae can hatch under a relatively proper environmental condition and can assist the growth of the larvae.
Disclosure of Invention
The invention aims to provide a method for researching symbiotic microorganisms in coleoptera insects aiming at the defects of the prior art.
The purpose of the invention is realized by the following technical scheme.
A method for extracting in-vivo symbiotic microorganisms from coleoptera insects comprises the following operation steps:
(1) preparing: selecting a plurality of sexually mature female coleoptera insects, turning the insects with the ventral surfaces upward, slightly extruding the rear section of the abdomen of the insects by fingers to enable the insects to extend out of female sexual organs, repeatedly operating the insects for several times, forcibly extruding the tail ends of the abdomen of the female insects again to enable the insects to extend out of more tissues, and at the moment, extending out of the front ends of the sexual organs to form an oval structure with a small cyst (the inside is mostly yellow or white tissues), wherein the tissues are coleoptera symbiotic bacteria cysts.
(2) Obtaining coleoptera insect fungus sacs: and (2) disinfecting the head of the tweezers by using a flame burning method, immersing the disinfected tweezers into a 1.5ml separation tube containing a PBS solution with a certain concentration for cooling in order to avoid the inactivation of insect tissues due to overhigh temperature of the tweezers, and then picking off symbiotic bacteria cysts extending out of the tail end of the abdomen of the female insect in the step (1) under a microscope by using the cooled tweezers and immersing the symbiotic bacteria cysts into the PBS solution under the condition of not damaging the ovary.
(3) And (3) purifying symbiotic bacteria: observing whether the female fungus sacs are completely treated under a microscope, judging whether the tissues picked in the step (2) contain ovaries or not, if so, separating the ovaries from the symbiotic fungus sacs under the microscope by using the picked tissues, then immersing the successfully picked fungus sacs into a 1.5ml separation tube containing a PBS solution with a certain concentration, rolling the picked fungus sacs by using a sterilized grinding rod to fully dissolve symbiotic bacteria contained in the fungus sacs into the solution, and diluting the solution at different concentrations to finally obtain the symbiotic fungus solution with a proper concentration.
(4) Culturing symbiotic bacteria: quantitatively transferring the symbiotic bacteria solution obtained in the step (3) to a culture dish containing YNB (YNB) -by using a quantitative rod, sealing the transferred culture dish in a sterile environment, recording the experiment time and the serial number of the sample content on a culture dish cover, and putting the culture dish subjected to the experiment operation into an incubator at a proper temperature (25 ℃) to carry out symbiotic bacteria culture.
(5) And (4) observation: and (4) after two to three days, taking out the culture dish in the step (4), placing the culture dish under a microscope, and observing whether white punctate or bar-shaped tissues appear.
(6) And (3) detection: and (3) extracting the white tissue observed in the step (5) by using a quantitative vessel, carrying out PCR detection, comparing the obtained results with the gene sequence of the known symbiotic bacteria, and if the white tissue belongs to the same clade, indicating that the bacteria observed in the step (5) are coleoptera insect symbiotic bacteria.
And (4) finishing the operations of dissection, sample adding and sampling in the steps (1) to (6) in an ultra-clean workbench in a sterile way.
The invention has the beneficial effects that: the method fills the blank of the method for extracting the symbiotic bacteria in the field of coleoptera insect symbiotic microorganisms, and can provide convenience for researching a fungus-insect symbiont system.
Detailed Description
The following examples are intended to illustrate the invention without limiting it, and any modifications and variations of the invention within the scope of the claims of the invention are within the scope of the invention.
Example (b):
the method comprises the following steps:
(1) preparing: a plurality of sexually mature female colored glaze spade beetles are selected, turned with the ventral surface upward, the middle and rear sections of the ventral parts of the female colored glaze spade beetles are lightly pressed by fingers to extend out of female sexual organs, the female colored glaze spade beetles are repeatedly operated for several times, the tail ends of the ventral parts of the female worms are forcibly pressed again to extend out more tissues, at the moment, an oval structure with a cyst type structure (the inside is mostly yellow or white tissues) can be extended out of the front ends of the sexual organs, and the tissues are coleopteran symbiotic bacteria cysts.
(2) Obtaining coleoptera insect fungus sacs: and (2) disinfecting the head of the tweezers by using a flame burning method, immersing the disinfected tweezers into a 1.5ml separation tube containing a PBS solution with a certain concentration for cooling in order to avoid the inactivation of insect tissues due to overhigh temperature of the tweezers, and then picking off symbiotic bacteria cysts extending out of the tail end of the abdomen of the female insect in the step (1) under a microscope by using the cooled tweezers and immersing the symbiotic bacteria cysts into the PBS solution under the condition of not damaging the ovary.
(3) And (3) purifying symbiotic bacteria: observing whether the female fungus sacs are completely treated under a microscope, judging whether the tissues picked in the step (2) contain ovaries or not, if so, separating the ovaries from the symbiotic fungus sacs under the microscope by using the picked tissues, then immersing the successfully picked fungus sacs into a 1.5ml separation tube containing a PBS solution with a certain concentration, rolling the picked fungus sacs by using a sterilized grinding rod to fully dissolve symbiotic bacteria contained in the fungus sacs into the solution, and diluting the solution at different concentrations to finally obtain the symbiotic fungus solution with a proper concentration.
(4) Culturing symbiotic bacteria: quantitatively transferring the symbiotic bacteria solution obtained in the step (3) to a culture dish containing YNB (YNB) -by using a quantitative rod, sealing the transferred culture dish in a sterile environment, recording the experiment time and the serial number of the sample content on a culture dish cover, and putting the culture dish subjected to the experiment operation into an incubator at a proper temperature (25 ℃) to carry out symbiotic bacteria culture.
(5) And (4) observation: and (4) after two to three days, taking out the culture dish in the step (4), placing the culture dish under a microscope, and observing whether white punctate or bar-shaped tissues appear.
(6) And (3) detection: and (3) extracting the white tissue observed in the step (5) by using a quantitative vessel, carrying out PCR detection, comparing the obtained results with the gene sequence of the known symbiotic bacteria, and indicating that the bacteria observed in the step (5) are azure stone spade symbiotic bacteria if the bacteria belong to the same clade.
And (4) finishing the operations of dissection, sample adding and sampling in the steps (1) to (6) in an ultra-clean workbench in a sterile way.
Drawings
FIG. 1 is a drawing of female adult azure beetles; FIG. 2 is a diagram of a female larva bacterial sac of a colored glaze spade A; FIG. 3 is a diagram showing the condition of a culture dish for culturing bacteria in an incubator for three days after extracting the substance; FIG. 4 is a diagram showing the condition of colonies on a selected culture dish transferred to a new culture dish for further culture.
FIGS. 1-4 illustrate the process of extracting symbiotic microorganisms from the body of borage beetles, exemplified by the female adult platycerurus hongwoonpyoi Imura et Choe, 1989 of borage beetles. FIG. 1 shows a female adult vitreous spade beetle; FIG. 2 shows genital and cyst tissues from which a borage beetle protrudes by squeezing the ventral end of a female adult; FIG. 3 shows that after the content of the sac of the shovel nail is extracted and transferred to a culture dish and cultured for three days under the condition of a constant temperature environment, a yeast type colony group appears on the surface of the culture dish; FIG. 4 is a schematic representation of 16 colonies selected from the yeast type colony population of FIG. 3 transferred to a new petri dish for continued culture in preparation for subsequent verification of the colonies as being PCR-tested for the desired symbiotic microorganisms.
Claims (2)
1. A method for extracting symbiotic microorganisms in vivo from insects of the order coleoptera, characterized in that said insects belong to the order coleoptera and in that it comprises the following operative steps:
(1) preparing insects: selecting a plurality of sexually mature female coleoptera insects, turning the insects with the ventral surfaces upward, slightly extruding the rear section of the abdomen of the insects by fingers to enable the insects to extend out of female sexual organs, repeatedly operating the insects for several times, forcibly extruding the tail ends of the abdomen of the female insects again to enable the insects to extend out of more tissues, and at the moment, extending out of the front ends of the sexual organs to form an oval structure with a small cyst (the inside is mostly yellow or white tissues), wherein the tissues are coleoptera symbiotic bacteria cysts.
(2) Obtaining coleoptera insect fungus sacs: and (2) disinfecting the head of the tweezers by using a flame burning method, immersing the disinfected tweezers into a 1.5ml separation tube containing a PBS solution with a certain concentration for cooling in order to avoid the inactivation of insect tissues due to overhigh temperature of the tweezers, and then picking off symbiotic bacteria cysts extending out of the tail end of the abdomen of the female insect in the step (1) under a microscope by using the cooled tweezers, and immersing the symbiotic bacteria cysts into a PBS solution vessel with a certain concentration.
(3) And (3) purifying symbiotic bacteria: observing whether the female fungus sacs are completely treated under a microscope, judging whether the tissues picked in the step (2) contain ovaries or not, if so, separating the ovaries from the symbiotic fungus sacs under the microscope by using the picked tissues, then immersing the successfully picked fungus sacs into a 1.5ml separation tube containing a PBS solution with a certain concentration, rolling the fungus sacs by using a sterilized grinding rod to fully dissolve symbiotic bacteria contained in the fungus sacs into the solution, diluting the solution for 2-3 times, and finally obtaining the symbiotic fungus solution with a proper concentration.
(4) Culturing symbiotic bacteria: quantitatively transferring the symbiotic bacteria solution obtained in the step (3) to a culture dish containing YNB (YNB) -by using a quantitative rod, sealing the transferred culture dish in a sterile environment, recording the experiment time and the serial number of the sample content on a culture dish cover, and putting the culture dish subjected to the experiment operation into an incubator at a proper temperature (25 ℃) to carry out symbiotic bacteria culture.
(5) And (4) observation: and (5) after two to three days, taking out the culture dish in the step (4), and placing under a microscope, if white punctiform or lumpy tissues of the jujubes appear, preliminarily indicating that the symbiotic microorganisms are successfully extracted.
(6) And (3) detection: and (3) extracting the white tissue observed in the step (5) by using a quantitative vessel, carrying out PCR detection, comparing the obtained results with the gene sequence of the known symbiotic bacteria, and if the white tissue belongs to the same clade, indicating that the bacteria observed in the step (5) are coleoptera insect symbiotic bacteria.
2. The method for extracting symbiotic microorganisms from coleopteran insects as recited in claim 1, wherein the steps (1) to (6) comprise the steps of dissecting, loading, and sampling, all performed in a clean bench.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105532581B (en) * | 2015-12-16 | 2019-04-05 | 中国计量大学 | A method of insect fungal component is studied in vitro through ovum vertical transmission |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105532581B (en) * | 2015-12-16 | 2019-04-05 | 中国计量大学 | A method of insect fungal component is studied in vitro through ovum vertical transmission |
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| 徐冬丽等: "白蜡虫肠道微生物分析及立克次氏体分子检测", 《林业科学研究》 * |
| 李迁等: "昆虫内共生菌-昆虫-植物互作关系研究进展", 《植物保护学报》 * |
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Application publication date: 20220329 |