A method for obtaining salivary glands of fall armyworm larvae in living form
By fixing and cutting the fall armyworm larvae on the anatomical dish to obtain their salivary glands, and cleaning and preserving them in the tissue expansion fluid, the problems of tissue damage and degradation in the prior art are solved, and the complete acquisition of salivary glands is achieved. and preservation provide technical support for the research of Fattish grassland.
Patent Information
- Application Number
- CN202210469948.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-04-30
- Publication Date
- 2024-12-13
- Estimated Expiration
- 2042-04-30
AI Technical Summary
The prior art is difficult to effectively obtain the salivary glands of the fall armyworm larvae. Conventional methods are prone to mechanical damage and degradation of tissues, and it is impossible to obtain complete tissue.
The larvae of Fattia meadow was washed in saline and fixed on the dissection dish. The insect body was cut from the side through ophthalmic dissection scissors, the salivary glands were removed using dissecting forceps, and cleaned and stored in tissue expansion fluid at low temperature. Maintain organizational integrity.
The integrity of the salivary glands and related tissues of the fall armyworm larvae are guaranteed to the greatest extent, avoid tissue degradation, provide raw materials for subsequent research, simplify the operation process and reduce material waste.
Smart Images
Figure CN114699116B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of insect anatomy, and in particular relates to a method for obtaining salivary glands of fall armyworm larvae in living form. Background Art
[0002] The fall armyworm is a global migratory pest belonging to the genus Spodoptera of the family Noctuidae in the order Lepidoptera. It has the characteristics of fast migration, great harm, great difficulty in prevention and control, and a wide host range. Since its invasion, it has caused significant economic losses to my country's food security.
[0003] Insect salivary glands are important organs in the insect digestive system. Their main function is to secrete saliva, which not only lubricates swallowed food, but also contains digestive enzymes to help digestion. The salivary glands of different insects have different shapes. Insect salivary glands are multicellular glands that open into the mouth. The structure of the salivary glands consists of a base membrane, a glandular cell layer, and an inner membrane. The fall armyworm has a chewing mouthpart type. The salivary glands are located in the insect's abdomen. They are two transparent long tubes that are close to the insect's intestines, and some even extend to the hindgut.
[0004] Larvae dissection is the basis for studying their internal structure, physiological and biochemical reactions, growth and development, as well as insect feeding adaptability and molecular biology research. Fall armyworm is a polyphagous pest. Using its salivary glands as research materials is of great significance to the green prevention and control and biological control of fall armyworm. The conventional method of dissecting the salivary glands of fall armyworm larvae is prone to mechanical damage to other tissues, and it is difficult to obtain the salivary glands after freezing, and it is impossible to obtain complete tissues, and it is easy to cause tissue degradation. Compared with the dissection method of the salivary glands of stink bugs, there is no need to cut off the insect's feet, antennae, and mouthparts, nor is there any need to add tissue fluid to the insect body. The dissection of the digestive organs of leafhopper insects will cause damage to the insect body. However, the method of the present invention is relatively simple to operate, has high feasibility, and this method can dissect other tissues at the same time, and the dissection of other tissues does not interfere with each other, and will not cause waste of experimental materials.
[0005] After searching existing literature at home and abroad, there is currently no method to obtain the salivary glands of the living larvae of the fall armyworm.
[0006] Conventional dissection method:
[0007] Improved with reference to Wu Shuang (Study on the defense mechanism of tomato mediated by salivary ATP hydrolase of Spodoptera exigua) and the second edition of General Entomology edited by Lei Chaoliang and Rong Xiulan. Bury the larvae in ice for about 15 minutes and take them out when they stop moving. Put them on the dissection board, fix the two ends of the insect body with a dissection needle, make a small cut near the end of the abdomen with dissection scissors, and then dissect upward along the midline of the abdomen to the end near the head. Take out the salivary glands with a dissection needle, wash them in ddH2O, and put them into centrifuge tubes respectively. Summary of the invention
[0008] 1. Problems to be solved
[0009] In view of the problems existing in the above-mentioned prior art, the present invention provides a method for obtaining the salivary glands of the fall armyworm larvae in vivo. Through the dissection method of the present invention, the integrity of the salivary glands and their related tissues can be guaranteed to the greatest extent, and tissue degradation can be avoided. At the same time, it also provides technical support for the acquisition of raw materials for subsequent related research on the salivary glands.
[0010] 2. Technical solution
[0011] To solve the above problems, the present invention adopts the following technical solutions.
[0012] A method for obtaining salivary glands of fall armyworm larvae in living form comprises the following steps:
[0013] (1) Wash the larvae of Fall Armyworm in saline solution and then use filter paper to absorb the surface moisture;
[0014] (2) placing the larvae cleaned in step (1) in a dissecting dish and fixing them in the horizontal direction of the larvae;
[0015] (3) Using ophthalmic dissecting scissors, cut the worm body fixed in step (2) from the side of the spiracle position from the tail to the head;
[0016] (4) Using dissecting forceps, remove the salivary gland tissue of the fall armyworm larva from the opening of the insect body cut in step (3);
[0017] (5) placing the salivary gland tissue obtained in step (4) in a tissue expansion solution to remove the fat bodies brought out, and washing it clean;
[0018] (6) Take out the salivary gland tissue from the tissue expansion solution in step (5), dry the surface water with filter paper, and put it into a centrifuge tube for cryopreservation. Specifically, cryopreservation is cryopreservation under liquid nitrogen quick freezing.
[0019] The low temperature is -80°C and stored for subsequent experiments.
[0020] In the above-mentioned method for obtaining salivary glands of fall armyworm larvae in living form,
[0021] The fall armyworm larvae described in step (1) are fall armyworm larvae of the third instar or above.
[0022] In the above-mentioned method for obtaining salivary glands of fall armyworm larvae in living form,
[0023] The mass percentage of the physiological saline in step (1) is 0.7%, wherein the physiological saline is sterilized by high pressure.
[0024] In the above-mentioned method for obtaining salivary glands of fall armyworm larvae in living form,
[0025] The dissecting dish described in step (2) is pre-autoclaved.
[0026] In the above-mentioned method for obtaining salivary glands of fall armyworm larvae in living form,
[0027] The surface of the dissecting dish described in step (2) is covered with a sterile transparent plastic film and adhered with alcohol.
[0028] In the above-mentioned method for obtaining salivary glands of fall armyworm larvae in living form,
[0029] The larvae cleaned in step (2) are placed horizontally in a dissecting dish using a dissecting needle.
[0030] In the above-mentioned method for obtaining salivary glands of fall armyworm larvae in living form,
[0031] In step (2), two insect pins are used to fix the larva along the horizontal direction.
[0032] In the above-mentioned method for obtaining salivary glands of fall armyworm larvae in living form,
[0033] The method for allowing the insect to fully stretch after fixation in step (2) is as follows:
[0034] Use an insect pin to first fix one end of the tail or head, stretch it horizontally to its normal size, and then use another insect pin to fix the other end.
[0035] In the above-mentioned method for obtaining salivary glands of fall armyworm larvae in living form,
[0036] The method of taking out in step (4) is as follows:
[0037] Reach in from the third or fourth abdominal segment away from the head to the position just below the intestine to obtain it.
[0038] In the above-mentioned method for obtaining salivary glands of fall armyworm larvae in living form,
[0039] The salivary gland tissue cleaned in step (5) is in the shape of a long tube with two transparent ends;
[0040] In step (5), the tissue expansion solution is physiological saline, DNA protection solution, RNA protection solution or tissue fixative solution.
[0041] 3. Beneficial effects
[0042] Compared with the prior art, the present invention has the following beneficial effects:
[0043] Compared with the traditional method for dissecting the salivary glands of lepidopteran larvae, the material of the present invention is a live dissection, and the dissected tissue is intact. Compared with other insects of the class Insecta, for example: Pentatomidae insects: dissecting the salivary glands destroys the insect's legs, antennae, and mouthparts, and it is necessary to add tissue fluid to the insect body; leafhopper insects: dissecting the digestive organs, the insect body is severely damaged during dissection, and the integrity of the material cannot be guaranteed. Through the dissection method of the present invention, the integrity of the salivary glands and related tissues can be guaranteed to the greatest extent, and the degradation of the tissues can be avoided. At the same time, it also provides technical support for the acquisition of raw materials for subsequent related research on salivary glands. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Figure 1 This is a complete salivary gland diagram of the fall armyworm larvae of the present invention;
[0045] Figure 2 This is a comparative diagram of the salivary gland anatomy of the fall armyworm in the present invention;
[0046] Figure 3 This is a distribution diagram of the salivary gland structure of the fall armyworm larvae in the present invention. DETAILED DESCRIPTION
[0047] The present invention is further described below in conjunction with specific embodiments.
[0048] The terms "include" and "comprising" used herein should be understood as inclusive and open-ended, rather than exclusive. Specifically, when the terms "include" and "comprising" and their synonyms are used in the specification and claims, they mean including the specified features, steps or components. These terms cannot be understood to exclude the existence of other features, steps or components.
[0049] Instruments and tools: stereo microscope, anatomical tweezers, dissecting tray, ophthalmic dissecting scissors, insect pins, filter paper.
[0050] The invention belongs to the field of insect anatomy, and specifically relates to a method for dissecting salivary glands of fall armyworms. The technical problem to be solved is to provide a method for dissecting salivary glands of fall armyworms, which can ensure the integrity of the salivary glands of fall armyworms to the greatest extent and is conducive to further research on saliva. The technical scheme adopted is: a method for obtaining salivary glands of fall armyworm larvae in vivo, comprising cleaning the fall armyworms, fixing them horizontally on a dissecting plate with a dissecting needle, cutting them from the side with dissecting scissors, and extending dissecting forceps under the intestine to quickly and gently pull out the salivary glands to obtain complete salivary glands.
[0051] Specifically, the present invention comprises the following steps: (1) washing the fall armyworm larvae in physiological saline, and then drying the surface moisture with filter paper; (2) placing the larvae washed in step 1) in a horizontal direction on a high-pressure sterilized dissection tray (a layer of sterile transparent plastic film is attached to the surface of the dissection tray and made to fit with alcohol) using a dissection needle, and fixing it in a horizontal direction with two insect pins to stretch it; (3) cutting the insect body fixed in step 2) from the side valve position from the tail to the head direction with ophthalmic dissection scissors; (4) Use dissecting forceps to reach into the insect body that has been untied in step 3) from the third or fourth abdominal segment away from the head to the position just below the intestine, and gently and quickly remove the complete salivary gland tissue of the fall armyworm. At this time, the fall armyworm larvae still have life characteristics; (5) Place the salivary gland tissue removed in step 4) in tissue expansion fluid to remove the fat body brought out, and wash it clean. The salivary gland is in the shape of a long tube with two transparent ends; (6) Take out step 5) from the tissue expansion fluid, absorb the surface moisture with filter paper, and place it in a centrifuge tube.
[0052] In the following three examples of the present invention, step 1) washing the larvae of fall armyworm over the third instar with physiological saline and then drying the surface moisture with filter paper is aimed at removing impurities on the surface of the insect body and avoiding contamination caused by impurities during the operation.
[0053] In the following three examples of the present invention, step 2) places the insect on a dissecting plate and fixes both ends of the insect body with insect pins to prevent shaking of the insect body during the operation and affecting the dissection. At the same time, since the salivary gland is located in the abdomen of the insect, cutting it from the side will not damage the target tissue and will not cause damage to other tissues.
[0054] In at least one example, step 3) places the obtained tissue into a tissue expansion solution to remove the fat on the surface, thereby ensuring the physiological activity of the tissue in a short period of time and obtaining a clean and complete salivary gland.
[0055] In at least one example, in step 3), the upper and lower ends of the epidermis of the fall armyworm larvae are turned over and fixed flat with insect pins to expose the intestine, and forceps are inserted under the intestine to separate the intestine and the salivary gland. This facilitates the dissection of salivary gland tissue while dissecting other digestive organs, and multiple tissues are obtained without affecting each other, and relatively complete salivary gland tissue can be obtained.
[0056] In at least one instance, step 3) is performed by using an insect pin to flip open the abdomen of the fall armyworm larva and lay it flat to expose the intestine, and then using forceps to separate the salivary glands attached to the insect abdomen or intestine. This operation makes it easier for unskilled anatomists to find and observe the salivary gland tissue located in the abdomen and accurately find the target salivary glands.
[0057] In the following three examples, the animal tissue fixative described in step a) is a liquid for maintaining the organ morphology of animal tissue, generally composed of a phosphate buffer composed of sodium dihydrogen phosphate and disodium hydrogen phosphate and glutaraldehyde, and is provided during morphological observation.
[0058] In the following three examples, the RNA protection solution described in step b) can delay tissue degradation and improve the success rate of salivary gland tissue in molecular experiments.
[0059] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
[0060] Example 1
[0061] 1. Wash the larvae of Spodoptera frugiperda that are over 3rd instar in saline. Use filter paper to absorb the moisture on the larvae.
[0062] 2. Place the insect on a dissecting dish, fix the head and tail with insect pins, place it under a dissecting microscope, and use dissecting scissors to cut horizontally from the tail to the head.
[0063] 3. Insert the tweezers under the insect intestine to separate the salivary gland from the insect body, place it in tissue expansion fluid to remove surrounding debris, and obtain a complete salivary gland ( Figure 1 ).
[0064] a. Morphological observation: transfer the intact salivary glands to a 1.5 ml centrifuge tube containing animal tissue fixative for morphological observation.
[0065] b. Molecular biology experiments: The intact salivary glands were quickly transferred to a 1.5 ml centrifuge tube containing RNA protection solution and placed in a -80°C refrigerator for use in molecular experiments.
[0066] Example 2
[0067] 1. Wash the larvae of fall armyworm that are over 3rd instar with physiological saline, and then use filter paper to absorb the moisture on the surface.
[0068] 2. Place the insect on a dissecting dish and fix the head and tail with insect pins. Cut the spiracles horizontally from the head under a dissecting microscope.
[0069] 3. Use an insect pin to flip open the upper and lower ends of the skin of the fall armyworm larvae and lay them flat to expose the intestine. Insert tweezers under the intestine to separate the intestine and salivary glands.
[0070] 4. Place the intact salivary gland in tissue expansion fluid to remove surrounding debris. Figure 1 ).
[0071] a. Morphological observation: transfer the intact salivary glands to a 1.5 ml centrifuge tube containing animal tissue fixative for morphological observation.
[0072] b. Molecular biology experiments: The intact salivary glands were quickly transferred to a 1.5 ml centrifuge tube containing RNA protection solution and placed in a -80°C refrigerator for use in molecular experiments.
[0073] Example 3
[0074] 1. Wash the larvae of fall armyworm that are over 3rd instar with physiological saline, and then use filter paper to absorb the moisture on the surface.
[0075] 2. Place the insect on a dissecting dish with its abdomen facing upwards and fix its head and tail with insect pins. Cut the tail horizontally along the spiracles under a dissecting microscope.
[0076] 3. Use an insect pin to turn over the abdomen of the fall armyworm larvae and flatten it to expose the intestines. Then use tweezers to separate the salivary glands attached to the insect's abdomen or intestines to obtain complete salivary glands.
[0077] 4. Place the intact salivary gland in tissue expansion fluid to remove surrounding debris. Figure 1 ).
[0078] a. Morphological observation: transfer the intact salivary glands to a 1.5 ml centrifuge tube containing animal tissue fixative for morphological observation.
[0079] b. Molecular biology experiments: The intact salivary glands were quickly transferred to a 1.5 ml centrifuge tube containing RNA protection solution and placed in a -80°C refrigerator for use in molecular experiments.
[0080] Comparison Example 1
[0081] 1. Wash the larvae of fall armyworm that are over 3rd instar with physiological saline, and then use filter paper to absorb the moisture on the surface.
[0082] 2. The insects were then frozen at -20°C for 15 minutes.
[0083] 3. Take out the worm body and place it on ice. Use a utility knife to cut it open from the abdomen. Because the worm body is twisted in the frozen environment, it is not suitable to cut the abdomen when it is slightly thawed, which will cause great damage to the worm body. When cutting, it is very easy to damage the worm body, causing the loss of body fluids and the leakage of contents, making it impossible to find the salivary glands. Figure 2 ).
[0084] Compared with Comparative Example 1, it can be seen that the dissection method of the present invention can ensure the integrity of the salivary glands of the larvae to the maximum extent without damaging the epidermis of the fall armyworm larvae, avoiding the leakage of body fluids and contents caused by mechanical damage and the contamination of other tissues in the body. In addition, the operation time of the present invention is greatly shortened compared with the traditional dissection method. Therefore, the dissection method of the present invention has obvious advantages in terms of operation time and the result of maintaining the integrity of the salivary gland specimens.
[0085] Basis for judging the integrity of salivary glands: salivary glands are a type of gland that opens in the mouth to secrete saliva to assist in feeding and digestion. The morphology and structure of salivary glands of different insects are different. Lepidoptera larvae generally use the labial glands as salivary glands and the labial glands as silk glands. The salivary glands of Lepidoptera insects are the simplest tubular structure, which consists of three parts: salivary tube, salivary duct and salivary gland. The salivary glands dissected by the present invention have a three-part structure ( Figure 3 ), so it is believed that the salivary glands dissected by the method of the present invention are intact. Specifically, the basis for judging the integrity of the salivary glands comes from the second edition of General Entomology edited by Lei Chaoliang and Rong Xiulan.
[0086] The above-described embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed. The dissection method of this experiment is not limited to the fall armyworm, but is also applicable to other types of Noctuidae insects.
[0087] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention are within the scope of the present invention.
Claims
1. A method for obtaining salivary glands of fall armyworm larvae in living form, characterized in that: The following steps are involved: (1) Wash the larvae of Fall Armyworm in saline solution and then use filter paper to absorb the surface moisture; (2) placing the larvae cleaned in step (1) in a dissecting dish and fixing them in the horizontal direction of the larvae; In step (2), two insect pins are used to fix the larva along the horizontal direction; The method for allowing the insect to fully stretch after fixation in step (2) is as follows: Use an insect pin to first fix one end of the tail or head, stretch it horizontally to its normal size, and then use another insect pin to fix the other end; (3) Using ophthalmic dissecting scissors, cut the worm body fixed in step (2) from the side of the spiracle position from the tail to the head; (4) Using dissecting forceps, remove the salivary gland tissue of the fall armyworm larva from the opening of the insect body cut in step (3); The method of taking out in step (4) is as follows: Reach from the third or fourth abdominal segment away from the head to the position just below the intestine to obtain; (5) placing the salivary gland tissue obtained in step (4) in a tissue expansion solution to remove the fat bodies brought out, and washing it clean; (6) Take out the salivary gland tissue from the tissue expansion solution in step (5), dry the surface moisture with filter paper, and store it in a centrifuge tube at low temperature.
2. The method for obtaining salivary glands of fall armyworm larvae from living bodies according to claim 1, characterized in that: The fall armyworm larvae described in step (1) are fall armyworm larvae of the third instar or above.
3. The method for obtaining salivary glands of fall armyworm larvae from living bodies according to claim 1, characterized in that: The mass percentage of the physiological saline in step (1) is 0.7%, wherein the physiological saline is sterilized by high pressure.
4. The method for obtaining salivary glands of fall armyworm larvae from living bodies according to claim 1, characterized in that: The dissecting dish described in step (2) is pre-autoclaved.
5. The method for obtaining salivary glands of fall armyworm larvae from living bodies according to claim 4, characterized in that: The surface of the dissecting dish described in step (2) is covered with a sterile transparent plastic film and adhered with alcohol.
6. The method for obtaining salivary glands of fall armyworm larvae from living bodies according to claim 1, characterized in that: The larvae cleaned in step (2) are placed horizontally in a dissecting dish using a dissecting needle.
7. The method for obtaining salivary glands of fall armyworm larvae from living bodies according to claim 1, characterized in that: The salivary gland tissue cleaned in step (5) is in the shape of a long tube with two transparent ends; In step (5), the tissue expansion solution is physiological saline, DNA protection solution, RNA protection solution or tissue fixative solution.