Method for collecting hemolymph of diaphorina citri

Abstract

The invention discloses a method for collecting hemolymph of diaphorina citri. The method comprises the following steps: anesthetizing diaphorina citri bodies, disinfecting the diaphorina citri bodies, pinching off one forefoot of each diaphorina citri body, and collecting hemolymph; the hemolymph is collected by pinching off one forefoot of the diaphorina citri, compared with other methods, the success rate is higher, the collected hemolymph is higher in content, lower in impurity rate and higher in protein concentration, and the blood volume and purity of the collected diaphorina citri hemolymph are guaranteed to the maximum extent. Meanwhile, according to the method, the integrity and activity of other tissues and organs in the diaphorina citri are not damaged, other tissues and organs can still be collected after the hemolymph is collected, and other subsequent experimental operations are not excessively influenced. The method provided by the invention can quickly and efficiently collect the hemolymph of the diaphorina citri, and compared with a general hemolymph collection technology, the method provided by the invention is short in time consumption, simple to operate and low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of insect physiology and biochemistry, and in particular relates to a method for collecting hemolymph of citrus psyllid. Background Art

[0002] Insect hemolymph is a mixture of blood and lymph, which circulates in the open circulatory system of insects. It is the place where all substances and metabolites necessary for insect physiological activities converge, and plays a key role in insect nutrient storage, material transportation, metabolism, detoxification, immunity, and response to damage. Since hemolymph has extremely complex components and characteristics and undertakes multiple physiological functions, the study of insect hemolymph has attracted more and more attention from insect physiologists.

[0003] Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter spp. (CLas), has become a global epidemic and has caused damage in more than 50 countries and regions in America, Asia, and Africa. It has become the "number one killer" of the citrus industry, bringing a devastating blow to the development of the world's citrus industry. Asian citrus psyllid (Diaphorina citri Kuwayam) is an important insect vector that spreads and spreads citrus Huanglongbing (HLB) in nature. It is distributed in many countries and regions, directly threatening the development of the global citrus industry. At present, the methods for controlling citrus psyllid include chemical control, physical control, and agricultural control. However, chemical control is easy to cause ecological pollution, and long-term use will lead to increased drug resistance in citrus psyllid. Agricultural control has certain limitations and regionality, and physical control will cost too much labor and cost. Therefore, it is urgent to find a green, efficient, and sustainable control method. Hemolymph is one of the commonly used research materials in physiological and biochemical research in the current process of searching for efficient prevention and control methods for citrus psyllids. However, due to the small size of citrus psyllids and the low hemolymph content, hemolymph exposed to the air in vitro is prone to melanization and coagulation, which greatly increases the difficulty of collection.

[0004] There are many methods for collecting hemolymph, including the commonly used methods of cutting, wax disc, centrifugation, dorsal blood vessel sampling, squeezing, homogenization, perfusion, puncture, etc. However, these methods are mainly used to collect hemolymph from medium and large insects, and the collection of hemolymph from small insects usually destroys the internal structure of the small insects, resulting in a low success rate of collected hemolymph, insufficient hemolymph content, impurity or contamination, causing inconvenience and difficulty for subsequent research. Summary of the invention

[0005] The object of the present invention is to overcome the above defects and shortcomings in the prior art and provide a method for collecting hemolymph of citrus psyllid.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The invention firstly provides a method for collecting hemolymph of citrus psyllid, which comprises anesthetizing the citrus psyllid insect body, disinfecting the insect body, clipping off one front leg of the insect body, and collecting hemolymph.

[0008] The method for collecting hemolymph of citrus psyllid provided by the present invention collects hemolymph by pinching off one of the front legs of the insect body. Compared with the puncture method commonly used in the art, the method provided by the present invention has a higher success rate for collecting hemolymph, and at the same time, the collected hemolymph has a higher content, a lower impurity rate, and a higher protein concentration, and can guarantee the blood volume and purity of the collected hemolymph of citrus psyllid to the greatest extent, thereby overcoming the problems of low success rate of collected hemolymph, low hemolymph content, and low purity of collected hemolymph due to a small number of citrus psyllid individuals.

[0009] Furthermore, the citrus psyllid includes 1st to 5th instar nymphs and adults.

[0010] Furthermore, the hemolymph contains lymph fluid, serum and blood cells.

[0011] Furthermore, the method specifically comprises the following steps:

[0012] S1. Anesthetize the citrus psyllid;

[0013] S2. Disinfect the body surface of the citrus psyllid anesthetized in S1;

[0014] S3. Fix the citrus psyllid disinfected in S2 with its abdomen facing upward, clip off one of the front legs of the citrus psyllid, collect hemolymph directly or press its abdomen lightly before collecting hemolymph to obtain citrus psyllid hemolymph.

[0015] Furthermore, the anesthesia in step S1 is carried out at -18 to -20°C for 6 to 10 minutes.

[0016] Preferably, the anesthesia is carried out by placing the animal at -20°C for 10 minutes.

[0017] Furthermore, the disinfection in step S2 is to immerse the citrus psyllid in 70-75% ethanol by volume for 3-5 minutes.

[0018] Preferably, the disinfection is carried out by soaking the citrus psyllid in 75% by volume ethanol for 3 minutes.

[0019] Preferably, after the body surface is disinfected in step S2, the insect body is rinsed with PBS, and then the surface of the insect body is absorbed with clean filter paper to keep it dry.

[0020] Furthermore, the fixing in step S3 is to fix the citrus psyllid on a stereo microscope with its abdomen facing upwards.

[0021] Furthermore, the hemolymph is collected in step S3 by using a micro capillary tube.

[0022] Furthermore, the hemolymph of the citrus psyllid obtained in step S3 is placed in a centrifuge tube, centrifuged, and the supernatant is collected and stored at -18 to -20°C.

[0023] Preferably, the supernatant is stored at -20°C.

[0024] Furthermore, the centrifuge tube is pretreated with 0.1% phenylthiourea solution before the citrus psyllid hemolymph is placed in it.

[0025] Furthermore, the centrifugation conditions are 2-4°C, 10000-12000 r / min, and 10-15 min.

[0026] Preferably, the centrifugation condition is 4°C, 12000r / min for 10min.

[0027] The present invention also provides application of any of the above methods in physiological and biochemical research of citrus psyllid.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The method of the present invention collects hemolymph by pinching off one of the front legs of the citrus psyllid. The average success rate of hemolymph collection is 94%, the average hemolymph content is 0.85 μL, the average impurity rate is 4.02%, and the average protein concentration is 17.915 mg / mL. Compared with other methods, the success rate is higher, the collected hemolymph content is higher, the impurity rate is lower, and the protein concentration is higher, which maximizes the blood volume and purity of the collected citrus psyllid hemolymph. At the same time, the method does not damage the integrity and activity of other tissues and organs in the citrus psyllid. Other tissues and organs can still be collected after collecting hemolymph, and there is no excessive impact on other subsequent experimental operations;

[0030] (2) The method for collecting hemolymph of citrus psyllid provided by the present invention also anesthetizes the insects at low temperature to temporarily incapacitate them, thereby preventing the insects from being stimulated by external factors and causing changes in their physiological environment, thereby facilitating subsequent operations; and disinfecting and cleaning the insects to prevent foreign matter or impurities on the insects' body surface from contaminating the hemolymph and affecting subsequent research;

[0031] (3) Compared with the general hemolymph collection technology, the method for collecting hemolymph of citrus psyllid provided by the present invention is time-saving, simple to operate, low-cost, and the amount of hemolymph collected is relatively stable. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the specification and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] Example 1

[0035] A method for collecting hemolymph of citrus psyllid comprises the following steps: anesthetizing the insect body, disinfecting the insect body, clipping off one front leg of the insect body with tweezers, and collecting the overflowed hemolymph with a capillary tube.

[0036] (1) Place the adult citrus psyllids in a -20°C refrigerator for 10 min;

[0037] (2) Soak the citrus psyllid adults in 75% ethanol for 3 min, rinse twice with PBS, and then use clean filter paper to absorb the insect surface to keep it dry;

[0038] (3) placing the anesthetized citrus psyllid adult with its abdomen facing upward under a stereo microscope, using forceps to clip off one of the citrus psyllid's forelegs, and collecting hemolymph directly with a microcapillary tube, or by lightly pressing its abdomen before collecting;

[0039] (4) The collected hemolymph was placed in a 1.5 mL centrifuge tube pretreated with 0.1% phenylthiourea solution, centrifuged at 4°C, 12,000 rpm for 10 min, and the supernatant was aspirated and stored at -20°C.

[0040] Example 2

[0041] A method for collecting hemolymph of citrus psyllid comprises the following steps: anesthetizing the insect body, disinfecting the insect body, clipping off one front leg of the insect body with tweezers, and collecting the overflowed hemolymph with a capillary tube.

[0042] The specific steps include:

[0043] (1) Place the first-instar nymphs of citrus psyllids in a -18°C refrigerator for 6 min;

[0044] (2) Soak the first-instar nymphs of citrus psyllid in 70% ethanol for 5 min, rinse twice with PBS, and then absorb the surface of the insect body with clean filter paper to keep it dry;

[0045] (3) placing the anesthetized first-instar citrus psyllid nymph with its abdomen facing upward under a stereo microscope, using forceps to clip off one of the forelegs of the first-instar citrus psyllid nymph, and collecting the hemolymph using a capillary tube after it overflows;

[0046] (4) The collected hemolymph was placed in a 1.5 mL centrifuge tube pretreated with 0.1% phenylthiourea solution, centrifuged at 2°C, 10,000 rpm for 15 min, and the supernatant was aspirated and stored at -18°C.

[0047] Example 3

[0048] A method for collecting hemolymph of citrus psyllid comprises the following steps: anesthetizing the insect body, disinfecting the insect body, clipping off one front leg of the insect body with tweezers, and collecting the overflowed hemolymph with a capillary tube.

[0049] The specific steps include:

[0050] (1) Place the second-instar nymphs of citrus psyllids in a -20°C refrigerator for 10 min;

[0051] (2) Soak the second-instar nymphs of citrus psyllid in 75% ethanol for 3 min, rinse twice with PBS, and then use clean filter paper to absorb the surface of the insect body to keep it dry;

[0052] (3) placing the anesthetized 2nd instar nymph of citrus psyllid with its abdomen facing upward under a stereo microscope, using forceps to clip off one of the forelegs of the 2nd instar nymph of citrus psyllid, and collecting the hemolymph using a capillary tube after the hemolymph overflows;

[0053] (4) The collected hemolymph was placed in a 1.5 mL centrifuge tube pretreated with 0.1% phenylthiourea solution, centrifuged at 4°C, 12,000 rpm for 10 min, and the supernatant was aspirated and stored at -20°C.

[0054] Example 4

[0055] A method for collecting hemolymph of citrus psyllid comprises the following steps: anesthetizing the insect body, disinfecting the insect body, clipping off one front leg of the insect body with tweezers, and collecting the overflowed hemolymph with a capillary tube.

[0056] The specific steps include:

[0057] (1) Place the third-instar nymphs of citrus psyllids in a -20°C refrigerator for 10 min;

[0058] (2) Soak the third-instar nymphs of citrus psyllid in 75% ethanol for 3 min, rinse twice with PBS, and then absorb the surface of the insect body with clean filter paper to keep it dry;

[0059] (3) placing the anesthetized third-instar nymph of citrus psyllid with its abdomen facing upward under a stereo microscope, using forceps to clip off one of the forelegs of the third-instar nymph of citrus psyllid, and collecting the hemolymph using a capillary tube after it overflows;

[0060] (4) The collected hemolymph was placed in a 1.5 mL centrifuge tube pretreated with 0.1% phenylthiourea solution, centrifuged at 4°C, 12,000 rpm for 10 min, and the supernatant was aspirated and stored at -20°C.

[0061] Example 5

[0062] A method for collecting hemolymph of citrus psyllid comprises the following steps: anesthetizing the insect body, disinfecting the insect body, clipping off one front leg of the insect body with tweezers, and collecting the overflowed hemolymph with a capillary tube.

[0063] The specific steps include:

[0064] (1) Place the 4th instar nymphs of citrus psyllids in a -20°C refrigerator for 10 min;

[0065] (2) Soak the 4th instar nymphs of citrus psyllid in 75% ethanol for 3 min, rinse twice with PBS, and then absorb the surface of the insect body with clean filter paper to keep it dry;

[0066] (3) placing the anesthetized 4th instar nymph of citrus psyllid with its abdomen facing upward under a stereo microscope, using forceps to clip off one of the forelegs of the 4th instar nymph of citrus psyllid, and collecting the hemolymph using a capillary tube after it overflows;

[0067] (4) The collected hemolymph was placed in a 1.5 mL centrifuge tube pretreated with 0.1% phenylthiourea solution, centrifuged at 4°C, 12,000 rpm for 10 min, and the supernatant was aspirated and stored at -20°C.

[0068] Example 6

[0069] A method for collecting hemolymph of citrus psyllid comprises the following steps: anesthetizing the insect body, disinfecting the insect body, clipping off one front leg of the insect body with tweezers, and collecting the overflowed hemolymph with a capillary tube.

[0070] The specific steps include:

[0071] (1) Place the fifth-instar nymphs of citrus psyllids in a -20°C refrigerator for 10 min;

[0072] (2) Soak the fifth-instar nymphs of citrus psyllid in 75% ethanol for 3 min, rinse twice with PBS, and then use clean filter paper to absorb the surface of the insect body to keep it dry;

[0073] (3) placing the anesthetized fifth-instar nymph of citrus psyllid with its abdomen facing upward under a stereo microscope, using forceps to clip off one of the forelegs of the fifth-instar nymph of citrus psyllid, and collecting the hemolymph using a capillary tube after it overflows;

[0074] (4) The collected hemolymph was placed in a 1.5 mL centrifuge tube pretreated with 0.1% phenylthiourea solution, centrifuged at 4°C, 12,000 rpm for 10 min, and the supernatant was aspirated and stored at -20°C.

[0075] Comparative Example 1

[0076] (1) Place the adult citrus psyllids in a -20°C refrigerator for 10 min;

[0077] (2) Soak the citrus psyllid adults in 75% ethanol for 3 min, rinse twice with PBS, and then use clean filter paper to absorb the insect surface to keep it dry;

[0078] (3) Place the anesthetized citrus psyllid adult with its abdomen facing upward under a stereo microscope, use forceps to gently clamp the abdomen of the citrus psyllid, then pierce the cheek below the compound eyes of the citrus psyllid with a sterile dissecting needle, and collect hemolymph with a microcapillary tube;

[0079] (4) The collected hemolymph was placed in a 1.5 mL centrifuge tube pretreated with 0.1% phenylthiourea solution, centrifuged at 4°C, 12,000 rpm for 10 min, and the supernatant was aspirated and stored at -20°C.

[0080] Test Example 1 Comparison of the efficiency of collecting citrus psyllid hemolymph by different methods

[0081] Citrus psyllid hemolymph was collected by two different methods, Example 1 and Comparative Example 1, and the work efficiency of collecting citrus psyllid hemolymph under different methods was compared. Each method was repeated 5 times, and each repeat was 30 citrus psyllids. The results are shown in Table 1. The results show that the average success rate and failure rate of collecting citrus psyllid hemolymph by Example 1 were 94% and 6%, respectively, and its success rate was significantly higher than the average success rate (76.66%) of collecting citrus psyllid hemolymph by Comparative Example 1, and the failure rate was significantly lower than the average failure rate (23.34%) of collecting citrus psyllid hemolymph by Comparative Example 1. It can be seen that the work efficiency of collecting citrus psyllid hemolymph by Example 1 is higher than that of Comparative Example 1.

[0082] Table 1 Comparison of the efficiency of collecting hemolymph of citrus psyllids by different methods

[0083]

[0084] Test Example 2 Comparison of different methods for collecting hemolymph content of citrus psyllids

[0085] The hemolymph of citrus psyllids was collected by two different methods, Example 1 and Comparative Example 1, and the contents of hemolymph of citrus psyllids collected by different methods were compared. Each method was repeated 5 times, and each repeat was 30 citrus psyllids. The results are shown in Table 2. The results showed that the average content of hemolymph of citrus psyllids collected by Example 1 was 0.85 μL, and the average content of hemolymph of citrus psyllids collected by Comparative Example 1 was 0.69 μL. The content of hemolymph of citrus psyllids collected by Example 1 was higher than that of Comparative Example 1.

[0086] Table 2 Comparison of hemolymph content of citrus psyllid collected by different methods (unit: μL)

[0087]

[0088] Test Example 3 Comparison of different methods for collecting impurity rates of citrus psyllid hemolymph

[0089] The impurities in the hemolymph of citrus psyllid collected in Example 1 and Comparative Example 1 were compared. Five repetitions were set for each method, and 30 citrus psyllids were collected in each repetition. The results are shown in Table 3. The results show that the average impurity rate of the hemolymph of citrus psyllid collected in Example 1 was 4.02%, and the average impurity rate of the hemolymph of citrus psyllid collected in Comparative Example 1 was 11.32%. The hemolymph of citrus psyllid collected in Example 1 was clearer than that in Comparative Example 1.

[0090] Table 3 Comparison of impurity rates of hemolymph collected from citrus psyllids by different methods

[0091]

[0092] Test Example 4 Determination of hemolymph protein concentration in citrus psyllids collected by different methods

[0093] Citrus psyllid hemolymph was collected by two different methods, Example 1 and Comparative Example 1, and a BCA protein detection kit was used. An appropriate volume of the collected citrus psyllid hemolymph was added to 200 μL BCA working solution, and the absorbance at A562nm was measured by an ELISA instrument after culturing at 60°C for 40 minutes. The protein concentration was calculated from the standard curve, and 2 replicates were set for each method. The results are shown in Table 4. The results showed that the average protein concentration of the citrus psyllid hemolymph collected by Example 1 was 17.915 mg / mL, and the average protein concentration of the citrus psyllid hemolymph collected by Comparative Example 1 was 15.64 mg / mL, indicating that the protein concentration of the citrus psyllid hemolymph collected by Example 1 was higher.

[0094] Table 4 Comparison of hemolymph protein concentrations of citrus psyllids collected by different methods

[0095]

[0096]

[0097] The above embodiments are preferred implementation modes of the present invention, but the above implementation modes of the present invention can only be regarded as explanations of the present invention and cannot limit the invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for collecting hemolymph of citrus psyllid, characterized in that: The method includes anesthetizing the citrus psyllid, disinfecting the insect body, clipping off one of the insect's front legs, and collecting hemolymph.

2. The method according to claim 1, characterized in that: The hemolymph contains lymph fluid, serum and blood cells.

3. The method according to claim 1, characterized in that: The following steps are involved: S1. Anesthetize the citrus psyllid; S2. Disinfect the body surface of the citrus psyllid anesthetized in S1; S3. Fix the citrus psyllid disinfected in S2 with its abdomen facing upward, clip off one of the front legs of the citrus psyllid, collect hemolymph directly or press its abdomen lightly before collecting hemolymph to obtain citrus psyllid hemolymph.

4. The method according to claim 3, characterized in that: The anesthesia in step S1 is to place the mixture at -18 to -20°C for 6 to 10 minutes.

5. The method according to claim 3, characterized in that: The disinfection in step S2 is to immerse the citrus psyllid in 70-75% ethanol by volume for 3-5 minutes.

6. The method according to claim 3, characterized in that: The fixing in step S3 is fixing on a stereo microscope.

7. The method according to claim 3, characterized in that: In step S3, the hemolymph is collected by using a micro capillary tube.

8. The method according to claim 3, characterized in that: The hemolymph of the citrus psyllid obtained in step S3 is placed in a centrifuge tube, centrifuged, the supernatant is collected, and stored at -18 to -20°C.

9. The method according to claim 8, characterized in that: The centrifuge tube was pretreated with 0.1% phenylthiourea solution before the hemolymph of citrus psyllid was placed in it.

10. The method according to claim 8, characterized in that: The centrifugal conditions are 2-4° C., 10,000-12,000 r / min, and 10-15 min.

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