Parasitic wasp quality detection method

Abstract

The invention discloses a parasitic wasp quality detection method. According to the method, high-resolution multi-dimensional imaging is carried out on parasitic wasps in a host body through a micro-CT technology, and therefore the survival, the number, the size, the gender and the morphological characteristics of the parasitic wasps are judged. The method has the advantages of timeliness, high efficiency, accuracy and the like, and an effective method is provided for detecting the development condition of parasitic wasps, especially egg parasitic wasps, in hosts, so that the quality of a large number of propagated parasitic wasps is accurately evaluated, and smooth implementation of a biological control technology is guaranteed.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting the quality of parasitic wasps. Background Art

[0003] Parasitic wasps are an important type of natural enemy insects that can achieve the purpose of controlling pests by parasitizing pests. They have the advantages of being environmentally friendly, leaving no residue, harmless to non-targets, and environmentally friendly, and are known as "natural enemies of agricultural and forestry pests". Parasitic wasps have been put into large-scale agricultural applications as the most important biological control tool. However, during the large-scale production and application of natural enemy parasitic wasps, the parasitic wasps cannot be directly observed in the host, especially egg parasitic wasps, that is, parasitic wasps that parasitize insect eggs. The insects themselves are tiny, and the insect eggs are even smaller. Most insect eggs are less than 1mm. However, as an effective natural enemy product, the development of parasitic wasps needs to be monitored during production, and the quality of parasitic wasps, such as the number and survival of parasitic wasps, needs to be tested before the product is applied. At present, the existing technology can only dissect the parasitized tussah eggs and other alternative host eggs under a body dissecting microscope, and determine the development of the parasitic wasps after taking out the parasitic wasps. However, since the eggs of tussah silkworms and rice moths are very small as hosts, the dissection is difficult and requires skilled professional technicians to operate. At the same time, the survival, surviving gender, and number of parasitic wasps in host eggs can only be determined by sampling the parasitized host eggs in a clean and non-toxic culture dish, placing them in suitable temperature, humidity and light environment conditions and culturing until they emerge, so as to determine the gender and number of parasitic wasps. However, culturing to emergence requires specific environmental conditions and a long time, and energy is consumed and time is wasted in the process of waiting for their development. Therefore, a fast and efficient quality detection method is urgently needed to evaluate the quality of parasitic wasps, so as to ensure the smooth progress of biological control.

[0004] In order to overcome the defects of existing parasitic bee quality detection technology, the present invention proposes a new parasitic bee quality detection technology based on micro-CT. It has the characteristics of timeliness, high efficiency, accuracy, etc., and can quickly obtain the internal structure image and morphological parameters of its host egg without complicated operations. Through micro-CT technology, the size, developmental morphology, quantity and survival information of parasitic bees can be accurately measured without waiting for the parasitic bees to emerge as adults, which greatly shortens the detection time. And provide reliable data such as parasitic bee development, survival and quantity, providing effective information for evaluating the quality of parasitic bees. Summary of the invention

[0005] The present invention aims to provide a timely, convenient and efficient method for detecting the quality of parasitic wasps. The method is safe, reliable, scientific and reasonable, and suitable for detecting the quality of parasitic wasps.

[0006] A first aspect of the present invention relates to a method for detecting the quality of parasitic wasps, which comprises detecting parasitic wasp samples in host eggs by using a micro-tomography scanner.

[0007] In some embodiments, the parasitic wasp includes at least one of Japanese flat-bellied wasp, striated stink bug flat-bellied wasp, oriental flat-bellied wasp, yellow-eyed trichogrammatid wasp, pine caterpillar trichogrammatid wasp, rice borer trichogrammatid wasp, brown-winged stink bug groove egg wasp, yellow-footed egg jumping wasp, and black egg wasp of noctuid. Its host eggs include eggs of at least one of insects such as tussah silkworm, rice moth, brown-winged stink bug and Spodoptera litura.

[0008] In some embodiments, the parasitic wasp quality detection method of the present invention includes a sample pretreatment step, i.e., sample fixation, staining, dehydration, and drying. First, fix with 2.5% glutaraldehyde for 24 hours and stain with 5% potassium iodide. Next, dehydrate with ethanol gradient, in order of 70%, 80%, 90%, 100% ethanol, each treatment for 10-30 minutes. Finally, perform carbon dioxide critical point drying.

[0009] In some embodiments, the parasitic wasp quality detection method of the present invention includes a sample fixation step, in which the host eggs and the parasitic samples are placed on a sample table and fixed with paraffin.

[0010] In some embodiments, the parasitic wasp quality detection method of the present invention includes a sample scanning step, using a micro-tomography scanner to collect sample images.

[0011] In some embodiments, a microtomography machine is used to scan the sample and collect 2D images. The microtomography machine includes at least one of SkyScan 1172, Bruker SkyScan 1276, Bruker SkyScan 1176, BrukerSkyScan 1272, Bruker SkyScan 1275, Bruker SkyScan1278, Scanco Medical VivaCT 80, Scanco Medical VivaCT 40, Scanco MedicalμCT 50, Scanco MedicalμCT 100, PerkinElmer Quantum GX2, ZEISS Xradia620Versa, ZEISS Xradia 520Versa, Rigakunano3DX, Nikon Metrology XT H 225ST, Nikon Metrology XT V 130, TESCAN UniTOM HR, TESCAN DynaTOM, GE Phoenix nanotom m, and Thermo Scientific Heliscan. By adjusting the specific scanning parameters of the micro-tomography machine, the imaging contrast is improved to obtain clear 2D tiff images.

[0012] In some embodiments, Bruker micro-CT NRecon software is used to reconstruct a 2D tiff image into a 2D bmp format image.

[0013] In some embodiments, 2D bmp format images are synthesized into 3D images through Ctvox, NRecon, CTAn, CTVox, DataViewer and Dragonfly, preferably Ctvox, and data analysis is performed.

[0014] In some embodiments, the present invention includes a parasitic wasp quality judgment step, in which the survival, quantity, size, sex and morphological characteristics of the parasitic wasp are judged by reconstructing the 3D image.

[0015] The morphological characteristics described in the present invention refer to the shape and / or structural characteristics of at least a part of the parasitic wasp.

[0016] Another aspect of the present invention relates to a parasitic wasp quality detection method with improved image quality and data accuracy.

[0017] Testing and optimizing scanning parameters are very important for the contrast and clarity of the final 3D image. Biological samples, with their complex microstructures and highly sensitive soft tissues, are very sensitive to scanning parameters. Appropriate scanning parameters (such as resolution, contrast, exposure time, scanning speed, and voltage) can ensure high-quality images that accurately reflect the size, shape, and internal structure of the sample. If the parameters are not set correctly, it may lead to blurred images, increased noise, and artifacts. In addition, different biological samples may have different densities and absorption characteristics, so scanning parameters need to be adjusted according to the specific sample characteristics. By systematically testing and optimizing these parameters, the image quality and data accuracy can be maximized, thus providing a reliable basis for subsequent dimensional measurement, morphological analysis, and quality assessment. This is crucial to ensure the credibility of research results and their effectiveness in application areas such as biological control.

[0018] In some embodiments, the scanning source current of the parasitic wasp quality detection method is 180-250uA, such as 240uA, 230uA, 220uA, 210uA, 200uA, 190uA, 180uA; preferably 190-210uA, most preferably 200±5uA;

[0019] In some embodiments, the scanning source voltage of the parasitic wasp quality detection method is 35-42 kV; preferably 36-40 kV, most preferably 37±1 kV;

[0020] In some embodiments, the resolution of the mass detection method of the parasitic wasp is 0.55 microns to 3 microns, preferably 0.68 microns to 1.76 microns, and most preferably 1.69 ± 0.1 microns;

[0021] In some embodiments, the scanning exposure time of the parasitic wasp quality detection method is 400-1100 ms, preferably 500-600 ms, and most preferably 550 ms;

[0022] In some embodiments, the parasitic wasp quality detection method described above, Bruker micro-CTNRecon software reconstructs Tiff images, dragonfly is used for 3D imaging, and data analysis is performed.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The invention has the advantages of being rapid, timely, and highly efficient, and provides a good basis for quality detection of egg parasitic wasps.

[0025] The present invention can clearly observe the survival, quantity, size, sex and morphological characteristics of parasitic wasps on eggs in hosts.

[0026] The use of micro-tomography technology to detect the quality of parasitic wasps not only overcomes the shortcomings of traditional methods, which are time-consuming and inefficient, but also improves the accuracy and reliability of the test results. This innovative method helps to grasp the timing of prevention and control in a timely manner, improve the effectiveness of biological control, reduce dependence on chemical pesticides, reduce environmental burden, and promote sustainable development of agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Reconstructed 2D images and constructed 3D images

[0028] Figure 2 The yellow-legged egg-jumping wasp parasitizing the eggs of the brown-winged stink bug

[0029] Figure 3 Trichogramma chinensis parasitizing on eggs of the silkworm DETAILED DESCRIPTION

[0030] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased from the market.

[0031] Example 1

[0032] The survival, quantity and size of the yellow-legged egg-jumping wasp in the eggs of the tea-winged stink bug are detected. The specific steps are as follows:

[0033] S11. Test insects:

[0034] The yellow-legged egg-jumping wasp parasitizing the eggs of the tea-winged stink bug was cultured in an insect-raising room at 25°C, relative humidity 50%, and L:D=16:8.

[0035] S12. Multiple yellow-legged egg-jumping wasps can develop and successfully emerge simultaneously in the large host. A 7-day-old parasitized egg of the brown-winged stink bug was randomly selected for scanning.

[0036] S13 Microtomoscope Selection: Imaging was performed using a SkyScan 1172 desktop high-resolution microtomoscope.

[0037] S14. Set scanning parameters:

[0038] The scanning parameters were set as follows: voxel size was 1.76um; source voltage was 37kV; source current was 220uA; exposure time was 550ms; image rotation step was 0.20°; 360° rotation scan was performed; and no filter was used.

[0039] S15, reconstruct the 2D image and construct a 3D image ( Figure 1 ).

[0040] S16. The data were analyzed by dragonfly. The results showed that there were 5 yellow-legged egg jumping wasps that had entered the prepupal stage and survived in a single tea-winged stink bug egg. The boundaries between the individuals were clear and they had obvious biological characteristics of the prepupal stage (Figure 2). Figure 2 ). The largest individual is about 857.96um long, and the smallest individual is about 641.11um long.

[0041] Example 2

[0042] The survival of a kind of trichogrammatid wasp in the eggs of tussah silkworms was detected, and the steps are as follows:

[0043] S11. Test insects:

[0044] The Trichogramma yellow-eyed wasp parasitizing in the eggs of the silkworm was stored at room temperature for 6 months.

[0045] S12. Multiple Trichogrammatus chinensis bees can develop and successfully emerge simultaneously inside the eggs of tussah silkworms. We randomly selected tussah silkworm eggs that had been parasitized by Trichogrammatus chinensis during routine bee breeding for 6 months and scanned them.

[0046] S13 Microtomoscope Selection: Imaging was performed using a SkyScan 1172 desktop high-resolution microtomoscope.

[0047] S14. Set scanning parameters:

[0048] The scanning parameters were set as follows: voxel size was 1.76um; source voltage was 37kV; source current was 220uA; exposure time was 550ms; image rotation step was 0.20°; 360° rotation scan was performed; and no filter was used.

[0049] S15, reconstruct the 2D image and construct a 3D image

[0050] S16. Data analysis was performed using dragonfly. The results showed that the randomly selected tussah eggs were the remaining parasitized eggs from the reproduction of Trichogramma chinensis half a year ago. The scan results showed that all Trichogramma chinensis had developed into pupae and died, accumulating at the bottom of the tussah eggs, and the contents at the top of the tussah eggs had been eaten up ( Figure 3 ).

[0051] Although the present invention has been illustrated and described with specific embodiments, it will be appreciated that many other changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, it is intended to include all such changes and modifications within the scope of the present invention in the appended claims.

Claims

1. A method for detecting the quality of parasitic wasps, characterized in that: Detection of the quality of parasitoids inside host eggs using microtomography.

2. The parasitic wasp quality detection method according to claim 1, characterized in that: The quality detection of parasitic wasps in the host body using micro-tomography technology includes obtaining multi-dimensional images using high-resolution micro-tomography technology to determine at least one of the survival, quantity, size, sex and morphological characteristics of the parasitic wasps.

3. The parasitic wasp quality detection method according to claim 1 or 2, characterized in that: The parasitic wasps include at least one of Japanese flat-bellied wasp, crimson-striped stink bug flat-bellied wasp, oriental flat-bellied wasp, yellow-eyed trichogrammatid wasp of stem borer, pine caterpillar trichogrammatid wasp, rice borer trichogrammatid wasp, tea-winged stink bug groove egg wasp, yellow-legged egg jumping wasp and noctuid black egg wasp.

4. The parasitic wasp quality detection method according to claim 1 or 2, characterized in that The host eggs include at least one of tussah eggs, rice moth eggs, tea winged stink bug eggs, Spodoptera litura eggs and artificial host eggs.

5. The parasitic wasp quality detection method according to any one of claims 1 or 2, characterized in that: The specific steps include: 1) Fix the sample on the sample stage with paraffin or plasticine; 2) Use a high-resolution micro-tomography machine to collect 2D images of the sample; 3) Reconstruct a 3D image based on the collected 2D images, and judge the quality of the parasitic wasp according to the 3D image.

6. The parasitic wasp quality detection method according to claim 5, characterized in that: The parasitoid wasp detection includes fixing, staining, dehydrating and drying the parasitoid wasp samples in the host eggs before step 1).

7. The parasitic wasp quality detection method according to claim 5, characterized in that: In step 2), the imaging contrast is improved by adjusting the specific scanning parameters of the micro-tomography scanner to obtain a clear 2D image.

8. The parasitic wasp quality detection method according to claim 5, characterized in that: In step 2), the Brukermicro-CT NRecon software is used to reconstruct the acquired 2D images into images that can be used to synthesize 3D images.

9. The parasitic wasp quality detection method according to claim 5, characterized in that: In step 3), the 2D images are synthesized into 3D images through Ctvox, NRecon, CTAn, CTVox, DataViewer or Dragonfly.

10. The parasitic wasp quality detection method according to claim 5, characterized in that: Based on the 3D images, determine the survival status of parasitic wasps, count the number of parasitic wasps, measure the size of parasitic wasps, identify their gender, and describe their morphological characteristics.

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