Phytophagous insect recipe identification method based on insect condition forecasting lamp and bar code

Abstract

The invention belongs to the technical field of agricultural pest monitoring, and particularly relates to a phytophagous insect recipe identification method based on pest situation forecasting lamps and bar codes. A method for identifying a recipe of phytophagous insects comprises the following steps: trapping and collecting the phytophagous insects, and treating the phytophagous insects by using far infrared heating; extracting the DNA of the intestinal contents of the phytophagous insects after the far infrared heating treatment; the method comprises the following steps: synthesizing a pair of primers with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, and carrying out PCR (Polymerase Chain Reaction) amplification by taking intestinal content DNA (Deoxyribonucleic Acid) as a template to obtain a trnL sequence; performing high-throughput sequencing on the trnL sequence; and comparing the high-throughput sequencing result with a DNA bar code database to obtain the phytophagous insect recipe. The method provided by the invention can be used for accurately identifying the variety of plants fed by the phytophagous insects, so that the accuracy of recipe analysis is improved, and the recipe variety of the phytophagous insects is expanded.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural pest monitoring, and in particular relates to a method for identifying the diet of herbivorous insects based on insect monitoring lamps and bar codes. Background Art

[0002] Plants contain all the nutrients required for the growth and development of herbivorous insects, including carbohydrates, amino acids, sterols, phospholipids, fatty acids, vitamins, minerals, trace elements and water. Identifying the plant species that herbivorous insects feed on and analyzing the nutritional relationship between the two will not only help us understand their functions in the biological community, but also enrich the theory of co-evolution between herbivorous insects and plants, and have important significance for the prevention and control of herbivorous insect pests on the basis of protecting ecological species diversity.

[0003] Traditional analysis of herbivorous insect diets relies on manual capture and direct observation. By observing the activity habits of herbivorous insects in the natural environment, including their habitats and food sources, we can preliminarily determine their possible food chain positions and food types. This method is not suitable for herbivorous insects with a wide range of activities and nocturnal activities. It is not only time-consuming and labor-intensive, but also greatly affected by the subjective judgment of the observer. For those complex environments with multiple potential prey that are difficult to separate in space, it is difficult to accurately identify the plant species that the insects feed on. It is also inefficient and difficult to obtain accurate feeding information. At the same time, this method may interfere with the ecosystem and cannot be carried out on a large scale and quickly.

[0004] The most common method for analyzing the diet of herbivorous insects is the microhistological method, which uses morphological microscopic identification of plant epidermal fragments in feces or intestinal contents. This method requires researchers to have high morphological expertise, has low accuracy in identifying closely related species with similar morphology, and has a very limited number of herbivorous insect food species.

[0005] In summary, there is an urgent need to provide a new method for accurately identifying the diet of herbivorous insects. Summary of the invention

[0006] In order to accurately obtain the diet of herbivorous insects, the present invention provides a method for identifying the diet of herbivorous insects based on insect monitoring lights and bar codes.

[0007] The technical solution adopted by the present invention is that the present invention provides a method for identifying a diet of herbivorous insects, comprising the following steps:

[0008] Trapping and collecting herbivorous insects, and treating the herbivorous insects with far-infrared heating; extracting the intestinal content DNA of the herbivorous insects after the far-infrared heating treatment; synthesizing a pair of primers, the primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, using the intestinal content DNA as a template, performing PCR amplification to obtain the trnL sequence; performing high-throughput sequencing on the trnL sequence; comparing the results of the high-throughput sequencing with a DNA barcode database to obtain the herbivorous insect diet.

[0009] Preferably, the far-infrared heating treatment lasts for 10 to 15 minutes.

[0010] Preferably, the temperature of the far-infrared heating treatment is 80°C to 85°C.

[0011] Preferably, the process of trapping and collecting herbivorous insects is: installing an insect monitoring lamp in a field, turning on the insect monitoring lamp to trap the herbivorous insects, and using a collection device provided with the insect monitoring lamp to collect the herbivorous insects.

[0012] Preferably, the light source wavelength of the insect monitoring lamp is 365nm~400nm.

[0013] Preferably, the herbivorous insect is a scarab beetle.

[0014] Preferably, the beetle is a copper green beetle or a dark gill beetle.

[0015] Preferably, the high-throughput sequencing is performed using PE250 sequencing.

[0016] Compared with the prior art, the present invention has the beneficial effect that the present invention provides a method for identifying the diet of herbivorous insects, comprising the following steps: trapping and collecting herbivorous insects, and using far-infrared heating to treat the herbivorous insects; extracting the intestinal content DNA of the herbivorous insects after far-infrared heating treatment; synthesizing a pair of primers, the primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, using the intestinal content DNA as a template, PCR amplification, and obtaining the trnL sequence; performing high-throughput sequencing on the trnL sequence; comparing the results of the high-throughput sequencing with the DNA barcode database to obtain the diet of herbivorous insects. The present invention effectively retains the DNA integrity of the intestinal contents of insects by non-contact infrared heating treatment. This treatment method avoids the problem of rapid DNA degradation that may be caused by traditional methods, so that the DNA information from the intestinal contents of insects can be kept in its original state for a long time, providing a more reliable and rich data basis for the types of insects feeding. The method of the present invention can accurately identify the plant species fed by herbivorous insects, improve the accuracy of diet analysis, and expand the diet types of herbivorous insects.

[0017] At the same time, the present invention also improves the collection efficiency of herbivorous insect samples and simplifies the sample processing process. In addition, the identification method of the present invention can quickly and large-scale analyze the diet of herbivorous insects, provide a scientific basis for ecological research and pest management, avoid interference with the ecosystem, and reduce direct damage to the ecosystem and biodiversity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The PCR amplification results of the trnL sequence of the intestinal content DNA of the beetles in Example 1 and Example 2 are shown in Figure 1. The lanes in A are the PCR amplification results of 14 dark-gilled beetles and 10 copper green beetles from left to right; lane B is the PCR amplification result of 4 copper green beetles and the negative control H2O. DETAILED DESCRIPTION

[0019] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0020] The inventive concept of the present invention is as follows: In the prior art, insect monitoring lamps have shown high efficiency in trapping insects, but how to quickly and losslessly convert captured scarab beetles into biological information remains a major problem. Existing processing methods often focus on quantitative statistics and prediction of pests, while ignoring deeper biological characteristic analysis, such as food preferences and nutritional status of trapped insects.

[0021] The present invention utilizes the infrared technology of insect monitoring lamps, which can not only efficiently trap insects, usually pests, but also is unique in that it effectively preserves the DNA integrity of the insect's intestinal contents through non-contact infrared heating treatment. This treatment method avoids the problem of rapid DNA degradation that may be caused by traditional methods, so that the DNA information from the insect's intestinal contents can be kept in its original state for a long time, providing a more reliable and rich data basis for the types of insects that feed. In addition, the present invention amplifies and sequences the plant DNA barcode trnL gene fragment based on the intestinal content DNA, and uses resources such as the plant species information system website, the national basic discipline data cloud website, and the GenBank public database to quickly identify the plant species that herbivorous insects feed on and analyze the pest feeding habits, providing a new way to analyze the diet of beetles, and its application in the identification of plant species that beetles feed on is currently a blank.

[0022] In contrast, for live pests captured directly in the field, DNA extracted from the intestines of live pests may face a higher risk of degradation, especially under conditions such as improper sample handling or changes in ambient temperature. The application of insect monitoring lamps not only simplifies the pest collection process, but also provides a more stable and efficient way to preserve and extract DNA through its advanced infrared processing technology, significantly reducing the risk of DNA degradation, thereby improving the accuracy and reliability of research results.

[0023] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1

[0025] A method for identifying a diet of herbivorous insects, the method being as follows:

[0026] S1. Trapping and collecting herbivorous insects, treating the herbivorous insects with far-infrared heating, and extracting DNA from the intestinal contents of the herbivorous insects treated with far-infrared heating. The process is as follows:

[0027] High-efficiency insect monitoring lamps are deployed in the farmland ecosystem to trap and collect dark gill beetles, which are treated using far-infrared heating to ensure the integrity of the DNA in the intestinal contents, and the DNA in the intestinal contents of the dark gill beetles after far-infrared heating treatment is extracted.

[0028] DNA was extracted by silica gel column purification according to the DNA Extraction Kit procedure. The extracted DNA was measured for quality and concentration using Nanodrop 2000 and stored in a -20°C refrigerator for later use.

[0029] S2. Synthesize a pair of primers, the primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, use the intestinal content DNA as a template, perform PCR amplification, and obtain a trnL sequence that can be used for analysis.

[0030] The trnL sequence is a plant DNA barcode. The primer sequences of SEQ ID NO.1 and SEQ ID NO.2 are based on the highly conserved region of the trnL gene, ensuring the specificity and efficiency of amplification.

[0031] Among them, the intestinal content DNA was PCR amplified using the following PCR primers and amplification conditions.

[0032] The primers used for PCR amplification are trnL-g and trnL-h, and the primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0033] SEQ ID NO. 1: trnL-g: 5'-GGGCAATCCTGAGCCAA-3'.

[0034] SEQ ID NO. 2: trnL-h: 5'-CCATTGAGTCTCTGCACCTATC-3'.

[0035] PCR uses DNA Polymerase, 20 μL reaction system, the reaction system is shown in Table 1:

[0036] Table 1 PCR reaction system

[0037] 5× Buffer 4μL 2.5 mM dNTPs 2μL 5 μM forward primer 0.8μL 5 μM reverse primer 0.8μL Pfu polymerase 0.4μL Template DNA 10ng <![CDATA[Make up to ddH2O]]> 20μL

[0038] PCR reaction parameters:

[0039] a. 1 cycle: 5 min, 5°C.

[0040] b. 35 cycles: 30 sec, 95°C; 30 sec, 58°C; 45 sec, 72°C.

[0041] c. 1 cycle: 10 min, 72°C, store at 10°C after completion.

[0042] The PCR products of the same sample were mixed and tested by 2% agarose gel electrophoresis. The PCR products were recovered by gel extraction kit, eluted with Tris-HCl, and tested by 2% agarose gel electrophoresis. Figure 1 As shown, lanes 1 to 14 are 14 dark gill beetles, lanes 15 to 28 are 14 copper green beetles, M represents DNA Marker, and CK represents blank control H2O.

[0043] S3. Perform high-throughput sequencing on the trnL sequence. The process is as follows:

[0044] High-throughput sequencing uses PE250 sequencing. The PE250 sequencing sequence first needs to obtain valid sequences for all samples based on the barcode barcode; then the quality of the reads is quality-controlled and filtered; then the paired reads are spliced ​​into a sequence based on the overlap relationship between the paired-end reads; finally, the high-quality sequence of each sample is obtained by splitting according to the barcode and primer sequence, and the sequence direction is corrected and chimeras are removed in the process according to the forward and reverse barcode and primer directions. Operational Taxonomic Units and OTU clustering are performed for non-repetitive sequences without single sequences at 97% similarity. Chimeras are removed during the clustering process to obtain representative sequences of OTUs, and OTU sequences are clustered.

[0045] S4. Compare the results of high-throughput sequencing with the DNA barcode database to obtain the diet of herbivorous insects, as follows:

[0046] First, the DNA barcode data of plants were obtained from the National Basic Disciplines Data Cloud website. For DNA barcode data, please refer to "Wang Yuhua. Chinese Plant DNA Barcode Database. (V1). Kunming Institute of Botany, Chinese Academy of Sciences [Creating Institution], 2023-09-05. National Basic Disciplines Public Science Data Center [Issuing Institution], https: / / cstr.cn / 16666.11.nbsdc.joqudavj"; secondly, the OTU sequences obtained by high-throughput sequencing were subjected to BLAST comparison analysis, and the uclust algorithm was used to perform taxonomic analysis on the OTU representative sequences with a similarity level of 97%. The community composition of each sample was statistically analyzed at each classification level, including class, order, family, and species, to obtain the diet of herbivorous insects.

[0047] Example 2

[0048] A method for identifying the diet of herbivorous insects.

[0049] The herbivorous insects trapped and collected were Anomala cooperi, and other experimental conditions were exactly the same as those in Example 1.

[0050] The data in Tables 2 and 3 list in detail the various plant DNA fragments detected in the intestinal samples of the two beetles in the examples of the present invention. These fragments not only cover a wide range of plant families and genera, but are also accurate to the specific species level, reflecting the powerful analytical ability of high-throughput sequencing technology in the study of complex ecosystems.

[0051] The diet preference of the copper green beetle includes hardwood species such as poplar and persimmon, fruit trees such as Prunus and cherry, and economic crops such as corn, which indicates that it may play a variety of roles in different ecosystems. It may be a pest in forest ecosystems, or it may cause economic impacts in agricultural ecosystems. In contrast, the diet of the dark gill beetle is more extensive, including important food crops such as rice and corn, as well as oil crops such as soybeans and forest resources such as elm, which reflects its strong ability to adapt to multiple environments and multiple hosts. This difference in the diet of the two beetles not only reflects their different strategies for utilizing food resources in the ecological environment, but may also be closely related to their respective biological characteristics, life history characteristics and the ecological environment in which they live.

[0052] In addition, the present invention also found that these sequencing results are highly consistent with the host plant species of the two beetles previously identified based on morphological characteristics, ecological observations and traditional molecular biological methods. This is not only a strong verification of the accuracy of the sequencing and bioinformatics analysis methods used in the present invention, but also shows that the method described in the present invention has extremely high sensitivity and specificity in identifying insect feeding habits and revealing insect-plant interactions. Compared with traditional methods, the present invention can not only quickly and efficiently identify a large number of plant species, but also capture species information that may be overlooked due to unclear morphological characteristics, thereby providing more comprehensive and detailed feeding information.

[0053] The plant species identified by the method of the present invention can cover the plant species identified by the traditional method; and the plant species not identified by the traditional method can also be obtained by the identification method of the present invention. At the same time, this embodiment is only the result of one location. Combining the identification results of different habitats, the types of plants that the scarab beetle pest feeds on can be fully understood.

[0054] Table 2 Types of plant DNA detected in the intestinal contents of A. corydalis

[0055]

[0056]

[0057] According to the reference document "A Concise Identification Manual for Major Crop Diseases and Insect Pests, edited by Cao Keqiang: Apple Volume", that is, "Cao Keqiang. A Concise Identification Manual for Major Crop Diseases and Insect Pests. Apple Volume [M]. Hebei Science and Technology Press, 2012.", the host plants of the copper green beetle include apple, hawthorn, crabapple, pear, apricot, peach, plum, apricot, persimmon and walnut.

[0058] Table 3 Types of plant DNA detected in the intestinal contents of Holotrichia obesa

[0059]

[0060]

[0061] According to the reference "Xiao Gangrou. Chinese Forest Insects. 2nd Edition (Revised Edition) [M]. China Forestry Publishing House, 1992," the host plants of the dark-gilled beetle are elm, willow, poplar, walnut, mulberry, apple, pear, chrysanthemum, sunflower and soybean.

[0062] From the sequencing results and bioinformatics analysis, the identification method of the present invention shows significant advantages in the identification of plant DNA in the intestinal contents of scarab beetles. This detailed taxonomic analysis provides a solid foundation for a deep understanding of the feeding habits of scarab beetles. From the sequencing results and subsequent in-depth bioinformatics analysis, the present invention not only reveals the richness of plant DNA species in the intestinal contents of scarab beetles, but also through careful comparison, clarifies the difference in feeding habits between the copper green scarab beetle and the dark gill scarab beetle, which provides valuable information for understanding their niche differentiation and potential plant-insect interactions.

[0063] The method of the present invention can systematically study the habitat, food source, etc. of the beetles, and preliminarily determine their possible food chain position and food types. Identifying the plant species that the herbivorous beetles feed on and analyzing the nutritional relationship between the two not only helps to understand their functions in the biological community; the identification method of the present invention not only accurately and efficiently identifies the plant DNA species in the intestinal contents of the beetles, but also reveals the differences in the feeding habits of the two beetles and their ecological significance through in-depth data analysis. The feeding preferences of the copper green beetle and the dark gill beetle not only reflect their different utilization strategies for food resources, but are also closely related to their biological characteristics, life history characteristics and the ecological environment in which they are located. This information is of great significance for enriching the theory of co-evolution between herbivorous insects and plants, and for the prevention and control of beetle pests on the basis of protecting ecological species diversity.

[0064] The application of this method is not limited to scarab beetles, but can also be extended to the identification of the feeding habits of other herbivorous insects. By collecting intestinal content samples of various herbivorous insects in different ecosystems and using this method for high-throughput sequencing and bioinformatics analysis, we can fully understand the feeding habits and ecological niche differentiation of these insects, providing strong technical support for pest management, ecological protection and biodiversity research.

[0065] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0066] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for identifying a diet of vegetative insects, characterized in that: The following steps are involved: Trapping and collecting herbivorous insects, and treating them with far-infrared heating; Extraction of DNA from the intestinal contents of herbivorous insects after far-infrared heating treatment; A pair of primers were synthesized, the primer sequences were shown in SEQ ID NO.1 and SEQ ID NO.2, and the intestinal content DNA was used as a template for PCR amplification to obtain the trnL sequence; High-throughput sequencing of trnL sequences; The results of high-throughput sequencing were compared with the DNA barcode database to obtain the diet of herbivorous insects.

2. The identification method according to claim 1, characterized in that: The far-infrared heating treatment takes 10 to 15 minutes.

3. The identification method according to claim 1, characterized in that: The temperature of the far-infrared heating treatment is 80°C to 85°C.

4. The identification method according to claim 1, characterized in that: The process of trapping and collecting herbivorous insects is as follows: Install the insect monitoring lamp in the field, turn on the insect monitoring lamp to trap herbivorous insects, and use the collection device that comes with the insect monitoring lamp to collect the herbivorous insects.

5. The identification method according to claim 4, characterized in that: The light source wavelength of the insect monitoring lamp is 365nm-400nm.

6. The identification method according to claim 1, characterized in that: The herbivorous insect is a scarab beetle.

7. The identification method according to claim 6, characterized in that: The beetle is a copper green beetle or a dark gill beetle.

8. The identification method according to claim 1, characterized in that: The high-throughput sequencing was performed using PE250 sequencing.

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