Application of wild soybean variant No.4 in promoting growth and development of clanis bilineata tsingtauica and / or improving nutritional quality
By using Wild Soybean Mutant No. 4 as an alternative feed for soybean hawk moth, the problem of anti-nutritional factors in plant leaves inhibiting insect growth was solved, thereby improving the growth, development, and nutritional quality of soybean hawk moth and providing a stable method for producing insect protein sources.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-10
- Publication Date
- 2026-04-03
AI Technical Summary
In existing technologies, anti-nutritional factors such as protease inhibitors and phytic acid in plant leaves inhibit the digestion of insect proteins, leading to poor growth and development of the bean hawk moth and reduced nutritional value. Furthermore, pests, diseases, and photoperiod changes increase production instability, making it difficult to provide a safe and stable source of insect protein.
Using Wild Soybean Mutant No. 4 as an alternative feed for soybean hawk moth, the growth and development of soybean hawk moth were promoted by reducing trypsin inhibitor activity and increasing phytic acid content, and the nutritional quality was improved by preparing feed products containing Wild Soybean Mutant No. 4 leaves.
It significantly increased the body weight and nutritional indicators of fifth-instar larvae of the soybean hawk moth, enhanced the nutritional quality of the soybean hawk moth, provided a stable method for producing insect protein sources, and reduced the impact of anti-nutritional factors.
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Figure CN121773997A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of edible insect breeding technology, specifically relating to the application of Wild Soybean Mutation No. 4 in promoting the growth and development of soybean hawk moth and / or improving its nutritional quality. Background Technology
[0002] Protein is one of the most essential nutrients and the building block of life. Insects have short rearing cycles, high food conversion efficiency, and are rich in protein, with protein being their primary nutrient in considerable quantities. Edible insects are a potential sustainable source of animal protein, capable of meeting the growing demand for novel protein sources.
[0003] The bean hawk moth (Clanis bilineata tsingtauica), belonging to the family Sphingidae in the order Lepidoptera, is an edible insect. This insect is widely considered to have extremely high nutritional value; its larvae are rich in protein, unsaturated fatty acids (UFAs), and vitamins. These nutrients play a role in promoting brain development, preventing cell degeneration, and maintaining endocrine balance. Plant nutrients play a crucial role in insect growth and development. However, protease inhibitors are commonly found in plant leaves, providing significant defense against insects. When insects ingest these protease inhibitors, these compounds form stable complexes with intestinal proteases, thereby inhibiting the activity of protein-digesting enzymes. Simultaneously, the complex of proteases and protease inhibitors may act as a negative feedback signal, further inhibiting insect feeding. This dual effect reduces the efficient utilization of dietary protein and decreases total food intake, ultimately hindering insect development and leading to protein deficiency and death. In addition, phytic acid in plant tissues also plays a role in defending against herbivorous insects. Furthermore, challenges such as pests and diseases, and changes in photoperiod can also reduce the nutritional value of bean hawk moth larvae, posing a significant obstacle to providing safe, stable, and nutritious products to the food industry. Therefore, there is an urgent need to provide an alternative feed that meets the growth and development needs of the soybean hawk moth, so that the production of new protein source products can be controlled, predictable, safe and efficient. Summary of the Invention
[0004] To address the aforementioned technical problems, the primary objective of this invention is to provide the application of Wild Soybean Mutant No. 4 in promoting the growth and development of the soybean hawk moth. Using Wild Soybean Mutant No. 4 as feed, the fifth-instar larvae of the soybean hawk moth showed a 12.9% increase in body weight, a 21.10% increase in crude protein content, and a significant increase in soluble sugar content, indicating that Wild Soybean Mutant No. 4 can serve as a substitute feed for the soybean hawk moth.
[0005] The second objective of this invention is to provide the application of Wild Soybean Mutation No. 4 in improving the nutritional quality of soybean hawk moth.
[0006] A third objective of this invention is to provide the application of the leaves of Wild Soybean Mutant No. 4 in the preparation of feed products that promote the growth and development of soybean hawk moth and / or improve the nutritional quality of soybean hawk moth.
[0007] The fourth objective of this invention is to provide a method for raising soybean hawk moths.
[0008] The fifth objective of this invention is to provide a method for the large-scale cultivation of soybean hawk moth.
[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of wild soybean variant 4 in promoting the growth and development of soybean hawk moth.
[0010] This invention also provides the application of Wild Soybean Mutation No. 4 in improving the nutritional quality of soybean hawk moth.
[0011] As one implementation method, the wild soybean variant 4 can reduce trypsin inhibitor activity and increase phytic acid content.
[0012] The present invention also provides the application of the leaves of wild soybean variant No. 4 in the preparation of feed products that promote the growth and development of soybean hawk moth and / or improve the nutritional quality of soybean hawk moth.
[0013] This invention also provides a method for raising soybean hawk moth, comprising the following steps: inoculating soybean hawk moth eggs onto leaves of wild soybean variant 4, allowing them to hatch naturally; the inoculation amount of soybean hawk moth eggs is 30-50 eggs / m². 2 .
[0014] This invention also provides a method for large-scale rearing of the soybean hawk moth, comprising the following steps: planting wild soybean variant 4, inoculating soybean hawk moth eggs onto soybean leaves, and allowing them to hatch naturally; the inoculation amount of the soybean hawk moth eggs is 30-50 eggs / m². 2 .
[0015] As one implementation method, soybean hawk moth eggs were inoculated onto the underside of the leaves of the wild soybean variant 4.
[0016] In one implementation method, the inoculation time is 30 to 50 days after planting Wild Soybean Mutant No. 4.
[0017] In one implementation method, the row spacing for planting the wild soybean variant 4 is 0.4-0.8 m, and the plant spacing is 0.1-0.2 m.
[0018] As one implementation method, the steps also include covering the planting area of Wild Soybean Mutant 4 with insect-proof netting one week before inoculation.
[0019] The advantages of this invention compared to existing technologies are as follows: This invention provides the application of Wild Soybean Mutation 4 in promoting the growth and development of the soybean hawk moth, clarifying its potential as an alternative feed for the soybean hawk moth. This invention compares and analyzes the nutrient composition and anti-nutritional factor content of leaves from cultivated soybean and Wild Soybean Mutation 4, and compares the nutritional characteristics of the two soybean varieties when feeding soybean hawk moth larvae. From the perspectives of feed composition and larval quality, this invention confirms the feasibility of Wild Soybean Mutation 4 as an alternative feed. Furthermore, this invention, through sequencing and correlation analysis of the phyllosphere microorganisms of cultivated soybean and Wild Soybean Mutation 4, as well as the gut microorganisms of soybean hawk moth larvae, reveals the differences in phyllosphere microbial recruitment between the two soybean varieties, laying the foundation for subsequent research on the microbial regulation of soybean hawk moth quality. Attached Figure Description
[0020] Figure 1 For the cultivation of cultivated soybeans and wild soybean variant 4, and for the rearing of soybean hawk moth larvae; Figure 2 The main anti-nutritional factors in the leaves of cultivated soybean and wild soybean variant 4 are: A, trypsin inhibitor content, and B, phytic acid content. express P <0.05, express P <0.01; Figure 3 Soybean hawk moth larvae reared on leaves of cultivated soybean and wild soybean variant 4. A compares average individual weight, and B compares morphology. express P <0.05; Figure 4 The study included the phyllodes microbiota of cultivated soybean and wild soybean variant 4, as well as the gut microbiota of soybean hawk moth larvae fed on both soybean species. A represents principal component analysis, and B represents cluster heatmap analysis. Gm_L represents the leaves of cultivated soybean, Gs_L represents the leaves of wild soybean variant 4, Gm_Gut represents the gut microbiota of soybean hawk moth larvae fed on cultivated soybean, and Gs_Gut represents the gut microbiota of soybean hawk moth larvae fed on wild soybean variant 4. Figure 5 Phylogenetic tree of differences in the phyllosphere microbiota between cultivated soybean and wild soybean variant 4; Figure 6 Phylogenetic tree of differences in gut microbiota between cultivated soybean and wild soybean variant 4 larvae of soybean hawk moth. Figure 7 Network analysis of the correlation between the phyllosphere microbiota and the gut microbiota of the bean hawk moth. Detailed Implementation
[0021] This invention provides the application of wild soybean variant 4 in promoting the growth and development of soybean hawk moth.
[0022] This invention also provides the application of Wild Soybean Mutation No. 4 in improving the nutritional quality of soybean hawk moth.
[0023] In this invention, the wild soybean strain is variant 4, which has been disclosed in non-patent literature (Li Mengliang, Comparative Study of Wild Soybean Variations in Wuhe, Journal of Anhui University of Science and Technology, 2017, 31(6):15-20). This wild soybean variant 4 can alter plant anti-nutritional factors, reduce trypsin inhibitor activity, and increase phytic acid content, thereby improving the weight and nutritional indicators of soybean hawk moth larvae.
[0024] Based on the fact that Wild Soybean Mutant No. 4 can promote the growth and development of the soybean hawk moth and improve its nutritional quality, this invention also provides the application of Wild Soybean Mutant No. 4 leaves in the preparation of feed products that promote the growth and development of the soybean hawk moth and / or improve its nutritional quality. The feed product of this invention uses Wild Soybean Mutant No. 4 leaves as the main active ingredient. As an optional implementation, the feed product also includes one or more of the following: carbohydrates, protein source supplements, enzyme preparations, mineral premixes, vitamin premixes, preservatives, binders, and antioxidants. In this invention, the dosage form of the feed product includes powder, granules, wet form, paste, or gel.
[0025] This invention also provides a method for raising soybean hawk moth, comprising the following steps: inoculating soybean hawk moth eggs onto leaves of wild soybean variant 4, allowing them to hatch naturally, wherein the inoculation amount of soybean hawk moth eggs is 30-50 eggs / m². 2 .
[0026] This invention also provides a method for the field rearing of soybean hawk moth, which involves planting wild soybean variant 4, inoculating soybean hawk moth eggs onto soybean leaves, and allowing them to hatch naturally; the inoculation amount of soybean hawk moth eggs is 30-50 eggs / m². 2 .
[0027] In this invention, the row spacing for planting Wild Soybean Mutation No. 4 is 0.4–0.8 m, and the plant spacing is 0.1–0.2 m. 30–50 days after planting Wild Soybean Mutation No. 4, soybean hawk moth eggs are inoculated onto the leaves of the plant, preferably 35, 40, or 45 days later. One week before inoculation, the planting area of Wild Soybean Mutation No. 4 is covered with insect-proof netting. As an optional implementation, soybean hawk moth eggs are inoculated onto the underside of the leaves of Wild Soybean Mutation No. 4; the inoculation amount of soybean hawk moth eggs is 30–50 eggs / m². 2 Preferably 35 grains / m 2 40 grains / m 2 or 45 grains / m 2 After inoculation, natural hatching occurs. The soybean hawk moth eggs used in this invention are from commercially available products.
[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of this invention.
[0029] Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available. Unless otherwise specified, they are generally used under conventional conditions or under conditions recommended by the company.
[0030] Test materials: Cultivated soybean: Qihuang 34.
[0031] Wild soybean variant 4: disclosed in non-patent literature (Li Mengliang, Comparative study of variant strains of wild soybean in Wuhe, Journal of Anhui University of Science and Technology, 2017, 31(6):15-20).
[0032] Example 1 This invention was tested in early June 2024 at the experimental field of the Yellow River Delta Modern Agriculture Research Institute, Shandong Academy of Agricultural Sciences. A plot planting model was adopted, with cultivated soybean (Qihuang 34) and wild soybean variant 4 sown separately, with a row spacing of 0.6 meters and a plant spacing of 0.15 meters. Each plant type was planted in an area of approximately 50 square meters, and conventional field management was employed. One week before inoculation with soybean hawk moth eggs, insect-proof netting was used to cover both cultivated soybean and wild soybean variant 4 plots to prevent predation of soybean hawk moth larvae by natural enemies. Forty days after sowing, when the plants reached sufficient biomass, commercially purchased soybean hawk moth eggs were attached to the undersides of the leaves of both cultivated soybean and wild soybean variant 4, with one egg mass (containing 30-50 eggs) placed per square meter, allowing the eggs to hatch naturally (e.g., ...). Figure 1 As shown), from Figure 1 The comparison between C and D shows that, compared to cultivated soybeans, wild soybean variant 4 has a greater number of larvae (also known as bean moths) on its leaves, making it more suitable for the growth of the bean hawk moth.
[0033] The growth status of the soybean hawk moth was monitored in the plots. Observations revealed that the soybean hawk moth mainly feeds on fully unfolded young leaves. When about 70% of the larvae in the field reached the fifth instar, leaves were collected from areas with high larval density on the upper part of the fully unfolded young leaves of cultivated soybean and wild soybean variant 4 plants in both plots. Three sampling points were set up in each plot for nutrient composition analysis and foliar microbial sequencing. Immediately after sampling, the leaves were frozen in liquid nitrogen and stored at -80℃ for later use.
[0034] Three days after leaf collection, fifth-instar larvae and their excrement were collected from the corresponding areas of cultivated soybean and wild soybean variant plot 4. Six to ten larvae were collected from each sampling point, and fresh excrement was collected from the same locations where the larvae were sampled. After collection, the larvae and excrement were immediately frozen in liquid nitrogen at -80°C for later use.
[0035] In this invention, each analytical parameter was measured three times to ensure the reliability and reproducibility of the experimental data. All data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, Illinois, USA) with one-way ANOVA, and Tukey's post-hoc test was used. P <0.05 or P Compare the differences in means at the <0.01 level.
[0036] 1. Nutrient composition of leaves of cultivated soybean and wild soybean variant 4 Leaf nutrient composition is a key indicator for evaluating feed source quality. To clarify the differences between wild soybean variant 4 and cultivated soybean, the leaf nutrient composition was analyzed. The nutrient composition (crude fat, crude protein, crude fiber, ash, neutral detergent fiber, acid detergent fiber, phosphorus, and calcium) in the leaves of cultivated soybean and wild soybean variant 4 were measured respectively.
[0037] The crude fat content was determined using a fully automated fat analyzer; the crude protein content was determined using a fully automated Kjeldahl nitrogen analyzer; the crude fiber, neutral detergent fiber, and acid detergent fiber content were determined using the Pantheon washing method; the crude ash content was determined using the muffle furnace ignition method; and after digestion, phosphorus and calcium were determined using inductively coupled plasma mass spectrometry.
[0038] The experimental results of the nutrient composition of leaves of cultivated soybean and wild soybean variant 4 are shown in Table 1.
[0039] Table 1 Comparison of nutrient components in leaves of cultivated soybean and wild soybean variant 4
[0040] Note: express P <0.01; express P <0.05.
[0041] Table 1 shows that the crude fat content of cultivated soybean leaves is 2.17%, while the crude fat content of wild soybean variant 4 leaves is significantly higher, reaching 2.58 times that of cultivated soybean leaves. Furthermore, the ash, neutral detergent fiber, and calcium content of wild soybean variant 4 leaves are significantly higher than those of cultivated soybean leaves. P <0.01), and the phosphorus content was also significantly higher than that of cultivated soybean leaves ( P<0.05); while the crude fiber content of cultivated soybean leaves was significantly higher than that of wild soybean variant 4 leaves ( P <0.01); there was no significant difference in crude protein and acid detergent fiber content in the leaves of the two varieties. These results indicate that the nutritional composition of the leaves of wild soybean variant 4 is relatively stable, and it is slightly better than cultivated soybean leaves in more nutritional indicators.
[0042] 2. Analysis of the main anti-nutritional components in the leaves of wild soybean variant 4 and cultivated soybean. Antinutritional factors directly interfere with the digestion and absorption of nutrients from the plants consumed by the soybean hawk moth. This invention quantitatively analyzed two key antinutritional factors (trypsin inhibitor and phytic acid) in the leaves of two soybean species, wild soybean variant 4 and cultivated soybean.
[0043] Phytic acid determination: Accurately weigh 5g of leaf samples from cultivated soybean and wild soybean variant 4, respectively, add 40mL of sodium sulfate-hydrochloric acid extraction solution, and extract on a shaker for 4 hours. After centrifuging the extract at 8000 rpm for 5 minutes, take the supernatant and dilute to 50mL with sodium sulfate-hydrochloric acid solution, then filter to obtain the phytic acid extract. Take 2mL of the phytic acid extract, add 2mL of 15% trichloroacetic acid (TCA) solution, mix well, and incubate at 4℃ for 2 hours. After centrifugation, take 2mL of the supernatant, adjust the pH to 6.0-6.5 with 1mol / L sodium hydroxide, and dilute to 30mL with distilled water. The preparation of the standard curve and sample determination were performed according to the national standard (GB5009.153-2016).
[0044] Trypsin inhibitor activity assay: Accurately weigh 1 gram of leaf sample from cultivated soybean and wild soybean variant 4, add 50 mL of 0.01 mol / L sodium hydroxide solution, adjust the pH to 9.5 ± 0.1 with 0.1 mol / L hydrochloric acid solution, and incubate at 4℃ for 24 hours. Remove the extract and equilibrate to room temperature (25℃), add water to a final volume of 100 mL, shake well, let stand for 15 minutes, dilute, and then perform the inhibitory activity assay using trypsin working solution and sample extract according to the national standard method (GB 5009.224-2016).
[0045] Experimental results are as follows Figure 2 The results showed that the trypsin inhibitor activity in cultivated soybean leaves was 254.10±11.11 TIU / g, while that in wild soybean variant 4 leaves was 228.53±8.17 TIU / g. The trypsin inhibitor level in wild soybean variant 4 leaves was significantly lower than that in cultivated soybean. P <0.01, Figure 2(A). The phytic acid content in the leaves of wild soybean variant 4 was 4.33±0.10 mg / g, while the phytic acid content in the leaves of cultivated soybean was 3.90±0.21 mg / g. The phytic acid content in the leaves of wild soybean variant 4 was significantly higher than that in cultivated soybean. P <0.05, Figure 2 (B). These results indicate that the two soybean species have different anti-nutritional properties and may have different effects on the bioavailability of nutrients when used as feed ingredients.
[0046] 3. Weight and nutritional analysis of soybean hawk moth larvae The source of feed has a significant impact on the final weight and nutritional value of soybean hawk moth larvae. This invention determined the weight and nutritional indicators (including crude protein, crude fat, soluble sugar, soluble protein, and potassium) of soybean hawk moth larvae reared on the leaves of two soybean species: wild soybean variant 4 and cultivated soybean. The methods for detecting crude protein and crude fat were the same as above; soluble sugar was determined using the anthrone colorimetric method; soluble protein was determined using the Coomassie Brilliant Blue G-250 staining method; after sample digestion, potassium was determined using inductively coupled plasma mass spectrometry.
[0047] From the experimental results Figure 3 It can be seen that the average weight of the fifth instar larvae of the soybean hawk moth fed with wild soybean variant 4 was 6.846 grams, significantly higher than that of the fifth instar larvae fed with cultivated soybean (6.066 grams), and the growth rate increased by 12.8% (e.g., ...). Figure 3 (As shown in A) Figure 3 Figure B shows a morphological comparison of soybean hawk moth larvae cultured on the leaves of wild soybean variant 4 and cultivated soybean.
[0048] The effects of leaves from different feed sources on the nutritional value of soybean hawk moth larvae are shown in Table 2. The crude protein content of larvae in the wild soybean variant 4 feeding group (672.14 g / kg) was significantly higher than that in the cultivated soybean feeding group (555.02 g / kg). P <0.01%, an increase of 21.10%; conversely, the crude fat content of larvae in the wild soybean variant 4 rearing group (187.44 g / kg) was lower than that in the cultivated soybean rearing group (205.82 g / kg), a decrease of 8.93%. P <0.05). Regarding soluble components, the total soluble sugar content of the larvae in the wild soybean variant 4 rearing group (21.27 mg / g) was significantly higher than that in the cultivated soybean rearing group (8.96 mg / g). P <0.01%, an increase of 137%; the total soluble protein content of larvae in the wild soybean variant 4 feeding group (26.35 mg / g) was slightly higher than that in the cultivated soybean feeding group (24.71 mg / g), and the difference was statistically significant. P<0.05); In addition, the potassium content of larvae in the wild soybean variant 4 rearing group (2.26 g / kg) was significantly lower than that in the cultivated soybean rearing group (2.44 g / kg). P <0.01). Nutritional analysis of soybean hawk moth larvae from different feed sources further showed that feed source significantly affected the nutritional composition of fifth instar larvae of soybean hawk moth.
[0049] Table 2. Comparison of nutritional components of fifth-instar larvae of soybean hawk moth reared from cultivated soybean and wild soybean variant 4.
[0050] Note: express P <0.01; express P <0.05.
[0051] 4. Microbial sequencing and related analysis Microbial sequencing analysis was performed on leaves of cultivated soybean and wild soybean variant 4, as well as excrement from fifth-instar larvae of the soybean hawk moth feeding on these two plants, to investigate the differences in phyllomicron recruitment between the two host plants and their potential impact on larval growth and quality. Total genomic DNA of microorganisms was extracted from leaf and excrement samples using the EasyPure® Genomic DNA Extraction Kit (TransGold, EE101-01) according to the manufacturer's instructions. The V5-V6 region of the bacterial 16S rRNA gene was amplified using primer pairs SEQ ID No. 1: 799F (5'-AACMGGATTAGATACCCKG-3') and SEQ ID No. 2: 1193R (5'-ACGTCATCCCCACCTTCC-3'). The amplified PCR products were mixed in equimolar amounts, and a DNA library was constructed using the SMRTbell prep kit 3.0 (Pacific Biosciences, California, USA) according to the PacBio instructions. Library sequencing was performed by Shanghai Meiji Biomedical Technology Co., Ltd. on a PacBio Sequel IIe system (Pacific Biosciences, California, USA). Circular consensus sequencing was performed using SMRT Link v11.0 software to obtain high-fidelity (HiFi) reads from subreads. Using UPARSE 7.1 software, the optimized HiFi reads were clustered into operational taxonomic units (OTUs) based on 97% sequence similarity, and the most abundant sequence in each OTU was selected as the representative sequence. The OTU table was manually filtered to remove chloroplast sequences from all samples. RDP Classifier version 2.2 was used to perform taxonomic analysis of each OTU representative sequence against a 16S rRNA gene database with a confidence threshold of 0.7. Based on the OTU representative sequences, PICRUSt2 was used to predict metagenomic functions. Bioinformatics analyses of the phyllosphere and gut microbiota were performed on the MajorBio platform (https: / / cloud.majorbio.com).
[0052] A total of 423 OTUs classifiable to the genus level were obtained from all samples. Principal component analysis (PCA) scores are as follows: Figure 4 The results from the A-level analysis showed that the gut microbiota of soybean hawk moth larvae feeding on the two soybean species and the phyllodes microbiota of the two soybean leaves were clearly distinguishable, indicating that the data had good discriminative ability and could be used for subsequent analysis. The heatmap analysis of the top 50 dominant microbial groups at the genus level is shown below. Figure 4 The results showed that there were significant differences in the phyllosphere microbiota of cultivated soybean and wild soybean variant 4 leaves, as well as the gut microbiota of soybean hawk moth larvae feeding on the two soybeans. This indicates that there are significant differences in the composition of the microbiota recruited by the leaves of the two soybeans, and this difference further leads to differences in the composition of the gut microbiota of larvae from different feed sources.
[0053] 5. Comparative analysis of the foliar microbiota of wild soybean variant 4 and cultivated soybean. Phyllostachyal microorganisms colonize the surface of plant leaves, forming a symbiotic relationship with the host. They are crucial for plant development and stress resistance, playing a key role in suppressing potential pathogens and promoting nutrient and carbon cycling. This invention uses linear discriminant analysis (LEfSe) with an LDA threshold > 2.0 to identify differentially enriched taxa in the phyllostachyal microbiota of wild soybean variant 4 and cultivated soybean.
[0054] Analysis results as follows Figure 5 The results showed that, compared with cultivated soybean leaves, the leaves of wild soybean variant 4 were significantly enriched with more diverse microorganisms at multiple taxonomic levels. Specifically, the leaves of wild soybean variant 4 were significantly enriched with 2 phyla (Bacteroidota and Proteobacteria), 2 classes (Alphaproteobacteria and Bacteroidia), 9 orders (such as Acetobacterales, Enterobacterales, and Sphingomonadales), 10 families (such as Acetobacteraceae, Enterobacteriaceae, and Sphingomonadaceae), 14 genera (such as Aureimonas, Brevundimonas, and Sphingomonas), and 13 species (such as Chryseobacterium indologenes, Cronobacter sakazakii, and Sphingomonas hankookensis).
[0055] In contrast, the microbial communities enriched in cultivated soybean leaves are relatively narrow, including 1 phylum (Firmicutes), 1 class (Bacilli), 3 orders (Burkholderiales, Kineosporiales, Lactobacillales), 7 families (such as Burkholderiaceae and Lactobacillaceae), 9 genera (such as Bradyrhizobium, Lactobacillus, and Ralstonia), and 16 species (such as Bacillus velezensis and Lactobacillus plantarum).
[0056] The analysis results show that the phylum of wild soybean variant 4 is significantly enriched with unclassified or uncultured microbial groups. Starting from the class level, the leaves of wild soybean variant 4 contain unclassified groups (such as unclassified p-Proteobacteria), and this trend is present at the order (2 unclassified orders), family (2 unclassified families), genus (5 unclassified genera), and species (15 unclassified or uncultured species). In contrast, cultivated soybean leaves contain fewer such unclassified groups, which only appear at the genus level (1 group) and the species level (3 groups).
[0057] 6. Comparative analysis of the gut microbiota of soybean hawk moths feeding on wild soybean variant 4 and cultivated soybean. The bacterial colonization process during the development of the soybean hawk moth is complex and influenced by rearing conditions, especially feed. Differences in feed composition can lead to variations in palatability and nutritional quality, which may affect the colonization of native gut bacteria. This is a key factor in determining whether beneficial bacteria or probiotics can successfully colonize the digestive tract.
[0058] Linear discriminant analysis (LEfSe) with an LDA threshold > 2.0 was used to identify differentially enriched groups in the gut microbiota of soybean hawk moths feeding on two soybean species. The experimental results are as follows: Figure 6The results showed that the gut microbiota of larvae feeding on the leaves of wild soybean variant 4 was significantly enriched with a wider range of taxa, including 2 phyla (Bacteroidota and Firmicutes), 2 classes (Bacilli and Bacteroidia), 5 orders (Cellvibrionales, Flavobacteriales, Lactobacillales, Sphingobacteriales, and Staphylococcales), 7 families, 10 genera (including Sphingobacterium, which is shared with the leaf microbiota of wild soybean variant 4), and 8 species. Among them, the Bacteroidota, Bacteroidia, and Sphingobacteriales groups in the larval gut microbiota were consistent with the leaf microbiota of wild soybean variant 4. In contrast, the gut microbiota of larvae feeding on cultivated soybean leaves is more limited in its enrichment of taxa, including only Proteobacteria, Gammaproteobacteria, Peptostreptococcales-Tissierellales, 4 families (Alcaligenaceae, Burkholderiaceae, Lactobacillaceae, Streptococcaceae), 5 genera (including Lactobacillus and Ralstonia), and 3 species (including Ralstonia solanacearum). Among these, the Lactobacillus, Ralstonia, and Ralstonia solanacearum species in the larval gut microbiota are consistent with the foliar microbiota of cultivated soybean.
[0059] The analysis results show that a significant enrichment of unclassified or uncultured taxa was observed in the gut of larvae feeding on wild soybean variant 4, and this enrichment was present at all taxonomic levels: 1 unclassified phylum (p_unclassified_d_Bacteria), 1 unclassified class, 2 unclassified orders, 3 unclassified families, 4 unclassified genera, and 10 unclassified or uncultured species. In contrast, such unclassified taxa were much less abundant in the gut of larvae feeding on cultivated soybeans, present only at the genus level (2) and the species level (3).
[0060] 7. Potential transmission and synergistic effects between the gut microbiota of the soybean hawk moth and the phyllosphere microbiota of wild soybean variant 4. To investigate the potential transmission relationship between the gut microbiota of the soybean hawk moth and the phyllosphere microbiota of wild soybean variant 4, this invention further grouped the gut microbiota of the soybean hawk moth from different feed sources with the phyllosphere microbiota of the leaves it ingested, and conducted correlation network analysis. The network diagram results are shown below. Figure 7 The results showed a significant correlation between the phyllosphere microbiota and the gut microbiota at the OTU level. These interacting microbes were divided into 5 modules: Module 1 contained 17 nodes, of which 5 were phyllosphere OTUs and 12 were gut OTUs; Module 2 contained 25 nodes, of which 5 were phyllosphere OTUs and 20 were gut OTUs; the remaining 3 modules each contained 2 nodes.
[0061] In Module 2, positive correlations were predominantly observed. Strong positive correlations were found between L_OTU370_o_Enterobacterales and OTU131_o_Enterobacterales, and between L_OTU48_s_Deinococcus antarcticus and OTU189_s_uncultured_Deinococcales_bacterium, suggesting that these bacterial strains may be transferred from feed to the insect gut at the OTU level.
[0062] Conversely, Module 1 primarily exhibits negative correlations, with these antagonistic relationships mainly occurring between phyllodes-phyllodes or gut-gut OTUs, potentially reflecting competition between microorganisms in the same ecological niche. Meanwhile, the correlations between phyllodes and gut microbiota are mostly positive, suggesting a potential synergistic or co-enrichment effect between the phyllodes microbiota of wild soybean variant 4 leaves and the insect gut microbiota.
[0063] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.
Claims
1. Application of Wild Soybean Mutation No. 4 in promoting the growth and development of soybean hawk moth.
2. Application of Wild Soybean Mutation No. 4 in improving the nutritional quality of soybean hawk moth.
3. The application according to claim 1 or 2, characterized in that, The described wild soybean variant 4 can reduce trypsin inhibitor activity and increase phytic acid content.
4. Application of leaves of wild soybean variant No. 4 in the preparation of feed products that promote the growth and development of soybean hawk moth and / or improve the nutritional quality of soybean hawk moth.
5. A method for raising soybean hawk moths, characterized in that, Includes the following steps: Soybean hawk moth eggs were inoculated onto leaves of wild soybean variant 4 and allowed to hatch naturally; the inoculation density was 30-50 eggs / m². 2 .
6. A method for large-scale rearing of the soybean hawk moth, characterized in that, Wild soybean variant 4 was planted, and soybean hawk moth eggs were inoculated onto soybean leaves, where they hatched naturally; the inoculation amount of soybean hawk moth eggs was 30-50 eggs / m². 2 .
7. The field aquaculture method according to claim 6, characterized in that, Soybean hawk moth eggs were inoculated on the underside of leaves of the aforementioned wild soybean variant 4.
8. The field aquaculture method according to claim 6 or 7, characterized in that, The inoculation time is 30-50 days after planting Wild Soybean Mutant No.
4.
9. The field aquaculture method according to claim 6 or 7, characterized in that, The row spacing for planting the wild soybean variant 4 was 0.4-0.8 m, and the plant spacing was 0.1-0.2 m.
10. The field aquaculture method according to claim 6 or 7, characterized in that, The steps also include covering the planting area of Wild Soybean Mutant 4 with insect-proof netting one week before inoculation.