Method for improving cold resistance of telenomus sp. based on three-level nutrition transmission relationship

Abstract

The present application belongs to the technical field of biological control, and provides a method for improving the cold resistance of noctuid black tachinid based on a three-level nutrition transmission relationship of "artificial feed-host-parasitic wasp", which is to use exogenous additives to prepare artificial feed to feed the host Spodoptera litura and make it lay eggs, and then let the parasitic wasp noctuid black tachinid lay eggs in the host using the eggs of Spodoptera litura as the host, so as to break through the industrial problems of weak cold resistance and short shelf life of noctuid black tachinid, and finally realize the sustainable control of the major invasive pest Spodoptera frugiperda. The results show that the addition of 1% trehalose / sucrose in the feed has no obvious inhibitory effect on the survival and development of Spodoptera litura, and can significantly improve the finite rate of increase and intrinsic rate of increase of Spodoptera litura, and shorten the generation cycle. At the same time, the cold resistance of noctuid black tachinid parasitizing in the host eggs can be improved, so as to prolong the shelf life of noctuid black tachinid products and reduce the cost of large-scale breeding.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology and relates to a black egg wasp of the noctuid moth, specifically a method for improving the cold resistance of the black egg wasp of the noctuid moth based on a three-level nutrient transfer relationship. Background Technology

[0002] The fall armyworm (Spodoptera frugiperda) (JESmith, 1979), also known as the autumn armyworm, is a major migratory agricultural pest that newly invaded China in 2019. It possesses biological characteristics such as high reproductive capacity, wide host range, rapid spread, strong adaptability, and high degree of damage, posing a significant threat to maize production and food security in my country. In light of this, my country's agricultural authorities have listed the fall armyworm as the top pest in the "List of Class A Crop Diseases and Pests."

[0003] Research has found that biological control technologies centered on protecting and utilizing natural enemy resources are an effective strategy for the sustainable control of fall armyworm. Among these, the black-egg wasp (Anoplophora spp.) has become the dominant egg parasitoid for controlling fall armyworm due to its high reproductive rate and unique advantage of efficiently parasitizing the inner egg masses of noctuid moths. For example, in Brazil, a flood release of 90,000-120,000 wasps per hectare achieved a parasitism efficiency of up to 74% on fall armyworm eggs in maize fields. In China, the combined application of the predatory natural enemy, the black-egg wasp, and the control effect on fall armyworm reached 86.61%-93.33%, with plant damage rate and leaf damage registration decreasing by 35% and 43.04% respectively compared to the control, recovering approximately 30%-60% of maize yield loss.

[0004] Currently, through preliminary research on alternative host selection, propagation conditions, storage conditions, and product quality control, a propagation system for the parasitic wasp *Spodoptera litura* using *Spodoptera litura* eggs as an alternative host has been established. However, we have also found that the relatively weak cold resistance of *Spodoptera litura* is one of the main factors leading to its short shelf life, high production costs, and limited application scope, representing a significant bottleneck in the large-scale breeding and promotion of this parasitic wasp. Furthermore, through omics analysis and determination of cold-resistant substance content, we discovered that the starch and sucrose metabolic pathways of *Spodoptera litura* play a crucial regulatory role under low-temperature stress. Significant accumulation of trehalose-6-phosphate and sucrose metabolites was observed in these pathways. Therefore, a suitable method to enhance the cold resistance of *Spodoptera litura* is particularly important for improving the efficiency of large-scale breeding and reducing production costs. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the cold resistance of the black egg wasp of the beet armyworm based on a tertiary nutrient transfer relationship. Without affecting the normal growth and development of the beet armyworm and the parasitic ability of the black egg wasp, the cold resistance of the natural enemy insect can be effectively improved by appropriately increasing the amount of exogenous metabolites. This invention provides important experimental evidence and theoretical support for the field of insect biological control, which is of great significance for improving the storage efficiency of biological control agents in low-temperature environments.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a method for improving the cold resistance of the black-egg parasite *Ichthyophthirius multifiliis* based on a three-level nutrient transfer relationship. The method involves adding exogenous metabolites as cold-resistant substances to artificial feed, and transferring the cold-resistant substances step by step through the three-level nutrient transfer relationship of artificial feed-host-parasitic wasp to improve the cold resistance of the black-egg parasite.

[0008] Preferably, the three-level nutrient transfer process of artificial feed-host-parasitic wasp is as follows:

[0009] (1) Prepare artificial feed containing exogenous metabolites;

[0010] (2) Feed the host beet armyworm with the artificial feed from step (1) and induce it to lay eggs;

[0011] (3) The female black egg wasp of the beet armyworm lays its eggs in the host using the beet armyworm eggs from step (2). The female wasp is removed, and the beet armyworm eggs continue to develop under the same conditions until the offspring emerge.

[0012] Preferably, the mass percentage of the exogenous metabolite is 0.1% to 5%.

[0013] Preferably, the exogenous metabolite accounts for 1% of the total mass.

[0014] Preferably, the Spodoptera litura eggs in step (3) are obtained by feeding with artificial feed with or without the addition of exogenous metabolites.

[0015] Preferably, the exogenous metabolite is trehalose or sucrose.

[0016] Preferably, the trehalose or sucrose is transferred through an artificial feed-host secondary relationship chain and eventually accumulates in the eggs of the beet armyworm.

[0017] This invention also provides the role of the above-described method in improving the periodic growth rate and intrinsic growth rate of the beet armyworm.

[0018] This invention also provides the role of the above-mentioned method in shortening the generation cycle of the beet armyworm and improving the population's reproductive capacity.

[0019] The present invention also provides the role of the above-described method in improving the cold resistance of the black egg wasp of the noctuid moth.

[0020] The beneficial effects of this invention are:

[0021] This invention is based on the three-level nutrient transfer relationship of "artificial feed-host-parasitic wasp". By adding exogenous metabolites, it achieves the stepwise delivery of cold-resistant substances, which can improve the cold resistance of the black-egg wasp of the noctuid moth. This is of great significance for extending the shelf life of black-egg wasp products and reducing the cost of large-scale breeding. Attached Figure Description

[0022] Figure 1 This invention describes the developmental stages of Spodoptera litura larvae after consuming artificial feeds with different proportions of trehalose (CK is the control group of conventional artificial feed without added exogenous metabolites; different letters indicate significant differences between groups).

[0023] Figure 2 This invention describes the developmental stages of Spodoptera litura larvae after consuming artificial feeds with different proportions of sucrose (CK is the control group of conventional artificial feed without added exogenous metabolites; different letters indicate significant differences between groups).

[0024] Figure 3 This refers to the mortality rate of Spodoptera litura larvae after consuming artificial feed supplemented with trehalose in this invention (CK is the control group of conventional artificial feed without the addition of exogenous metabolites; different letters indicate significant differences between groups).

[0025] Figure 4 This refers to the mortality rate of Spodoptera litura larvae after consuming artificial feed supplemented with sucrose in this invention (CK is the control group of conventional artificial feed without the addition of exogenous metabolites; different letters indicate significant differences between groups).

[0026] Figure 5 These are the population parameters of *Spodoptera litura* after consuming different formulated artificial feeds in this invention (A is the weekly growth rate; B is the intrinsic growth rate; C is the net proliferation rate; D is the generation cycle; CK is the control group of conventional artificial feed without added exogenous metabolites); * P < 0.05 ** P < 0.01, *** P < 0.001 (ns = no difference compared with the CK group);

[0027] Figure 6 This invention describes the performance of the black egg wasp parasitizing three types of host eggs (A represents the number of parasitized eggs; B represents the emergence rate; C represents the female ratio; CK represents the control group of conventional artificial feed without added exogenous metabolites; different letters indicate significant differences between groups).

[0028] Figure 7The mortality rate of first-instar larvae of the black egg wasp of the noctuid moth under low temperature stress in this invention (CK is the control group of conventional artificial feed without the addition of exogenous metabolites); * P < 0.05 ** P < 0.01, *** P < 0.001 (compared with the Control group). Detailed Implementation

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] Application Examples

[0033] Based on the three-level nutrient transfer relationship of "artificial feed-host-parasitic wasp", the cold resistance of the black-egg wasp can be improved by adding exogenous metabolites to achieve the stepwise delivery of cold-resistant substances. This is of great significance for extending the shelf life of the black-egg wasp product and reducing the cost of large-scale breeding.

[0034] 1 Experimental Methods

[0035] 1.1 Effects of exogenous metabolite addition on the growth and development of the host plant, *Spodoptera litura*

[0036] Based on the biological phenomenon observed in previous studies of the accumulation of trehalose-6-phosphate and sucrose metabolites in the beet armyworm *Spodoptera litura* under low-temperature stress, this invention utilizes a three-tiered nutrient transfer mechanism—artificial feed-host-parasitic wasp—to deliver cold-resistance substances step-by-step. This ensures that the beet armyworm has sufficient cold-resistance substances to withstand low-temperature stress, thereby enhancing its cold resistance. Within organisms, trehalose and trehalose-6-phosphate have a close biochemical relationship, and their metabolic pathways are interconnected. Trehalose-6-phosphate is a key intermediate in the trehalose metabolic pathway; trehalose can be converted to trehalose-6-phosphate by enzymes, and trehalose-6-phosphate can also be converted back to trehalose. Furthermore, trehalose is cheaper than trehalose-6-phosphate, making its use more cost-effective in large-scale experiments or production. For these reasons, this invention uses trehalose and sucrose as exogenous metabolites to clarify their effects on the growth and development of the host plant, *Spodoptera litura*.

[0037] Trehalose and sucrose were purchased from Shanghai Yuanye Biotechnology Co., Ltd. and Beijing Kulaibo Technology Co., Ltd., respectively. In this invention, exogenous metabolites were added at five different ratios (mass ratios): 0.1%, 0.5%, 1%, 3%, and 5%. Artificial feeds for *Spodoptera litura* containing different proportions of exogenous metabolites were prepared. When the temperature dropped to approximately 40°C, the corresponding proportion of exogenous metabolites was added, and the mixture was quickly stirred to ensure thorough mixing with the feed. After packaging, the mixture was allowed to cool and solidify at room temperature and then stored in a 4°C refrigerator for later use. A conventional artificial feed without added exogenous metabolites was used as a control (Table 1).

[0038] Table 1. Addition ratios of trehalose and sucrose in artificial feeds and corresponding formulation names.

[0039]

[0040] Larvae of the beet armyworm hatched on the same day were selected, and each larva was inserted individually into a finger-shaped tube (diameter × height: 2.5cm × 10cm) containing 60g of artificial feed. The tube was sealed with a sponge plug and placed in a rearing room to develop until pupation. During the rearing process, the survival status, developmental progress, and pupation date of the larvae were recorded daily. After pupation, the pupation rate, pupal period, and weight of male and female pupae were recorded. After adult emergence, the larvae were paired at a 1:1 female-to-male ratio and reared in rearing cages until the female moths died. Parameters such as emergence rate, sex ratio, egg production, and survival time were recorded. Each treatment was repeated 6 times, with 12 test larvae observed in each replicate. By comprehensively analyzing the survival, development, and reproductive parameters of the beet armyworm after consuming feed containing different proportions of exogenous metabolites, the optimal proportion of exogenous metabolites could be determined.

[0041] 1.2 Effects of exogenous metabolite addition to host feed on the parasitism ability of black-egg wasps in noctuid moths

[0042] Based on the optimal addition ratio of exogenous metabolites screened in the above studies, the effect of host eggs produced by *Spodoptera litura* after feeding on an artificial diet containing 1% trehalose (formula c) and 1% sucrose (formula h) on the parasitism of *Spodoptera litura* larvae wasp was evaluated. Egg cards containing approximately 200 *Spodoptera litura* eggs (<24h) were placed in transparent finger-shaped tubes with a small amount of 20% honey water smeared on the inner wall. A female *Spodoptera litura* larvae that had emerged within 12 hours and had mated was then introduced into the tube. The tube was sealed with a sponge plug and placed in a rearing chamber for development. After 24 hours, the female wasp was removed, and the egg cards continued to develop under the same conditions until the offspring emerged. During this process, hatched *Spodoptera litura* larvae were promptly removed to prevent them from feeding on the parasitized eggs. The number of parasitized eggs and the number of male and female offspring wasps were counted. Each treatment was repeated 5 times, with 6 female wasps observed in each replicate.

[0043] 1.3 Effects of exogenous metabolite addition to host feed on cold resistance of black-egg wasp *Hemiberlesia lataniae*

[0044] The exogenous metabolite addition protocol was based on the findings in section 1.2. A 0.5cm diameter hole was made in the center of a 2cm x 2cm square card. Fresh, plump *Spodoptera litura* eggs were evenly attached to the hole using double-sided tape to create egg cards. These parasitized egg cards were cultured at 26±1℃, 70±5% relative humidity, and a photoperiod of 14L:10D for 48, 72, 96, and 120 hours, respectively, until they developed into 1st instar larvae, 2nd instar larvae, prepupae, and pupae. At this stage, the *Spodoptera litura* wasps at each developmental stage were placed in thin-walled centrifuge tubes, sealed, and then placed in a water bath circulator at -5℃ for 4, 8, 12, and 24 hours. After the low-temperature treatment, the wasps were transferred to room temperature for rearing, and the number of adult wasps that emerged after 12 days was counted. Each treatment was repeated 10 times.

[0045] 1.4 Data Analysis

[0046] Tukey's Honestly Significant Difference (HSD) test was used to analyze the significant differences in developmental duration and larval mortality of different developmental stages of *Spodoptera litura* after feeding with different feed formulations (P < 0.05). This method was also used to analyze the significant differences in parasitized egg quantity, offspring emergence rate, sex ratio, and survival rate after low-temperature treatment of the *Spodoptera litura* wasp on different host eggs (P < 0.05). SPSS 19.0 was used for data analysis in this experiment.

[0047] Based on the age-stage sex life table theory, TWOSEX-MSChart software was used to analyze life table parameters of the beet armyworm population, including net proliferation rate (R0), periodic growth rate (λ), intrinsic growth rate (r), and generation period (T). The bootstrap technique (100,000 iterations) was used to estimate the standard errors of the life table parameters, and the paired bootstrap test was used for significance analysis.

[0048] 2 Experimental Results

[0049] 2.1 Effects of exogenous metabolite addition on the growth and development of the host plant, *Spodoptera litura*

[0050] The developmental stages of Spodoptera litura larvae after consuming feed containing different proportions of trehalose are as follows: Figure 1 As shown in the figure. The results indicated that the larval stage after consuming feed formula e was the longest, reaching 27.7 days; conversely, the larval stage after consuming feed formula c was only 18.6 days. There was no significant difference in the larval stage of the beet armyworm after consuming feed formulas a, b, c, and CK, but it was significantly shorter than that after consuming feed formulas d and e.

[0051] Figure 2The developmental period of Spodoptera litura larvae after feeding on artificial feeds containing different proportions of sucrose was shown. It can be seen that the larval stages of Spodoptera litura larvae fed on feeds containing g and h were 19.4 days and 18.6 days, respectively, which were not significantly different from the control (20.7 days), but were significantly shorter than those fed on feeds containing f (24.4 days), j (26.1 days), and k (32.2 days).

[0052] An evaluation of the biological characteristics of *Spodoptera litura* after feeding on diets containing different proportions of trehalose revealed that the larval mortality rate was as high as 44.44% after feeding on diet formula e, which was significantly different from the mortality rates after feeding on diets formulas CK, a, b, and c. Figure 3 ).

[0053] The mortality rates of Spodoptera litura larvae after feeding on feeds formulated with J and K were 41.67% and 81.94%, respectively, significantly higher than the mortality rate after feeding on feed formulated with CK (6.94%). Figure 4 ).

[0054] Based on the above assessment of the developmental period, mortality rate, and basic biological characteristics of *Spodoptera litura* after feeding on different feed formulations, a comprehensive analysis concluded that the growth, development, and survival of *Spodoptera litura* fed on feed formulations d, e, j, and k were significantly worse than the control, failing to meet the normal survival and development requirements of *Spodoptera litura*. Therefore, these four formulations were excluded. To better enrich the cold-resistance substances required by the black-egg wasp *Spodoptera litura* in the host eggs of *Spodoptera litura*, formulations c and h were selected as candidate formulations. This was to further evaluate the parasitic performance and cold resistance level of *Spodoptera litura* eggs bred using these feed formulations.

[0055] Analysis of life table parameters showed that the net proliferation rate (R0) of *Spodoptera litura* fed with c and h formulated diets was not significantly different from the control, while the finite growth rate (λ) and intrinsic growth rate (r) were significantly higher, and the generation cycle (T) was significantly shorter. This indicates that c and h formulated diets not only meet the normal developmental needs of *Spodoptera litura*, but also significantly enhance its population reproduction. Figure 5 ).

[0056] 2.2 Effects of exogenous metabolite addition to host feed on the parasitism ability of black-egg wasps in noctuid moths

[0057] Using eggs laid by *Spodoptera litura* after feeding on control, c-formula, and h-formula diets as hosts (hereinafter referred to as control eggs, c-formula eggs, and h-formula eggs, respectively), the performance of the black egg wasp on parasitized eggs was evaluated. The results showed no significant differences in the number of parasitized eggs and the female-to-female ratio among the three egg types; the number of parasitized eggs was 65.1, 61.4, and 62.2, respectively, with female-to-female ratios of 79%, 78.7%, and 77.2%, respectively. There were significant differences in the emergence rate of the offspring wasps on the three host eggs, with the highest being 94.2% on the control and the lowest being 90.3% on the c-formula eggs. Figure 6 ).

[0058] 2.3 Effects of exogenous metabolite addition to host feed on cold resistance of black-egg wasp *Hemiberlesia lataniae*

[0059] The mortality rates of black ovipositor wasps parasitizing eggs treated with different temperatures at various developmental stages are as follows: Figure 7 As shown in the figure. The results indicated that the mortality rates of first-instar larvae of the noctuid moth parasitizing c-formula eggs after 8, 12, and 24 hours of low-temperature treatment were 9.36%, 13.57%, and 26.30%, respectively, significantly lower than the control mortality rate, with reductions of 29.31%, 29.54%, and 14.72%, respectively. The mortality rate of first-instar larvae parasitizing h-formula eggs after 24 hours of low-temperature treatment was significantly lower than the control, decreasing by 13.88%. These results show that the mortality rates of noctuid moth parasitizing c-formula and h-formula eggs decreased to varying degrees after low-temperature stress compared to the control, with reductions ranging from 13.88% to 29.54%.

[0060] In conclusion, based on the three-tiered nutrient transfer relationship between artificial feed, host, and parasitic wasp, the addition of 1% trehalose / sucrose to the feed did not inhibit the survival and development of *Spodoptera litura*, and significantly increased its cyclical growth rate and intrinsic growth rate, shortening the generation cycle. The parasitic ability of *Spodoptera litura* was also not significantly affected. Further evaluation of the mortality rate of *Spodoptera litura* parasitizing the host eggs after low-temperature stress revealed a decrease in mortality compared to the control, ranging from 13.88% to 29.54%. In conclusion, the addition of 1% trehalose / sucrose to the host feed effectively enhances the cold resistance of *Spodoptera litura*.

[0061] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for improving the cold resistance of the black egg wasp of the noctuid moth based on a tertiary trophic transport relationship, characterized in that, The method involves adding exogenous metabolites as cold-resistant substances to artificial feed, and transferring the cold-resistant substances step by step through a three-level nutrient transfer relationship between artificial feed, host, and parasitic wasp to improve the cold resistance of the black egg wasp of the noctuid moth; the exogenous metabolites are trehalose or sucrose. The three-tiered nutrient transfer process of artificial feed-host-parasitic wasp is specifically as follows: (1) Prepare artificial feed containing exogenous metabolites; (2) Feed the host beet armyworm with the artificial feed from step (1) and induce it to lay eggs; (3) The female black egg wasp of the beet armyworm lays its eggs in the host using the beet armyworm eggs from step (2). The female wasp is removed, and the beet armyworm eggs continue to develop under the same conditions until the offspring emerge.

2. The method according to claim 1, characterized in that, The mass percentage of the exogenous metabolites is 0.1% to 5%.

3. The method according to claim 2, characterized in that, The mass percentage of the exogenous metabolite is 1%.

4. The method according to claim 1, characterized in that, The Spodoptera litura eggs mentioned in step (3) were obtained by feeding the animals with artificial feed with or without the addition of exogenous metabolites.

5. The method as described in claim 1, characterized in that, The trehalose or sucrose is transferred through a secondary relationship between artificial feed and host, and is eventually enriched in the eggs of the beet armyworm.

6. The effect of the method as described in any one of claims 1-4 in increasing the periodic growth rate and intrinsic growth rate of the host plant *Spodoptera litura*.

7. The effect of the method described in any one of claims 1-4 in shortening the generation cycle of the beet armyworm and improving population reproductive capacity.

8. The effect of the method as described in any one of claims 1-4 in improving the cold resistance of the black egg wasp of the noctuid moth.

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