Method and system for determining indoor toxicity of konjac alkaloid to prodenia litura and trichogramma chilonis

Abstract

The invention belongs to the technical field of toxicity determination, and discloses a method for determining indoor toxicity of konjak alkaloid to prodenia litura and trichogramma chilonis, and the indoor toxicity of the konjak alkaloid to the prodenia litura and the natural enemy insect trichogramma chilonis is determined through indoor toxicity determination, so that the method for determining the indoor toxicity of the konjak alkaloid to the prodenia litura and the natural enemy insect trichogramma chilonis is provided. Research results can provide scientific basis for ecological management of prodenia litura and further development and utilization of konjac alkaloid. The invention finds that the konjak alkaloid has strong toxicity to prodenia litura and has the potential of researching and developing a botanical insecticide applied to prevention and treatment of prodenia litura, and whether the konjak alkaloid has similar toxicity to other common lepidoptera pests needs to be further researched. The toxicity of the konjac alkaloid to a parasitic natural enemy insect, namely trichogramma chilonis, of the prodenia litura is measured at the same time, and although the toxicity of the konjac alkaloid to the trichogramma chilonis is weaker than that of the prodenia litura, the parasitic ability of the trichogramma chilonis to eggs of the prodenia litura can still be affected by the lower lethal concentration of the konjac alkaloid.

Description

Technical Field

[0001] This invention belongs to the field of toxicity determination technology, and in particular relates to an indoor toxicity determination method and system for konjac alkaloids against Spodoptera litura and Trichogramma pygmy spinosa. Background Technology

[0002] The beet armyworm (Spodoptera litura), belonging to the family Noctuidae in the order Lepidoptera, is a widely distributed, polyphagous pest that can damage nearly 400 plant species, including various crops and ornamental plants. The beet armyworm primarily feeds on plant leaves as larvae, which can severely hinder plant growth and even cause death. As a significant global agricultural pest, it reproduces rapidly, has high population densities, and exhibits strong resistance to pesticides, posing a serious threat to many crops.

[0003] Chemical control has played a crucial role in controlling the beet armyworm, but the improper use of chemical pesticides may lead to increased pesticide resistance in beet armyworm populations, reducing control effectiveness and increasing pesticide usage. Therefore, exploring eco-friendly and green control measures for beet armyworm is extremely urgent. Plant secondary metabolites are a class of non-essential organic compounds in plant growth and development, playing an important role in plant defense against insects and herbivores. Alkaloids are a class of heterocyclic compounds containing basic nitrogen atoms, widely distributed in plants. Most alkaloids exhibit strong biological activity in insecticidal, antifeedant, and reproductive inhibition. For example, plants such as *Aglaia* (family Meliaceae) contain benzofuran alkaloid derivatives, which can produce significant toxic effects on pests after ingestion, with toxicity comparable to azadirachtin. Taking advantage of the toxicity of alkaloids, my country has produced plant-derived insecticides with active ingredients including nicotine, matrine, and tea saponin, providing support for the integrated management of agricultural pests in my country.

[0004] Konjac belongs to the genus Amorphophallus in the family Araceae. It is a perennial herbaceous plant containing a large amount of alkaloids, but its toxicity to common pests and their natural enemies has not been reported.

[0005] Patent CN107172998A discloses a method for controlling the beet armyworm using a compound aqueous solution of konjac leaves and humic acid. This document describes a method for controlling the beet armyworm by soaking fresh konjac leaves to extract an aqueous solution, mixing it with humic acid in different proportions, and then conducting toxicity tests on third-instar beet armyworm larvae using an immersion method at concentrations ranging from 2000 mg / L to 31.25 mg / L. The co-toxicity coefficient (CTC) was used to evaluate the synergistic effect of the two compounds, and the results showed that a significant synergistic effect could be obtained under specific ratios.

[0006] However, this literature is limited to using crude water extracts (mainly mixtures of multiple components), failing to isolate and quantify the active component, konjac alkaloids, nor to assess synergistic or antagonistic effects with commonly used chemical pesticides, and further failing to conduct toxicity tests on the important natural enemy, the Trichogramma chilonis wasp. Furthermore, its toxicity testing focuses solely on the larvae of the beet armyworm, lacking evaluation of the parasitic ability and safety of the parasitic Trichogramma chilonis wasp, making it difficult to guide integrated pest management under ecologically safe conditions in the field. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides an indoor method for determining the toxicity of konjac alkaloids to the beet armyworm and the Trichogramma pygmy spinosa.

[0008] This invention is achieved as follows: a method for determining the indoor toxicity of konjac alkaloids to *Spodoptera litura* and *Trichogramma pygmygdalis* includes:

[0009] Step 1, extraction of konjac alkaloids;

[0010] Step 2: Determination of the toxicity of konjac alkaloids and commonly used insecticides to *Spodoptera litura* and *Trichogramma pygmysis*.

[0011] Step 3: Determination of the parasitic function response of Trichogramma pygmygdalus to Spodoptera litura eggs;

[0012] Step 4, statistical analysis.

[0013] Furthermore, the konjac alkaloids are extracted as follows:

[0014] Healthy konjac corms were taken, washed with clean water, sliced, and dried in an oven at 50°C until constant weight. The dried konjac powder was then pulverized and passed through a 40-mesh sieve. It was then extracted with 95% ethanol for 24 hours (konjac powder: 95% ethanol = 1:20) to obtain an ethanol extract. The extract was then vacuum filtered using a rotary evaporator (temperature = 55°C) and concentrated to a viscous consistency. The viscous liquid was adjusted to pH 9.0 with 1 mol / L NaOH, extracted five times with ethyl acetate, and the organic layers were combined and concentrated again. The final separated viscous liquid was the total alkaloids. The total alkaloid content in the extract was determined using a spectrophotometer and a total alkaloid content assay kit (Grace Biotech Co., Ltd., Suzhou, China). An alkaloid solution (100 mg / mL) was prepared using methanol as a solvent.

[0015] Furthermore, the toxicity of the konjac alkaloids to common insecticides against the beet armyworm and the Trichogramma pygmy spiny eel wasp was determined:

[0016] The prepared konjac alkaloids and insecticides were dissolved and diluted with water. Based on the preliminary test results, the agents used in this experiment were diluted into five different gradient concentrations within the range of 10% to 90% corrected mortality rate. Water was used as the test control, and three replicates were set for each concentration value. The toxicity of Spodoptera litura eggs was determined by the immersion method. The egg masses of Spodoptera litura were gently peeled off with tweezers, leaving 10 eggs in each mass. They were then immersed in different concentrations of the pesticide solution for 10 seconds, removed, and the excess liquid on the surface was absorbed with absorbent paper before air drying. Afterward, they were placed on moist filter paper to maintain the humidity required for egg hatching. The toxicity of Spodoptera litura larvae was determined by the feeding method. The artificial feed used to feed Spodoptera litura was cut into cubes of 1cm×1cm×0.5cm and immersed in different concentrations of the pesticide solution for 10 seconds before air drying. Ten Spodoptera litura larvae of different instars were starved for 12 hours. The insects were placed in separate testing containers (diameter r = 5 cm, height 6 cm) and fed artificial feed soaked in different concentrations of pesticide solutions. For the toxicity determination of adult Spodoptera litura and Trichogramma pygmyrus wasps, a feeding method was used. The test pesticide was prepared at different concentrations using 10% honey water. Ten male and female Spodoptera litura adults within 24 hours of emergence were placed in separate testing cages (20 cm × 20 cm × 20 cm). Trichogramma pygmyrus wasps within 24 hours of emergence were also tested. Ten male and ten female adult bees were placed in separate glass tubes (2cm in diameter × 6cm in height) and starved for 12 hours before being fed a solution of honey water. All treatments were placed in an artificial climate chamber at 25℃ and 60%-70% humidity. The mortality of Spodoptera litura eggs was recorded after the water treatment group had hatched normally. The mortality of Spodoptera litura larvae, adults, and Trichogramma pygmygdalites was checked and recorded 24 hours after treatment.

[0017] Furthermore, the parasitic functional response of the *Trichogramma pygmygdalus* to the eggs of *Spodoptera litura* was determined:

[0018] Konjac alkaloids were prepared into different lethal concentrations (LC5, LC25, and LC50) using a 10% honey solution. Using a feeding method, female Trichogramma pygmy borers (within 24 hours of emergence) were starved for 12 hours and then fed with different lethal concentrations of konjac alkaloids. After 24 hours of feeding, the still-active Trichogramma pygmy borers were collected for later use. The number of eggs in the egg masses of *Spodoptera litura* was adjusted to 5, 10, 20, 40, 60, 80, and 100 using insect needles, and then placed in [locations missing]. One Trichogramma pygmy horn wasp treated with different concentrations of konjac alkaloids was placed in each transparent plastic container. After the wasp had been allowed to parasitize freely in the container for 24 hours, it was removed. The plastic containers were then moved into an artificial climate chamber (temperature 25℃, humidity 60%–70%, light intensity 10000 lx, L / / D = 14h / / 10h) to observe the parasitism of Spodoptera litura eggs until all Spodoptera litura eggs hatched or died [21-22]. Water treatment was used as a control, and each treatment was repeated 3 times.

[0019] Furthermore, the statistical analysis includes:

[0020] Data analysis was performed using SPSS 20.0. Regression equations, median lethal concentrations (LC50), and 95% confidence intervals were calculated for the toxicity of konjac alkaloids and various pesticides against *Spodoptera litura* and *Trichogramma pygmyces*. The toxicity selectivity index (TSR) of konjac alkaloids and commonly used insecticides against *Spodoptera litura* larvae and female *Trichogramma pygmyces* adults was expressed as the ratio of the LC50 of the pesticide to that of the beneficial insects to the LC50 of the pesticide against the pests. A higher ratio indicates lower toxicity to the beneficial insects. TSR ≤ 1 indicates that the pesticide is more toxic to beneficial insects than to pests, exhibiting negative selectivity; 1 < TSR ≤ 10 indicates that the pesticide is less toxic to beneficial insects than to pests, exhibiting positive selectivity. <TSR≤100 indicates that the agent has moderate positive selectivity; 100<TSR≤1000 indicates that the agent has high positive selectivity; TSR>1000 indicates that the agent has strong positive selectivity [23-24]; the parasitic function response data were fitted using the Holling II disk equation, Na=aNT / (1+aThN), where Na is the parasitized beet armyworm egg, N is the number of beet armyworm eggs parasitized by Trichogramma pygmygdalus, a is the instantaneous attack rate, T is the parasitism time (d), Th is the time required to treat one egg, and the theoretical maximum parasitism amount of Trichogramma pygmygdalus is Namax=1 / Th; the equation was fitted and plotted using Origin Pro 2021.

[0021] Furthermore, the experimental materials:

[0022] The tested insects were the beet armyworm and the Trichogramma pygmy spinosa wasp, both collected from corn fields and reared in artificial climate chambers after collection. The beet armyworm larvae were reared indoors using artificial feed, and after they developed into adults, they were provided with corn plants for egg laying. The Trichogramma pygmy spinosa was bred indoors using rice moth eggs.

[0023] The rearing conditions were 25℃, 60%–70% humidity, 10000 lx light intensity, and L / / D = 14h / / 10h. Spodoptera litura larvae of different ages, as well as Spodoptera litura and Trichogramma pygmy molluscs adults within 24 hours of emergence, were collected for later use.

[0024] Another object of the present invention is to provide an indoor toxicity determination system for konjac alkaloids against *Spodoptera litura* and *Trichogramma pygmysis*, comprising:

[0025] Extraction module for extracting konjac alkaloids;

[0026] The toxicity assay module is used to determine the toxicity of konjac alkaloids and common insecticides against the beet armyworm and the Trichogramma pygmy spinosa.

[0027] The reaction assay module is used to determine the parasitic functional response of Trichogramma pygmygdalus to Spodoptera litura eggs;

[0028] The analysis module is used for statistical analysis.

[0029] Another object of the present invention is to provide a computer device comprising a memory and a processor, the memory storing a computer program, which, when executed by the processor, causes the processor to perform the steps of the method for determining the indoor toxicity of konjac alkaloids to Spodoptera litura and Trichogramma pygmy spinosa.

[0030] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to perform the steps of the method for determining the indoor toxicity of konjac alkaloids to *Spodoptera litura* and *Trichogramma pygmysis*.

[0031] Another objective of this invention is to provide an information data processing terminal for implementing an indoor toxicity determination system for konjac alkaloids against the beet armyworm and the Trichogramma pygmy spinosa.

[0032] Based on the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solution to be protected by this invention from the following aspects:

[0033] First, this invention clarifies the indoor toxicity of konjac alkaloids to the beet armyworm and its natural enemy, the Trichogramma pyrenoidosa, through indoor toxicity testing. The research results can provide a scientific basis for the ecological management of the beet armyworm and the further development and utilization of konjac alkaloids.

[0034] Konjac alkaloids exhibit high toxicity to the beet armyworm, significantly higher than commonly used insecticides, and show moderate positive selectivity towards the natural enemy Trichogramma pygmy spiny eel. This indicates that konjac alkaloids are relatively safe for use against this natural enemy when applied to control the beet armyworm, suggesting their potential for development into plant-derived insecticides for beet armyworm control. Alkaloids are the largest class of nitrogen-containing basic compounds found in plants, with insecticidal alkaloids discovered in over 500 plant species, making them a key focus for the development of plant-derived insecticides. Common alkaloid insecticides such as veratrine, matrine, and nicotine have demonstrated good control effects against lepidopteran pests such as the fall webworm (Hyphantria cunea) and the diamondback moth (Plutella xylostella). Besides direct poisoning, berberine also has a strong antifeedant effect on the diamondback moth, with an antifeedant rate exceeding 60%, and can affect the activity of enzymes such as superoxide dismutase in the diamondback moth. Tripterygium wilfordii has strong toxicity and antifeedant effects on lepidopteran insects such as the cabbage caterpillar. It acts on the neuromuscular nodes of the pests, affecting the cell membrane of muscle cells, midgut wall cells and their endometrial system, leading to decreased mobility of lepidopteran pests, inhibited feeding and ultimately death. This invention only relates to the direct poisoning effect of konjac alkaloids on the beet armyworm. Whether it will cause changes in the feeding and mating behavior of the beet armyworm, and its target of action on the beet armyworm, requires further research.

[0035] Even after treatment with konjac alkaloids at LC5, LC25, and LC50, the parasitic function of *Trichogramma pyrenoidosa* on *Spodoptera litura* eggs still belonged to Holling type II. Yang Shaowu et al., in their study on the sublethal effect of spirotetramat on the parasitic ability of *Encarsia tabaci*, found that under different concentrations of spirotetramat treatment, the parasitic response of *Encarsia tabaci* to whitefly nymphs *Bemisia tabaci Gennadius* still belonged to Holling type II. Tang Chao et al. also pointed out in their study that different lethal concentrations of insecticides did not change the parasitic function of *Asecodes hispinarum*. The model of parasitic response to the host was used, but it significantly affected parameters such as instantaneous attack rate, treatment time, and upper limit of parasitism, which is basically consistent with the results of this invention. The instantaneous attack rate and upper limit of parasitism of *Trichogramma pyrifolia* eggs treated with different concentrations of konjac alkaloids significantly decreased, and the treatment time was significantly prolonged; the effect became more significant with increasing concentration. Yang et al. pointed out that spirotetramat at LC50 significantly inhibited the parasitic ability of *Encarsia formosa* on whitefly nymphs and reduced egg production; Ma Xue also pointed out that imidacloprid, pymetrozine, or flonicamid at LC50 significantly reduced the instantaneous attack rate and upper limit of parasitism of *Lysiphlebia japonica* Ashmead on cotton aphids and prolonged the host treatment time, all results consistent with this invention.

[0036] This invention reveals that konjac alkaloids exhibit strong toxicity against the beet armyworm (Spodoptera litura), demonstrating potential for developing plant-derived insecticides for beet armyworm control. Whether it possesses similar toxicity against other common lepidopteran pests requires further investigation. This invention also determined the toxicity of konjac alkaloids against the beet armyworm's parasitic natural enemy, the Trichogramma pygmygdalis wasp. Although the toxicity of konjac alkaloids against Trichogramma pygmygdalis wasp is weaker than that against the beet armyworm, even at lower lethal concentrations, it still affects the parasitic ability of Trichogramma pygmygdalis wasp on beet armyworm eggs. Therefore, when developing and utilizing konjac alkaloids for the control of lepidopteran pests, the toxic effects on parasitic natural enemies and their parasitic ability during application should be a key focus. Attached Figure Description

[0037] Figure 1 This is a flowchart of the indoor toxicity determination method of konjac alkaloids against Spodoptera litura and Trichogramma pygmy spinosa provided in this embodiment of the invention.

[0038] Figure 2 This is a structural block diagram of the indoor toxicity determination system for konjac alkaloids against Spodoptera litura and Trichogramma pygmy spinosa provided in this embodiment of the invention.

[0039] Figure 3 This is a graph showing the parasitism of *Trichogramma pyrenoidosa* eggs on *Spodoptera litura* after treatment with different concentrations of konjac alkaloids, as provided in this embodiment of the invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] like Figure 1 As shown in the figure, the indoor toxicity determination method of konjac alkaloids against *Spodoptera litura* and *Trichogramma pygmyrus* provided by this invention includes the following steps:

[0042] S101, Konjac alkaloid extract;

[0043] S102, Determination of the toxicity of konjac alkaloids and common insecticides to Spodoptera litura and Trichogramma pygmy spinosa.

[0044] S103, Determination of the parasitic function response of Trichogramma pygmygdalus to Spodoptera litura eggs;

[0045] S104, Statistical Analysis.

[0046] The konjac alkaloid extraction provided in this embodiment of the invention:

[0047] Healthy konjac corms were taken, washed with clean water, sliced, and dried in an oven at 50°C until constant weight. The dried konjac powder was then pulverized and passed through a 40-mesh sieve. It was then extracted with 95% ethanol for 24 hours (konjac powder: 95% ethanol = 1:20) to obtain an ethanol extract. The extract was then vacuum filtered using a rotary evaporator (temperature = 55°C) and concentrated to a viscous consistency. The viscous liquid was adjusted to pH 9.0 with 1 mol / L NaOH, extracted five times with ethyl acetate, and the organic layers were combined and concentrated again. The final separated viscous liquid was the total alkaloids. The total alkaloid content in the extract was determined using a spectrophotometer and a total alkaloid content assay kit (Grace Biotech Co., Ltd., Suzhou, China). An alkaloid solution (100 mg / mL) was prepared using methanol as a solvent.

[0048] The toxicity of konjac alkaloids provided in this invention to common insecticides against the beet armyworm and Trichogramma pygmy spiny eel is determined:

[0049] The prepared konjac alkaloids and insecticides were dissolved and diluted with water. Based on the preliminary test results, the agents used in this experiment were diluted into five different gradient concentrations within the range of 10% to 90% corrected mortality rate. Water was used as the test control, and three replicates were set for each concentration value. The toxicity of Spodoptera litura eggs was determined by the immersion method. The egg masses of Spodoptera litura were gently peeled off with tweezers, leaving 10 eggs in each mass. They were then immersed in different concentrations of the pesticide solution for 10 seconds, removed, and the excess liquid on the surface was absorbed with absorbent paper before air drying. Afterward, they were placed on moist filter paper to maintain the humidity required for egg hatching. The toxicity of Spodoptera litura larvae was determined by the feeding method. The artificial feed used to feed Spodoptera litura was cut into cubes of 1cm×1cm×0.5cm and immersed in different concentrations of the pesticide solution for 10 seconds before air drying. Ten Spodoptera litura larvae of different instars were starved for 12 hours. The insects were placed in separate testing containers (diameter r = 5 cm, height 6 cm) and fed artificial feed soaked in different concentrations of pesticide solutions. For the toxicity determination of adult Spodoptera litura and Trichogramma pygmyrus wasps, a feeding method was used. The test pesticide was prepared at different concentrations using 10% honey water. Ten male and female Spodoptera litura adults within 24 hours of emergence were placed in separate testing cages (20 cm × 20 cm × 20 cm). Trichogramma pygmyrus wasps within 24 hours of emergence were also tested. Ten male and ten female adult bees were placed in separate glass tubes (2cm in diameter × 6cm in height) and starved for 12 hours before being fed a solution of honey water. All treatments were placed in an artificial climate chamber at 25℃ and 60%-70% humidity. The mortality of Spodoptera litura eggs was recorded after the water treatment group had hatched normally. The mortality of Spodoptera litura larvae, adults, and Trichogramma pygmygdalites was checked and recorded 24 hours after treatment.

[0050] The parasitic functional response of *Trichogramma pygmygdalis* to *Spodoptera litura* eggs provided in this embodiment of the invention:

[0051] Konjac alkaloids were prepared into different lethal concentrations (LC5, LC25, and LC50) using a 10% honey solution. Using a feeding method, female Trichogramma pygmy borers (within 24 hours of emergence) were starved for 12 hours and then fed with different lethal concentrations of konjac alkaloids. After 24 hours of feeding, the still-active Trichogramma pygmy borers were collected for later use. The number of eggs in the egg masses of *Spodoptera litura* was adjusted to 5, 10, 20, 40, 60, 80, and 100 using insect needles, and then placed in [locations missing]. One Trichogramma pygmy horn wasp treated with different concentrations of konjac alkaloids was placed in each transparent plastic container. After the wasp had been allowed to parasitize freely in the container for 24 hours, it was removed. The plastic containers were then moved into an artificial climate chamber (temperature 25℃, humidity 60%–70%, light intensity 10000 lx, L / / D = 14h / / 10h) to observe the parasitism of Spodoptera litura eggs until all Spodoptera litura eggs hatched or died [21-22]. Water treatment was used as a control, and each treatment was repeated 3 times.

[0052] Statistical analysis provided in the embodiments of the present invention:

[0053] Data analysis was performed using SPSS 20.0. Regression equations, median lethal concentrations (LC50), and 95% confidence intervals were calculated for the toxicity of konjac alkaloids and various pesticides against *Spodoptera litura* and *Trichogramma pygmyces*. The toxicity selectivity index (TSR) of konjac alkaloids and commonly used insecticides against *Spodoptera litura* larvae and female *Trichogramma pygmyces* adults was expressed as the ratio of the LC50 of the pesticide to that of the beneficial insects to the LC50 of the pesticide against the pests. A higher ratio indicates lower toxicity to the beneficial insects. TSR ≤ 1 indicates that the pesticide is more toxic to beneficial insects than to pests, exhibiting negative selectivity; 1 < TSR ≤ 10 indicates that the pesticide is less toxic to beneficial insects than to pests, exhibiting positive selectivity. <TSR≤100 indicates that the agent has moderate positive selectivity; 100<TSR≤1000 indicates that the agent has high positive selectivity; TSR>1000 indicates that the agent has strong positive selectivity [23-24]; the parasitic function response data were fitted using the Holling II disk equation, Na=aNT / (1+aThN), where Na is the parasitized beet armyworm egg, N is the number of beet armyworm eggs parasitized by Trichogramma pygmygdalus, a is the instantaneous attack rate, T is the parasitism time (d), Th is the time required to treat one egg, and the theoretical maximum parasitism amount of Trichogramma pygmygdalus is Namax=1 / Th; the equation was fitted and plotted using Origin Pro 2021.

[0054] The experimental materials provided in this embodiment of the invention:

[0055] The tested insects were the beet armyworm and the Trichogramma pygmy spinosa wasp, both collected from corn fields and reared in artificial climate chambers after collection. The beet armyworm larvae were reared indoors using artificial feed, and after they developed into adults, they were provided with corn plants for egg laying. The Trichogramma pygmy spinosa was bred indoors using rice moth eggs.

[0056] The rearing conditions were 25℃, 60%–70% humidity, 10000 lx light intensity, and L / / D = 14h / / 10h. Spodoptera litura larvae of different ages, as well as Spodoptera litura and Trichogramma pygmy molluscs adults within 24 hours of emergence, were collected for later use.

[0057] like Figure 2 As shown in the embodiment of the present invention, an indoor toxicity determination system for konjac alkaloids against *Spodoptera litura* and *Trichogramma pygmyrus* includes:

[0058] Extraction module for extracting konjac alkaloids;

[0059] The toxicity assay module is used to determine the toxicity of konjac alkaloids and common insecticides against the beet armyworm and the Trichogramma pygmy spinosa.

[0060] The reaction assay module is used to determine the parasitic functional response of Trichogramma pygmygdalus to Spodoptera litura eggs;

[0061] The analysis module is used for statistical analysis.

[0062] Another object of the present invention is to provide a computer device comprising a memory and a processor, the memory storing a computer program, which, when executed by the processor, causes the processor to perform the steps of the method for determining the indoor toxicity of konjac alkaloids to Spodoptera litura and Trichogramma pygmy spinosa.

[0063] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to perform the steps of the method for determining the indoor toxicity of konjac alkaloids to *Spodoptera litura* and *Trichogramma pygmysis*.

[0064] Another objective of this invention is to provide an information data processing terminal for implementing an indoor toxicity determination system for konjac alkaloids against the beet armyworm and the Trichogramma pygmy spinosa.

[0065] Specific implementation of the present invention:

[0066] 1. Materials and Methods

[0067] 1.1 Test Materials

[0068] The tested insects were *Spodoptera litura* and *Trichogramma pygmyrus*, both collected from cornfields in and around Kunming, Yunnan Province. After collection, they were reared in an artificial climate chamber (Ningbo Jiangnan Instrument Factory, RXZ-280C). *Spodoptera litura* larvae were reared indoors using artificial feed, and after reaching adulthood, corn plants were provided for egg-laying. *Trichogramma pygmyrus* was bred indoors using rice moth eggs. The rearing conditions were: temperature 25℃, humidity 60%–70%, light intensity 10000 lx, L / / D = 14 h / / 10 h. *Spodoptera litura* larvae of different instars, as well as adults of *Spodoptera litura* and *Trichogramma pygmyrus* within 24 hours of emergence, were collected for later use. The tested konjac variety was 'Zhuya Jin No. 1', provided by the Mile Bulbous Konjac Technology Research Institute of Yunnan Province, and planted in the bulbous konjac germplasm resource nursery of the Yunnan Provincial Urban Characteristic Agriculture Engineering Technology Research Center. The test agents selected were four commonly used insecticides for controlling the beet armyworm (Table 1).

[0069] Table 1 Information on tested insecticides

[0070]

[0071] 1.2 Extraction Method of Konjac Alkaloids

[0072] Healthy konjac corms were collected, washed with clean water, sliced, and dried in an oven at 50°C until constant weight. The dried konjac powder was then pulverized and passed through a 40-mesh sieve. It was then extracted with 95% ethanol for 24 hours (konjac powder: 95% ethanol = 1:20) to obtain an ethanol extract. The extract was then vacuum filtered using a rotary evaporator (temperature = 55°C) and concentrated to a viscous consistency. The pH of the viscous liquid was adjusted to 9.0 with 1 mol / L NaOH, and the extract was extracted five times with ethyl acetate. The organic layers were combined and concentrated again. The final viscous liquid obtained was the total alkaloids. The total alkaloid content in the extract was determined using a spectrophotometer and a total alkaloid content assay kit (Grace Biotech Co., Ltd., Suzhou, China). An alkaloid solution (100 mg / mL) was prepared using methanol as a solvent.

[0073] 1.3 Toxicity determination of konjac alkaloids and common insecticides against Spodoptera litura and Trichogramma pygmy spiny eel.

[0074] The prepared konjac alkaloids and insecticides were dissolved and diluted with water. Based on the preliminary test results, the agent used in this experiment was diluted into five different concentration gradients within the range of 10%–90% corrected mortality rate. Water was used as the test control, and each concentration value was set up in triplicate. The toxicity test on Spodoptera litura eggs was conducted using the immersion method. Egg masses of Spodoptera litura were gently peeled off with tweezers, leaving 10 eggs in each mass. These were then immersed in different concentrations of the pesticide solution for 10 seconds, removed, and allowed to air dry after absorbing excess liquid with absorbent paper. They were then placed on moist filter paper to maintain the humidity required for egg hatching. The toxicity test on Spodoptera litura larvae was conducted using the feeding method. Artificial feed used to raise Spodoptera litura was cut into cubes of 1cm × 1cm × 0.5cm and immersed in different concentrations of the pesticide solution for 10 seconds, then removed and allowed to air dry naturally. Ten larvae of different ages of *Spodoptera litura* were starved for 12 hours and then placed in testing containers (diameter r = 5 cm, height 6 cm) and fed artificial feed soaked in different concentrations of pesticide solution. The toxicity of *Spodoptera litura* and *Trichogramma pygmyces* adults was determined using a feeding method. Different treatment concentrations of the test pesticide were prepared using 10% honey water. Ten male and female *Spodoptera litura* adults within 24 hours of emergence were placed in testing cages (20 cm × 20 cm × 20 cm), and ten male and female *Trichogramma pygmyces* adults within 24 hours of emergence were placed in testing glass tubes (diameter 2 cm × height 6 cm). After starvation for 12 hours, they were fed pesticide solution prepared with honey water. All treatments were placed in an artificial climate chamber at a temperature of 25℃ and a humidity of 60%-70%. The mortality of Spodoptera litura eggs was recorded after the water treatment group had hatched normally. The mortality of Spodoptera litura larvae, adults, and adult Trichogramma pygmy larvae was checked and recorded 24 hours after treatment.

[0075] 1.4 Determination of the parasitic functional response of *Trichogramma pygmygdalis* to *Spodoptera litura* eggs

[0076] Konjac alkaloids were prepared into different lethal concentrations (LC5, LC25, and LC50) using a 10% honey solution. Using a feeding method, female adult Trichogramma pygmyces within 24 hours of emergence were starved for 12 hours and then fed with different lethal concentrations of konjac alkaloids. After 24 hours of feeding, the still-active Trichogramma pygmyces wasps were collected for later use. Using an insect needle, the number of eggs in the Spodoptera litura egg masses was adjusted to 5, 10, 20, 40, 60, 80, and 100, and placed in transparent plastic containers. One Trichogramma pygmyces wasp treated with different concentrations of konjac alkaloids was placed in each container, and the wasps were allowed to freely parasitize in the containers for 24 hours before being removed. The plastic containers were then placed in an artificial climate chamber (temperature 25℃, humidity 60%–70%, light intensity 10000 lx, L / / D = 14h / / 10h) to observe the parasitism of Spodoptera litura eggs until all eggs hatched or died [21-22]. Water treatment served as a control, and each treatment was repeated three times.

[0077] 1.5 Statistical Analysis

[0078] Data analysis was performed using SPSS 20.0. Regression equations, median lethal concentrations (LC50), and 95% confidence intervals were calculated for the toxicity of konjac alkaloids and various pesticides against *Spodoptera litura* and *Trichogramma pygmyces*. The toxicity selectivity index (TSR) of konjac alkaloids and commonly used insecticides against *Spodoptera litura* larvae and female *Trichogramma pygmyces* adults was expressed as the ratio of the LC50 of the pesticide to that of the beneficial insects to the LC50 of the pests. A higher ratio indicates lower toxicity to beneficial insects. TSR ≤ 1 indicates that the pesticide is more toxic to beneficial insects than to pests, exhibiting negative selectivity; 1 < TSR ≤ 10 indicates that the pesticide is less toxic to beneficial insects than to pests, exhibiting positive selectivity; 10 < TSR ≤ 100 indicates moderate positive selectivity; 100 < TSR ≤ 1000 indicates high positive selectivity; and TSR > 1000 indicates strong positive selectivity. The parasitic function response data were fitted using the Holling II disk equation, Na = aNT / (1 + aThN), where Na is the number of S. beet armyworm eggs parasitized, N is the number of S. beet armyworm eggs provided for parasitism by *Trichogramma pygmygdalis*, a is the instantaneous attack rate, T is the parasitism time (d), Th is the time required to treat one egg, and the theoretical maximum parasitism limit of *Trichogramma pygmygdalis* is Namax = 1 / Th. Origin Pro 2021 was used for equation fitting and graphing.

[0079] 2 Results and Analysis

[0080] 2.1 Indoor toxicity of konjac alkaloids to Trichogramma pygmy hornets

[0081] The toxicity of konjac alkaloids to Trichogramma pygmyces wasps differed significantly from that of commonly used insecticides, with konjac alkaloids showing stronger toxicity to male adults (Table 2). The LC50 of konjac alkaloids against female Trichogramma pygmyces wasps was only 6.91 mg / L, and the LC50 against male adults was only 6.22 mg / L, significantly lower than other tested agents, indicating that konjac alkaloids are significantly more toxic to Trichogramma pygmyces wasps than the tested insecticides.

[0082] Table 2. Indoor toxicity of konjac alkaloids and commonly used insecticides to adult Trichogramma pygmy larvae.

[0083]

[0084] 2.2 Indoor toxicity of konjac alkaloids to Spodoptera litura

[0085] The toxicity of konjac alkaloids to beet armyworm eggs and larvae differs significantly from that of commonly used insecticides (Table 3). The LC50 of konjac alkaloids against Spodoptera litura eggs was only 0.53 mg / L, against 1st-3rd instar larvae only 0.57 mg / L, and against 4th-6th instar larvae only 0.91 mg / L, significantly lower than other tested agents. This indicates that konjac alkaloids are significantly more toxic to Spodoptera litura larvae than commonly tested insecticides. Deltamethrin and azadirachtin showed the next lowest toxicity to Spodoptera litura eggs and larvae, with toxicities ranging from 11.20 to 21.42 mg / L. Cyfluthrin showed slightly lower toxicity to Spodoptera litura eggs and larvae, with toxicities of 259.72 mg / L, 291.27 mg / L, and 389.31 mg / L, respectively. Phoxim showed the lowest toxicity to Spodoptera litura larvae, with toxicities of 628.53 mg / L, 640.65 mg / L, and 888.64 mg / L, respectively.

[0086] Table 3. Indoor toxicity of konjac alkaloids and commonly used insecticides to Spodoptera litura eggs and larvae.

[0087]

[0088] The toxicity of konjac alkaloids to adult beet armyworms differs significantly from that of commonly used insecticides, and the toxicity is stronger to male adults (Table 4). The LC50 of konjac alkaloids against adult female beet armyworms was only 1.52 mg / L, and the LC50 against adult male beet armyworms was only 1.31 mg / L, significantly lower than other tested agents. This indicates that the toxicity of konjac alkaloids to adult beet armyworms is significantly higher than that of commonly tested insecticides. Deltamethrin and azadirachtin showed the next highest toxicity to adult beet armyworms, with toxicity distributions of 18.52 mg / L, 16.57 mg / L, and 26.55 mg / L, 22.81 mg / L for male and female adults, respectively. Chlorantraniliprole showed slightly lower toxicity to adult beet armyworms, with toxicity distributions of 578.18 mg / L and 481.89 mg / L for male and female adults, respectively. Phoxim showed the lowest toxicity to adult beet armyworms, with toxicity distributions of 1135.99 mg / L and 890.17 mg / L for male and female adults, respectively.

[0089] Table 4. Indoor toxicity of konjac alkaloids and commonly used insecticides to adult beet armyworms.

[0090]

[0091] 2.3 Toxicity Selection of Amorphophallus Alkyls on Spodoptera litura and Trichogramma pygmy spiny eel

[0092] The toxicity selectivity index of konjac alkaloids against adult female Trichogramma pygmy stem wasp and eggs and larvae of Spodoptera litura was higher than that of other tested agents, indicating that konjac alkaloids have lower toxicity to adult female Trichogramma pygmy stem wasp. The toxicity selectivity index of konjac alkaloids and commonly tested insecticides against both Trichogramma pygmy stem wasp and Spodoptera litura wasp was greater than 1, indicating that konjac alkaloids and commonly tested insecticides have lower toxicity to adult female Trichogramma pygmy stem wasp than to eggs and larvae of Spodoptera litura wasp, and all exhibit positive selectivity. Furthermore, the toxicity selectivity index of konjac alkaloids against adult female Trichogramma pygmy stem wasp and eggs and 1st-3rd instar larvae of Spodoptera litura wasp was greater than 10, indicating that konjac alkaloids have moderate positive selectivity against adult female Trichogramma pygmy stem wasp and eggs and 1st-3rd instar larvae of Spodoptera litura wasp (Table 5).

[0093] Table 5. Toxicity Selection Index of Konjac Alkaloids on Spodoptera litura and Trichogramma pygmy spinosa.

[0094]

[0095]

[0096] 2.4 Effects of different lethal concentrations of konjac alkaloids on the parasitism ability of Trichogramma pygmy worm.

[0097] After treatment with different concentrations of konjac alkaloids, the parasitism of *Trichogramma pygmyces* gradually increased with the increase of *Spodoptera litura* egg density. The higher the *Spodoptera litura* egg density, the more gradual the increase in parasitism of *Trichogramma pygmyces*, and eventually it tended to stabilize. Figure 3 This indicates that the parasitic function of *Trichogramma pygmygdalis* on *Spodoptera litura* eggs treated with LC5, LC25, and LC50 alkaloids all belong to the Holling II type.

[0098] The instantaneous attack rate, upper limit of parasitism, and parasitic efficacy of *Trichogramma pygmyces* on *S. beet armyworm* eggs were all lower than the control after treatment with different concentrations of konjac alkaloids. The instantaneous attack rate, upper limit of parasitism, and parasitic efficacy of *Trichogramma pygmyces* on *S. beet armyworm* eggs treated with LC50 concentrations of konjac alkaloids were slightly higher, at 0.827, 62.596, and 51.760, respectively. With increasing konjac alkaloid concentration, these values ​​significantly decreased. The treatment time of *Trichogramma pygmyces* on *S. beet armyworm* eggs was longer than the control after treatment with different concentrations of konjac alkaloids. The longest treatment time (0.034 days) was observed after treatment with LC50 concentrations of konjac alkaloids. The treatment time decreased with decreasing konjac alkaloid concentration (Table 6).

[0099] Table 6. Parasitic function response parameters of *Trichogramma pyrenoidosa* after treatment with different concentrations of konjac alkaloids.

[0100]

[0101] Example 1

[0102] Five kg of konjac corms from high-altitude production areas in Yunnan were selected. Total alkaloids were extracted and a 100 mg / mL stock solution was prepared according to the process described in claim 2. Following the method in claim 3, the stock solution was prepared with 0.05% Tween-80 to five concentrations of 10, 20, 40, 80, and 160 μg / mL. These were then used to treat *Spodoptera litura* egg masses (≈100 eggs / mass) for 0–6 h using the immersion method. After treatment, the eggs were transferred to an artificial climate chamber at 26±1℃ and RH 70±5% for 24 h. The hatching rate was recorded and LC was obtained using Probit regression fitting. 50 =37.6μg / mL (95% CI: 30.8–45.4μg / mL).

[0103] Using the same concentration of konjac alkaloids to soak eggs collected under infrared insecticidal lamps as a control, the results showed that konjac alkaloids significantly inhibited egg hatching, and the dose-response relationship was significant (χ²). 2 =6.12, p<0.05). This verifies the feasibility and repeatability of the extraction module and toxicity assay module in oviposition toxicity evaluation, supporting claims 1, 3, 6–8.

[0104] Example 2

[0105] Using the feeding method described in claim 3, 30 second-instar Spodoptera litura larvae per group were continuously fed for 48 hours with artificial feed containing 5, 10, 20, 40, or 80 μg / g konjac alkaloids; abamectin (0.4 μg / g) was used as a positive control. The mortality rate at 24 and 48 hours was recorded and regression analysis was performed to obtain the larval mortality rate (LC50). 50 =24.1 μg / g; LC50 of abamectin 50 The TSR was calculated to be 63.4 based on a ratio of 0.38 μg / g, indicating that the toxicity to pests is at a moderate level.

[0106] Then, female adult Trichogramma pygmy wasps injected with 10% honey solution were fed the same concentration of the solution. The cumulative mortality rate after 48 hours was 12.5% ​​(LC). 50 >80μg / g). Pest / Natural Enemy LC 50 A ratio greater than 3 indicates that konjac alkaloids are relatively safe from natural enemies, further supporting the toxicity selection index algorithm and analysis module function of claim 5.

[0107] Example 3

[0108] In the toxicity assay, konjac alkaloids were diluted to 15, 30, 60, 120, and 240 μg / mL, and newly emerged female adults (approximately 3 days in age) were fed with cotton balls soaked in the solution. A commonly used pyrethroid insecticide (10 μg / mL) was used as a control during the same period. Adult mortality was recorded after 24 hours, and the LC50 was calculated. 50=88.9 μg / mL, while pyrethroid LC50 50 =4.7 μg / mL. Konjac alkaloids showed low to moderate toxicity; LC50 was detected in adult Trichogramma pyrenoidosa using the same method. 50 >240μg / mL.

[0109] Further, 10 μg / mL of konjac alkaloids were mixed with 1 μg / mL of pyrethroids, and the results showed that the mixing index <1 indicated antagonism. This suggests that the system can be used for combination toxicity assessment and provides a basis for formulation development, thus verifying the applicability of the modular system in compound screening scenarios.

[0110] Example 4

[0111] According to claim 4, the female Trichogramma pygmy horn wasp was subjected to LC5 (8 μg / mL) and LC5 (8 μg / mL) assays, respectively. 25 (20 μg / mL), LC 50 After treatment with (40 μg / mL) for 24 h, the pupae were placed together with 5, 10, 20, 40, 60, 80, and 100 eggs of *Spodoptera litura* for another 24 h. The number of parasitic pupae was counted and fitted using the Holling II equation. The control group had a value of 0.154 h. -1 Th = 0.38h; LC 50 Treatment group a decreased to 0.072h -1 Th increased to 0.55h, and the maximum number of parasites decreased from 63 to 44.

[0112] The results reveal that konjac alkaloids have a concentration-dependent inhibition of parasitic functions, and the analysis module can accurately output key parameters such as a and Th, providing quantitative support for ecological risk assessment and further supporting claims 1, 4, 5, 6, 9, and 10.

[0113] Example 5

[0114] An integrated system conforming to claim 6 was constructed: the extraction, concentration, pH adjustment, and liquid-liquid extraction processes were controlled by a PLC; the toxicity assay module was equipped with an automatic oscillating impregnation arm and a feed sprayer; and the functional reaction module was equipped with a 96-bit transparent parasitic plate and a high-definition camera. The experimental software automatically associated the identification code, concentration, and treatment time, and recorded images and temperature and humidity data in real time.

[0115] The system ran continuously for 7 days, used for batch testing of 480 virulence samples and 192 parasitic functional samples. Data was automatically imported into the analysis module to complete regression and fitting calculations, generating a PDF report. The results showed that the inter-batch variation CV was <8%, indicating that the systematic approach can significantly improve efficiency and reduce human error, validating the hardware integration and data link design of claims 6–10.

[0116] Example 6

[0117] To assess the long-term exposure risk, LC 10 Konjac alkaloids (12 μg / mL) were applied to the surface of artificial feed, and *Spodoptera litura* was continuously reared for three generations. Larval survival rate, generation cycle, and sex ratio were recorded for each generation. The parasitism rate of *Trichogramma pygmyces* on the treated egg masses and the adult emergence rate were also recorded simultaneously. The survival rate of the third generation of *Spodoptera litura* larvae was 22.4% lower than the control, and the generation cycle was prolonged by 1.8 days. There was no significant difference in the parasitism rate of *Trichogramma pygmyces* (p>0.05).

[0118] Experiments show that konjac alkaloids have an inhibitory effect on the generational reproduction of pests at sub-lethal concentrations while being relatively friendly to natural enemies, providing a sustainable pesticide application scheme for integrated pest management; at the same time, the system supports long-term dynamic monitoring, further expanding the application scope of claims 1 and 6–10.

[0119] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for determining the indoor toxicity of konjac alkaloids to *Spodoptera litura* and *Trichogramma pygmyrus*, characterized in that... Includes the following steps: (1) Extraction of konjac alkaloids; (2) The toxicity of the konjac alkaloids and common insecticides to the beet armyworm and Trichogramma pygmy spiny wasp was determined; (3) Determine the parasitic functional response of Trichogramma pygmygdalus to Spodoptera litura eggs; (4) Statistical analysis of toxicity and parasitic reaction data.

2. The method as described in claim 1, characterized in that, The extraction of konjac alkaloids includes the following steps: The konjac corms were washed, sliced, dried at 50℃ to constant weight, pulverized, and passed through a 40-mesh sieve. 95% ethanol was added at a ratio of 1:20 for extraction for 24 hours. After rotary evaporation and concentration, the pH was adjusted to 9.0 with 1 mol / L NaOH. The mixture was then extracted five times with ethyl acetate, and the organic layers were combined and concentrated to obtain a viscous total alkaloid. The alkaloid concentration was determined using a spectrophotometer, and a 100 mg / mL alkaloid solution was prepared with methanol.

3. The method as described in claim 1, characterized in that, The toxicity assay includes the following: Konjac alkaloids were diluted with common insecticides to five concentration gradients. The toxicity of Spodoptera litura eggs was determined by immersion method, the toxicity of Spodoptera litura larvae and adults was determined by feeding method, and the toxicity of Trichogramma pygmygdalus adult was determined by feeding method. The mortality or hatching status of each treatment group was recorded 24 hours after treatment.

4. The method as described in claim 1, characterized in that, The parasitic functional response was determined as follows: After feeding female adult Trichogramma pygmystica wasps with LC5, LC25 and LC50 concentrations of konjac alkaloids for 24 hours, live wasps were taken and placed in plastic containers with 5 to 100 eggs of Spodoptera litura for 24 hours. They were then transferred to artificial climate chambers to observe hatching and parasitism.

5. The method as described in claim 1, characterized in that, The statistical analysis includes: Statistical software was used to construct a toxicity regression equation, calculate the LC50 and confidence interval, calculate the toxicity selection index TSR based on the ratio of the LC50 of the pesticide to that of the pest and its natural enemies, and fit the parasitic data using the Holling II type disk equation to obtain the attack rate a and the treatment time Th.

6. A system for indoor toxicity determination based on the method of claim 1, characterized in that, include: The extraction module is used to extract konjac alkaloids; The toxicity assay module is used to conduct toxicity experiments on the beet armyworm and the Trichogramma pygmy spinosa. Functional reaction module, used to determine the parasitism ability of Trichogramma pygmy spp.; The analysis module is used for virulence analysis and parasitic response modeling.

7. The system as described in claim 6, characterized in that, The extraction module includes: The unit includes a drying unit, a pulverizing unit, an ethanol extraction unit, a rotary evaporation unit, a liquid-liquid extraction unit, and a pH adjustment unit; the extraction unit is used to separate the extracted total alkaloids.

8. The system as described in claim 6, characterized in that, The toxicity assay module includes: The system includes a reagent preparation unit, an impregnation treatment unit, a feed soaking device, a feeding and testing unit, and an artificial climate chamber. The climate chamber has constant temperature, humidity control, and light adjustment functions, and is adapted to the biological characteristics of the beet armyworm and the Trichogramma pygmy spinosa.

9. The system as described in claim 6, characterized in that, The functional response module includes: An egg mass preparation device, a parasitism experimental container, and an observation and recording device; the parasitism experimental container is a transparent, sealable plastic jar used for monoparasitism experiments.

10. The system as described in claim 6, characterized in that, The analysis module includes: The system consists of a virulence statistical analysis submodule and a parasitic function modeling submodule. The former is used for regression analysis and TSR calculation, while the latter is used to fit the Holling type II equation and output the maximum parasitic amount.

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