Application of mannitol in promoting the rigidification of bat moth larvae

Abstract

The present invention discloses the application of mannitol in promoting the rigidification of bat moth larvae. The present invention, through control experiments, finds that mannitol can quickly promote bat moth larvae to enter the rigidification period after being injected into the bat moth larvae. It is therefore concluded that the preparation promoting the rigidification of bat moth larvae can be prepared using the metabolite mannitol of Cordyceps sinensis. Usage S1, select 5-instar bat moth larvae as the infection object, select Cordyceps sinensis spores as the infection strain, and inoculate the infected bacteria by acupuncture; S2, select 8-instar infected bat moth larvae, inject 15%-20% mannitol 20μL from the larvae stomata, S3, 72H larvae begin to rigidify in large quantities after injection, and the test hardness value changes in 6.8HC-92.6HC. The beneficial effects of the present invention include: the bat moth larvae injected with mannitol can quickly enter the rigidification period, can accelerate the rigidification process of the bat moth larvae for about 7 days, and do not affect the quality of Cordyceps sinensis.

Description

Technical Field

[0001] The invention relates to the technical field of cordyceps cultivation, and in particular to application of mannitol in promoting the rigidification of bat moth larvae and rapid acquisition of cordyceps by mannitol injection. Background Art

[0002] Cordyceps sinensis is a valuable traditional Chinese medicine with pharmacological properties including antitumor, antioxidant, antimicrobial, and immunomodulatory effects. The formation of Chinese Cordyceps sinensis is the result of parasitism, in which the larvae of the lepidopteran bat moth become infected by the fungus Ophiocordyceps sinensis (Ophiocordyceps sinensis), ultimately forming a rigid host carcass containing the fruiting body of the entomopathogenic fungus. Both the bat moth and Ophiocordyceps sinensis are psychrophilic, and the distribution of Cordyceps sinensis is limited to the Qinghai-Tibet Plateau at altitudes between 3,000 and 5,000 meters. The scarcity and high medicinal value of Cordyceps sinensis result in its extremely high price. Artificial cultivation has the potential to increase Cordyceps sinensis production, but success has been limited due to the difficulty in establishing laboratory models of the entomopathogenic fungus and its host, and the lack of clarity surrounding the mysterious life cycle of Cordyceps sinensis.

[0003] Existing research has mostly focused on the fungus and its host, such as the isolation and genetic identification of different strains of Os, the extraction and pharmacological mechanisms of active ingredients, and large-scale fermentation of Os. However, little research has focused on the interaction between the fungus and its host. While some progress has been made in understanding how the fungus invades its host and develops into fruiting bodies, the mechanisms by which the fungus kills its host and completes the ossification process remain largely unresolved, impacting the quality of Cordyceps sinensis. Improving Cordyceps sinensis yields and shortening its cultivation cycle are also crucial research topics. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides the use of mannitol in promoting the rigidification of bat moth larvae, which accelerates the rigidification cycle of the bat moth larvae and solves the problem of the long cultivation cycle of Cordyceps sinensis.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention has found through control experiments that mannitol can quickly promote the rigor mortis of bat moth larvae after being injected into the body of the bat moth larvae, and therefore it is concluded that mannitol, a metabolite of Cordyceps sinensis, can be used to prepare a preparation for promoting the rigor mortis of the bat moth larvae.

[0007] The present invention also aims to provide a method for promoting the rapid rigidification of bat moth larvae, which is characterized by comprising the following steps:

[0008] S1. Select the fifth-instar larvae of the bat moth as the target of infection, select Cordyceps sinensis as the infection strain, and inoculate the infection by acupuncture;

[0009] S2. Select 8th-instar larvae of the fungus-infected bat moth and inject 20 μL of 15%-20% mannitol through the larvae's stomata.

[0010] S3. After injection of 15%-20% mannitol, larvae showed a large amount of rigidity compared with the control group at 72 hours.

[0011] Furthermore, the bat moth is the small golden bat moth.

[0012] Furthermore, in step S1, the inoculation dose of each bat moth larvae was 2 μl and the concentration was 3 × 10 6 blastospores / ul of fungal suspension.

[0013] Furthermore, the bat moth larvae are reared at a temperature of 15°C, a humidity of 80%, and fed with carrots.

[0014] Furthermore, the 8th instar fungus-susceptible bat moth larvae include larval hemolymph fungi in the form of capsule-shaped spore larvae and pod-shaped spore larvae.

[0015] Furthermore, step S2 can be replaced by regularly applying a certain amount of 15%-20% mannitol to the susceptible bat moth larvae or feeding them with a feed mixed with 15%-20% mannitol.

[0016] The beneficial effects of the present invention include: the bat moth larvae injected with mannitol can quickly enter the rigidification period, can accelerate the rigidification process of the bat moth larvae by about 7 days, and have no effect on the quality of Cordyceps sinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a comparison diagram of the morphology of the 8th instar uninfected golden bat moth larvae and the 8th instar infected larvae and the dead insects at different stages of the present invention;

[0018] Figure 2 This is a schematic diagram of the hardness test of the dead insect of the present invention;

[0019] Figure 3 This is a graph showing changes in the hardness of the dead insect of the present invention;

[0020] Figure 4 This is a diagram showing the developmental stages of the hemocoel Os of the larvae and dead insects of the small golden bat moth of the present invention at different stages;

[0021] Figure 5 This is a comparison chart of the rigidity rate in the injection control experiment of the present invention;

[0022] Figure 6 Schematic diagram of the microinjection system structure of the present invention;

[0023] Figure 7 It is a schematic diagram of the injection process of the present invention;

[0024] Figure 8 This is a diagram showing the morphological changes of a dead insect injected with 20% mannitol according to the present invention;

[0025] Figure 9 This is a graph showing changes in the hardness of dead insects injected with 20% mannitol according to the present invention;

[0026] Figure 10 This is a diagram of the development of Os in the hemocoel of the larvae and dead insects of the golden bat moth injected with 20% mannitol according to the present invention;

[0027] Figure 11 This is a classification diagram of the developmental stages of the Cordyceps sinensis fungus of the present invention in the body cavity of the larvae and dead insects of the small golden bat moth;

[0028] Figure 12 This is a comparison chart of the rigidification rate of the capsule-shaped spore larvae of the present invention after injection of 20% mannitol;

[0029] Figure 13 This is a comparison chart of the rigidification rates of the pod-shaped spore larvae of the present invention after injection of 20% mannitol. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0031] Example 1

[0032] Test hosts: H. xiaojinensis pupae were collected from the alpine meadows of Xiaojin County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province. They were allowed to eclode, mate, and lay eggs indoors in Chongqing. They were then reared on carrots in an artificial room at a temperature of 15°C and a humidity of 80%. The H. xiaojinensis population used in this experiment had been bred for three generations in the laboratory.

[0033] Cordyceps sinensis was isolated and purified from Cordyceps sinensis in Xiaojin County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province, and fermented in liquid glucose-peptone medium at 14 degrees Celsius to produce blastospores. The blastospores were then cultured in a 2 μl diluted fungal suspension (3 × 10 6 Fifth-instar larvae (head capsule 2-2.5 mm, body length 2.8-3.3 cm, body weight 0.20-0.26 g, reared for 3 months) were inoculated using a glass capillary tube containing blastospores / ul.

[0034] Injected larvae samples included: 100 8th instar infected larvae (random samples not screened by hemolymph), 100 larvae with capsule-shaped spores by hemolymph microscopy, and 100 larvae with pod-shaped spores by hemolymph microscopy.

[0035] Preparation of injection drugs: Erythritol 20%, Mannitol 20%, 15%, 10%, 5%, Sorbitol 20%, sterile water, and Figure 6 The microinjection system shown (brand: Bell Tower, model 0.33mm, produced by the Instrument Factory of West China University of Medical Sciences). Figure 7 Larvae (3.5-4 mm head capsule, 4-5 cm body length, 0.85-0.90 g body weight) were injected with 20 μl of 20%, 15%, 10%, and 5% mannitol solutions, along with sorbitol, erythritol, and water, through their stomata using the technique shown. No injection served as a control. Rigorization rates were measured 24, 72, 120, and 240 hours after injection. Hemolymph samples were collected before, 24, 72, 120, and 240 hours after injection, and the percentage of pseudohyphae present was recorded under a microscope. Subsequently, 20 μl of hemolymph of 8th instar infected larvae of each experimental sample at 24 h, 72 h, 120 h, and 240 h after injection was randomly selected and diluted into 1 ml of 0.1% Tween 80 solution. 20 μl of the diluted solution of each mixture was dropped onto a glass slide and pressed. The bacterial morphology ratio of hemolymph samples at different periods was counted under an optical microscope (model: BS203 biological microscope) with 10X (eyepiece) and 40X (objective).

[0036] Hardness test after injection: 8th instar infected larvae, 8th instar uninfected larvae, 0h, 24h, 72h, 120h, 240h, 600h, 960h, 1320h, 1920h after injection, and Cordyceps sinensis were tested using a digital display Shore hardness tester, Model: LXD-C; NO: 21065697; hardness value range 0~100HC, such as Figure 2 As shown, the procedure is as follows: Place the specimen on a solid surface. Hold the durometer with the tester panel at a direct viewing angle to the operator, and keep the indenter at least 12 mm from the surface of the test specimen. Press the durometer steadily into the specimen until the presser foot makes full contact with the specimen. Read the durometer within 1 second of full contact. Test points are located at the first, third, and fifth pores on the sides of the larvae, dead insects, and Cordyceps sinensis, corresponding to the five legs. Each test point is 6 mm apart. Each worm constitutes one experimental group, and a total of eight worms are tested. After testing, take the average value.

[0037] Post-injection bacterial assay: 20 μl of hemolymph from 6th-8th instar infected larvae with uninflated spindle-shaped blastospores, inflated blastospores, and pseudohyphae was diluted into 1 ml of 0.1% Tween 80 solution. 8th instar uninfected larvae served as controls. 20 mg of the blood lipid mixture from 0, 24, 72, and 120 h post-injection rigor mortis was diluted into 1 ml of 0.1% Tween 80 solution and mixed well. 20 μl of each dilution was dropped onto a glass slide and pressed under an optical microscope (model: BS203 biological microscope, brand: CHONGQING OPTICAL & ELECTRICALINSTRUMENT). CO., LTD) 10X (eyepiece) and 40X (objective) magnifications were used to observe the gross changes in the appearance of the fungus in infected larvae and dead worms. The proportion of fungal morphology was also counted in samples from different periods. Each worm constituted an experimental group, and three slides were prepared and counted for each experimental group. A total of 8 worms were counted as 8 experimental groups.

[0038] Observation results: The morphology and hardness of the larvae of the golden bat moth changed during the rigidification process;

[0039] After Os infection, the morphology of the larvae of the small golden bat moth was recorded by visual comparison and digital camera at the 8th instar (when the 8th instar larvae entered the rigid phase), the newly formed rigid phase (the critical point of larval death), and the completely rigid phase (about 5 days after the larvae died). Figure 1 As shown, no obvious changes were found between the larvae of the golden bat moth and the infected larvae. Then when the larvae entered the rigidification period, they showed slow movement, less feeding and body rigidity. Finally, the larvae of the golden bat moth were completely rigidified 5 days after death. Figure 2 The hardness measurements of the dead insects are shown. Figure 3 As shown, the hardness values of newly dead and infected larvae are similar (approximately 1.4 HC), but once the rigor mortis process begins, the hardness increases rapidly. During the early stages of rigor mortis, the hardness increases from 2.9 HC on day 1 to 8.0 HC on day 3, then from 18.9 HC on day 5 to 56.8 HC on day 40. Finally, the hardness slowly reaches 68.9 HC until the formation of cordyceps on day 80.

[0040] Development of Os in the hemocoel of the gypsy moth during ossification

[0041] like Figure 4As shown, the development of Os in the hemocoel of the small golden bat moth can be divided into three stages: blastospores, prehyphae, and hyphae. Only dark, short, spindle-shaped blastospores are observed in the hemocoel of sixth-instar larvae. After 2-3 months of reproduction, at the eighth instar, some of these thalluses develop into enlarged blastospores. When the larvae enter the death stage on day 0 of ossification, a small number of blastospores develop into pseudohyphae. The proportion of pseudohyphae in the hemocoel rapidly increases from 15.3% on day 1 to 32.8% on day 3. Finally, on day 5 after ossification, some pseudohyphae germinate into elongated hyphae (8.5%).

[0042] like Figure 5 As shown in Figure 2, injection of 20% mannitol significantly increased the rigidification rate of 8th instar larvae 24 hours after injection, and the rigidification rate was higher than that of injections of other concentrations of mannitol or similar injections. The rigidification rate quickly reached a peak of about 15% 72 hours after injection of 20% mannitol, and growth almost stopped 120-240 hours after injection. Figure 5 As shown in the results, injection of 15% mannitol also increased the rigidification rate of larvae, but this was lower than that of 20% mannitol. In addition, the rigidification rate of larvae injected with low concentrations of mannitol (5% and 10%), sorbitol, erythritol, and sterile water was not different from that of the control group.

[0043] Therefore, it can be preliminarily concluded that the injection of mannitol can promote the rigidification of Cordyceps sinensis.

[0044] Further research showed that mannitol injection had no significant effect on the quality of Cordyceps militaris.

[0045] like Figure 8 As shown in the figure, we observed the morphology and hardness of Helicoverpa larvae injected with 20% mannitol, as well as the development of Os in the hemocoel. The results showed that Helicoverpa larvae still had normal morphology after injection of mannitol 24 hours, 72 hours, 120 hours, and 240 hours after rigidification. Figure 9 As shown in Figure 1, the hardness value on day 0 was also around 1.4 HC. In the early stages of rigidification, it soared from 2.6 HC on day 1 to 6.8.0 HC on day 3, continued to increase from 16.3 HC on day 5 to 48.6 HC on day 40, and slowly reached 68.6 HC on day 80. Figure 10 As shown, in the development of Os in the hemocoel, more than half of the blastospores were observed to be expanded by the eighth instar. When the larvae entered the moribund stage on day 0 of ossification, 6.1% of the blastospores grew into pseudohyphae, a proportion that rapidly increased from 11.9% on the first day of ossification to 36.4% on the third day of ossification. Finally, when the larvae were fully ossified on day 5, 6.8% of the pseudohyphae germinated into elongated hyphae during ossification (8.5%).

[0046] like Figure 11As shown, in this study, we further divided the development of blastospores into four stages. In order to verify whether mannitol is a promoter of spore rigidification at specific developmental stages, we further tested the host larvae in the capsule spore stage and the pod spore stage by injecting mannitol.

[0047] The results show that Figure 12 As shown, at 24 h, 72 h, 120 h and 240 h after injection of 20% mannitol, the rigidification rates (%) of the capsule-shaped spores were 21.1±0.6 (control group 8.0±0.0), 85.9±1.4 (control group 23.7±1.6), 93.2±0.4 (control group 49.0±1.7) and 96.3±0.4 (control group 94.0±1.3), respectively, indicating that the injection of 20% mannitol into the small golden bat moth with capsule-shaped spores can accelerate the rigidification process by about 7 days.

[0048] like Figure 13 As shown in the figure, the rigidification rate (%) of pod-shaped spores was higher than that of capsule-shaped spores 24 hours, 72 hours, 120 hours and 240 hours after injection of 20% mannitol, which were 64.7±0.8, 92.6±0.9, 96.0±0.03 and 97.3±0.5 respectively, indicating that the rigidification rate of pod-shaped spore larvae injected with mannitol was faster than that of capsule-shaped spore larvae.

[0049] Draw conclusions

[0050] Mannitol is a key regulatory factor in the rigidification process of Cordyceps sinensis. Injecting 20% mannitol into the hemolymph of the larvae of the small golden bat moth in the capsule spores can accelerate the rigidification process by about 7 days without affecting the quality of Cordyceps sinensis.

[0051] Based on the conclusions drawn from the above research, the present invention also experimented with promoting the rapid rigidification of the larvae of the small golden bat moth by regularly applying 15% and 20% mannitol with excipients to the larvae. The results showed that rigidification can also be promoted by applying, and the rigidification process of capsule-shaped spore larvae and pod-shaped spore larvae can be accelerated.

[0052] In addition, the present invention also experiments that feeding artificial feed mixed with 15% and 20% mannitol to the infected larvae can also promote the rigidification process of the small golden bat moth larvae. The results show that feeding the artificial feed with added mannitol during the period of insect and fungus coexistence shortens the period of long-term coexistence of insects and fungi.

[0053] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. Application of mannitol in promoting the rigidification of Cordyceps sinensis.

2. The use according to claim 1, characterized in that: The invention comprises the application of mannitol in preparing a preparation for promoting the rigidification of Cordyceps sinensis.

3. A method for promoting rapid rigidification of bat moth larvae, characterized in that: The following steps are included: S1. Select the fifth-instar larvae of the bat moth as the target of infection, select Cordyceps sinensis as the infection strain, and inoculate the infection by acupuncture; S2. Select 8th-instar larvae of the fungus-infected bat moth and inject 20 μL of 15%-20% mannitol through the larvae's stomata. S3. 72 hours after injection of 15%-20% mannitol, the larvae showed a large amount of rigidity compared with the control group.

4. The method for promoting rapid rigidification of bat moth larvae according to claim 3, characterized in that: The bat moth is the small golden bat moth.

5. The method for promoting rapid rigidification of bat moth larvae according to claim 3, characterized in that: The bat moth larvae are reared at a temperature of 15°C and a humidity of 80%, and are fed with carrots as feed.

6. The method for promoting rapid rigidification of bat moth larvae according to claim 3, characterized in that: The 8th instar infected bat moth larvae include larval hemolymph fungus bodies in the form of capsule-shaped spore larvae and pod-shaped spore larvae.

7. The method for promoting rapid rigidification of bat moth larvae according to claim 3, characterized in that: The inoculation dose for each bat moth larva in step S1 was 2 μl with a concentration of 3 × 10 6 blastospores / μL of fungal suspension.

8. The method for promoting rapid rigidification of bat moth larvae according to claim 3, characterized in that: Step S2 is replaced by regularly applying a certain amount of 15%-20% mannitol to the susceptible bat moth larvae or feeding them with a feeding feed mixed with 15%-20% mannitol.

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