Low temperature storage method of mulberry dominant parasitic wasp

Abstract

The application discloses a low-temperature storage method for dominant parasitic wasps in mulberry gardens, uses female wasps of Cremastocharenaeformis as parasitic mother wasps, parasitizes Galleriamellonella, obtains offspring wasps, and stores the offspring wasps at low temperature; selects pupae and adult wasps of the offspring wasps at different growth and development stages for low-temperature storage; places the wasp pupae and adult wasps fed with honey in a low-temperature environment at 4 DEG C for low-temperature storage; after the low-temperature storage is completed, the wasp pupae and adult wasps are taken out and are subjected to transition treatment in a transition temperature environment; and the wasp pupae and adult wasps subjected to the above treatment are bred for standby use. The low-temperature storage method is simple in operation, low in cost, realizes effective low-temperature storage of parasitic wasps, prolongs the shelf life of the parasitic wasps, and lays a good foundation for large-scale release in the wild.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for agricultural pests, specifically relating to a low-temperature storage method for dominant parasitic wasps in mulberry orchards. Background Technology

[0002] Mulberry trees are susceptible to a wide variety of pests, including the mulberry moth (Glyphodes pyloalis), the mulberry geometrid moth (Phthonandria atrineata), and the mulberry leech (Rondotia menciana), all of which are lepidopteran pests that occur year-round in mulberry orchards. Severe infestations can lead to total crop failure, seriously threatening the development of the sericulture industry (Wu Fu'an, 2015). Due to long-term improper use of chemical pesticides, pesticide resistance in mulberry pests is becoming increasingly prominent (Xu Jinsong, 2006). In particular, commonly used insecticides in mulberry orchards frequently cause silkworm poisoning incidents, making the development of new methods for controlling mulberry pests an urgent priority.

[0003] Biological control using parasitic wasps is an important component of green pest management systems and a key development direction for mulberry pest control. Although mulberry pests have abundant natural enemies, biological control practices using parasitic wasps for mulberry pests remain extremely limited. The parasitic wasps currently being promoted for mulberry pest control are still in the large-scale breeding stage, and the long-term stable supply of these wasps is still under exploration. Low-temperature refrigeration is a common method for preserving parasitic wasps, effectively extending the shelf life of their products, reducing production costs, and improving field control efficiency. Research has been conducted on various parasitic wasp groups, including Trichogramma, Aphididae, and Parasitic Wasps, with some success. However, research on parasitic wasps for mulberry orchard pests has not yet been carried out, significantly limiting the widespread application of biological control for mulberry pests.

[0004] *Iseropus kuwanae* (Vlereck), belonging to the family Ichthyophthidae in the order Hymenoptera, is a major parasitic wasp of the mulberry leech, a pest of mulberry trees. *Iseropus kuwanae* has a wide host range, including important lepidopteran pests such as the mulberry leech, mulberry stem borer, rice leaf roller, and large wax borer. Under laboratory conditions, *Iseropus kuwanae* can parasitize and lay eggs year-round. In the wild, it overwinters as a pupa within the cocoons of the third-generation mulberry leech in late November. While indoor parasitism and propagation techniques for this parasitic wasp have been gradually mastered, long-term storage remains a challenge, leading to high breeding costs and limited wild release. Therefore, there is an urgent need to develop an effective low-temperature storage method for *Iseropus kuwanae* to extend its shelf life without altering its primary parasitic characteristics, thus providing a solution to fully utilize its biocontrol potential. Summary of the Invention

[0005] Technical problem solved: In response to the above-mentioned technical problems, this invention provides a low-temperature storage method for dominant parasitic wasps in mulberry orchards, which extends the shelf life of parasitic wasp products, ensures the parasitism index of parasitic wasps, and improves the solution for biological control of natural enemies of mulberry tree pests.

[0006] Technical solution: Application of feeding honey to extend the shelf life of female mulberry leafhopper ichneumon wasps and improve their parasitism rate during low-temperature storage.

[0007] Preferably, the low-temperature storage temperature of the above-mentioned female mulberry leaf worm is 4°C, and the storage time is 5-15 days.

[0008] The aforementioned female mulberry worm wasp were adult females obtained from 5-10 day old mother wasps.

[0009] Preferably, the aforementioned female mulberry leafminer wasp is a female adult obtained from a 5-day-old mother wasp.

[0010] The aforementioned female adults were female *Ichthyophthirius multifiliis* wasps that emerged 1-12 hours after their emergence.

[0011] The honey mentioned above is honey water with a concentration of 5 wt.%-30 wt.%.

[0012] Preferably, the concentration of the above-mentioned honey water is 5 wt.%.

[0013] The application also includes a step of aging at 8-24℃ for 6 hours after low-temperature storage.

[0014] The above placement temperature is 16℃.

[0015] A method for low-temperature storage of dominant parasitic wasps in mulberry orchards: (1) Using 5-10 day old female mulberry leech wasps as parasitic mother wasps, parasitizing large wax moths to obtain offspring wasps that have emerged 1-12 hours ago, which are then used for low-temperature storage; (2) Selecting 5-7 day old female pupae and adults that have emerged 1-12 hours ago for storage at 4℃ for no more than 15 days; (3) After the low-temperature storage is completed, the pupae and adults are taken out and placed at 8-24℃ for 6 hours for later use.

[0016] Beneficial Effects: This invention targets the biological characteristics of parasitic wasps that infest mulberry orchards. It selects different developmental stages of the mulberry leafminer wasp (Ichthyophthirius multifiliis) suitable for low-temperature storage, achieving multi-stage low-temperature storage of a single parasitic wasp. The parasitic wasps successfully recover their development after low-temperature storage, with no impact on lifespan or reproductive capacity. The low-temperature storage method used in this invention is simple to operate, low-cost, and achieves effective low-temperature storage of parasitic wasps, extending their shelf life and laying a solid foundation for large-scale release in the wild. Detailed Implementation

[0017] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0018] Example 1: Low-temperature storage of *Ichthyophthirius multifiliis*

[0019] Five-day-old female pupae, five-day-old male pupae, female adults 12 hours after emergence, and male adults 12 hours after emergence were used as subjects for cryogenic storage for 5 days, 15 days, and 30 days, respectively, in a 4°C refrigerator. The pupae were placed in 1.5 mL centrifuge tubes (5 pupae / tube), and the adults were placed in 50 mL centrifuge tubes (20 pupae / tube). After cryogenic storage, the pupae or adults were removed and placed in a 20°C incubator for 6 hours. The survival rate of pupae and adults, as well as the parasitism rate of female pupae, were then investigated. The parasitism rate of refrigerated female pupae was investigated after emergence. The results are shown in Table 1. The survival rate of female pupae, male pupae, female adults, and male adults was highest after 5 days of cryogenic storage. The survival rate of female adults after 15 days of cryogenic storage was slightly lower than that after 5 days. The parasitism rate of female adults after 15 days of storage was lower than that after 5 days, but still within acceptable limits. Therefore, under the above conditions, female adult insects can still maintain a high survival rate and parasitism rate within 15 days of low-temperature storage.

[0020] Table 1. Effects of different low-temperature storage times on the survival rate and parasitism rate of *Ichthyophthirius multifiliis*.

[0021]

[0022] Different lowercase letters indicate significant differences (p<0.05).

[0023] Example 2: Low-temperature storage of *Ichthyophthirius multifiliis*

[0024] Female *Ichthyophthirius multifiliis* larvae at 5, 10, and 15 days of age were used as parasitoid wasps to obtain adult wasps and pupae for cryogenic storage. Five-day-old female and male pupae, and female and male adults 12 hours after emergence, were used for cryogenic storage for 15 days at 4°C. Pupae were placed in 1.5 mL centrifuge tubes (5 wasps / tube), and adults were placed in 50 mL centrifuge tubes (20 wasps / tube). After cryogenic storage, the pupae or adults were removed and placed in a 20°C incubator for 6 hours. The survival rate of pupae and adults, as well as the parasitism rate of female wasps, were then investigated. The parasitism rate of refrigerated female pupae was investigated after emergence. The results are shown in Table 2. The survival rates of male pupae obtained from parasitized female wasps at 5 and 10 days of age showed no significant difference after low-temperature storage. The survival rates of female pupae, female adults, and male adults decreased slightly but were significantly higher than those obtained from 15-day-old female wasps. The parasitism rate of female wasps obtained from 5-day-old female wasps after cold storage was slightly different from that of 10-day-old female wasps but significantly higher than that of 15-day-old female wasps. After low-temperature storage and molting into female wasps, the parasitism rate of female pupae obtained from 5-day-old female wasps was significantly higher than that obtained from 10-day-old and 15-day-old female wasps.

[0025] Table 2. Effects of different-day-old female bees on the survival rate and parasitism rate of *Ichthyophthirius multifiliis* after low-temperature storage.

[0026]

[0027] Different lowercase letters indicate significant differences (p<0.05).

[0028] Example 3: Low-temperature storage of *Ichthyophthirius multifiliis*

[0029] Five-day-old female *Ichthyophthirius multifiliis* were used as parasitic wasps to obtain wasp pupae for cryogenic storage. Five-day, seven-day, and nine-day-old male and female pupae of *Ichthyophthirius multifiliis* were used for cryogenic storage for 15 days at 4°C. The pupae were placed in 1.5mL centrifuge tubes (5 pupae / tube), and the adults were placed in 50mL centrifuge tubes (20 pupae / tube). After cryogenic storage, the pupae or adults were removed and placed in a 20°C incubator for 6 hours. The survival rate of the pupae and the parasitism rate of the female wasps were then investigated. The results are shown in Table 3. The survival rate of male and female pupae aged 5–7 days showed little difference, but was significantly higher than that of male and female pupae aged 9 days. After the female pupae of 5 days and 7 days emerged as adults, the parasitism rate showed little difference, but was significantly higher than that of female pupae aged 9 days.

[0030] Table 3. Effects of different ages on the survival rate and parasitism rate of *Ichthyophthirius multifiliis* pupae after low-temperature storage.

[0031]

[0032] Different lowercase letters indicate significant differences (p<0.05).

[0033] Example 4: Low-temperature storage of *Ichthyophthirius multifiliis*

[0034] Five-day-old female *Ichthyophthirius multifiliis* were used as parasitic wasps to obtain wasp pupae for cryogenic storage. Adult *Ichthyophthirius multifiliis*, both male and female, were used for cryogenic storage at 1 hour, 12 hours, 24 hours, and 48 hours after emergence. The storage period was 15 days, and the wasps were stored at 4°C. The pupae were placed in 1.5 mL centrifuge tubes (5 wasps / tube), and the adults were placed in 50 mL centrifuge tubes (20 wasps / tube). After cryogenic storage, the pupae or adults were removed and placed in a 20°C incubator for 6 hours. The survival rate of the adults and the parasitism rate of the female wasps were then investigated. The results are shown in Table 4. The survival rates of male and female adults at 1 hour and 12 hours after emergence during cryogenic storage were not significantly different, but were significantly higher than those at 24 hours and 48 hours after emergence. Female adults that emerged 1 hour and 12 hours after being stored at low temperature showed no significant difference in parasitism rate after the refrigeration was lifted, but were significantly higher than female wasps that were stored at low temperature for 24 hours and 48 hours.

[0035] Table 4. Effects of adult storage at different times after emergence on the survival rate and parasitism rate of adult *Ichthyophthirius multifiliis*.

[0036]

[0037] Different lowercase letters indicate significant differences (p<0.05).

[0038] Example 5: Low-temperature storage of *Ichthyophthirius multifiliis*

[0039] Five-day-old female *Ichthyophthirius multifiliis* were used as parasitic wasps to obtain wasp pupae for cryopreservation. Adult male and female *Ichthyophthirius multifiliis*, 12 hours after emergence, were used for cryopreservation. Before storage, the adults were fed honey water at concentrations of 5%, 15%, and 30% (w / w), respectively. Storage lasted 15 days in a 4°C refrigerator. Adults were placed in 50mL centrifuge tubes (20 wasps / tube). After cryopreservation, the parasitic wasps were removed and placed in a 20°C incubator for 6 hours. The survival rate of adults and the parasitism rate of female wasps were then investigated. The results are shown in Table 5. Feeding with different concentrations of honey water before cryopreservation had no significant effect on the survival rate of both male and female adults. Feeding with 30% honey water resulted in the highest survival rates for both female and male wasps. Feeding female bees with different concentrations of honey water before storage and then storing them showed no significant difference in parasitism rate after the end of refrigeration. The female bees fed with 5% honey water and then refrigerated had the highest parasitism rate.

[0040] Table 5. Effects of feeding different concentrations of honey water before low-temperature storage on the survival rate and parasitism rate of *Ichthyophthirius multifiliis*.

[0041]

[0042] Different lowercase letters indicate significant differences (p<0.05).

[0043] Example 6: Low-temperature storage of *Ichthyophthirius multifiliis*

[0044] Five-day-old female *Ichthyophthirius multifiliis* were used as host wasps to obtain wasp pupae for cryogenic storage. Adult male and female *Ichthyophthirius multifiliis*, 12 hours after emergence, were used for cryogenic storage. Before storage, they were fed 5% honey water and stored for 15 days at 4°C. The pupae were placed in 1.5mL centrifuge tubes (5 wasps / tube), and the adults were placed in 50mL centrifuge tubes (20 wasps / tube). After cryogenic storage, the pupae or adults were removed and placed in incubators at 8°C, 16°C, and 24°C for 6 hours each. The survival rate of adults and pupae, as well as the parasitism rate of female wasps, were then investigated. The results are shown in Table 6. The highest survival rates were observed in both male and female adults and pupae after cryogenic storage, and the highest parasitism rates were also observed in female adults and pupae that emerged after cryogenic storage and 16°C bridging.

[0045] Table 6. Effects of different transition temperatures after low-temperature storage on the survival rate and parasitism rate of *Ichthyophthirius multifiliis*.

[0046]

[0047] Different lowercase letters indicate significant differences (p<0.05).

Claims

1. The application of feeding honey in extending the shelf life and increasing the parasitism rate of female *Ichthyophthirius multifiliis* larvae stored at low temperatures, characterized in that... Five-day-old female *Ichthyophthirius multifiliis* was used as the parasitic wasp to obtain wasp pupae for low-temperature storage. Adult male and female *Ichthyophthirius multifiliis* 12 hours after emergence were used as the low-temperature storage subjects. Before storage, they were fed honey water with a mass fraction of 5% and stored for 15 days in a refrigerator at 4℃. After low-temperature storage, the adult parasitic wasps were removed and placed in an incubator at 16℃ for 6 hours as a transition.

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