CA3171644C - Method for precise indoor mass rearing of the rice striped stem borer [chilo suppressalis (walker)] on rice seedlings - Google Patents

Abstract

The present invention relates to method for precise indoor mass rearing of the rice striped stem borer, Chilo suppressalis (Walker) using rice seedlings. The procedure involves cleaning and panning of rice seeds, sterilizing the resultant rice seeds, germinating the seeds to obtain rice sprouts and then germinating seeds to obtain rice seedlings, using rice seedlings for controlled larval rearing, allowing pupation and collecting pupae, allowing mating and egg-laying by adults, and collecting and treating the obtained egg masses. The invention precisely quantifies the operational steps of the preparation of rice seedlings as natural feed, larval rearing, pupation and pupa collection, adult mating, and egg mass collection, forming a set of mass rearing procedures that save rearing time, cost, and labor.

Description

METHOD FOR PRECISE INDOOR MASS REARING OF THE RICE STRIPED STEM
BORER ICHILO SUPPRESSALIS (WALKER)1 ON RICE SEEDLINGS
Field of the Invention The present invention is related to a precise indoor mass rearing method for the rice striped stem borer, Chilo suppressalis (Walker), which belongs to the field of artificial rearing technology for insects.
Background of the Invention To the best of the inventor's knowledge, the rice striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae) is a polyphagous economic pest that harms rice production, which is distributed in Asia, the Middle East, and southern Europe.
Specifically, the rice production in Yangtze River Basin and southern regions in China are affected by this pest. Due to the promotion of hybrid rice and the reform of farming system, the occurrence of C. suppressalis has increased year by year. The larvae of C. suppressalis feed on rice stems and leaves through boring, resulting in severe yield losses. This pest can cause damage at all stages of rice growth.
At the tillering stage, borer larvae feed on rice seedlings, leading to withering. At the booting and heading stages, the larvae feed on the rice stalks, causing booting failure and resulting in the development of dead withered or white ears. At the filling and milk stages, the larvae will feed on rice stalks, hampering grain filling and resulting in the development of semi-withered ears and damaged plants, which ultimately increases the number of blighted grains.
In addition to rice, this pest harms water bamboo, sugar cane, sorghum, corn, wheat, foxtail millet, arrowhead, broad beans, rape, cockspur grass, and clubhead cutgrass. According to statistics, the area annually affected by C. suppressalis in China reaches 14 million hectares, causing annual economic losses of 11.5 billion yuan. At present, the control of C.
suppressalis mainly relies on chemical pesticides to prevent outbreak However, C. suppressalis have developed resistance to various commonly used insecticides, prompting the use of excess and frequent doses, increasing the cost of control, and exerting management pressure on the environment. In studies on the control and management of C. suppressalis, stable indoor populations of insects and large-scale artificial rearing can ensure a stable source of pests at specific times and ages for experiments on integrated control trials, drug resistance, mechanistic attributes, pesticide screening, sex pheromones, transgenic insect-resistant cultivars, and parasitic wasps, among others. Therefore, Date Recue/Date Received 2022-08-29 improvements in artificial rearing methods for C. suppressalis will promote prevention and control.
Since the 1970s, Chinese and overseas studies on artificial breeding of C.
suppressalis have been performed successively. The common artificial rearing methods for this insect are divided into two types: natural and artificial diet rearing.
In natural diet feeding, rice seedlings or rice stems are widely used. Water bamboo, water chestnut, and rice are commonly used as feed based on existing methods. The effects of these natural feeds on the growth, development, and fertility of C. suppressalis have been analyzed. C.
suppressalis could complete its life cycle and reproduction by feeding on all of these three natural feeds (Meng FX, Wu KM, Gao XW. Evaluation of few-flower wildrice, chufa and rice as foods for rearing the striped stem borer, Chinese Bulletin of Entomology 2003, 40(5):469-472).
However, the use of these feeds is limited by season, and they cannot consistently provide sufficient forage.
In addition to being a food source, natural feeds provide a habitat for C.
suppressalis.
However, the host plants used for rearing the insects are susceptible to decay and mold due to the limitations of feed preservation technology. Rotten or moldy host plants lead to malnutrition and infection of larvae, resulting in their death and decreasing the insect population. This is not favorable for large-mass rearing. In natural diet rearing, the number and sex of C. suppressalis are not uniform and the growth progress is inconsistent (Shang ZZ, Wang YS, Zou YH. Studies on successive mass rearing method of the rice stem borer Chilo suppressalis Walker. Acta Entomologica Sinica. 1979, 22(2): 164-167; Lu YR, Su JK, Ji CHM, Liu Q, Zhang CM, Liu HA.
Preliminary research on the method of rice stem borer culture in room. Journal of Anhui Agricultural Sciences. 2003, 31(2): 270-271; Zheng FS, Qiang CK, Dong HX, Shen Y.
Collection and indoor-feeding of Chilo suppressalis Walker. Journal of Anhui Agricultural Sciences. 2009, 37(17):7837-7838].
Artificial feed rearing of C. suppressalis started with the development of Shijin and Fuye feeds. Studies on artificial feeds for C. suppressalis were conducted successively. In 1984, through preliminary studies on practical artificial feeds for Asiatic rice borer, the problem of insect source demand could be solved to some extent. However, this feed had a single composition and could not meet the life cycle requirements of insects.
Furthermore, the feed decays easily and must be replaced more frequently. To address these issues, the artificial feed

2 Date Recue/Date Received 2022-08-29 for C. suppressalis was further optimized, and the survival rate, pupation, and emergence rates of insects were improved; nonetheless, the growth rate remained was slow and inconsistent, which affected large-mass rearing (Hu Y, Zheng YL, Cao GL, Fu QA. A technique for rearing Chilo suppressalis in the large scale with an oligidic diet in laboratory. Chinese Journal of Rice Science.
2013,27(5): 535-538; Liu HM, Li SH, Wang MQ, Zhang GA. The optimized artificial diet of the rice stem borer, Chilo suppressalis. Chinese Bulletin of Entomology. 2008, 45(2): 310-314; Li B, Han LZ, Peng YF. Development of a standardized artificial diet and rearing technique for the striped stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae).
Chinese Journal of Applied Entomology. 2015, 52(2):498-503).
In addition, artificial feed cannot replace the habitat provided by natural feed, and the experiments with artificial and natural feeds yielded different results.
Moreover, the raw materials for artificial feed are relatively difficult to obtain, and the cost of feed is high. In addition, the preparation procedure is cumbersome, requiring feed processing equipment and incurring high labor costs. Finally, although artificial feed is subjected to preservation treatment, it still requires refrigeration, which increases equipment cost.
Our search found that the invention patent application no. CN201910423574.2 and application publication no. CN110024751A describe a rearing device and a rearing method of C.
suppressalis, respectively. Although these techniques use natural feed to rear C. suppressalis, they have disadvantages, such as complicated device production and tedious operation. In addition, the invention patent application no. CN201510086108.1 and application publication no.
CN104604814A describe a method for efficient rearing of C. suppressalis.
Although the rearing device is simplified, disadvantages, such as the complexity of operation and limitations in the scale of rearing, remain.
Application no. CN201210068466.6 and application publication no. CN102578051A
describe a method for indoor artificial rearing of C. suppressalis. This method mainly uses small self-sealing bags commonly used in laboratories as C. suppressalis egg beds.
The materials easy to obtain, and egg mass collection is simplified. In addition, the whole egg-laying device requires less space than that required for rice seedlings grown in plastic boxes measuring 20x 15 x15 (cm).
Although this method uses rice seedlings as natural feed, the problems of rice seedling rotting or mold development as well as the inconsistent developmental progress of insects persist. More importantly, it does not focus on large-scale mass rearing of C. suppressalis but rather on

3 Date Recue/Date Received 2022-08-29 simplifying the rearing process. It is difficult to increase the scale of rearing of C. suppressalis based on technical solutions alone; thus, a sufficient population of insects cannot be sustained.
The invention patent application no. CN201911073784.X and application publication no.
CN110604102A describes a standardized rearing and management method for C.
suppressalis, including artificial feed preparation and a 36-day larvae rearing and adult rearing process. The egg collection cycle from indoor populations lasts 5 days. Pupal collection lasts 4 days. The larval rearing cycle from the inoculation of individual batches of egg masses to the pupation stage lasts 36 days. This patent establishes a new system of multi-batch and overlapping cyclic feeding through standardized management of operation for different insect stages. However, this technical solution uses artificial feed instead of natural feed, thus lacking its advantages. In addition, the operation steps are cumbersome, and the entire process of rearing egg masses from inoculation to pupation must be performed on a sterile operating table, requiring specialized operating environment and operators. Thus, this method is not favorable for large-scale mass rearing.
In conclusion, natural feed rearing of C. suppressalis offers advantages that cannot be achieved by the current artificial feed rearing methods. Thus, the natural feed rearing method for C. suppressalis must be improved to simplify the rearing operation, save cost and labor, and ensure insect supply for large-scale experiments.
Summary of the Invention The key objectives of the present invention are to overcome the problems of prior techniques and provide a method for precise indoor mass rearing for C.
suppressalis using rice seedlings, which can accurately quantify the rearing process of this insect, save time and rearing costs, solve the problems of rotting and deterioration due to mold, reduce the mortality rate of larvae, improve the pupation rate, and ensure the scale of the indoor population of C.
suppressalis, which can be reproduced in successive generations.
The technical protocol of the present invention to solve the current technical problems is summarized below.
The proposed precise indoor mass rearing method for C. suppressalis comprises the following steps.

4 Date Recue/Date Received 2022-08-29 Step 1: Washing and selection of rice seeds, followed by sterilization In the said disinfection process, sodium hypochlorite solution is mixed with tap water at a volume ratio of 1 0.2:100. Next, rice seeds are soaked in this solution for 24 2 h and then washed with tap water until sodium hypochlorite is removed. Next, the seeds are soaked in tap water for 24-48 h and then washed with double-distilled water. The sodium hypochlorite solution used in this step contains active chlorine at a mass ratio of 9 1%.
Step 2: In step 2, the rice seeds obtained in step 1 are germinated to obtain rice sprouts, which are further grown to obtain rice seedlings. The resulting seedlings are grown in sterilized jars. An average of 50 5 g of rice sprouts per jar is used. When the seedlings grow over 3 cm but no more than 4 cm, the jars are used in step 3.
Step 3: In step 3, sterilized C. suppressalis egg masses are placed on rice seedlings in jars obtained in step 2. Five to seven egg masses are placed in each jar on average. For sterilization, fumigation is performed with formaldehyde solution. Each egg mass contains 200-300 eggs on average, and the number of eggs in each jar is 1,000-21,000 on average. A
disinfected black cloth is used to seal the jar tightly, and the jars are incubated in an artificial climate chamber at 28 C. 1 C under 70-80% relative humidity. Double-distilled water is added to jars along the walls once every 2 days to moisten the roots of the rice seedlings. From the placed egg masses, the first instar larvae hatch within 2-4 days. Development from first to second instar larvae requires 6-7 days. When the larvae reach the mid-to-late second instar stage, they are transferred to rice seedlings in new jars, with an average of 900-2,000 mid-second instar larvae per jar.
Development from second to third instar larvae requires 3-4 days. When the larvae reach the end of the third instar stage, they are transferred to rice seedlings in new jars, with an average of 800-1,900 third-instar larvae per jar. Development from third to fourth instar larvae requires 3-4 days. When the larvae reach the fourth instar stage, they are transferred to rice seedlings in new jars, with an average of 700-1,800 larvae per jar. Development from fourth to fifth instar requires 2-3 days. When the larvae reach the mid-fifth instar stage, they are transferred to rice seedlings in new jars, with an average of 600-1,700 larvae per jar. Rearing is continued until the late fifth instar larvae enter the pupation stage after 3-4 days.
Date Recue/Date Received 2022-08-29 Step 4: Sterile papers rolls are cut and folded into fans. When the larvae at the end of the fifth instar stage start metamorphosing to form pupae, fans are placed on rice seedlings so that the larvae can move into the gaps or folds of the fans to pupate. Then, the jars are sealed and tied with sterilized black cloths. After 2 days, the fans are removed using sterilized instruments. First, sterile paper sheets are laid at the bottom of culture plates. Using sterilized instruments, pupae are collected from the fans and placed onto the paper in culture plates.
Thereafter, the paper sheets are moistened with double-distilled water to maintain humidity for developing pupae.
Step 5: Sterile paper moistened with double-distilled water is placed at the bottom of culture plates. Pupae collected in step 4 are transferred to culture plates, with an average of 50 5 pupae per plate for emergence and mating. Bottoms of several insect cages are lined with sponges, which are moistened with double-distilled water. Two pots of rice seedlings for egg laying are placed in each cage in a diagonal manner, and then two culture plates containing pupae are introduced. The insect cages are placed in an artificial climate chamber at 28 C 1 C and relative humidity of 70-80%. After 3-4 days of emergence, adult insects emerge from pupae and mate in the cage. After mating, they will lay eggs on rice seedlings in the jars.
Step 6: After 6-7 days of adult emergence, the rice seedlings were removed from the cages. The leaves of the rice seedlings containing egg masses were cut using sterilized instruments and placed onto culture dishes lined with moistened filter paper at the bottom. An average of 100-150 egg masses per cage were obtained. The culture dishes were fumigated with formaldehyde solution to sterilize the egg masses, and the resulting eggs were used for the next cycle of breeding.
In this method, the disinfection of rice seeds can prevent subsequent rotting and deterioration due to mold, thus solving the problems of spoilage of natural feed and death of a large number of insects. The quantity of rice sprouts per jar is strictly limited to allow precise quantitation of the rearing process and build a foundation for continuous mass rearing of C.
suppressalis. The time windows of rice seedling transfer operations from egg mass incubation to the end of the fifth instar are stringently controlled to achieve precise quantitation of the rearing process and ensure a higher viability of larvae for larger-scale rearing.
During the pupation Date Recue/Date Received 2022-08-29 period, pupae can be collected easily using a simple carrier of fan-folded paper, rendering the rearing process simple. During the adult mating and egg laying periods after emergence, rice seedlings are used as the natural substrate for adults to lay eggs, which improves the egg-laying rate and facilitates subsequent mass rearing. Rearing can be continued in cyclically, thus providing multiple batches with consistent developmental progress (morphology, age, individual size, health status, susceptibility, and so on) to study C. suppressalis control, ensuring the accuracy of repeat experiments, and shortening the research duration. The process is simple, and only one person can complete all rearing operations.
The technical solution for further refinement of the proposed invention is summarized below.
Preferably, the jars containing larvae in step 3, the culture dishes containing pupae in step 4, or the egg masses obtained in step 6 are placed at 4 C 2 C for a predetermined time to control their developmental progress.
Using the preferred solution, the growth and development of eggs, larvae, and pupae of C.
suppressalis can be further controlled by regulating the temperature, which enables consistent developmental progress and meets the time window requirements of the developmental stages of the insect source for the corresponding control studies.
Preferably, in the first step, the specific process of cleaning and plating the selected rice seeds is as follows. The rice seeds are placed into the basin, which is filled with tap water to submerge the seeds. The seeds are left standing for several minutes so that the rice husks or blighted seeds float on the water surface, while the saturated rice seeds sink to the bottom of the basin. Rice husks or blighted rice seeds are discarded into the basin together with water, leaving only saturated rice seeds at the bottom of the basin. This process is repeated until the saturated rice seeds sunk to the bottom of the basin are washed and the water becomes clear. The average weight of seeds added in each basin is >1,000 g. The average weight of saturated rice seeds per pot is 93 3% of the weight of added rice seeds.
Using the above preferred solution, the specific technical details of the first step can be further optimized and simplified. Simultaneously, the use of a large amount of rice seeds facilitates the subsequent continuous provision of rice seedlings as natural feed.
Preferably, in the second step, the specific process of germination to obtain rice shoots is as follows. The rice seeds obtained in the first step are collected and water is drained before they Date Recue/Date Received 2022-08-29 are placed into the basin. Then, the basin is sealed with a clean black plastic bag. The bag is tightened and placed in an artificial climate chamber at 28 C PC under 70-80%
relative humidity to accelerate seed exposure. Then, the seeds are turned once at 24 2 h, such that their temperature is uniform and consistent and successful germination is facilitated. At 48 2 h, the seeds are fully germinated, and rice sprouts can be obtained.
Preferably, in the second step, the specific process of obtaining rice seedlings by accelerating growth is as follows. The rice sprouts are washed with double-distilled water, and the water is discarded. The bottom of the clean jar is lined with sterile paper sheets as a substrate for the of germination rice seeds. The paper sheets are moistened using double-distilled water, and the rice sprouts are transferred to ajar sealed with cling film. Holes are punched on the film with a needle to ensure smooth oxygen flow, and the jars are placed in an artificial climate chamber at 28 C 1 C under 70-80% relative humidity to accelerate seedling emergence. Every 48 2 h, the rice seeds are rinsed with 100 10 mL of double-distilled water, and then the double-distilled water is decanted to maintain the moisture required for seedling growth.
Using this above preferred solution, the specific technical details of the second step can be further optimized for further simplification and production of a large number of rice seedlings with consistent growth in each batch, providing sufficient natural feed for subsequent rearing cycles.
Preferably, in the third step, forceps sterilized with medical alcohol are used. Specifically, the forceps are burned over an alcohol lamp and cooled naturally. When transferring the larvae from a jar to rice seedlings in a new jar, they are first picked up with forceps and placed on the first sheet of clean paper. Then, the larvae are picked up from these rice seedlings and placed on the second sheet of clean paper. Finally, the larvae on the second sheet of clean paper are placed directly onto rice seedlings in the new jar. At this time, if any larva escapes from the clean paper, they are picked up with forceps and placed on rice seedlings in a new jar.
Thereafter, the jars are sealed with a sterilized black cloth, tied tightly, and incubated in the artificial climate chamber at 28 C 1 C under 70-80% relative humidity. In the third step, the larvae are incubated in the artificial climate chamber under the same environment throughout the rearing period.
Using the above preferred solution, the specific technical details of the third step can be further optimized to ensure that the larvae do not die on a large scale due to changes in seedlings.
A uniform rearing environment ensures that the larvae enter each developmental stage Date Recue/Date Received 2022-08-29 appropriately.
Preferably, in the fourth step, a folded paper is added into each sterilized glass jar. After removing the paper from each jar, the subsequent operation is conduced.
Forceps sterilized with medical alcohol are used. The diameter of the culture plate used is 90 mm. In the fifth step, the diameter of the plate dish used is 90 mm. The sponge is moistened with 100+10 mL of double-distilled water.
Using the above preferred solution, the specific technical details of the fourth and fifth steps can be further optimized to achieve better technical results.
Preferably, in the fifth step, the process of cultivating rice seedlings for egg laying is as follows. Rice sprouts obtained following germination in the second step are collected, sown on the soil surface in the preset pot, and covered with a thin layer of soil.
Then, the pot is covered with a clean black plastic bag and placed in an artificial climate chamber at 28 C 1 C under 70-80% relative humidity until the rice seedlings reach 10 cm in height, that is, the rice seedlings are appropriate for egg laying. In the fifth step, a cage is used. In the fifth step, a culture plate (diameter 60 mm) containing sterilized cotton balls dipped in 10 1% honey water by volume is placed in the cage. The cotton balls are replaced every 2 days.
Using this preferred solution, additional specific technical details in the fifth step can be further optimized, in which the rice seedling cultivation process for egg laying is precisely defined to provide a better egg laying environment. Honey water can supplement the nutrition for emerged adults and promote egg laying. The use of sterilized cotton balls can prevent the adults from falling into the honey water and dying.
Preferably, in the sixth step, the specific process of fumigation is as follows. The culture plate containing the egg mass is fumigated in a dry cylinder with formaldehyde solution at the bottom for 15+1 min and then immediately removed. The disinfected instrument described is sterilized scissors. The culture plate diameter is 90 mm. The concentration of formaldehyde solution is 35+1% by volume.
Using the above preferred solution, the specific technical details of the sixth step can be further optimized, in which the fumigation process can eliminate parasitic bacteria on the surface of the egg masses and improve the survival rate of larvae during the subsequent rearing process.
Preferably, the said rice seeds are japonica; the said jar has a diameter of 6 cm and a height of 10 cm; the said insect cage size is 45 cm x 45 cm x 55 cm and the number of jars used in the Date Recue/Date Received 2022-08-29 third step is >20.
The invention precisely quantifies the operational steps of the preparation rice seedlings are natural feed, larval rearing, pupation and pupae collection, adult mating, and egg mass collection, forming a set of mass rearing methods that save rearing time, cost, and labor.
Compared with the prior techniques, the major advantages of the present invention are as follows. (1) Relevant disinfection and anti-bacterial measures are performed throughout the process of egg-larvae-pupae-adults-eggs to comprehensively control the infection of pathogenic bacteria and ensure the development of generations and population size. (2) The preparation of natural feed includes cleaning, sterilization, germination, and sowing of rice seeds, which effectively controls decay and deterioration due to mold, provides sufficient nutrition to larvae, optimizes the habitat for larvae, reduces the number of natural feed replacements, and improves the survival rate of larvae. (3) The proposed invention offers a new method of pupal collection;
specifically, in this method, older larvae are allowed to form cocoons and pupate in folds of a paper, providing a safe and concealed place with suitable moisture for pupation. This increases the number and weight of pupae, and the formed pupae can be easily found pupae and transferred. Overall, this method is suitable for large-scale consecutive rearing. (4) The egg-laying environment for the rice striped stem borer adults is optimized through the use of a suitable cage for mating and egg laying in a sterile environment under constant temperature and humidity, which improves the egg-laying rate. Moreover, rice seedlings used as natural feed provide an egg-laying substrate to mimic the natural habitat of the rice striped stem borers, which improves the egg-laying rate. (5) Finally, the biological characteristics of the rice striped stem borers reared following to proposed method of invention are close to those of wild insects, with little deviation from the results of related previous studies.
According to one aspect of the invention, there is provided a method for precise indoor mass rearing of rice striped stem borer, Chilo suppressalis (Walker) using rice seedlings, said method comprising the following steps:
step (1): clean and select rice seeds, and sterilize the rice seeds, wherein sterilization involves mixing sodium hypochlorite solution with tap water at a volume ratio of 1 0.2:100, soaking the rice seeds in this solution for 24 2 h, washing the rice seeds with tap water until sodium hypochlorite is removed, and washing the rice Date Recue/Date Received 2024-02-15 seeds with double-distilled water after soaking the rice seeds in tap water for 24-48 h, said sodium hypochlorite solution contains active chlorine at a mass ratio of 9 1%, step (2): germinate the rice seeds obtained in step (1) to obtain rice sprouts, grow the rice sprouts to obtain rice seedlings; put the rice seedlings in sterilized jars, wherein on average 50 5 g of rice sprouts per jar is used, and when the rice seedlings have grown to over 3 cm but below 4 cm, the jars are used in step (3), step (3): place sterilized C. suppressalis egg masses on the rice seedlings in the jars obtained in step (2), on average five to seven egg masses are placed in each jar, sterilization treatment of the C. suppressalis egg masses involves fumigation with formaldehyde solution, on average each egg mass contains 200-300 eggs, and on average the number of eggs in each jar is 1,000-21,000, seal each of the jars with a disinfected cloth tightly, and incubate the jars in an artificial climate chamber at 28 C PC under 70-80% relative humidity, add double-distilled water to the jars along the walls once every 2 days to moisten roots of the rice seedlings, the egg masses hatch into first instar larvae in 2-4 days, from the first instar larvae to second instar larvae requires 6-7 days, when the larvae reach mid- to late second instar stage, transfer the larvae to the rice seedlings in new jars obtained in step (2), with an average of 900-2,000 mid-second instar larvae per jar, development from the second instar larvae to third instar larvae requires 3-4 days, when the larvae reach an end of the third instar stage, transfer the larvae to the rice seedlings in new jars obtained in step (2), with an average of 800-1,900 third instar larvae per jar, development from the third instar larvae to fourth instar larvae requires 3-4 days, 10a Date Recue/Date Received 2024-02-15 when the larvae reach the fourth instar stage, transfer the larvae to the rice seedlings in new jars obtained in step (2), with an average of 700-1,800 fourth instar larvae per jar, development from the fourth instar larvae to fifth instar larvae requires 2-3 days, when the larvae reach mid-fifth instar stage, transfer the larvae to the rice seedlings in new jars obtained in step (2), with an average of 600-1,700 fifth instar larvae per jar, rearing is continued, and late fifth instar larvae enter pupation stage after 3-4 days, step (4): cut sterile rolls of papers and fold into fans, when the larvae are at an end of the fifth instar stage start metamorphosing to form pupae, place the fans on the rice seedlings so that the larvae can move into gaps or folds of the fans to pupate, seal and tie each jar with a sterilized cloth, after 2 days, remove the fans using a first sterilized instrument, place sterile paper sheets at bottom of a first set of culture plates, collect the pupae from the fans using the first sterilized instrument and place the pupae on the sterile paper sheets in the first set of culture plates, and moisten the sterile paper sheets with double-distilled water to maintain humidity for developing the pupae, step (5): place sterile paper moistened with double-distilled water at bottom a second set of culture plates, transfer the pupae collected in step (4) to each of the second set of culture plates, on average, 50 5 pupae are placed in each of the second set of culture plates for emergence and mating, line bottom of a set of insect cages with sponges moistened with double-distilled water, place two pots of rice seedlings for egg laying in each of the set of insect cages, and introduce two of the second set of culture plates containing the pupae, 10b Date Recue/Date Received 2024-02-15 place the insect cages in an artificial climate chamber at 28 C PC under 70-80% relative humidity, after 3-4 days, adult insects emerge from the pupae and mate in the insect cages, after mating, the adult insects lay eggs on the rice seedlings in the jars, step (6): remove the rice seedlings from the insect cages after 6-7 days of adult emergence using a second sterilized instrument, cut leaves of the rice seedlings containing egg masses and place in a third set of culture plates lined with moist filter paper at bottom, on average 100-150 egg masses per cage are obtained, fumigate the third set of culture plates with formaldehyde solution to sterilize the egg masses, the egg masses resulting are used for a next cycle of breeding.
Brief Description of Drawings Figure 1 shows a schematic diagram of the preparation of natural feed for Chilo suppressalis in Embodiment 1 of the present invention.
Figure 2 shows a schematic diagram of Chilo suppressalis larvae reared using japonica rice seedling in Embodiment 2 of the present invention.
10c Date Recue/Date Received 2024-02-15 Figure 3 shows a schematic diagram of Chilo suppressalis pupation and pupa collection in Embodiment 3 of the present invention.
Figure 4 shows a schematic diagram of the mating and egg-laying of Chilo suppressalis adult in Embodiment 4 of the present invention.
Figure 5 shows a schematic diagram of the collection and preservation of Chilo suppressalis egg masses in Embodiment 5 of the present invention.
Detailed Description The present invention is described in further detail below in conjunction with embodiments.
Note, however, that the present invention is not limited to the embodiments shown. The reagents involved in the present invention are commercially available.
Embodiment 1: Preparation of natural feed for Chilo suppressalis In this embodiment, the natural feed for C. suppressalis is the japonica rice variety Nangeng 46, which is the major variety promoted in the Jiangsu Province and widely planted in various cities and counties therein. In this invention, different rice varieties, as local ones, can be selected as natural feed both to facilitate local material collection and match the actual living environment of native C. suppressalis.
(1) Briefly, 1,500 g of Nangeng 46 seeds is weighed with a balance. This weight of japonica seeds can fulfill the needs for large-scale reproduction of C. suppressalis and provide food and habitat for rearing progeny.
The weighed Nangeng 46 seeds are placed into a 10 L plastic basin, and 5 L of tap water is pour into the basin with a measuring cup. The seeds are stirred vigorously in the tap water, and allowed to stand for several minutes. Then, the residual rice husk or blighted seeds are discarded, leaving saturated rice seeds at the bottom of the basin. Using a measuring cup, 5 L of tap water is poured into a plastic basin, and the rice seeds are stirred vigorously to remove the residual rice husks or blighted seeds. This panning step is repeated five times to remove the rice husk or blighted seeds.
The weight of japonica seeds remaining at the bottom of the plastic basin is approximately 1,400 g. Using a measuring cup, 3 L of tap water is poured into the plastic basin to soak the Date Recue/Date Received 2022-08-29 japonica seeds for subsequent disinfection.
(2) According to the 1:100 ratio of sodium hypochlorite solution to tap water, 3 L of tap water is added to 0.03 L of sodium hypochlorite solution (containing 9% by weight of active chlorine; the same applies below) and thoroughly mixed. Rice seeds are soaked in this solution. After 24 h, the solution is discarded, and the rice seeds are thoroughly washed with tap water until the strong odor of sodium hypochlorite vanishes.
Using a measuring cup, 3 L of tap water is poured into the plastic basin to soak the japonica rice seeds. The criteria for adequate soaking of seeds are that the hulls are transparent, grains are visible, and grains are easy to break without sounds when the chaffs are peeled off and can be crushed by hand, indicating that the seeds have absorbed sufficient water. After 24 h, tap water used to soak the seeds is decanted.
Using a measuring cup, 2 L of double-distilled water is poured to clean the soaked japonica rice seeds. Water is discarded after sufficient cleaning. The cleaning process is repeated three times. This operation step removes the trace chemical components in tap water to allow successful germination and avoid toxication of C. suppressalis.
(3) After draining the rice seeds, the plastic pots are sealed with black plastic bags, and the mouth of the bags is tightened. These bags allow heat preservation and are conducive to the germination of japonica rice seeds. The pots are placed in the RGC-1000C
artificial climate chamber (Hefei Youke Instruments & Equipment Co., Ltd.), set at 28 C under 80%
relative humidity and a 16/8 h light/dark cycle. Germination temperature must be no less than 25 C to prevent high temperature seed burning. Seeds lose their germination ability at temperatures exceeding 40 C. The seeds are turned over once every 24 h. When turning, residual water at the bottom of the plastic pots is re-mixed with the seeds. The pots are aerated and oxygenated to accelerate the growth of seed shoots.
(4) After 48 h, the germinated rice seeds are gently washed with 2 L of double-distilled water added using a measuring cup. This process is repeated two times, without breaking the rice sprouts during washing. Water is fully drained. Commercially available sterile rolls of paper are cut into 13.9 cm long x 10.2 cm wide pieces and used as substrates for the growth of japonica Date Recue/Date Received 2022-08-29 rice seeds. The paper sheets are placed at the bottom of a 500 mL jar with a diameter of 6 cm and a height of 10 cm and then moistened by pouring 10 mL of double-distilled water with a measuring cylinder. Next, 50 g of soaked japonica rice sprouts are weighed and sown in the jar.
Ordinary cling film is cut to the size of the mouth of the jar and used to seal it. Thereafter, a 0#
insect needle is used to make 10-15 holes in the cling film to ensure oxygen flow. Overall, 1,400 g of japonica rice sprouts are planted in 28 jars and placed in the RGC-1000C
artificial climate chamber (Hefei Youke Instruments & Equipment Co., Ltd.) at 28 C under 80%
relative hi tn. dity and a 16/8 h light/dark cycle. To ensure the emergence of japonica rice shoots, the temperature must be maintained at no less than 25 C. At 72 h, the rice shoots grew to 1-2 cm. To prevent the drying of rice shoots, 5 mL of double-distilled water is poured along the inner wall of the jar.
During the emergence process, the rice seedlings are rinsed with 100 mL of double-distilled water every 48 h, and then the water is decanted. This step ensures moisture required for the growth of rice seedlings.
The above process is presented in Figure 1.
Embodiment 2. Feeding of C. suppressalis larvae (1) First instar larval rearing When the japonica rice seedlings in jars grow above 3 cm, five sterile egg masses of the same size were introduced. The introduced egg masses are those from which the larvae are about to hatch. The selection criteria are that the egg masses must appear black and have been placed in the artificial climate chamber for 2 days. Egg masses were introduced in all 28 jars. The egg mass used in this embodiment measure 15 mm in length and 2 mm in width. The number of eggs in a mass is 200-300, and the number of eggs in the jar is 1,000-1,500, totaling 28,000-42,000 eggs in a generation. The jars are sealed with a black cloth and tied with a rubber band (as shown in Figure 2). The pots are placed in the RGC-1000C artificial climate chamber (Hefei Youke Instruments & Equipment Co., Ltd.) at 28 C under 80% relative humidity and a 16/8 h light/dark cycle. The egg masses hatch as first instar larvae after 2 days. To moisten the roots of rice seedlings, 10 mL of double-distilled water is added along the wall of the jar every 48 h during larval growth. At the first instar larval rearing stage, the same number of jars of japonica rice seedlings required for larval rearing are prepared as described in Embodiment 1.

Date Recue/Date Received 2022-08-29 (2) Second instar larval rearing The first instar larvae of C. suppressalis grow into second instar larvae in 7 days. The japonica rice seedlings need not be replaced at this stage, and the rice seedlings should grow with sufficient water. When the larvae enter the mid- and late second instar stage, their feeding capacity starts to increase as they increase in size, and the japonica rice seedlings must be replaced. First, forceps are taken from 75% alcohol, held over the flame of an alcohol lamp for sterilization, and then placed on a sterile roll of paper for natural cooling.
A sheet of A4 copy paper is laid on the experimental bench to temporarily place the japonica rice seedlings removed from the jars, and another sheet is laid to temporarily place the second instar larvae picked up from the seedlings. The second instar larvae on the paper are directly transferred to jars containing fresh japonica rice seedlings, and escaped larvae are picked up using the forceps and placed in the jars. Each jar contains 900-1,400 second instar larvae. The jars are sealed with a black cloth, tied with a rubber band, and placed in the RGC-1000C artificial climate chamber (Hefei Youke Instruments & Equipment Co., Ltd.) at 28 C under 80% relative humidity and 16/8 h light/dark cycle.
The black cloth used for rearing the first instal larvae is placed in a plastic basin. Using a measuring cup, 2 L of tap water is added to the basin. According to a 1:100 ratio of sodium hypochlorite solution to water, 20 mL of sodium hypochlorite solution is added to tap water in the basin. After 24 h of immersion, tap water is added until the pungent odor of sodium hypochlorite vanishes. Next, several drops of cold water degreasing detergent are added to the plastic basin. The black cloth is soaked and cleaned under tap water, rinsed well, wrung, and placed in a WGL-125B electric blast dryer (Tianjin Taiste Instrument Co., Ltd.) at 50 C. Using a measuring cup, 500 mL of tap water is poured into jars used for rearing the first instar larvae.
The required amount of sodium hypochlorite solution is 5 mL according to a 1:100 ratio of sodium hypochlorite solution to water. The solution is poured into the jar.
After 24 h of immersion, the jars are rinsed well under tap water until the strong odor of sodium hypochlorite vanishes. Next, 1 to 2 drops of cold water degreasing detergent are added to the jar, the jar is washed under tap water, and the foam is rinsed off. The jars are placed in a digital display blast dryer and baked at high temperature (200 C) for 6 h.
After baking, the jars are cooled naturally. The black cloth for sealing and jars is placed in the artificial climate chamber and sterilized under UV lamp for 30 min, followed by wind blowing for 10 min. Finally, Date Recue/Date Received 2022-08-29 the black cloth and jars is removed and set aside.
(3) Third instar larval rearing The mid-second instar larvae of C. suppressalis develop into mid-third instar larvae in 4 days. The third instar larval rearing stage requires a change of japonica rice seedlings. First, forceps are taken from 75% alcohol, held over the flame of an alcohol lamp for sterilization, and then placed on a sterile rolled paper for natural cooling. A sheet of A4 copy paper is laid on the experimental bench to temporarily place the japonica rice seedlings removed from the jar, and another sheet is laid to temporarily place the third instar larvae picked up from the japonica rice seedlings. The third instar larvae on the copy paper are directly transferred to jars containing fresh japonica rice seedlings, and the escaped larvae are picked up using forceps and placed into the jars. Each jar contains 900-1,300 third instar larvae. After the transfer is completed, the jars are sealed with a black cloth, tied with a rubber band, and placed in the RGC-1000C artificial climate chamber (Hefei Youke Instruments & Equipment Co., Ltd.) at 28 C under 80% relative humidity and a 16/8 h light/dark cycle. The black cloth used for sealing and the jars used for rearing are sterilized following the steps described in Embodiment 2 (2). The japonica rice seedlings required for larval rearing are grown according to the procedure described in Embodiment 1, and the number of jars is the same.
(4) Fourth instar larval rearing At the end of the third instar stage, the larvae enter the "heavy feeding period" until the end of the fourth instar stage. During the period from the end of the third instar stage to the end of the fourth instar stage, the rice seedlings are replaced once. Forceps are taken from 75% alcohol, held over the flame of an alcohol lamp for sterilization, and then placed on a sterile roll of paper to cool naturally. A sheet of A4 copy paper is laid on the experimental bench to temporarily place the japonica rice seedlings removed from the jar, and another sheet is laid to temporarily place the fourth instar larvae picked up from the japonica rice seedlings. The fourth instar larvae on the copy paper are directly transferred to jars containing fresh japonica rice seedlings, and the escaped larvae are picked up using forceps and placed into the jars. Each jar contains 700-1,200 fourth instar larvae. After the transfer is completed, the jars are sealed with a black cloth, tied with a rubber band, and placed in the RGC-1000C artificial climate chamber (Hefei Youke Date Recue/Date Received 2022-08-29 Instruments & Equipment Co., Ltd.) at 28 C under 80% relative humidity and a 16/8 h light/dark cycle. The black cloth used for sealing and the jars used for rearing is sterilized following the steps described in Embodiment 2 (2). The japonica rice seedlings required for larval rearing are grown according to the procedure described in Embodiment 1, and the number of jars was the same.

(5) Fifth instar larval rearing As larvae at the end of the fourth instar stage enter the fifth instar stage, their food intake decreases, and they start to form cocoons and pupate. Rice seedlings are replaced once at mid-fifth instar stage. Forceps are taken from 75% alcohol, held over the flame of an alcohol lamp for sterilization, and then placed on a sterile roll of paper to cool naturally. A
sheet of A4 copy paper is laid on the experimental bench to temporarily place the japonica rice seedlings removed from the jar, and another sheet is laid to temporarily place the fifth instar larvae picked up from the japonica rice seedlings. The mid-fifth instar larvae on the copy paper are directly transferred to jars containing fresh japonica rice seedlings, and the escaped larvae are picked up using forceps and placed into the jars. Each jar contains 600-1,100 mid-fifth instar larvae.
After the transfer is completed, the jars are sealed with a black cloth and tied with a rubber band.
Next, six pieces of paper (13.9 cm long x 10.2 cm wide) cut from sterile rolls are used, folded into fans, and placed on top of the japonica rice seedlings. The jars are sealed with a black cloth, tied with a rubber band, and placed in the RGC-1000C artificial climate chamber (Hefei Youke Instruments &
Equipment Co., Ltd.) 28 C under 80% relative humidity and a 16/8 h light/dark cycle. The larvae reared in this embodiment are placed at a constant temperature, humidity, and illumination environment. The larvae enter the pupation stage at the end of the fifth instar.
Embodiment 3. Pupation and pupa collection of Chilo suppressalis (1) C. suppressalis pupation (Figure 3) The fifth instar larvae of C. suppressalis enter the pupation stage after 3 days. The larvae crawl toward the gaps of folded paper to pupate and hardly feed at the end of the fifth instar stage. Thus, we only need to wait for the larvae to pupate at this stage. In glass jars (18 cm in diameter and 9 cm in height) prepared for placing the fan-folded paper, 2 L of tap water is added using a measuring cup, to which 20 mL of sodium hypochlorite solution is added according to a Date Recue/Date Received 2022-08-29 1:100 ratio of sodium hypochlorite solution to water. After 24 h of immersion, the jars are rinsed well under tap water until the strong odor of sodium hypochlorite vanishes. A
few drops of cold water degreasing detergent are added to the glass jars, they are washed under tap water, and foam is rinsed off. The glass jars are placed in the GZX-9140 ME digital display blast dryer and baked at high temperature (200 C) for 6 h. After baking, the jars are cooled naturally. Then, the glass jars are sterilized under UV light in an artificial climate chamber for 30 min, followed by wind blowing for 10 min. The glass culture plates (dimeter = 90 mm) are placed in a plastic basin. Tap water is poured, and a few drops of cold water degreasing detergent are added.
Tap water is stirred thoroughly, and the culture dishes are cleaned using a brush and allowed to dry after thorough cleaning. Thereafter, 10 culture plates per stack are wrapped in newspaper and placed in a D-1 automatic steam sterilizer (Beijing Fa'en Technology Trade Co., Ltd.) for sterilization at 120 C for 20 min. After sterilization, the plates are placed in the WGL-125B
electric blast oven (Tianjin Taiste Instrument Co., Ltd.) and dried at 50 C. After drying is completed, the dishes are removed. This procedure allows the reuse of culture plates, which reduces the cost.
(2) C. suppressalis pupa collection (Figure 3) Forceps are taken from 75% alcohol, held over the flame of an alcohol lamp to sterilize, and placed on a sterile roll of paper to cool naturally. After 2 days, folded papers containing pupae are removed with forceps and placed in sterilized glass jars. Six pieces of paper (13.9 cm long x 10.2 cm wide) cut from sterile rolls are used, folded into fans, and placed on top of the japonica rice seedlings. The jars are sealed with a black cloth and tied with a rubber band to allow the larvae to continue pupating. The jars are placed in the RGC-1000C
artificial climate chamber (Hefei Youke Instruments & Equipment Co., Ltd.) at 28 C under 80%
relative humidity and a 16/8 h light/dark cycle. After 2 days, a sheet of A4 copy paper is laid on the experimental bench, and the folded papers are clamped to the A4 photocopy papers with forceps. The folded fan is pulled apart using 2 forceps, and the pupae in the folded paper are transferred into 90 mm glass culture plates. The bottom of the culture plate is lined with sterile paper moistened with double-distilled water to maintain humidity for developing pupae.
Embodiment 4. Chilo suppressalis mating and egg laying (1) Preparation of insect cages Date Recue/Date Received 2022-08-29 The insect cages (45 cm x 45 cm x 55 cm) prepared for mating and egg-laying of C.
suppressalis adults are first rinsed with tap water. Then, 5 L of tap water is added into a plastic basin using a measuring cup. According to the sodium hypochlorite solution to water ratio of 1:100, the required volume of sodium hypochlorite solution is 50 mL, which is poured into the plastic basin and mixed with tap water to prepare the disinfection solution.
The cages prepared for mating and egg-laying are scrubbed and disinfected using an ordinary brush dipped in this disinfectant solution. Thereafter, the cages are rinsed under running water until the strong odor of sodium hypochlorite vanishes. The cages are dried at room temperature.
(2) Preparation of japonica rice seedlings for egg-laying The rice shoots required for spawning are prepared according to the operation steps in Embodiment 1. Briefly, sterilized rice shoots are sown at 50 g in plastic pots (17.5 cm in diameter and 15.5 cm in height). Seeds sprout are spread on the soil surface of the pots and covered with a thin layer of soil. The pots are placed in an artificial air-conditioned climate chamber with temperature controlled at 28 C under a 16/8 h light/dark cycle (controlled with a timer) and 80% relative humidity. Black plastic bags are used to cover the top of the plastic pots for insulation. After 7 days, rice shoots emerge from the plastic pots, which are used for spawning. The seedlings are 10 cm tall.
(3) Adult mating and egg laying (Figure 4) The collected pupae are transferred to 90 mm glass culture plates. The bottom of the plate is lined with sterile paper moistened with double-distilled water. Fifty pupae are placed in each culture plate for mating and egg laying. First, disinfected insect cages are placed in the RGC-1000C artificial climate chamber (Hefei Youlce Instruments & Equipment Co., Ltd.) at 28 C
under 80% relative humidity and a 16/8 h light/dark cycle. An absorbent sponge of suitable size is placed at the bottom of the cage, and the sponge is moistened with 100 mL
of double-distilled water. Two pots of rice seedlings are placed in the cage in a diagonal arrangement for egg-laying, and then two culture plates with pupae are placed in the cage.
Using a 1,000 1.1L pipette, 10 mL of pure honey is transferred into a disposable cup, and 90 mL of double-distilled water is added using a measuring cylinder. The solution is mixed thoroughly with an iron medicine spoon to prepare 10% honey water. Next, 20 mL
of 10% honey Date Recue/Date Received 2022-08-29 water is poured into a 60 mm culture plate. A sterilized cotton ball dipped into honey water is provided as a feeding carrier for adults, preventing them from falling into the honey water and dying. A culture plate with honey water is placed in the cage to supplement the emerged adults and promote egg laying. Honey water in the culture plate is changed once every 2 days to ensure freshness and prevent mold and decay. The pupae are placed in an artificial climate chamber at 28 C. The pupae emerged after 4 days, and the emerged adults mate and lay eggs in the insect-rearing cages. The seedlings used for spawning are 20 cm tall.
Embodiment 5: Collection and preservation of Chilo suppressalis egg masses After 7 days of emergence, pots of japonica rice seedlings are removed from the artificial climate chamber and adult borers still attached to the rice seedlings, which are less active at this stage, are removed. The emerged adults lay eggs on the surface of rice leaves, and the eggs coalesce to form egg masses. Scissors soaked in 75% alcohol are removed and wiped with a sterile paper for disinfection. First, a sheet of A4 copy paper is laid on the experimental bench.
Rice seedlings with egg masses are cut from the roots using sterilized scissors and temporarily placed on an A4 copy paper. The bottom of a 90 mm culture plate is lined with a piece of sterile paper moistened with double-distilled water. Rice leaves containing egg masses are cut with sterilized scissors and placed in 90 mm culture plates lined with a moist paper at the bottom.
From two pots of egg-laying seedlings, 100 japonica rice leaves containing 100-150 egg masses can be obtained.
Using a 5 mL pipette, 35 mL of formaldehyde is added to a disposable cup.
Next, 65 mL of double-distilled water is added using a measuring cylinder to prepare 35%
formaldehyde solution.
Culture plates with egg masses are placed in a glass drying dish with formaldehyde solution at the bottom for 15 min of fumigation to remove parasitic bacteria on the surface of egg masses and removed immediately afterward (Figure 5).
Thereafter, the egg masses are placed in an artificial climate chamber at 28 C
and used for the next cycle of incubation and rearing for 0 to 2 days. Alternatively, the egg masses are placed in a refrigerator at a low temperature (4 C) to ensure consistent developmental progress of the eggs and control their hatching time.
In addition to the ones mentioned above, the invention may cover other embodiments. Any Date Recue/Date Received 2022-08-29 technical solution obtained through equivalent substitution or transformation falls within the scope of protection claimed by the present invention.
Date Recue/Date Received 2022-08-29

Claims (10)

Claims:

1. A method for precise indoor mass rearing of rice striped stem borer, Chilo suppressalis (Walker) using rice seedlings, said method comprising the following steps:
step (1): clean and select rice seeds, and sterilize the rice seeds, wherein sterilization involves mixing sodium hypochlorite solution with tap water at a volume ratio of 10.2:100, soaking the rice seeds in this solution for 24 2 h, washing the rice seeds with tap water until sodium hypochlorite is removed, and washing the rice seeds with double-distilled water after soaking the rice seeds in tap water for 24-48 h, said sodium hypochlorite solution contains active chlorine at a mass ratio of 91%, step (2): germinate the rice seeds obtained in step (1) to obtain rice sprouts, grow the rice sprouts to obtain rice seedlings; put the rice seedlings in sterilized jars, wherein on average 505 g of rice sprouts per jar is used, and when the rice seedlings have grown to over 3 cm but below 4 cm, the jars are used in step (3), step (3): place sterilized C. suppressalis egg masses on the rice seedlings in the jars obtained in step (2), on average five to seven egg masses are placed in each jar, sterilization treatment of the C. suppressalis egg masses involves fumigation with formaldehyde solution, on average each egg mass contains 200-300 eggs, and on average the number of eggs in each jar is 1,000-21,000, seal each of the jars with a disinfected cloth tightly, and incubate the jars in an artificial climate chamber at 28°C1°C under 70-80% relative humidity, add double-distilled water to the jars along the walls once every 2 days to moisten roots of the rice seedlings, the egg masses hatch into first instar larvae in 2-4 days, from the first instar larvae to second instar larvae requires 6-7 days, when the larvae reach mid- to late second instar stage, transfer the larvae to the rice seedlings in new jars obtained in step (2), with an average of 900-2,000 mid-second instar larvae per, jar, development from the second instar larvae to third instar larvae requires 3-4 days, when the larvae reach an end of the third instar stage, transfer the larvae to the rice seedlings in new jars obtained in step (2), with an average of 800-1,900 third instar larvae per, jar, development from the third instar larvae to fourth instar larvae requires 3-4 days, when the larvae reach the fourth instar stage, transfer the larvae to the rice seedlings in new jars obtained in step (2), with an average of 700-1,800 fourth instar larvae per, jar, development from the fourth instar larvae to fifth instar larvae requires 2-3 days, when the larvae reach mid-fifth instar stage, transfer the larvae to the rice seedlings in new jars obtained in step (2), with an average of 600-1,700 fifth instar larvae per, jar, rearing is continued, and late fifth instar larvae enter pupation stage after 4 days, step (4): cut sterile rolls of papers and fold into fans, when the larvae axe at an end of the fifth instar stage start metamorphosing to form pupae, place the fans on the rice seedlings so that the larvae can move into gaps or folds of the fans to pupate, seal and tie each jar with a sterilized cloth, after 2 days, remove the fans using a first sterilized instrument, place sterile paper sheets at bottom of a first set of culture plates, collect the pupae from the fans using the first sterilized instrument and place the pupae on the sterile paper sheets in the first set of culture plates, and moisten the sterile paper sheets with double-distilled water to maintain humidity for developing the pupae, step (5): place sterile paper moistened with double-distilled water at bottom a second set of culture plates, transfer the pupae collected in step (4) to each of the second set of culture Date Recue/Date Received 2024-02-15 plates, on average, 50 5 pupae are placed in each of the second set of culture plates for emergence and mating, line bottom of a set of insect cages with sponges moistened with double-di stilled water, place two pots of rice seedlings for egg laying in each of the set of insect cages, and introduce two of the second set of culture plates containing the pupae, place the insect cages in an artificial climate chamber at 28 C 1 C under 70-80% relative humidity, after 3-4 days, adult insects emerge from the pupae and mate in the insect cages, after mating, the adult insects lay eggs on the rice seedlings in the jars, step (6): remove the rice seedlings from the insect cages after 6-7 days of adult emergence using a second sterilized instrument, cut leaves of the rice seedlings containing egg masses and place in a third set of culture plates lined with moist filter paper at bottom, on average 100-150 egg masses per cage are obtained, fumigate the third set of culture plates with formaldehyde solution to sterilize the egg masses, the egg masses resulting are used for a next cycle of breeding.

2. The method according to claim 1, wherein the jar containing the larvae in step (3), the culture plates containing the pupae in step (4), or the egg mass obtained in step (6) are placed in an environment of 4 C 2 C for a preset duration to control developmental progress.

3. The method according to claim 1 or claim 2, wherein in step (1), cleaning and selection of the rice seeds is as follows:
(a) place the rice seeds into a basin filled with tap water, (b) let the rice seeds stand for several minutes such that rice husks or blighted seeds float to water surface, while saturated rice seeds sink to bottom of the basin, (c) discard the rice husks or the blighted seeds together with the tap water, leaving only the saturated rice at the bottom of the basin, repeat (a) to (c) until the saturated rice seeds sunk to the bottom of the basin are washed, Date Recue/Date Received 2024-02-15 and the water becomes clear, the average weight of the rice seeds added to each basin is >1,000 g, the average weight of the saturated rice seeds per basin is 93 3% of the weight of added seeds.

4. The method according to claim 3, wherein in step (2) germination of the rice seeds is as follows:
from the rice seeds obtained in step (1), drain water before placing the rice seeds into the basin, seal the basin with a clean plastic bag, and tighten the plastic bag, place the basin in an artificial climate chamber at 28 C 1 C under 70-80% relative humidity to accelerate seed germinati on, the rice seeds are turned over once every 24 2 h to ensure that temperature of the rice seeds is uniform and consistent, at 48 2 h, the rice seeds germinate to produce the rice sprouts.

5. The method according to claim 4, wherein in step (2) seed germination to obtain rice seedlings is as follows:
wash the rice sprouts with double-distilled water, and discard the double-distilled water, line a bottom of clean jars with sterile paper sheets as substrate for seed germination, moisten the sterile paper sheets using double-distilled water, and sow the rice sprouts in the jars, seal each of the clean jars with a cling film, and punch holes in the cling film with a needle to ensure oxygen flow, place the clean jars in an artificial climate chamber at 28 C 1 C under 70-80% relative humidity to accelerate the emergence of rice shoots, rinse the rice seeds with 100 10 mL using double-distilled water every 48 2 h, and discard the double-distilled water to maintain moisture required for growth of the rice seedling.

6. The method according to claim 5, wherein in step (3) larval rearing is as follows:
forceps sterilized with medical alcohol, flamed with an alcohol lamp, and cooled naturally are used, transfer the larvae from their current jar to a new jar, rice seedlings in the current jar are Date Recue/Date Received 2024-02-15 first picked up using the forceps and placed on a first sheet of clean paper, the larvae on the rice seedlings are picked up and placed on a second sheet of clean paper, the larvae on the second sheet of clean paper are directly transferred to the rice seedlings in the new jar, if some larvae escape from the second sheet of clean paper, the escaped larvae are picked up with the forceps and placed on the rice seedlings in the new jars, each of the new jars are then sealed with a sterilized cloth and tied tightly, and incubated in an artificial climate chamber at 28 C 1 C under 70-80% relative humidity, in step (3) the larvae are maintained in the artificial climate chamber under the same environment throughout a period of rearing.

7. The method according to claim 5, wherein in step (4) pupation is as follows:
place fan-folded papers into each sterilized glass jar to allow the pupation, remove the fan-papers following the pupation, wherein the first sterilized instrument is medical forceps sterilized with medical alcohol, wherein in step (5), each of the second set of culture plates used is 90 mm in diameter, 100 10 mL of double-distilled water is used to moisten the sponge.

8. The method according to claim 5, wherein in step (5) cultivating rice seedlings for egg laying is as follows:
The rice sprouts obtained by germination in step (2) are sown on soil surface in preset pots and covered with a thin layer of soil, each of the pots are covered with a clean plastic bag and placed in an artificial climate chamber at 28 C 1 C under 70-80% relative humidity until the rice seedlings reach 10 cm in height when the rice seedlings can be introduced for egg laying, wherein in step (5), placed in the insect cage, a fourth set of culture plates each having a diameter of 60 mm and containing sterilized cotton balls dipped in 10 1% honey water by volume, the cotton balls are replaced every 2 days.

9. The method according to claim 5, wherein in step (6) fumigation is as follows:
fumigate the third set of culture plates containing the egg masses in a drying cylinder with formaldehyde solution at bottom of the drying cylinder for 15 1 min, the second sterilized instrument is sterilized scissors, Date Recue/Date Received 2024-02-15 the third set of culture plates each having a diameter of 90 mm, concentration of formaldehyde solution is 35 1% by volume.

10.
The method according to claim 5, wherein the rice seeds are of the japonica type, the clean jars each having a diameter of 6 cm and height of 10 cm, the insect cages each with a size of 45 x 45 x 55 cm3, the number of jars used in step (3) is >20.

Date Recue/Date Received 2024-02-15